Recent Advances in Chemical Sciences and

Recent Advances in Chemical Sciences and Biotechnology

Recent Advances in Chemical Sciences and Biotechnology

Edited By Dr. Ashok Kumar Jha (Associate Professor) University Department of Chemistry T.M. Bhagalpur University, Bhagalpur-812007 Ujjwal Kumar (Ph.D Scholar) Post Graduate Department of Biotechnology T.M. Bhagalpur University, Bhagalpur-812007

NEW DELHI PUBLISHERS New Delhi: Kolkata

Recent advances in Chemical Sciences and Biotechnology edited by Dr. Ashok Kumar Jha and Ujjwal Kumar published by New Delhi Publishers, New Delhi.

© Publishers

First Edition 2019

ISBN: 978-93-85503-63-4

All rights reserved. No part of this book may be reproduced stored in a retrieval system or transmitted, by any means, electronic mechanical, photocopying, recording, or otherwise without written permission from the publisher and authors.

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Foreword

Chemistry is the science of materials which finds application in human life. Major trust of chemical science is restriction of contamination in a specific domain and effective methods for welfare of mankind. Needless to say that chemical sciences have played a pivotal role in all round development of society. Impressive progress in chemical sciences and biotechnology has provided the opportunities to devise better ways and means of agriculture production, food security and nanodrugs. With an aim to achieve the goals of environmental protection and sustainable development, principles of green chemistry ought to be adopted by industries. The study of biological molecules has become easier with the advances in chemical science. I congratulates the editors to being out this volume by compiling original research articles from various institutions and universities. Each paper has been written in scientific manner having latest references. I am sure that this book will help researchers to great extent all other the world. Dr. D. C. Mukharjee Professor (Retd.) President Indian Chemical Society, Kolkata

Preface Chemical Sciences have been playing an important role in the developed and developing world namely in field of energy, water, agriculture and health. These areas need new materials with specific properties which chemical sciences satisfy. Chemical Science forms a base for nano technology providing polymers, clusters and nanoparticles. Although the application of nanotechnology is gaining popularity, environmentalists have warned about the health hazard of nanoparticls. Significant advances have been made in harnessing energy from biomass and solid waste. The waste after gasification may be used as useful purposes. Use of biodiesel is an energy saver and pollution reducer too. This is being produced from jatropha which grows in wasteland needing little water. The sun provides enormous energy to the earth which has been converted to thermal energy. Some success has been reported in artificial photo synthesis e.g. photo oxidation of water using nano crystals to produce oxygen, protons and electrons. They have also succeeded to reduce CO2 to CO which could be changed to CH3OH using technology. To mitigate CO2 crystalline sponge has been used. A new class of materials called Metal organic frame works have been utilized for carbon capture. During recent years many methods of removal of heavy metals and other pollutants have been developed. Bioremediation of arsenic is a low cost alternative to other traditional methods. Biotechnology has played an important role in identifying arsenic mitigating bacteria by identifying its gene sequences. In addition to this, DNA based sensors have been designed to detect and quantify pollutants. Chemical sciences have been useful in agriculture and horticulture e.g. extraction of flavors, herbal medicines and cholesterol-free foods. Simpler analytical techniques for soil analysis e.g. detection of arsenic and heavy metals in soil have been developed. Sustainable development without causing any damage to the eco-system can be achieved by the principles of green chemistry. Chemists around the world have laid stress on green chemistry and advised industries to enhance R and D fund on green chemistry as a core research area. I would like to extend my sincere thanks to the publishers for publishing the book in a very shot time.

Ashok Kumar Jha

Ujjwal Kumar

Contents 1.

Arsenic contamination in groundwater and soil and its mitigation approach.......................................................................................................11 Ashok Ghosh, Jajati Mandal and Ranjit Kumar

2.

Introduction to biological databases......................................................... 25 Anil Kumar

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Foodomics with perspective on nutritional and health aspects of fast food............................................................................................................... 53 Ruchi Kumari, Mamta Kumari and Ravi S Singh

4.

Phytofabricated nanoparticles using Artocarpus heterophyllus: A multifunctional approach....................................................................... 63 Archita Gupta1 and Devendra K. Singh

5.

Studies on the changes in nitrogenous excretory products of pebrine infected tasar silkworm, antheraea mylitta drury (daba tv).................. 75 R. K. Verma, P. K. Roy and S. P. Roy

6.

Role of association mapping in crop improvement................................. 83 Rahul Singh, Renu, Niranjan Kumar Chaurasia, Rohit kumar, S.P. Singh, Satyendra and Dharamsheela Thakur

7.

Role of plant biotechnology in crop improvement................................... 97 Ravi Kumar, Varsha Kumari, Komal Shekhawat and Swarnlata Kumawat

8.

Role of tissue culture techniques in social forestry.................................113 Komal Shekhawat, P. C. Gupta, Ravi Kumar, Anil Kumar, Rekha Kumari and Swarnlata Kumawat

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Tissue culture: An approach toward effective cost management......... 125 Renu, Rahul Singh and Niranjan Kumar Chaurasia

10. Prospect of molecular markers in precision plant breeding................. 131 Anjani Kumar, Swapnil, Shahina Perween, R.S. Singh and D.N. Singh 11. A review on hydroponic system: hope and hype.................................... 143 Ankita Jain Nidhi Kumari and Vikash Kumar Jha

12. Single-cell techniques for stress tolerance in breeding plants.............. 151 Niranjan Kumar Chaurasia, Rahul Singh, Renu and Praveen Kumar 13. Marker assisted backcrossing (MAB) an approach for selection by using molecular markers.......................................................................... 161 Shahina Perween, Anjani Kumar, N. Swathi Rekha, Swapnil, Priyanka Kumari, Surya Prakash 14. Characteristics features of diara and tal land of some different blocks of Bhagalpur district Bihar...................................................................... 173 Sumitap Ranjan and Rajkishore Kumar 15. Allelopathy: a natural and an environment-friendly unique alternative tool and their effect on fruit crops........................................................... 179 Kanchan Bhamini and Anjani Kumar 16. Marker assisted selection: a new tools of plant breeding...................... 189 Swapnil, Priyanka Kumari, Jenny Priya Ekka, Anjani Kumar, Shahina Perween, Krishna Prasad and Ekhlaque Ahmad 17. Program cell death in plant: a sustainable tool for crop improvement....... 199 Rekha Kumari, Komal Shekhawat, Ravi S. Singh, Ankita Sinha and Kunwar Satyendra Singh 18. Biotic stresses on wheat: Issues and Management................................ 209 Pankaj Kumar Singh, Girish Chandra Pandey, Varsha Rani and Sunita Singh 19. Genetically modified food: a revolutionary change............................... 215 Priyanka Kumari, Swapnil, Jenny Priya Ekka, Anjani Kumar, Shahina Perween and S.K. Tirkey 20. A Beam on nanoparticles: features and future...................................... 225 Prerna Kumari, Rohit kumar and Vikash Kumar Jha 21. Molecular techniques for improvement of agricultural yield............... 231 Anil Kumar, N. K. Sharma, Komal Shekhawat, Rekha Kumari and Swarnlata Kumawat 22. Potential approach of bacterial biosorption of arsenic and chromium contamination from soil and water resources........................................ 251 Ujjwal Kumar, Ankita Sinha, Ghanshyam K. Satyapal, Sailesh Kumar and Ashok K. Jha

Chapter -

1

Arsenic contamination in groundwater and soil and its mitigation approach Ashok Ghosh1, Jajati Mandal2* and Ranjit Kumar1 Mahavir Cancer Institute and Research Centre, Phulwarisharif, Patna, 801505 2 Department of Soil Science and Agricultural Chemistry, Bihar Agricultural University, Sabour-813210 *Corresoponding author: [email protected]

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Abstract Arsenic (As) is a natural pollutant, toxin and carcinogen. It is generally found in rocks of the earth’s crust in varying concentrations. Its natural leaching from rocks results in dissolution of arsenic in groundwater. The cause of elevated As contamination of groundwater is not well known. Human beings are more vulnerable to arsenic toxicity in groundwater due to their biochemical changes. Now arsenic was also reported in soil and agriculture products like cereals, pulses and vegetables. Arsenic is a non-essential element for human beings. According to the World Health Organization, the safe level of arsenic in blood is less than 1 ppb. The level of toxicity depends on the chemical form of arsenic in the environment. Arsenite and arsenate are the major forms found in groundwater. While arsenite is more toxic than arsenate. In groundwater, arsenic is present in various forms such as H3AsO3, H2AsO3, HAsO3, H3AsO4, H2AsO4 and HAsO4. The arsenic concentration in groundwater varies with geographical locations. Ore of arsenic like H3AsO3, is more prominent in West Bengal (India) and Bangladesh, while HAsO4 and H2AsO4 are more prominent in Arizona (USA) and Korea (Saxena et al, 2004).

Arsenic contamination in groundwater in the ganga delta basin The groundwater arsenic concentration (50–1600 μgl-1), reported from the affected areas of Indo-Gangetic Plain of Uttar Pradesh, Bihar, West Bengal, parts of Assam and also alluvial aquifers of Punjab varying from 4 to 688 μgl-1 (Sanyalet. al., 2015) are several orders of magnitude higher than the stipulated Indian standard for the permissible limit in drinking water (50 μgl-1, which is also the maximum acceptable concentration (MAC) for drinking water in Bangladesh, India and several other countries), as well as the WHO guideline value (10 μgl-1). The effect of ingestion of inorganic arsenic in drinking water and the health effects in adults has also been well established (GuhaMazumdaret. al., 1998).The main focus of attention, until recently, has been exclusively on arsenic contamination in groundwater-derived drinking water. However, since groundwater is also used extensively (more than 90%) for crop irrigation in the arsenic belt of Ganga delta basin the possibility of a build-up of arsenic concentration in agricultural soils and agronomic produce was anticipated. Indeed,

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Recent advances in Chemical Sciences and Biotechnology

arsenic uptake by crop plants grown in soils contaminated with high concentration of arsenic, and irrigated with such arsenic contaminated groundwater has been reported by several workers(Sanyalet. al., 2015). Such findings call for an immediate attention since what remains essentially a point and fixed source of arsenic contamination as for drinking water (e.g., a tube well discharging contaminated water), may well become a diffuse and uncertain source of contamination when arsenic finds its way into the food web, accompanied with possible bio-magnification up in the food chain. This assumes added significance in view of the reported finding of higher (than permissible) level of arsenic in the urine samples of some people having no history of consuming arsenic contaminated drinking water (Guha Mazumdar et. al.,2013, 2014). Interestingly, the surface water bodies, located in the affected belt, have remained largely free of arsenic. This tends to suggest that the soil, which receives arsenic-contaminated water, acts as an effective sink to contain the toxin (as stated earlier), thereby preventing the surface run-off to carry it to the adjoining water systems (Sanyal, 2005).

Chemistry of arsenic in soil and groundwater Arsenic in groundwater is generally present as dissolved, deprotonated/protonated oxyanions, namely arsenites (AsIIIO33-; Hn AsIIIO3(3-n)-, with n = 1,2) or arsenate (AsVO43, HnAsVO4(3-n)-, with n = 1,2), or both, besides the organic forms. The toxicity of arsenic compounds in groundwater/soil environment depends largely on its oxidation state, and hence on redox status and pH, as well as whether arsenic is present in organic combinations. The toxicity follows the order : arsine [AsH3; valence state of arsenic (As): -3]> organo-arsine compounds >arsenites (As3+form) and oxides (As3+ form)> arsenates (As5+ form) >arsonium metals (+1)> native arsenic metal (0) (Sanyal et. al., 2015).The arsenites are much more soluble, mobile, and toxic than arsenates in aquatic and soil environments. At pH 6-8, in most aquatic systems, both H2AsVO4and HAsVO42- ions (pentavalent arsenic forms) occur in considerable proportions in an oxidized environment ( redox potential, Eh= 0.2-0.5V), while the arsenous acid, H3AsIIIO3, is the predominant species (trivalent arsenic form) under reduced conditions (Eh = 0-0.1V) (Sadiq, 1997). Reduction of As (V) to As (III) would be accompanied by mobilization of arsenic in aquatic system. The arsenites are much more soluble, mobile, and toxic than arsenates in aquatic and soil environments. The organic forms, namely dimethyl arsinic acid (DMA) or cacodylic acid, which on reduction (e.g., inanoxic soil conditions) forms di- and trimethyl arsines, are also present in soil. Another organic form present in groundwater and soil is monomethylarsonic acid (MMA). At pH 6-8, and in an aerobic oxidized environment (redox potential, Eh= 0.2-0.5V), arsenic acid species andarsenate oxyanions, that is, HnAsVO4(3-n)- ions, with n= 1, 2), (pentavalent arsenic forms) occur in considerable proportions in most aquatic systems, whereas under mildly reducing conditions (such as one encountered in flooded paddy soils with Eh= 0 - 0.1V), the arsenous acid, H3AsIIIO3, andarsenite oxyanion species (arsenic in trivalent form) are the predominant species. Furthermore, As (III) is more prevalent in soils of neutral pH range(and in most groundwater), as in the soils of the affected belt of West Bengal, India and Bangladesh, than otherwise thought, and hence is ofconcern. This

Arsenic contamination in groundwater and soil and its mitigation approach

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is primarily because As (III) exists as a neutral, uncharged molecule, namely arsenous acid, H3AsIIIO30 (pKa= 9.2), at the pH of the neutral soils and most natural groundwater as one would expect based on the Henderson’s equation (Sanyalet. al., 2015), and is thus less amenable to retention by the charged mineral surfaces in soils and sediments. It ought to be emphasized that groundwater or soil solution, which is subject to affluxes and influxes, as well as circulation and also to man-made perturbations of groundwater due to its withdrawal, cannot be expected to remain in thermodynamic equilibrium, it being very much of an open system (thermodynamically speaking). Thus, more often than not, the ratio of concentrations of arsenic species, namely the ratio, [(AsIII)/ (AsV)], in field soils does not quite agree with the ones computed from the observed redox potential (Eh) and the application of the Nernst’s equation (at 250C) to the equilibrium redox reaction, namely: AsVO43- + 2H+ + 2e = AsIIIO33- + H2O Eh = Eh0 – 0.0295 log [(AsO33-)]/(AsO43-)] – 0.059 pH Where the (Eh) terms refer to the equilibrium concentrations of the respective ionic species in dilute soil solution, and Eh0 is the standard redox (reduction)potential of the AsVO43- / AsIIIO33- redox couple at 250C. It is evident from the above equation that the proportion of AsIII, and hence soluble arsenic level in soil, should increase substantially with diminishing Eh and increasing pH. Furthermore, at a high pH, the OH- ion concentration would increase, causing displacement of AsIII and AsV species from their binding sites through competitive ligand exchange reactions. The dependence of arsenic sorption on pH of the sorption medium is governed largely by the nature of the soil colloidal fraction. A fall of arsenate adsorption was noted within creasing pH, but only at lower arsenate concentrations, which got reversed at a higher arsenate equilibrium concentration. This trend was explained in terms of the varying electrostatic potential of the variable-charge soil colloidal surfaces with pH, solubility product principles, and buffering action of the arsenic salt used (Majumder and Sanyal, 2003).

Need of mitigation method A wide range of arsenic related human illnesses have been reported across various parts of the world. Several methods for removing arsenic from groundwater are available but they are more amenable to industrial usage. There is no easily usable solution for arsenic removal for individual end consumers. The incidence of arsenic related health issues is steadily rising, particularly in the Ganges-Brahmaputra region of northern India. Alternative for free water sources Deep groundwater It was observed that arsenic rich water occurs mainly in the shallow groundwater, whereas groundwater from deeper aquifers is almost completely free from arsenic. The British Geological survey revealed that only 5% of the deep tube well below

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150 meters had arsenic concentrations above 10 ppb and 1% exceeded the 50 ppb (BGS, 2004); thus, water supply relying on manually operated deep tube wells could be an appropriate source. However, the depth to arsenic free aquifers differs between the locations. The major restriction to the deep water extraction option is its costly installation. This leads to its applicability only on community basis. Few drawbacks to this option include availability of the arsenic free deep aquifer, the uncertainty of the groundwater recharge mechanism (Hoque, 2012). Shallow groundwater (Well Switching) The arsenic contamination widely varies in the shallow groundwater (Smedley, 2002). The study by British Geological Survey in Bangladesh (BGS, 2004) and Chakraborti et. al. (2004) revealed that in Ganga-Meghna-Brahmaputra plain, the proportion of arsenic contamination in tube wells is in the range started from 20% to greater than 50%. It is often possible to get uncontaminated tube wells in many areas within reasonable distances and well switching to an uncontaminated shallow tube well will be a suitable option. Among the various tried mitigation strategies, well switching to shallow tube wells has been found as most preferred strategy (29%) (Ahmed et al, 2006). The major drawback to well switching option is the degree of the spatial and temporal variation in arsenic level in groundwater. Which makes it difficult and unpredictable for its reliably. Many studies reveal that the As concentration in the tube wells changes over time, and it is high during the monsoon period as compared to dry winter season (Rahman and Ishiga, 2003; Rahman et al, 2003). In this way the monitoring of each and every well may be required and, long term analysis is needed to guarantee that the tube wells will remain arsenic free. Dug well water The open wells are generally called dug wells with large diameters; arsenic free safe drinking water can be obtained from arsenic contaminated shallow aquifers. Dug wells used were one of the alternative sources of water supply in the Bengal delta, before the installation of tube wells (Ahmed and Rahman, 2003). Many studies suggests that the arsenic level in most of the dug wells was very low (Warner et al, 2008; Benett et al, 2010) due to prevailing oxidative environment and precipitation of Fe or due to groundwater recharge of the dug wells with rainwater (Hira-Smith,2007). Dug wells have been suggested as the preferable alternatives of safe drinking water by The National Policy for arsenic Mitigation in areas marked with high concentration of As in Bangladesh (DPHE, 2004). The evaluation of dug well performance in early stages of implementation establishes that these options are appropriate (Joya, 2006). Prolonged studies report that tube wells will be the preferred choice over dug wells (Milton et al, 2007). The reasons for the unpopularity of the dug wells are obnoxious smell, taste, turbidity, and distance and time bound limitations to fetch water (Hoque et al, 2004). Bacteriological contamination is the principal problem associated with the use of dug wells water. The use of drinking water from these sources without appropriate treatment may lead to diseases like diarrhoea, cholera, typhoid, dysentery, and hepatitis. The frequency of microbial contamination of dug wells with thermo


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tolerant coliforms has been found as high as 94% with seasonal variation with higher contamination during the monsoon compared to the dry season (Ahmed, 2005). Surface Water Ponds, lakes and rivers are generally low or free from arsenic and can be introduced in affected areas as a source of safe drinking water. Most of the arsenic affected areas are in the vicinity of a large river and these rivers can serve as sustainable mitigation option for long run, over decades. The major limitation of using ponds, lakes and river is the risk of potential bacteriological contamination. This was also the main reason behind non replacement of surface water with groundwater as the drinking water source. ReIntroduction of surface water as a source of safe drinking water would require antimicrobial treatment like incorporation of disinfectants and use of pond sand filters (Yokota et al, 2001), or combined surface water treatment units. The use of pond sand filters is preferred by The National Policy for arsenic Mitigation for its application in arsenic-affected regions in Bangladesh (DPHE, 2004). About 95% pond sand filters have been found microbially contaminated with elevated levels of thermo tolerant coliforms in the monsoon season as compared to the dry season (Ahmed et al, 2005). Rainwater harvesting The rainwater harvesting has been widely used practice throughout the world since ancient times as a potential method of utilizing rainwater for domestic water use (WHO, 2011). Rainwater harvesting is widely used method at household level globally. It was widely accepted method at larger community level. The rainwater is safe if it is hygienically maintained and this technology is feasible in areas with average rainfall is above 1600 mm/year (DPHE, 2008). In coastal areas, rainwater is the main source of drinking water because presence of high salinity in shallow and deep tube wells there. Rainwater is preserved in large ponds in these areas (Islam et al, 2011) and the experience from these areas can be transferred to other arsenic affected regions. One of the critical limitations of grass root implementation of rain water harvesting technology is its high installation cost in building special roofs and large storage tanks for storage and collection of rain water due to the unequal rainwater precipitation throughout year. Microbial contamination is also another limitation (Karim et al, 2010).

Removal of arsenic Removal of arsenic mainly depends on the composition and chemistry of the arsenic contaminated water. Arsenic occurs as AsIII in most of the major reported cases and oxidation of AsIII to AsV is considered as necessary to obtain satisfactory arsenic removals. Oxidation The oxidation converts soluble AsIII to AsV, which is followed by precipitation of AsV. This is essential for anoxic groundwater, since AsIII is the prevailing format near neutral pH (Masscheleyn et al, 1991). AsV adsorbs more easily onto solid surfaces than AsIII. Thus, oxidation followed by adsorption is deemed to be effective for the removal of arsenic (Leupin and Hug, 2005). Several oxidants have been utilized for

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the oxidation. The reaction kinetics of O3, H2O2, Cl2, NH2Cl, and ferrate are of first order reactions with reference to both AsIII and its oxidants. The concentrations of AsIII and the oxidant are the critical parameters for effective removal of arsenic from aqueous solution. The reaction is very fast for permanganate, chlorine, and ozone as compared to hydrogen peroxide and chloramine when applied for oxidation of AsIII to AsV (Dodd et al, 2006). Bajpai and Chaudhuri 1999 have reported that 54–57% of AsIII can be oxidized to AsV in contaminated groundwater using air and pure oxygen whereas complete oxidation of AsIII can be obtained with ozone. Coagulation-Flocculation The incorporation of a coagulant followed by the formation of a floc is a potential method used for arsenic removal from groundwater. Positively charged cationic coagulants decrease the negative charge of the colloids and, larger particles are formed due to aggregation of particles (Choong et al, 2007). The flocs formed in flocculation process are due to polymeric bridging between the flocculent particles. Which later agglomerate to form larger mass particle. Soluble As is precipitated onto the flocs and thus eliminated from aqueous solution. Arsenic removal requires, Fe and Al based coagulants (McNiell and Edward, 1995). However, the critical limitation of the coagulation process is the production of a huge amount of sludge with a considerable arsenic concentration. The management of this contaminated sludge is important for safeguarding the environment from secondary pollution which reduces the applicability of this method in field conditions. Adsorption Arsenic removal by adsorption onto activated or coated surfaces is popular because of its simple operation system and sludge free day to day operation. Different types of adsorbents can be regenerated and reused which are the key features of this technology. (Mohan and Pittman,2007). The removal of arsenic by adsorption techniques in general depends on pH and the speciation of arsenic with better AsV removals as compared to AsIII at pH lower than 7 (Kanematsu et al, 2013). Ferrihydrite, granular ferric hydroxide, and hydrous ferric oxide are the most widely explored iron oxides and hydroxides for the removal of arsenic yielding promising results for both AsIII and AsV removals (Guan et al, 2008). One of the major problems encountered with aforesaid adsorption methods is the presence of high iron content in groundwater, which emanates into clogging of the filter material and reducing the lifetime of the filter (Bamwsp wt al, 2001). Use of zero valent iron (ZVI) or Fe(0) for removal of As has been widely explored by several research groups in last decade both in the laboratory (Klas et al, 2013) and in the field (Khan et al, 2000). According to Hussam and Munir (2007), approximately 350,000 ZVI filters are operational in Bangladesh, Nepal, Pakistan, India, and Egypt and there are several studies showing promising results of arsenic removals in field (Neumann et al, 2013). Proper maintenance of these filters are required; otherwise, they are clogged and not reliable in removing arsenic.


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Latest advancements on arsenic removal by adsorption A wide spectrum of different materials have been explored for adsorption of arsenic from groundwater but iron oxides and oxy hydroxides are the most widely studied and their commercial products already dominate a major portion of the market for arsenic removal system (Mohan and Pittman, 2007). In water treatment plants, iron oxy hydroxides are used as mechanically resistant particles in fixed bed pressure columns. Use of iron oxy hydroxides is encouraged due to their cheap and easy production. The amorphous structure of these hydroxides provides high specific surface area values and their strong affinity and relative high selectivity for binding with the most frequently occurring arsenate species at natural pH-values of groundwater. Tresintsi et. al., 2012 synthesized various iron oxyhydroxides between the pH range 3–12 using the most common low cost iron salts (FeSO4 • H2O and FeCl2 • H2O) in a continuous flow kilogram-scale production reactor in intense oxidative conditions which serve as arsenic adsorbents. Synthesized iron oxyhydroxides at acidic condition (pH 4.0) and highly oxidizing one resulted in a very effective arsenic adsorbent.

Biological arsenic removal through microbes Bacteria play crucial role in geochemical cycling of arsenic by oxidation and reduction reactions as well as determining its speciation and mobility (Smedley and Kinniburgh, 2002). Arsenic pentavalent (AsV) reduction and arsenic trivalent (AsIII) oxidation are both detoxification mechanisms of microbes (Silver and Phung, 2005). Bacteria coupling anaerobic oxidation of organic substrates to the reduction of arsenates have been reported. Such bacteria are known as dissimilatory arsenate reducing bacteria or arsenate respiring bacteria (ARD),e.g : Geospirillum arsenophilus, Geospirillum barnesi, Desulfutomaculum auripigmentum, Bacillus arsenicoselenatis, and Crysiogenes arsenatis (Oremland et al, 2005). This bacterium uses AsV as a electron acceptor in their respiratory process. The oxidation of AsIII is carried out by the incorporation of chemical reagents such as ozone, chlorine, hydrogen peroxide, or potassium permanganate (Jackel, 1994). The use of chemical reagents in drinking water treatment is discouraged as it often leads to the formation of undesirable by products such as trihalomethanes (THMs) (Katsoyiannis et al, 2004). The biological oxidation of iron by two bacteria, Gallionella ferruginea and Leptothrix ochracea, has been found to be a very promising technology for effective removal of arsenic from groundwater. This process requires coating of iron oxide on filter medium, along with the microorganisms, which offer an ideal environment for arsenic to be adsorbed and removed from the water. Under optimum conditions, trivalent arsenic has been found to be oxidized by these bacteria, contributing to almost complete arsenic removal (up to 95%) even when initial arsenic concentrations were 200mg/L (Katsoyiannis and Zouboulis, 2004). The pentavalent arsenic content, under these experimental conditions, can be removed, leading to residual concentrations below the newly enforced limit of 10mg/L. This technology was efficient in removal of arsenic from groundwater and it offers several advantages as compared to conventional physicochemical treatment processes. This technology avoids the incorporation of

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chemical reagents for the oxidation of trivalent arsenic; therefore, it is a cost effective and eco-friendly method. In addition, it does not need monitoring of a breakthrough point, as in various sorption processes, because the sorbents (iron oxides) are consistently produced in situ.

Organic amendment and arsenic mitigation in soil Arsenic fractions in soil Arsenic is present in soils or sediments in various forms with varying degree of bioavailability, toxicity and mobility. In order to assess arsenic toxicity and impact, a good understanding of the chemical forms of the element is required (Shiowatanaet. al., 2001). The use of sequential extraction technique for fractionation of metals in solid materials and evaluation of their potential effects has been widely used and well recognized (Tessier et. al., 1979). The As fractionation studies, in general, revealed that the As affected soils were endowed with internally held arsenic followed by Fe and Al chemisorbed arsenic, Ca associated arsenic and freely exchangeable arsenic (McLaren et. al., 1998; Shiowatanaet. al., 2001). The fractions of arsenic varied mainly due to the mineralogical make-up of the soils, surface area, pH, total and Olsen extractable As, amorphous iron and, to a smaller extent, calcium and magnesium content of these soils (McLaren et. al., 1998). Arsenic species in soils The topic of elemental speciation is now a well-established area of research. Studying the chemical forms of elements helps elucidate themobility, biological availability distribution, and toxicity of the chemical element. The greatest arsenic(As) toxicity is attributed to inorganic arsenic (i-As), a non-threshold class-1human carcinogen (IARC, 2004). In arsenic-contaminated soils, simultaneous reactions of arsenic, such as adsorption/desorption, precipitation, and oxidation/reduction, might occur on mineral surfaces, which influences the arsenic solid-state speciation in the soil environment. The predominant inorganic As species in the oxic layer of soils is arsenate which has a similar chemical behaviour to phosphate with limited presence of arsenite . As(V) is the predominant form that exists in soils, in which the pH + pe> 10; in contrast, As(III) is the dominant form found in soils, in which the pH + pe is less than 6 (Sadiq, 1997). Under aerobic conditions, sulfides are easily oxidized, and as a consequence arsenic is released into the environment when soil pH is between 3 and 13, the major species found are H2AsO4- andHAsO42− . In reducing environments, arsenic is found as arsenite, the predominant species of which is H3AsO3.Arsenite is more mobile and more toxic than is arsenate. Highly reducing conditions can cause As co-precipitation with iron-sulfurs, such as arsenopyrite, or the formation of arsenic sulfides (AsS, As2 S3). During the oxidation of pyrite, Fe is oxidized from valence II to III, and arsenic isoxidized to arsenate. In contrast, under reducing conditions, Fe and Mnoxides are dissolved, releasing arsenate that is rapidly reduced to arsenite (Gräfe and Sparks, 2006).


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Organic amendment and arsenic mitigation A large number of studies explore the mitigation potential of soil amendments such as the application of inorganic fertilizer or organic manure which can immobilize, adsorb, bind or co-precipitate arsenic in situ. The overwhelming majority of studies found that fertilization (irrespective oftype) reduces available arsenic in soils. Das et. al. (2008) and Mukhopadhyay and Sanyal (2002) found that the arsenic content in soil markedly decreased, especially with farmyard manure application. Organic amendments such ascomposts and manures which contain a high amount of humified organic matter can decrease the bioavailability of heavy metals through adsorption and by forming stable complexes with humic substances (Sinha andBhattacharyya, 2012).In soil and organic manures, humic substances often represent a high fraction of dissolved organic matter due to their recalcitrance, and they havefunctional moieties with a variety of properties (Stevenson 1994). The chelation of cations can influence the presence of free metal ions and regulate their availability and mobility in soils and aquatic environments following the formation of metal–humate complexes (chelates) with different degrees of stability (Sinha and Bhattacharya 2011). The stability constant values exhibit a significant and positive correlation with total acidity which establishes the fact that the stability constant of the organo-metallic complexes are dependent on the presence of active functional groups (carboxylic and phenolic) of the HA and FA fractions of the organic amendments. The findings of Khalili (1990) and Stevenson (1991) supports the above fact of positive correlation between stability constant and total acidity. The stability constant values were conferring to the findings of (Schnitzer and Skinner 1966; Matsuda and Ito 1970; Stevenson 1976, 1977). Besides the stability constants have higher values in comparison to that observed in case of organo-metallic complexes with the root exudates consisting of low molecular weight organic acids (Mench et. al. 1988).Ghosh et. al. (2012) observed that HA/FA extracted from compost was found to be the better in scavenging arsenate in its matrices and more specifically Sinha and Bhattacharyya (2011) observed higher stability of As-HA/FA complexes with vermicompost rather than FYM or oil cakes. As per the configuration geometry of the adsorbate at the adsorbent surface there are two types of surface complexes. These include inner sphere due to presence of hydration sphere and outer-sphere which occurs in absence, of the hydration sphere of the adsorbate molecule upon interaction. Presence of at least one water molecule categorizes the surface complex as outersphere complex. Whereas when the ion is bound directly to the adsorbent without the presence of the hydration sphere, an inner-sphere complex is formed. The two-phase exchange of the oxy anions for As might be initially due to exchange from the loosely held sites i.e. from outer sphere complexes followed by the inner sphere complexes that are more strongly bonded. Conclusions Arsenic removal technologies are one of the mitigations options. However, because of costs and operational complexity it is preferable to seek other alternative safe water source. This is especially the case in developing countries. The mitigation options include shift to an alternative water source (that may include treatment techniques as

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the alternative source may not be safe) i.e : Rainwater, Surface-water (that requires some kind of treatment), arsenic safe groundwater and arsenic removal techniques. All treatment options are associated to additional management and continual costs by the consumer or distributor in order to avoid malfunction. Different safe drinking water options, e.g. As-removal filters, tablet reagents and deep tube wells (Argentina>Canada in 2017 (ISAAA, 2017). The most planted biotech crops in 2017 were soybean (94.1 mh)>Maize (59.7 mh)>Cotton (24.1 mh) and Canola (10.2 mh). GMO’s are those organisms that have been modified by the application of recombinant DNA technology or genetic engineering, a technique used for altering a living organism’s genetic material. In biotechnplpgy, a number of genetically modified (GM) crops or transgenic crop carrying novel traits have been developed and released for commercial agriculture production. These include pest resistant cotton, maize, canola (mainly Bt) herbicide glyphosate resistant soybean, cotton and viral disease resistant potatoes and papaya etc. Commercial cultivation of transgenic crops started in the early 1990’s. Herbicide tolerance and insect resistance are the main GM traits that are currently under commercial cultivation and the main crops are soybean, maize, cotton and canola. Herbicide resistance Herbicide tolerance is the inherent ability of a species to survive and reproduce after herbicide treatment. Generally herbicides inhibit either photosynthesis or the biosynthesis of an essential amino acid. For example glyphosate inhibits an enzyme (ESPS) involved in the biosynthesis of aromatic amino acid, Atrazine inhibits photosynthesis etc. ♦ The bromoxynil specific nitrilase (bxn) gene was used widely in the production of crops resistant to herbicide bromoxynil. The gene was originally isolated from the soil bacterium Klebsiella pneumonia sub sp. ozaenae and was successfully transformed into plant or appropriate crops of commercially interest. ♦ Certain herbicides inhibit amino acids biosynthesis e.g. Glyphosate marketed as RoundupR , while others are photosynthetic inhibitor’s (e.g. S-triazines like atrazine). There are two approaches to produce herbicide resistant transgenic plants;

Detoxification Transfer of gene whose enzyme product detoxifies the herbicide. In this approach the introduced gene produces an enzyme which degrades the herbicide sprayed on the plant. Introduction of bar gene cloned from bacteria streptomyces hygroscopicus, into plants makes them resistant to herbicides based on phosphinothricin (ppt). The

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bar gene produces an enzyme phosphinothricin acetyl transferase which degrades phosphinothricin into a non-toxic acetylated from plants engineered with bar gene were found to grown in ‘ppt’ at levels 4-10 times higher than normal field application. ♦ Bxn gene of Klebsiella ozaenae which produces nitrilase enzyme imparts resistance to plants against herbicide bromoxynil. Example: BXN-cotton a transgenic cotton variety released in USA. ♦ Roundup Ready corn, soybean, cotton and canola are resistant to the broad spectrum herbicide roundup that contains the active ingredient glyphosate. The most widely utilized herbicide tolerance crop in production today is the Roundup Ready soybean. ♦ Glufosinate tolerant crops:- Glufosinate herbicide’s contain the active ingredient phosphinothricin, which kills plants by blocking the enzyme responsible for nitrogen metabolism and for detoxifying ammonia, a byproduct of plant metabolism. Crops modified to tolerate glufosinate contain a bacterial gene that produces an enzyme that detoxifies phosphinothricin and prevents it from doing damage. ♦ In the velvet leaf weed Abutilon theophrasti increase in the enzyme glutathione S-transferase causes detoxification of atrazine herbicide and hence the weed gains resistance. ♦ Similarly, in Echinochloa colona, increased content of aryl-acylamidase detoxifies propanil herbicide.

Target modification Under this approach, a mutated gene is introduced into the plant, which produces a odified enzyme in the transgenic plant that is not recognized by the herbicide. Hence, the herbicide cannot kill the plant. For instance, a mutated aroA gene from Salmonella typhimurium has been used for developing tolerance to glyphosate herbicide. The target site of glyphosate is a chloroplast enzyme 5-enol pyruvylshikimic acid 3-phosphate synthase (EPSPS). Introduction of the mutated aroA gene produces modified EPSPS, not recognizable to glyphosate (Gosal et. al., 2010). Insect resistance Insect resistant transgenic varieties have been field tested and released in crops like tomato, potato, cotton and maize. Insect resistant transgenic plants can be created by Introduction of bacterial genes, Bt/synthetic Bt and Introduction of plant gene (s) for insecticidal proteins. By introducing bacterial gene (Bt) several insect resistant varieties are developed such as BollgardTM of cotton, MaximizerTM and Yield GardTM of maize, New LeafTM of potato etc. and released in different countries. Bacillus thuringiensis synthesis an insecticidal crystal protein which when ingested by insect larvae is solublized in the alkaline conditions of the midgut of lepidopteran insect and processed by midgut proteases to produce a protease resistant poly peptide toxic to the insect. The Bt, a lepidopteran specific gene from Bacillus thuringiensis sub sp. Kurstaki has been widely and successfully used in tobacco, tomato, potato, cotton, rice and maize for developing resistance against several lepidopteran insect pest. Whereas

Role of plant biotechnology in crop improvement

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several insecticidal proteins of plant origin such as lectins, amylase inhibitors and protease inhibitors can reduce insect growth and development when ingested at high doses. ♦ Genes like CpTI, PIN-I, PIN-II, αAI and GNA have been cloned and are being used in the transformation programmes aiming at the insect resistance. The cowpea trypsin inhibitor (CpTI) containing transgenic cotton lines have been found to be highly resistant to cotton bollworm (Li et. al., 1998). ♦ Expression of potato trypsin inhibitor gene confers resistance to insects in rice (Duan et. al., 1996). ♦ Accumulation of soybean Kunitz trypsin inhibitor (SKTI) in rice confers resistance to brown plant hopper (Lee et. al., 1999). ♦ Supernatant of vegetative Bacillus cereus culture have two compound: VIP1 & VIP-2, which have been shown to possess toxic effects to ward insects (Estruch et. al., 1997). Disease resistance A significant percentage of the potential harvest yield of most crops is lost each year due to diseases caused by viroids, viruses, bacteria, fungi and nematodes. Resistance to viral disease’s was one of the first practical applications of plant genetic engineering to improve important crops. Genetic engineering of virus resistant plants has exploited new genes derived from viruses themselves in a concept referred to as ‘pathogen derived resistance’ (PDR). Xa21 gene from rice exhibits activity against 29 distinct races of the bacterial blight pathogen Xanthomonas oryzae (Wang et. al., 1996) Accumulation of soybean Kunitz trypsin inhibitor (SKTI) in rice significantly increased resistance to C. suppressalis & Sesamai inferens (Xuet. al., 1996). Introduction of chitinase gene in tobacco and rice has been shown to enhance fungal resistance in plants (Nishizawa et. al., 1999). Chitinase enzyme degrades the major constituents of the fungal cell wall (chitin & a 1-3 glucan). Co-expression of chitinase & gluconase genes in tobacco and tomato plants confers a higher level of resistance than that imported by either gene alone. Transgenic potato plants expressing an H2O2 generating fungal gene for glucose oxidase were found to have elevated levels of H2O2 and enhanced levels of resistance both to fungal and bacterial pathogens particularly to Verticillium wilt. The Introduction of H2O2 gene has also improved fungal resistance in rice (Anthony et. al., 1997). Male sterility Engineered male sterility is an alternative method for developing hybrids in cases, where natural male sterility is not available. Numerous genes have been identified, in diverse range of plant species, which show anther specific expression. Interference with the expression of cytotoxic genes from an anther specific promoter has been used to obtain male sterility.

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♦ Mariani et. al. (1990) fused an RNase gene (barnase) to the promoter of a gene (TA29) expressed specifically in the tapetum. The tapetum is belived to play a crucial role in the maturation of microspores (Shivanna et. al., 1997). Expression of barnase leads to degradation of the tapetum and inhibition of pollen maturation. ♦ van der Meer et. al. (1992) achieved male sterility by depleting flavonoid pigments using a silencing strategy based on antisense chalcone synthase gene expression, driven by a modified cauliflower mosaic virus 35S promoter. Improved quality Plants are remarkable in their capacity to synthesize a variety of organic substances, such as vitamins, sugar’s starches and amino acids. The success of the genetic approach has been mostly restricted to improving protein quality in model plants with enriched Lys, Trp and Met production. The genes of a number of plant storage proteins have been cloned and expressed in transgenic plants for example the zein genes of maize, the β 1 hordein gene of barley, the glutenin gene’s of wheat and the glycinin and conglycinin genes of soybean. ♦ The Flavr SavrTM tomato has increased storage life through suppression of the tomato polygalacturonase (PG) gene, resulting from transformation of an antisense expression cassette of the PG cDNA (Sheehy et. al., 1988). ♦ Goto et. al. (1999) reported improvement of the iron content of rice by transferring the entire coding sequence of the soyabean ferritin gene into Japonica rice. The introduced ferritin gene was expressed under the control of a rice seed storage protein glutelin promoter to mediate the accumulation of iron specifically in the grain. The transgenic seeds stored up to three times more iron than the normal seeds. ♦ Lysine, an important essential but limiting amino acid in rice, which might promote the uptake of trace minerals, can be improved by genetic engineering. Introduction of two bacterial genes DHDPS (dihydrodipicolinic acid synthase) and AK (aspartokinase) enzymes encoded by the Corynebacterium dapA gene and a mutant Escherichia coli lysC gene has enhanced lysine about fivefold in canola, corn, and soya bean seeds (Falco et. al., 1995 & Mazur et. al., 1999). ♦ β-Carotene, a precursor of vitamin A (retinol), does not occur naturally in the endosperm of rice. Ye et. al. (2000) have reported transgenic rice that produces grain with yellow-coloured endosperm. Biochemical analysis confirmed that the colour represents β-carotene (provitamin A). Abiotic stress Transgenic development is straight forward technology to improve crop yield in abiotic stress affected land (Roy & Basu, 2009). The development of tolerant crops by genetic engineering requires the identification of key genetic determinants underlying stress tolerance in plants and introducing these genes into crops. The basic resources for biotechnology are genetic determinants of salt tolerance and yield stability. The extensive genetic diversity for salt tolerance found in plant texa


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is distributed over many genera. Most plants are sensitive or hypersensitive to salt called as glycophytes. These are different from halophytes which are native to saline environments. Some halophytes can tolerate extreme salinity owing to their special anatomical and morphological adaptations or avoidance mechanisms. A salt tolerance gene isolated from mangrove (Avicennis msrins) has been cloned and can be transferred into other plants (Swaminathan, 2000). Wheat productivity is severely affected by soil salinity due to Na+ toxicity to plant cells. Xue et. al. (2004) generated transgenic wheat expressing a vacuolar Na+/H+ antiport gene. AtNHX1. The transgenic wheat lines exhibited improved biomass production. The field trial revealed that the transgenic wheat lines produced higher grain yield and heavier and larger grains in the field of saline soil. Transformation of Chinese cabbage (Brassica campestris ssp. pekinesis) by over expression a B. napus Group 3 LEA (Late Embryogenesis- Abundant) gene enhanced tolerance to salinity & drought (Park et. al., 2005). Cold tolerance: Classical plant breeding has limited success in imparting cold hardiness to crop plants. Biotechnology with its powerful tools however may provide answer through the isolation of cold fighting genes and thus may help in the development of crop plants that can withstand freezing temperature. Sarad et. al. (2004) developed transgenic tomato with osmatin gene. Their preliminary tests revealed that the transgenic plants are more tolerate to cold than wild types. For instance, for frost protection an antifreeze protein gene from fish has been transferred into tomato & tobacco. Likewise, a gene coding for Glycerol-3-phosphate acyltransferase from Arabidopsis has been transferred to tobacco for enhancing cold tolerance.

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Chapter -

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Role of tissue culture techniques in social forestry Komal Shekhawat1*, P. C. Gupta1, Ravi Kumar2, Anil Kumar1, Rekha Kumari 3 and Swarnlata Kumawat1 1 Department of Genetics and Plant Breeding, S.K. Rajasthan Agriculture University, Bikaner- 334 006 2 Department of Plant Breeding and Genetics, SKN Agriculture University, Jobner 3 Department of Plant Breeding and Genetics, Bihar Agricultural University, Sabour, Bhagalpur-813 210 *Correspondence author: [email protected]

Introduction Forest resources are disappearing and unprecedented rate. An important component of forest ecosystems are trees, which provide food, fuel, construction and industrial products. In addition, trees are recognized as the critical elements in maintaining stability in the world’s atmosphere. Social forestry may be defined as the science and art of the management and protection of forests and afforestation on barren lands with the purpose of helping in the environmental, social and rural development. Social forestry is the practice of forestry on lands outside the conventional forest area for the benefit of the rural and urban communities. Social forestry is the greatest instrument of land transformation. Development of trees on agricultural and other waste lands has tremendous effect. The trees control sheet, rill and gully erosion, they retain moisture in soil, provide the farmer with fuel and timber for agricultural implements, improve the climate, provide recreation to people, save cow dung for manure and wood required for cremation which is scarce sometime.The termsocial forestry was coined by forest scientist named J. C. Westoby. In India the term, was first used in 1976 by The National Commission on Agriculture, Government of India. Types of social forestry Mainly 5 type of social forestry, which are discussed below: 1. Farm forestry Farm forestry is the management of trees for a specific purpose within a farming context. Typically these are timber plantations on private land. However, it can be applied to a range of enterprises utilizing different parts of the tree and managed in a variety of ways.

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Benefits Farm forestry can produce multiple benefits for the farm, the environment and the community. The benefits to the landholder include: i. Shelter for stock, pasture and crops. ii. Additional and diversified earnings. iii. Improved living environments. iv. A buffer against the cyclical downturns in prices and in drought, frost and flood. v. Improvement and maintenance of soil and water health through water table reduction. vi. Increase in capital value of the plantation. The benefits to the environment and community are: i. The creation of new jobs and industries. ii. Sustainable management of natural resources. iii. Increases in biodiversity. iv. It itself is an industry that easily fits around the activities of most agricultural enterprises. v. Prices of wood products are relatively stable compared to most agricultural products. vi. Long term productivity is not weather dependent. 2. Agro-forestry This is the combination of agriculture and tree growing in order to produce both agricultural products and tree products on a commercial basis. The purpose of this scheme is to gain positive interactions between the two systems at both the paddock level and the enterprise level.The two systems may be fully physically integrated, or treated as separate entities within a single business enterprise. It is therefore ideally suited to the landholder seeking to enter farm forestry on a small scale, whilst maintaining an existing agricultural enterprise. Benefits I. Agro-forestry systems can be advantageous over conventional agricultural and forest production methods through increased productivity, economic benefits, social outcomes and the ecological goods and services provided. II. Biodiversity in agro-forestry systems is typically higher than in conventional agricultural systems. III. Agro-forestry incorporates at least several plant species into a given land area and creates a more complex habitat that can support a wider variety of birds, insects, and other animals. IV. Agro-forestry also has the potential to help reduce climate change since trees take up and store carbon at a faster rate than crop plants.

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Extension Forestry Planting of trees on the sides of roads, canals and railways, along with planting on wastelands is known as ‘extension’ forestry. Extension forestry helps in increasing the boundaries of forests. Under this project, there has been creation of forests on the village common lands, government wastelands and panchayat lands.Schemes for afforesting degraded government forests that are close to villages are being carried out all over the country. Community forestry Community forestry is a village-level forestry activity, decided on collectively and implemented on communal land, where local populations participate in the planning, establishing, managing and harvesting of forest crops, and so receive a major proportion of the socio-economic and ecological benefits from the forest.Community forestry is a process of increasing the involvement of and reward for local people, of seeking balance between outside and community interests and of increasing local responsibility for the management of the forest resource. Also, like sustainable development, community forestry should be a learning experience for all involved parties. Silviculture or scientific forestry Silviculture is the art and science of controlling the establishment, growth, composition, health, and quality of forests to meet diverse needs and values of the many landowners, societies and cultures over all the parts of the globe that are covered by dry land. Silviculture lays great stress on replacement and replanting of new crops and trees. The objectives of silviculture are as follows: i. Deriving environmental benefits, regulating afforestation, ensuring soil conservation. ii. Raising species of more economic value and Introduction of exotics. iii. Production of plants of high quality timber species. iv. Increasing production per unit area. v. Reduction of rotation period. vi. Afforestation of blank areas. vii. Creation of plantations. viii. Increasing production of fuels and fodder quality. ix. Increasing raw materials for forest based industries. x. Increasing employment potential 3. Role of tissue culture in social forestry Currently, a major concern is the erosion of genetic variability for a given species. Therefore, both in situ and ex situ management is necessary for biodiversity, conservation and consequently for tree crop improvement (NRC, 1991). When grown as a crop, forest trees represent a renewable resource and although lagging behind

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agricultural crops, attempts have been made to improve several economic species by conventional breeding. Tree improvement involves managing genetic resources, and includes conservation, selection and breeding, and propagation (Cheliak and Rogers, 1990). Biotechnology has the potential to accelerate the genetic improvement of forest yield (Timmis et. al., 1987) and in the short term, this impact will revolve around the cloning of selected phenotypes. As in vitro methods become more integrated with breeding and selection programs, other aspects such as engineering trees for disease and herbivore resistance, fiber quality, drought tolerance and so on will be added to tree improvement programs. However, the applications of tissue culture technology to tree improvement, other than for micropropagation, have remained largely unfulfilled (Thorpe, 1983; Haissig et. al., 1987). Due to rapid deforestation and depletion of genetic stocks, concerted efforts must be made to evolve new methods for mass propagation and production of short duration trees with a rapid turnover of biomass and induction of genetic variability for the production of novel fruit and forest trees which are high yielding, resistant to pest and disease associated with increased photosynthetic efficiency. This required genetic manipulation to evolve vigorous and fast growing trees with a short reproductive cycle which can be mass propagated. It is envisaged that the technology of tissue culture is competent to meet this challenge. Tissue culture techniques have already revolutionized the mass scale propagation of many horticultural crops and several commercial laboratories have been set up in many parts of world for mass production of elite, cloned plant material. However, its exploitation for forest tree species has started only recently. The following are some of the areas of tissue culture which are of prime interest in forestry. They have the biotechnological potential not only from the basic fundamental research point of view, but also for direct application for the immediate improvement of trees and increased biomass production. Forest plants /trees are the major source of raw materials for industrial & domestic wood products and also provides renewable energy, fiber and timber. 4. Tissue culture techniques In recent years, the interest has, aroused in commercializing the in vitro propagation of forest trees. This will bring about refinement in the existing procedures to make micropropagation more cost effective. For betterment and improvement of tree plants of high economic value a break through in the forestry research has come with production of artificial seeds in Eucalyptus (Muralidharan & Mascarenhas, 1989) and genetic transformation and in vitro regeneration in conifers (Gupta, 1989). Moreover, micropropagation has been successfully done in many trees (Gupta et. al., 1980, 1981; Jaiswal & Narayan, 1985, Amin & Jaiswal, 1988 and Mascarenhas & Muralidharan, 1989). Utility of clonal propagation in trees: •

Production of quality planting stock.

•

Propagation of trees which having non-viable seeds or poor germination.

•

Maintenance of genetic uniformity.

Role of tissue culture techniques in social forestry •

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Production of disease free plants.

Limitations •

Expensive than seed propagation

•

Short lived compare to seed propagated trees.

I. Cell culture Plant tissue cultures can be divided in to five classes based primarily on the type of material used on the medium. a) Meristem culture: This term is often used loosely to refer to very small shoot apices dissected from terminal or lateral buds. It refers to the microscopic apical dome with only the smallest leaf primordial evident, usually less than 2 mm across. The advantage of using shoot meristem is that they are most likely to be free of internal pathogens. b) Callus culture: The undifferentiated groups of cell is called callus. Explants produce callus rather than new shoot growth particularly where high levels of hormones are applied. Callus may be induced intentionally because of its potential for mass production of new plantlets. The limiting factors are the difficulty in inducing the initiation of new shoot apices, especially in woody species. c) Cell suspension culture: This is essentially product of callus culture, i.e. callus usually refers to a mass of undifferentiated cells. Once these are separated in liquid culture, it becomes a cell suspension. This culture may be used to produce a product directly from these cells without regenerating new plants. These cells may be genetically engineered to increase the synthesis of different secondary metabolites. d) Protoplast culture: This is a next step beyond cell suspension culture were the cell walls of suspended cells are removed using enzymes to digest the cellulose to leave the isolated protoplast. With the cell wall removed, it is possible to insert or remove foreign materials including the basic genetic materials DNA and RNA or to fuse together cells from entirely different species. e) Organ culture: The culture of embryos, anthers, shoots, roots or other organs on a medium is called organ culture. II. Protoplast fusion Protoplast fusion has little apparent applicability to little known tropical hardwoods and non-industrial species, for which investigations of the possibilities for even traditional hybridization remain minimal. Many hybrids have been tested for established industrial plantation taxa such as Eucalyptus, Pinus, Picea, and Larix. A number of these hybrids are in commercial use, and it seems likely that hybrids will become of wider importance in industrial plantation forestry. Those currently in use are mostly reasonably easy to produce sexually. To be addressed then is the extent to which sexual incompatibility limits the production of other useful hybrids. This is difficult to assess. The Introduction of disease resistance into commercial poplar clones has been

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limited by interspecific incompatibility, but other solutions to the problem have been identified, e.g. the use of solvents to remove stigmatic recognition factors underlying incompatibility (Knox et. al., 1972). There are other instances where transfer of traits among distantly related species would be desirable - e.g. rust resistance in pines. Given the difficulties involved, however, and the fact that protoplast fusion research is empirical and likely to be long term anyway, genetic engineering may offer better hopes for the manipulation of such traits.3. III. Embryo rescue This technique has been used, particularly in fruit trees, to grow embryos that normally would abort due to incompatibility between ovule and embryo development, and also for the rescue of the zygotic embryos of apomictic species (Ramming 1983). Embryo rescue has been applied also to some forest tree species. The culture of embryos with associated gametophyte tissue was used to raise seedlings of a hybrid between Pinuslambertiana and P. armandii, a hybrid for which attempts at normal germination had been unsuccessful (Stone & Duffield 1950). Culture of entire ovules successfully rescued the embryos of a hybrid between Populussimonii and P. pyramidalis, in which endosperm typically degenerates at the free nucleate stage (Ho 1987). In French poplar breeding programmes, embryo rescue is used routinely in the production of hybrids of Populustrichocarpa × P. deltoides, to minimize the post-fertilization attrition of ovules usually seen in controlled crosses between these species (D. Cornu pers. com.). In Brazil, the in vitro rescue of embryos 90 days after pollination was used to improve the yield of hybrids between Eucalyptus pellita and E. cloeziana (T. De Assis, pers. com.). Field growth of the resultant plants, however, has not been good, suggesting that genetic incompatibility in the zygote may be a feature of at least a proportion of hybrid genotypes resulting from the cross-fertilization of these two species. Embryo rescue does not provide a solution to compatibility problems at this level. IV. Clonal propagation Forest and fruit tree species which have long generation cycles complicated with the problem of heterozygosity as result of wide crossing. For example forest trees like the eucalyptus, teak and fruit trees like cashew, coconut etc. never breed true to type. Methods of tissue culture are now available for rapidly multiplying “elite” teak and eucalyptus trees, growing in the forests of chandrapur and Tamil Nadu respectively. There are also other reports where tissue culture methods have been developed in India for forests trees Dalbergia sissoo, D.latifolia, Albizialebbeck, tamarind, sandal, rubber etc. V. Haploid cultures Induction of haploid plants thus does not have immediate application in forest tree improvement programmes. Such plants may be of some use in basic genetic studies, e.g. for studies of heterosis in forest tree species. As a long-term strategic research objective for industrial species, induction of haploid plants should be of low priority until such time as methods for early selection and the promotion of early flowering are available. Woody plants generally have been recalcitrant, and there have been


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few successes, particularly for forest tree species, among a large number of reported attempts over the last 40 years. Plantlets have been obtained from pollen culture of species of Hevea, Aesculus, Citrus, Vitis, Malus, Litchi, Euphorbia, Poncirus, Lycium and Camellia (Chen 1987). Hardwood forest tree species for which pollen cultures have produced plants are Betulapendula (Radojevic&Kovoor 1986) and 13 species or interspecific hybrids of Populus (Chen 1987). Some of the resultant trees are now several years old in the field, and have flowered (Chen 1987). Regeneration of plants has been achieved with haploid megagametophyte cultures of a number of gymnosperms (Rohr 1987). Mostly these are cycads (Cycas, Zamia, Ceratozamia) or Ephedra, but included are two conifers - Piceaabies and Sequoia sempervirens. VI. Micropropagation Micropropagation is used because it permits rapid multiplication to meet market demand, and in some cases to overcome difficulties in alternative methods such as propagation by cuttings. In pineapple, for example, release of new varieties typically took 25 years with traditional multiplication procedures, but can be accomplished in one year using micropropagation (Drew 1993). Micropropagation is now the basis of a large commercial plant propagation industry involving hundreds of laboratories around the world. For example, 65–70 laboratories produced over 53 million plants in 1988 in Holland alone (Wang & Charles 1991). Details of micropropagation technologies have been well reviewed in recent years (Durzan 1988, Durzan and Gupta 1988, Wann 1988, Thorpe 1988, Thorpe et. al. 1991, Le Roux & van Staden 1991, Tartorius et. al. 1991). To briefly summarize the alternatives comparatively, the axillary budding, adventitious budding and somatic embryogenesis approaches are characterized by progressively increasing potential multiplication rates, progressively lower numbers of species for which success has been reported, and increasing apparent restriction to juvenile material. Over 1 000 plant species can now be micropropagated (Bajaj 1991), including many forest tree species. Thorpe et. al. (1991) listed over 70 angiosperm and 30 gymnosperm tree species for which successful methods for the production of plantlets have been reported, and Le Roux and van Staden (1991) listed over 25 species of Eucalyptus alone. These lists include most of the major plantation species. It is reasonable to conclude that, with sufficient research effort, successful protocols could be developed for most forest tree species. In general, a research project of this type might be described as short to medium term with a moderately high expectation of success. Micropropagation systems such as axillary shoot multiplication and somatic embryogenesis, employed in plantation forestry is regarded as an imperative stratergy to achieve rapid genetic gain (Ritchie, 1996, Libby & Ahuja, 1993 and Haines & Martin, 1997). These techniques are currently in use for the large scale multiplication of important forest/tree species. In last years micropropagation has been applied extensively to forest tree species (Bonga & von Aderkas, 1992 and Ahuja, 1993). Among various Asian countries, the success in micropropagation of tree species, including bamboos, differs and depends on their breeding programmes. In India the major accomplishment has been made in the in vitro propagation of various plant

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species, including forest trees like teak, eucalypts, bamboos and sandal (Sharma, 2002). Indo American hybrid seeds and southern Petrochemical Industries Corporation, Agro Biotech Limited which have large micropropagation within Asia, have successfully adopted the micropropagation technique for mass multiplication of teak, bamboo and eucalypts. In India for mass propagation of forest species, the Department of Biotechnology, Government of India set up two tissue culture pilot plants in 1989 at The Energy and Resources Institute (TERI) at Gual Pahari, Gurgaon and National Chemical Laboratory (NCL) at Pune. In December 1997, the Gual Pahari plant was upgraded into an Micropropagation Technology Park (MTP) to give it a larger mandate mass produce economically important plant species, including forest trees, fruits and cash crops medicinal and aromatic plants etc. Micropropagation protocols for mass propagation are available at MTP for different forest tree species like Anogeissus pendula, A. latifolia, Bambusa bambos, Dendro calamusasper, D. strictus, Eucalyptus populous deltoids etc. Large scale field demonstrationa by TERI prove reassuring for forest officials, private growers, breeders, seed company officials etc The alternative approaches have been grouped into three categories (Lutz et. al. 1985, Thorpe et. al. 1991), namely: Axillary Budding Multiplication of plants through the sequential subculture of axillary bud explants has been achieved for a large number of plant species, and is the basis of most of the commercial systems. Included in this category is the eucalypt axillary budding system, although some of the multiple shoots arising from the culture of eucalypt axillary buds may be adventitious in origin (Le Roux & van Staden 1991). Although best results for most tree species concern young seedling material, the method has been applied successfully to older trees of certain species, through the culture of adult material in some cases, and through the propagation of coppice material for those species for which such can be obtained. Compared to other micropropagation approaches, multiplication rates are low - five to ten propagules per culture cycle in many of the commercial operations (Lutz et. al. 1985). Nevertheless, even a multiplication factor of this order can amount to rates of millions per year for many species (Wang & Charles 1991), and rates for eucalypts are frequently much higher (Le Roux & van Staden 1991). Favourable responses for pines are generally limited to young trees, and multiplication rates are lower (Thorpe et. al. 1991). Adventitious Budding This includes systems involving direct induction on explants and those involving adventitious budding in callus cultures (Lutz et. al. 1985). Well described protocols are the meristic nodule systems for poplar (McCown et. al. 1988) and radiata pine (Aitken-Christie et. al. 1988). These involve the formation, multiplication and ultimately regeneration of plants from spherical cell clusters which show tissue differentiation and some vascularization (McCown et. al. 1988). These are similar to systems reported for a number of herbaceous species (Aitken-Christie et. al.1988). In general, juvenile tissues are much more responsive than adult, and embryos have been


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the favoured explant, particularly for conifers (Thorpe et. al. 1991). For responsive species, multiplication rates through adventitious budding are commonly substantially higher than for the axillary budding route (Wang & Charles 1991). In radiata pine, for example, it was estimated that an embryo of one of the more responsive clones could yield 260 000 plants (ready for the field) in 2.5 years (Aitken-Christie et. al. 1988). Somatic Embryogenesis (SE) In flowering plants, the process of double fertilization involves a haploid sperm fertilizing a haploid egg cell to form a diploid zygote. Subsequently, the zygote undergoes a series of morphological, biochemical, and molecular events to develop into an embryo. This stage of development is referred to as embryogenesis (Goldberg et. al., 1994). Somatic embryogenesis can also be used for the micropropagation of forest trees. Regeneration through SE has been reported in more than 50 woody species encompassing over 20 angiosperm families and at least a dozen conifer species (Wann, 1988, Watt et. al., 1991 and Park et. al., 1998). Somatic embryogenesis is when a somatic cell dedifferentiates to a totipotent embryonic stem cell that can give rise to an embryo in vitro (Verdeil et. al., 2007 and Ikeuchi et. al., 2015). Tectona grandis (Krishnadas and Muralidharan, 2008), Anogeissus latifoglia and Bamboo spp. (Godbole et. al., 2004 and Shali and Muralidharan, 2008) have been multiplied through tissue culture on a commercial scale in India. 5. Application of tissue culture in social forestry Embryo culture production It has been successfully used for peach, plum, pear and apple cultivars. Another application of embryo culture is to overcome seed dormancy which with many trees take several years for germination under natural condition. Isolation of disease-free plants : This technique has been exploited to produce virus free plants of fruit trees like citrus, apples, etc. In general, meristem culture results in the production of completely disease free root stocks and other plants. The plants produced have been found to be healthier, more vigorous and to produce higher yields. Early induction of flowering to shorten to breeding cycle This problem is further aggregated in some trees such as bamboo, which may flower once in forty years. Thus the early induction of flowering by the application of growth regulators in vivo or their use in in vitro cultures would help to reduce the breeding cycle. Transformation through uptake of foreign genomes Genetic transformation using Agrobacterium mediated transfer is another important technique in which a gene or group of genes encoding for a specific trait can be isolated and cloned. Cryropreservation of germplasm of trees: The rapid rate of diminishing of the genetic resources has caused concern of great magnitude for the conservation of important and elite germplasm. For this,

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cryopreservation techniques have been evolved which involves freeze preservation of cells, tissue and organs as a meaningful tool for long term conservation (-196ºC), establishment of gene banks and international exchange of germplasm. Forest tree material for which successful cryopreservation has been reported includes embryos of Quercuspetraea, Fagussylvatica, Aesculushippocastanum (Jorgensen 1990), Araucaria excelsa, Castanea (Engelmann 1992), Artocarpus, and Juglans (Withers 1992); embryogenic cell lines of Piceaglauca, Acer pseudoplatanus (Withers 1992), Piceaabies (Durzan 1988, Bercetche et. al. 1990, Gupta et. al. 1987) and Pinustaeda (Gupta et. al. 1987, Durzan 1988), seeds of Abies alba, Sequoiadendrongiganteum, Larix decidua, Pseudotsugamenziesii, Piceaabies, Pinussylvestris (but not Quercuspetraea) (Jorgensen 1990), pollen of Betulapendula, B.pubescens, Larix decidua, L.kaempferi, Pseudotsugamenziesii, Piceaabies, Pinussylvestris, Quercuspetraea and Q. robur (Jorgensen 1990), and encapsulated shoot tips of Eucalyptus gunnii (Monod et. al. 1992).The expectation is that, once cryopreserved, material can be stored indefinitely in liquid nitrogen without further loss. Strawberry and peanut meristem were capable of regeneration after two years storage, and 1.5 years for oil palm, while potato and cassava meristem survived for four years (Engelmann 1991). Cell lines of Piceaglauca have shown no loss of viability after over two years of storage (Attree & Fowke 1991).

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Thorpe, T.A. (1988) In vitro somatic embryogenesis. ISI Atlas of Science: Animal and Plant Sciences 1: 81–88.

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Thorpe, T.A., Harry, I.S. & Kumar, P.P. (1991) Application of micropropagation to forestry. pp. 311–336 in Debergh, P.C. & Zimmerman, R.H (eds) “Micropropagation: Technology and application.” Kluwer Academic Publishers, Dordrecht/Boston/London.

39.

Timmis, R. & Ritchie, G.A. (1988) Epigenetic effects in clonal propagation. pp. 12–31 in Worrall, J., Loo-Dinkins, J. & Lester, D.P. (eds) Proceedings of the Tenth North American Forest Biology Workshop, Dept. Forest Science, Univ. British Columbia.

40.

Timmis, R., Ritchie, G.A. & Pullman, G.S. (1992) Age and position of origin and rootstock effects in Douglas fir plantlet growth and metabolism. Plant Cell, Tissue and Organ Culture 29, 179– 186.

41.

Verdeil, J. L., Alemanno, L., Niemenak, N., and Tranbarger, T. J. (2007). Pluripotent versus totipotent plant stem cells: dependence versus autonomy? Trends Plant Sci. 12, 245–252.

42.

Wann, S. R. (1988). Somatic embryogenesis in woody species. Hortic. Rev. 10: 153-181.

43.

Watt, M. P., Blackeway, F., Cresswell, C. F. and Herman, B. 1991. Somatic embryogenesis in Eucalyptus granis. S. Afr. Forest. J., 157: 59-65.

44. Withers, L.A. (1992) In vitro conservation. In F.Hammerschlag and R.E.Litz (eds) “Biotechnology of Perennial Fruit Crops”, CAB International.

Chapter -

9

Tissue culture: An approach toward effective cost management Renu1*, Rahul Singh2 and Niranjan Kumar Chaurasia2 Department of MBGE, Bihar Agricultural University, Sabour, Bhagalpur-813210 2 Department of Plant Breeding and Genetics, Bihar Agricultural University, Sabour, Bhagalpur-813210 *corresponding author : [email protected] 1

Abstract The basic application of tissue culture in plants is production of high quality planting material.This technology exhibits several advantages over conventional propagation techniques. Instead of highly efficient tissue culture system, per unit cost of micro propagule and plant production becomes costly. In regard to this shortcoming adoption of low-cost tissue culture technology lower the production cost without compromising the quality of planting material. Low cost tissue culture technology ultimately aims toward cost reduction which is achieved by improving efficiency and better utilization of resources. Low cost tissue culture technology is gaining priority even in developing countries in all these sectors agriculture, horticulture, forestry, and floriculture for production of high quality planting material. Introduction Plant tissue culture is one of the major focus areas in biotechnology as having wide range of applications. These applications are broadly classified into three categories : basic, environmental, and commercial applications. Basic application includes physiology and molecular pathways in plant cells, whereas environmental covers conservation strategies of elite germplasms. Thirdly, commercial applications is highly focused current research in plant tissue culture as it deals on topics such as crop improvement, secondary metabolite production, and gene transfer. Instead of this it is a known technique for the production of large numbers of genetically identical plantlets. However, few facilities are prerequisite for any tissue culture laboratory which include washing areas and media preparation, sterilization, storage, aseptic transfer, observation/data collection, and environmentally controlled incubators or culture rooms. Thus micropropagation technique is valuable only when available explant is limited tissue. The major advantage of this technique is rapid multiplication of plants which faces difficulty in propagation as in case of plants having very small seeds, including most orchid also rare genotypes. Apart from these all micropropagation technology is more expensive than the conventional plant propagation methods, as it is a costly and also requires several types of skills. During the early years of the technology, there were difficulties in marketing of tissue culture products because

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the conventional planting material was comparatively cheaper. Now this problem has been minimized by adopting reliable and cost effective tissue culture methods without compromising its quality. This requires a constant monitoring of the input costs of chemicals (carbon sources, gelling agents, inorganic and organic supplements, and growth regulators), labour and capital.Thus, the aim of this chapter is to discuss the development of low cost technique for micropropagation of plant through tissue culture technique. Low cost technology options Low cost options should reduce cost of production without compromising the quality of the micropropagules and plants. Apart from this it is important that plants produced must be vigorous having high field survival rate. In addition, they should maintain genetic uniformity, diseases free, and comparatively cheaper. Practically the private industries in developing countries mainly focus toward adoption of cost effective technology as many of them are uneconomic mainly due to the high cost of production and the absence of quality tests. Hence, low-cost tissue culture technology can be utilized in these sectors namely agriculture, horticulture, forestry, and floriculture. There are several ways to make tissue culture cost effective out of these few are listed belowAlternatives to Carbon Sources Sucrose is the most commonly used carbon source in plant tissue culture. It significantly adds to the media cost. Household sugar and other sugar (sugar cubes) sources can be used to reduce medium cost (Thorpe, 2007). Sucrose is made up of cane sugar having 99.98% sucrose and 0.01% reducing sugar. Household sugar made of cane syrup contain 96-97% sucrose and 0.75-1% reducing sugar. Sugar cubes are made from grains of refined crystalline sugar, and contain 99.5% sucrose and 0.03% reducing sugar (Tyagi et. al. 2007). Use of all these indicated that common sugarreduces the cost of the medium between 78 to 87%. Gebre and Sathyanarayana (2001) and Demo et. al. (2008) evaluated potato culture media as alternative cheap sources of carbon in order to reduce the overall cost of micro-propagation. Alternatives to Agar The growth of cultures is strongly influenced by thephysical consistency of the culture medium. Gelling agents are usually added to the culture medium to increase its viscosity as it ensures adequate contact between the plant tissue and the medium. Agar is mostly used in tissue culture media as it is biologically inert and has high gel clarity, stability and resistance to digestion by plant enzymes. Concentration of agar used depends on its purity and brand. Apart from cost saving, there are a number of other advantages in using low concentrations of gelling agents. Other cheaper alternatives to agar include various types of starches and plant gums (Nagamori and Kobayashi, 2001). The National Research Development Corporation,India (NRDC, 2002) has listed low cost agar alternatives, foruse in commercial micropropagation. The use of agar is eliminated by using liquid media. While other options include white flour, laundry starch, potato starch, semolina, starch-gelrite mixture, plant gum and

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rice powder. Kodym et. al. (2001) reported that gelrite can be replaced with starchgelrite mixture. The cost of gelling agent was reduced by 70-82% using laundry starch, potato starch and semolina in a ratio of 2:1:1. However, some gelling agents contain inhibitory substances that hinder morphogenesis and reduce the growth rate of cultures. In another study Mohamed et. al. (2010) reported that use of corn starch (CS) and potato starch (PS) significantly increased the number of shoots/explant. The highest number of shoots/explant was achieved in medium having 50 or 60 g/l of PS + 1 g/l of agar. While media with 50, 60 g/l of PS or 60 g/l of CS and 50 g/l of CS + agar at 1 g/l significantly enhanced percentage of dry weight. Propagation of vegetative plants was done using 0.5 g gelrite along with 50.0 g/l corn starch (Zimmerman et. al. 1995). Enhanced shoot proliferation was observed on corn starch-medium than on agar. However, it became difficult to detect the contamination as corn starch medium turned grayish-white(Smykalova et. al. 2001). Naik and Sarkar (2001) substituted agar on potato micropropagated medium with 13% of sago and found significantly higher number of shoots and leaves and root length as compared to the agar medium. Sago (obtained from the stem pith of Metroxylon) at 13% concentration was substituted for agar in MS medium for the multiplication of chrysanthemum. Isubgol derived from the seeds of Plantago ovata is a colloidal mucilaginous husk, having good gelling activity. ‘Isubgol’ at 3% in MS medium hasbeen used for the propagation of chrysanthemum (Babbar and Jain, 1998; Bhattacharya et. al.,1994).To overcome shortcomings by using gelling mixes having one gelling agent from among the alternative gelling agents and agar. Xanthagar, a mix of xanthan gum and agar, possesses all desirable properties comparable to agar and offers a substantial cost benefit over agar (Jain and Babbar, 2010). Use of liquid media and physical matrices Suspension cultures are commonly used for culturing callus, cell clusters, buds and somatic embryos. It allows greater contact between the explants and the medium. Moreover, agitation of such media is required to reduce the diffusion gradient in the nutrient supply. The high production costs can drastically be reduced if cheap and (or) reusable and biologically inert alternative support matrix alternatives (glass bead) could be used. The glass beads (1.5 mm diameter) prior to use were soaked overnight in chromic acid and washed with Teepol 1% and distilled water and dried in hot air oven (Goel et. al. 2007). Other alternative includes foam plastic, filter paper bridges, glass beads glass wool and rock wool in liquid media (MacLeod and Nowak, 1990). Alternatives to distilled water Water is prerequisite component of all plant tissue culture media. Usually in research, distilled or doubled distilled and de-ionized water, produced through electrical distillation is expensive. Alternative water sources such as autoclaved tap water free from heavy metals and contaminants can be substituted for distilled water (Sharifi et. al. 2010). Use of RO water for stock solutions and hormone preparations while media preparation by using distilled water will reduce the cost effectively.

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Low cost option for media making Media preparation itself is a time consuming process. Now a days many companies sell readymade media either in liquid or powder form. Although ready-made media save time, their relative cost is high. They are useful when small quantities of medium are required. However, for large-scale use, it is economical to prepare media by mixing the basic ingredients. Therefore, pros and cons of laboratory buying readymade media verses making should be considered. Alternative to conventional equipments An alternative is to replace autoclave by using pressure cooker. Contamination was not observed when pressure cooker instead of an autoclave was used for sterilizing the media and equipment. Jam jarsalso replaced culture bottles (Gitonga et. al. 2010). Irrespective of need for maintaining in vitro plants the containers used should be transparent tofacilitate illumination and easy inspection of plantlets. Prakash et. al. (2004) reported use of Gamma ray for sterilized non-autoclavable food containers, plastic bags and PVC pots are being used for large scale micropropagation. Low cost energy options Reduction in the cost of energy and labour is important to minimize the production cost of tissue cultured plantlets. A large portion of the electrical energy consumed in tissue culture is used for autoclaving, artificial lighting of the growth room, laminar-flow cabinets and air conditioning. In developing countries, electricity cost accountsup to 60% of the production costs. Also plantlets grown under artificial light of low intensity, have low reserves, and a poor root system. Culture of in vitro plantsunder natural light, during their last phase in liquid medium, based on half- or quarter-strengthbasal MS salts without sugar and vitamins, under either aseptic or non-aseptic conditions is used to circumvent negative effects related to artificial light (Kodymet. al., 2001). Other methods Surface sterilization was found to be easy using explants, obtained from the green house than that of field grown plants in term of contamination and also reduces the maintenance cost (Sudipta et. al. 2011). Jaisy and Ghai (2011) reported the use of charcoalother than rooting hormones will reduce the cost of the media. In the rooting media, addition of 2℅ charcoal instead of hormones showed 95 ℅ success. Another low cost technique during acclimatisation is using rice husks which are available and free of charge in the rice growing areas therefore, be used as an alternative resource to reduce costs. Few other approaches include bioreactor-based propagation of plants which enhances multiplication rate and growth of cultures and reduces space, energy and labour requirements in comparison to commercial micropropagation (Gross and Levin, 1999). Conclusions In this chapter, we have attempted to present the prominent approaches related to low-cost methods employed in plant tissue culture. As plant tissue culture techniques have a vast potential to produce superior quality plants, but this potential has not been

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fully utilized due to huge capital requirement. From this prospect to provide smooth and efficient operations low cost options can be incorporated to design building, laboratories, working areas, lighting etc. also selection of commercially important and rare genotypes to allow cash flow and optimal use of equipment and facilities. The uniformity and consistency of tissue-cultured plants in field is must for a good production system. However, low cost approach could be well integrated into large scale commercial micropropagation to achieve the ultimate aim of cost effectiveness in tissue culture. These techniques must be integrated without reducing the efficiency of plant propagation and compromising the plant quality.

References 1.

Babbar SB and Jain N. (1988). Isubgol as an alternative gelling agent in plant tissue culture media. Plant Cell Rep. 17: 318-322.

2.

Bhattacharya P, Dey S and Bhattacharya BC. (1994). Use of low-cost gelling agents and support matrices for industrial scale plant tissue culture. Plant Cell Tiss. Org. Cult. 37: 115-123.

3.

Demo P, Kuria P, Nyende AB and Kahangi EM. (2008). African Journal of Biotechnology. 7(15) 2578-2584.

4.

Gebre E and Sathyanarayana BN. (2001). Tapioca: A new and cheaper alternative to agar for direct in vitro regeneration and micro tuber production from nodal cultures of potato Ethiopian Agricultural research Organisation EARO Afr Crop Sci J 9 : 1-8.

5.

Gitonga NM, Ombori O, Murithi KSD and Ngugi M. (2010). Low technology tissue culture materials for initiation and multiplication of banana plants. African Crop Science Journal. 18: 243-251

6.

Goel MK, Kukreja AK and Khanuja SP. (2007). Cost-effective Approaches for in vitro mass Propagation of RauwolfiaserpentinaBenth. Ex Kurz. Asian Journal of Plant Sciences. 6: 957-961.

7.

Gross A. and Levin R. (1999). Design considerations for a mechanised micropropagation laboratory. In: Plant Biotechnology and In Vitro Biology in the 21st Century. A. Altman, M. Ziv and S. Izhar (Eds.) Kluwer Acad. Pub. Dordrecht. 633- 636.

8.

Jain RR and Babbar SB. (2010). Evaluation of Blends of Alternative Gelling Agents with Agar and Development of Xanthagar, A Gelling Mix, Suitable for Plant Tissue Culture Media. Asian Journal of Biotechnology. 3: 153-164.

9.

Jaisy RC and Ghai D. (2011). Development of low cost methodology and optimization of multiplication and rooting hormones in the micropropagation of Red banana in vitro.Plant Sciences Feed.1 (7): 84-87.

10. Kodym A, Hollenthoner S and Zapata-Arias FJ. (2001). Cost reduction in the micropropagation of banana by using tubular skylights as source for natural lighting. In Vitro Cell. Biol. Plant 37: 237-242. 11.

MacLeod K and Nowak J. (1990). Glass beads as an alternative solid matrix in plant tissue culture. Plant Cell Tiss. Org. Cult. 22: 113-117.

12.

Mohamed MAH, Alsadon AA and Mohaidib MS. 2010. Corn and potato starch as an agar alternative for Solanum tuberosum micropropagation. African Journal of Biotechnology.9: 012-016.

13.

Nagamori E and Kobayashi T. (2001). Viscous additive improves micropropagation in liquid medium. J. Biosci. Bioeng. 91: 283-287.

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14.

Naik PS and Sarkar D. (2001). Sago: An Alternative Cheap Gelling Agent for Potato In Vitro Culture. BiologiaPlantarum. 44(2): 293-296.

15.

NRDC. (2002). Low cost plant tissue culture. National Research Development Corporation. 20-22, Zamroodhpur Community Center, Kailash Colony Extension, New Delhi, India

16.

Prakash S, Haque MI and Brinks T. (2004). Culture media and containers In : Low cost option for tissue culture technology in developing countries Proceedings of a Technical Meeting Organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture and held in Vienna 26-30 August 2002 IAEATECDOC-1384 : 29-40

17.

Sharifi A, Moshtaghi N and Bagheri A. (2010). Agar alternatives for micropropagation of African violet(Saintpauliaionantha). African Journal of Biotechnology. 9 (54): 9199-9203.

18.

Smykalova I, Ortova M, Lipavska H and Patzak J. (2001). Efficient in vitro micropropagation and regeneration of Humuluslupulus on low sugar, starch gelrite media. Biologiaplantarum. 44: 7-12.

19. Sudipta KM, Kumara Swamy M, Balasubramanya S and Anuradha M. (2011). Cost effective approach for in vitro propagation of (Leptadeniareticulatawight&arn.) – A threatened plant of medicinal importance. Journal of Phytology. 3(2): 72-79. 20.

Thorpe T. History of plant tissue culture. J. Mol. Microbial Biotechnol. (2007). 37: 169180.

21.

Tyagi RK, Agrawal A MahalakshmiC,Hussain Z and Tyagi H. (2007). Low cost media for in vitro conservation of turmeric (Curcuma longa L.) and genetic stability assessment using RAPD markers. In Vitro Cell Dev Biol. 43:51-58.

22.

Zimmerman RH, Bhardwaj SV and Fordham IM. (1995). Use of starch gelled medium for tissue culture of some fruit crops. Plant cell, Tissue and Organ culture. 43: 207-213.

Chapter -

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Prospect of molecular markers in precision plant breeding Anjani Kumar1*, Swapnil1, Shahina Perween1, R.S. Singh2 and D.N. Singh1 1 Department of Genetics and Plant Breeding, BAU Kanke, Ranchi – 834006 2 Department of Plant Breeding and Genetics, BAU Sabour, Bhagalpur – 813210 *Corresponding Author: [email protected]

Abstract Molecular markers are useful for plant genome analysis and have now become an important tool in crop improvement. The improvement and application of molecular markers for the detection of DNA polymorphism is one of the best significant developments in the field of molecular genetics. In modern biotechnology permit to modernize significantly the traditional plant breeding programme. The codominant nature of molecular markers reduces significantly the quantity of breeding material and also promotes the selection of superior genotypes. Molecular markers are widely employed in plant breeding and also used for the acceleration of selection of plant with the help marker assisted selection. Plant breeding has made significant progress in increasing crop yields for over a period with the help of molecular marker technology. This chapter gives some idea about different types of mostly used molecular markers for example- hybridization based markers (RFLPs), PCR based markers (RAPD, SSRs, AFLP) and sequencing based markers (SNPs and DArT) in plant breeding programme. Keywords: Molecular marker, RFLP, RAPD, SSR, AFLP, SNP, DArT

Introduction The genetic variations existing in various plant populations and their structure and level can play a favorable role in the efficient utilization of plants (Cole et. al., 2003). Plant breeding generally focus to improve the agronomically relevant characters or otherwise remarkable traits, by combining characters present in different parental lines of cultivated species or their wild relatives. Traditional breeding programmes achieve this goal by generating of pooled or individual plants for the presence of the desirable trait. It is a time consuming and costly process of repeated backcrossing, selfing and testing. Through this process, the breeder depends on precise screening methods and the availability of genotypes with clear cut phenotypical characters. The investigation of the diversity and other important characteristics, various types of agronomic and morphological parameters have been used successfully. During the last three decades, the world has observed a rapid increase in the knowledge about the plant genome sequences and the physiological and molecular role of various plant

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genes, which has evolutionized the molecular genetics and its competence in plant breeding programmes. Cereals are the most important sources of food in the world. It can be consumed directly as food by humans, or indirectly as inputs to improve animal production. Millions of consumers and farmers in both the developing and the developed country depend on on cereals as their favored staple food. Over the past several decades, especially through the conventional breeding programme, severe attempts have been done for the improvement of a large number of cereal varieties. The genetics and breeding community found that there is an urgent need to introduce new techniques including molecular marker-assisted breeding in association with high-throughput and precision phenotyping (Gupta et. al., 2010). Genetic diversity play a great role in the crop species as means it is the gift of nature. The assessment of genetic relationship is useful for efficient germplasm management for determining the uniqueness and distinctness of the phenotypic and genetic constitution of genotypes for the persistence of protecting breeder’s intellectual property rights, selection of parents for hybridization programme and reducing the number of accessions needed to ensure sampling a broad range of genetic variability (Brown, 1989; Lee, 1995; Bretting and Widrelechner, 1996). The variability of cultivated species and their wild relatives together forms a prospective and continued basis for breeding new and improved crop varieties. In the crop improvement programme, assessment of genetic diversity between various genotypes is the first process for identification of superior genotypes at both the morphological and molecular level. The more diverse strain can be crossed to produce genotypes with resistance to abiotic and biotic stresses. The genetic diversity that is present in plants assessed by common morphological traits. Though, such traits are limited in number, affected by human selection, influenced by environment, did not produce enough polymorphism and might be controlled by epistatic and pleiotropic gene effects (Fufa et. al., 2005). For this reason, there is a prerequisite to go in for a highly reliable and precise method for assessment of genetic variability with no environmental effects. Assessment of genetic diversity with molecular markers overcomes this problem. The molecular markers are the only way to allow the direct assessment of genotypic variation at the DNA level. Molecular markers are nothing but a fragment or portion of DNA, which can be used to detect polymorphism between alleles of a gene for a particular sequence of DNA or different genotypes. Such fragments are linked to a definite location within the genome and may be detected by using certain molecular technology (Henry et. al., 2012). This molecular marker systems are now being progressively developed and also has moved from the first and second generation marker systems, including RFLPs, RAPDs, SSRs and AFLPs to the third and the fourth generation marker systems, which include SNPs, DArT assays. (Paux et. al., 2010, 2012). These improvements have motivated new interest in exploring the applications of genetic markers in plant breeding and allow plant breeders to separate complex traits without having to measure the phenotype, hence reducing the need for extensive field testing over time and space.

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A brief description about different types of markers Morphological markers Those markers which can visually differentiate one from another on the basis of flower colour, seed structure, growth habit, surface of various plant parts and other important agronomic traits. There are no requisite of specific instruments, any specialized biochemical and molecular technique in the case morphological markers. These types of marker are highly used by plant breeder in the breeding programmes for various crops. The major drawbacks of morphological markers are : they are limited in number, influenced by the plant growth stages and various environmental factors (Eagles et. al., 2001). This types of marker generally express late into the development of an organism, hence their detection is dependent on the development stage of the organism. The also major disadvantage of morphological markers are they exhibit pleiotropy, epistasis, dominance and sometimes they display deleterious effects. Then earliest times, humans have successfully used these types markers to investigate the variation for utilization in plant breeding (Karaköy et. al., 2014). In crop like rice, the examples of morphological marker may contain the presence or absence of awn, leaf sheath coloration, grain color, height, aroma etc. Cytological markers Cytological markers are those marker that are related with variations present in the numbers, banding patterns, shape, size, order and position of chromosomes. Euchromatin and heterochromatin region found in chromosomeare are easily identified by cytological marker with the help of banding pattern. For example G bands are produced by Giemsa stain. The slides of such chromosomes are examined for bnding pattern under light microscope. Q bands are produced by quinacrine hydrochloride and R bands are also produced by Giemsa stain. It is reverse of G bands. Cytological markers can also be used in the identification of linkage groups and in physical mapping (Jiang et. al., 2013). Biochemical markers Biochemical markers are related to variations present in protein and amino acid banding pattern. Biochemical markers are identified with the help of gel electrophoretic studies. These markers have been effectively applied in the detection of genetic diversity, population structure, gene flow and population subdivision (Mateu et. al., 2005). The nature of biochemical markers are co-dominant, easy to use and cost effective. However, this marker detect less polymorphism and they are affected by various extraction methodologies, tissues of plant and different plant growth stages (Mondini et. al., 2009). A gene that is segment of DNA encodes a protein that can be extracted and observed like, isozymes and storage proteins. Molecular markers or DNA markers The polymorphism present between the nucleotide sequences of different individuals can be detected by molecular markers. It is the sequence of nucleotide. The various types of polymorphism are found in the genome of organism like Insertion, deletion, point mutations duplication and translocation. In other words, DNA marker is a gene or

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other fragment of DNA whose location in the genome is known. It is a DNA sequence, present in proximity to the gene of interest. It can be recognized by a range of molecular techniques such as RFLPs, RAPDs, SNPs, AFLP, SSRs etc. Molecular markers are also known as DNA markers or genetic markers. At the present time, Several new marker technology and breeding strategies have been designed. DNA markers use in plant breeding is called marker-assisted selection (MAS) and It is a element of the new discipline of ‘molecular breeding’. DNA-based markers have developed to resolve the morphological markers. Generally ideal molecular markers should have the following properties a. They are highly polymorphic b. They amply occur throughout the genome. c. They show minimum pleiotropic effect d. Easy and rapid assay e. Co-dominant inheritance f. Insensitive to environment g. Easy data exchange between different laboratories h. Cost and time effective i. No epistasis Classification of molecular markers: Molecular markers are classified into many groups on the basis ofa) Mode of gene action (co-dominant or dominant markers) b) Method of detection (hybridization based molecular markers or polymerase chain reaction based markers) c) Mode of transmission (paternal organelle inheritance, maternal organelle inheritance, bi-parental nuclear inheritance or maternal nuclear inheritance) (Semagn et. al., 2006) Various types of molecular markers have been developed and positively applied in genetics and breeding activities in various agricultural crops. The following chapter provides some brief information about molecular markers. Comparisons of the important characteristics of most commonly used molecular markers in agricultural crops are shown in Table 1 and their comparision with morphological markers in Table 2. Table 1: Comparison of important most commonly used molecular markers Characteristics Abundance

Isozyme

RFLP

RAPD

AFLP

SSR

SNP

DArT

Low

Medium

Very high

High

Very high

Co-dominant/ Dominant

Co-dominant

Co-dominant

Very high Dominant

Dominant

Co-dominant

Co-dominant

Very high Dominant

Prospect of molecular markers in precision plant breeding Reproducibility Level of polymorphism

Medium

High

High

Low

Medium

Technical complexity

Medium

Automation

135

High

High

High

High

Intermediate High

High

High

High

High

Low

Medium

Low

Medium

Medium

Low

Low

Medium

High

High

-

Sequencing

-

Yes

Medium No

No

Yes

Yes

No

PCR requirement cost

No

No

Yes

Yes

Yes

Yes

No

Less

High

Less

High

High

high

less

Table 2: Comparision of Morphological and Molecular Markers S.No.

Particulars

Morphological markers

Molecular markers

1

Nature

Dominant

Codominant

2

Occurence

Low

Abundant

3

Polymorphism

Low

High

4

Epistasis

Present

Absent

5

Detection

Easy

Easy

6

Pleiotropic effect

High

Minimum

7

Environmental effect

High

Absent

Molecular markers may be generally divided into three classes based on the method of their detection. A. Hybridization based markers In hybridization based markers, Restriction fragment length polymorphism (RFLP) was the first molecular marker system based on hybridization. In this process DNA profiles are pictured by hybridizing the DNA fragment, which is digested by restriction endonuclease to a labeled probe. Probe is a known sequence of DNA fragment. DNA isolation is the first step in the RFLP methodology. This isolated DNA is properly mixed with restriction enzymes this enzyme is isolated from bacteria and these enzymes are used to cleave DNA at particular recognition sites. For this process huge number of fragments of DNA with different length are produced. After that agarose or polyacrylamide gel electrophoresis is applied for the separation of these DNA fragments by producing a series of bands. The main cause for the variation of RFLP pattern are mutations, base-pair deletions, inversions, translocations and transpositions. This is reported that a major dominant resistance gene for the cyst nematode Globodera schachtii was tagged with RFLP markers in potato crop (Barone et. al., 1990). The association between the components of the hexaploid genome of bread wheat and its ancestors has been revealed using RFLP markers (Gill et. al., 1991). The application of RFLP markers are time consuming and expensive, For this reason scientist developed PCR based marker systems.

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B. PCR based markers The PCR technique was developed by Cary Mullis in 1983, It is a technique where small quantity of DNA is required for amplification without the application of any living organisms (Mullis et. al., 1986). The most important steps involved in PCR reactions are denaturation, annealing and extension. The PCR based markers are listed below Simple sequence repeats (SSRs): Simple sequence repeats are also called microsatellites. They are randomly repeated mono, di, tri, tetra, penta and hexo nucleotide motifs. These are generally highly polymorphic. These are flanked with unique sequences that are highly conserved. These can thus be assayed with the help of PCR and act as co-dominant markers. Therefore, it can be easily differentiate between heterozygotes and homozygotes. These are also stated to as simple sequence length polymorphism (SSLP). As compared to RFLP and RAPD, the polymorphism level is very high in microsatellite markers in case of practical plant breeding. SSRs can also be used to study the relationship between inherited traits within a species (Dunn et. al., 2005). SSRs technique does not require any special instruments. Very small quantity of DNA samples can be needed for microsatellite assay. Random amplified polymorphic DNA (RAPD): The technique was proposed by Williams et al 1990. It is a PCR based molecular markers. The variation present within a species in the randomly amplified fragments of DNA generated by restriction endonuclease enzyme. Single DNA primer of arbitrary sequence are used for RAPD assay. RAPD markers are dominant markers because the polymorphism is due to presence or absence of a particular amplified fragment. These are simpler and less expensive to work with than hybridization based molecular markers (RFLPs) because no prior knowledge of sequences is required and there is no requirement for radioactive probes. Some factor affecting the reproducibility of RAPD markers are quantity and quality of DNA, PCR buffer, magnesium chloride concentration, annealing temperature and Taq DNA (Wolff et. al., 1993). This type of markers are widely used in diverse plant species for assessment of genetic variation in populations and species, fingerprinting and study of evolutionary relationships among species and subspecies (Gupta et. al., 1999). This technique is simple and quick as compared to RFLPs. It does not need special equipment. Only PCR is required. Amplified fragment length polymorphism (AFLPs): The technique was developed by Zabeau and Vas in 1993. Amplified Fragment Length Polymorphism is a vastly sensitive method for finger printing genomic DNA within any organism (Vos et. al., 1995). It is a PCR-based markers. This technique provides very high multiplex ratio and genotyping throughput. The boundaries found in the RAPD and RFLP molecular markers were overcome through the development of AFLP markers (Vos et. al., 1995). This marker combine the RFLP and PCR based molecular marker RAPD. The method of this marker systems involves three steps : Firstly, oligonucleotide adapters are ligated to both ends of the resulting restriction fragments and genomic DNA is digested. In this technique two restriction


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enzymes a frequent cutter and a rare cutter are used for the cutting of DNA. Adapters are really short, enzyme specific DNA sequences generally used for fishing an unknown DNA sequence (Vos et. al., 1995). The advantages of using this method are that it is cost effective, since a single assay allows detection of a large number of co-amplified restriction fragments and it requires moderate quantities of DNA. This technique is useful in detection of polymorphism between closely related genotypes. C. Sequence based markers Sequencing is a technique in which determination of nucleotide bases and their order is identified along the DNA strand and molecular markers which are depend on the identification of a particular sequence of DNA in a pool of unknown DNA are known as sequence based markers (Franca et. al., 2002).

Single nucleotide polymorphism (SNP) Single nucleotide polymorphism markers are known as the third generation molecular markers. These are currently extensively used in various genomic studies for individual genotyping. The variations which are found at a single nucleotide position are referred to as Single nucleotide polymorphism. The polymorphism generated in this molecular markers on the basis of deletion, substitution, insertion. It may be transitions (G/A or C/T) or transversions (C/G, A/T, C/A or T/G) on the basis of the nucleotides substitution. SNP molecular markers are highly polymorphic and mostly biallelic. This marker provide ultra-high throughput for genotyping. Single nucleotide polymorphism are existing abundance in plants and animals and the SNP frequency in plants ranges between 1 SNP in every 100–300 bp (Xu et. al., 2010). These markers are widely distributed within the genome and can be also found in coding or non-coding regions of genes or between two genes with different frequencies (Xu et. al., 2010). There are several methods are presently available for Single nucleotide polymorphism (SNPs) discovery either following are database approach, where SNPs are detected by following the experimental approach, or mining sequence databases. These molecular markers are probably to become the marker of choice for breeding in the near future especially as the full sequences of more plant genomes will become presented with the advantage of next generation sequencing technologies (Syvänen et. al., 2005). Diversity array technology (DArT): Diversity array technology is a high throughput DNA polymorphism analysis method. It can detect and type of DNA variation at several hundred genomic loci in parallel without sequence information. It is depend on microarray hybridizations that notice the presence versus absence of individual fragments in genomic region. In this method no prior sequence is required for genome coverage in organisms. Diversity array technology is a solid state open platform method for analyzing DNA polymorphism. This is a high throughput relatively low cost technique. These polymorphic markers within the genotyping arrays are generally used for genotyping (Huttner et. al., 2005). The major application of this techniques are genome profiling and diversity analysis and also identify QTLs in genome of organisms.

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Role of molecular markers in plant breeding Varietal Identification Molecular markers are the best technique to protect varieties developed by Plant Breeder’s. In order to achieve this work, DNA fingerprinting can be used for varietal identification. There are different types of molecular markers for example RAPDs, microsatellites and AFLPs, ISSRs used for the DNA fingerprinting. DNA fingerprinting is also very important tool for quantification of genetic diversity, characterization of accessions in germplasm of plant and also to protect germplasm mainly the CMS lines. Assessment of Diversity Molecular markers can be used for assessment of genetic diversity in cultivars, germplasm collection and advanced breeding material. This information can be used for germplasm characterization and developing varietal information system. The genetic diversity will help in selection of parental lines for producing high yielding hybrids. The high heterotic combinations can be developed through crosses between distantly related parents. Microsatellites are appropriate for purity control and differentiation of plant cultivars. Diversity array technology and SNPs markers are the most frequently used molecular markers for the assessment of genetic diversity in various crops (Baloch et. al., 2017). Evolutionary studies Molecular markers are useful in the study of crop evolution. It helps in tracing the genetic origin of crop plants. In the ancient time, The evolutionary studies were totally dependent on the geographical and morphological changes among the organisms. The development of molecular biology offer extended information related to the genetic structure (Slatkin et. al., 1987). Molecular studies depends on phylogeny are largely dependent on chloroplast genome sequence due to their simple and stable genetic nature, making them ideal markers in the evaluation of plant phylogeny (Dong et. al., 2012 and Wang et. al., 2016). Marker assisted selection Marker assisted selection refers to indirect selection for a desired plant phenotype on the basis of molecular marker or DNA markers. Sometimes also called markeraided selection, It is a new technique for plant breeding that is primarily based on the phenotypic selection of superior individuals among segregating progenies resulting from hybridization. In plant breeding, Selection of parents is an important step. The QTLs based selection results in increased efficiency of selection. It is very crucial for polygenic characters with low heritability. It is a technique in which phenotypic selection is complete on the basis of the genotype of a marker (Collard et. al., 2005). These are molecular breeding technique which supports to avoid the difficulties concerned with conventional plant breeding. Marker assisted selection totally changed the standard of selection (Mohan et. al., 1997 and Tabor et. al., 2002). Plant breeders mostly use this method for the identification of suitable dominant or recessive alleles across a generation and for the identification of the most favourable individuals across


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the segregating progeny (Francia et. al., 2005). The major steps present in marker assisted selection are described below (fig. 1).

. Fig. 1: Major steps involve in the Marker assisted selection

Heterosis breeding Heterosis may be defined as the superiority of an f1 hybrid over both its parents in terms of yield or some other character. It is also known as true heterosis or euheterosis. Mainly, heterosis is manifested as an increase in vigour, size, growth rate, yield or some other characteristics. If the effect in f1 is greater than that in its parents, such types of heterosis is called as positive heterosis while where the effect in f1 is lower than in its parents, such type of heterosis is known as negative heterosis (Comings et. al., 2000) Molecular markers like microsatellite (SSRs) have been used in the investigation of diversity and heterosis in rice (Wu et. al., 2013). This is reported that the DNA marker is used to select heterotic hybrid with phenotypic data set (Jordan et. al., 2003). Another example of molecular marker used in heterosis breeding are that RFLP analysis provide an alternative to field testing when attempting to assign maize inbred lines to heterotic groups (Lee et. al., 1989). QTL mapping A quantitative trait locus (QTLs) is a position in a chromosome that contains one or more polygenes involved in the determination of a quantitative trait. A quantitative trait is governed by polygenes and is markedy affected by the environment. The agricultural traits are mainly governed by polygenes and it is quantitative in nature. So, QTL mapping is a method in which molecular markers are utilized to locate the genes that affect the traits of interest. These traits are classified into two major groups one is quantitative traits and the second one is qualitative traits. discrete variations found in qualitative traits while continuous variation occurs in quantitative traits. Molecular

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markers are very important and considered as an ideal tool for the QTL analysis and marker assisted selection (Angaji et. al., 2009). The methodology of QTLs mapping are presented in flow diagram (fig. 2).

Fig. 2: QTL mapping methodology

Conclusions Molecular markers are useful in many parts of studying the genetic polymorphisms in plant breeding programme. The potential profits of using molecular markers that is linked to genes of interest in breeding programmes, thus moving from phenotype based towards genotype based selection. With the help of molecular markers it is possible to accelerate the plant breeding process because it is possible to generate high density linkage maps of traits. Molecular markers are widely used in MAS to become more widely applicable for plant breeding programmes. The continuous improvement in the molecular markers technique from RFLP to SNPs and a diversity array-technology (DArT) based markers are greatest achievement in the field of molecular biology. The coming centuries are likely to realize continued innovations in molecular marker techniques to make it more productive, precise and cost effective. References 1.

Angaji SA. (2009). QTL mapping: a few key points. Int J Appl Res Nat Prod. 2(2):1–3.

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Collard BC, Jahufer MZ, Brouwer JB, et. al. (2005). An Introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: the basic concepts. Euphytica. 142(1–2):169–196.

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Comings DE, MacMurray JP. (2000). Molecular heterosis: a review. Mol Gen Metab. 71(1):19–31.

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10. Dunn G, et. al. (2005). Microsatellites versus Single-Nucleotide Polymorphisms in Linkage Analysis for Quantitative and Qualitative Measures. BMC Genetics.6, S122. http://dx.doi.org/10.1186/1471-2156-6-S1-S122 11.

Eagles HA, Bariana HS, Ogbonnaya FC, et. al. (2001). Implementation of markers in Australian wheat breeding. Crop Pasture Sci. 52(12):1349–1356.

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Franca LT, Carrilho E, Kist TB. (2002). A review of DNA sequencing techniques. Q Rev Biophys. 35(2):169–200.

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Francia E, Tacconi G, Crosatti C, et. al. (2005). Marker assisted selection in crop plants. Plant Cell Tiss Org. 82 (3):317–342.

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15. Gill KS, Lubbers L, Gill BS, Raupp WJ, Cox TS. (1991). A genetic linkage map of Trificum fauschii (DD) and its relationship to the D genome of bread wheat (AABBDD). Genome. 34,362-372. 16.

Gupta M, Chyi YS, Romero-Severson J, et. al. (1994). Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theor Appl Genet. 89(7–8):998–1006.

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Gupta, P., et. al. (1999). Molecular Markers and Their Applications in Wheat Breeding. Plant Breeding.118, 369-390. http://dx.doi.org/10.1046/j.1439-0523.1999.00401.x

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Huttner E, Wenzl P, Akbari M, et. al. (2004). Diversity arrays technology: a novel tool for harnessing the genetic potential of orphan crops. In: Serageldin I, Persley GJ, editors. Discovery to delivery: BioVision Alexandria Proceedings of the 2004 Conference of the World Biological BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 281 Forum; 2004 Apr 3–6; Alexandria, Egypt. Wallingford: CABI; 2005. p. 145–155.

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Lee M, Godshalk EB, Lamkey KR, Woodman WW. (1989). Association of restriction fragment length polymorphisms among maize inbreds with agronomic performance of their crosses. Crop Sci. 29, 1067–1071.

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Lee M. (1995). DNA markers and plant breeding programmes. Adv. Agron. 55:265-344.

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Mateu-Andres I, De Paco L. (2005). Allozymic differentiation of the Antirrhinum majus and A. siculum species groups. Ann Bot. 95(3):465–473.

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Mohan M, Nair S, Bhagwat A, et. al. (1997). Genome mapping, molecular markers and marker-assisted selection in crop plants. Mol Breed. 3(2):87–103.

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Mullis K, Faloona F, Scharf S, et. al. (1986). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 51:263– 273.

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30. Paux, E., et. al. (2010) Insertion Site—Based Polymorphism Markers Open New Perspectives for Genome Saturation and Marker-Assisted Selection in Wheat. Plant Biotechnology Journal, 8, 196-210. http://dx.doi.org/10.1111/j.1467-7652.2009.00477.x 31.

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A review on hydroponic system: hope and hype Ankita Jain 1 Nidhi Kumari 2 and Vikash Kumar Jha 3* 1 Joura Government College, Morena Madhya Pradesh 2 Jiwaji University, Gwalior M.P. 3 Bihar Agricultural University, Bhagalpur Bihar. *Corresponding author : [email protected]

Abstract Hydroponic is a method of growing plants using mineral solution, in water, without soil. This method can be extremely helpful to countries that have poor land, which is not able to sustain agriculture. The purpose of this lab is to prove that hydroponic horticulture can be just as effective if not better than plants traditionally grown in soil. Due to rapid urbanization and industrialization as well as melting of icebergs (as an obvious impact of global warming), arable land under cultivation is further going to decrease. Again, soil fertility status has attained a saturation level, and productivity is not increasing further with increased level of fertilizer application. Besides, poor soil fertility in some of the cultivable areas, less chance of natural soil fertility buildup by microbes due to continuous cultivation, frequent drought conditions and unpredictability of climate and weather patterns, rise in temperature, river pollution, poor water management and wastage of huge amount of water, decline in ground water level, etc. are threatening food production under conventional soil-based agriculture, under such circumstances, in near future it will become impossible to feed the entire population using open field system of agricultural production only. Naturally, soilless culture is becoming more relevant in the present scenario, to cope-up with these challenges. In soil-less culture, plants are raised without soil. Improved space and water conserving methods of food production under soil-less culture have shown some promising results all over the World. Introduction Hydroponics isn’t a new practice; yet, it is somewhat of a new technology. This means that the principles behind hydroponics have been around for years, but the study of it has only been around recently. One of the Seven Wonders of the Ancient World, the Hanging Gardens of Babylon, was believed to have worked with some of the principles that are used in hydroponics.(“Hanging Gardens of Babylon.” Wikipedia) In India, Hydroponics was introduced in year 1946 by an English scientist, W. J. Shalto Duglas and he established a laboratory in Kalimpong area, West Bengal. He has also written a book on Hydroponics, named as ‘Hydroponics The Bengal System’. Later on during 1960s and 70s, commercial hydroponics farms were developed in Abu Dhabi,

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Arizona, Belgium, California, Denmark, German, Holland, Iran, Italy, Japan, Russian Federation and other countries. During 1980s, many automated and computerized hydroponics farms were established around the world. Home hydroponics kits became popular during 1990s. Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops. The term Hydroponics was derived from the Greek words Hydro’ means water and ponos’ means labour.(Beibel, et.al.,1960) It is a method of growing plants using mineral nutrient solutions, without soil. (Beibel, et.al.,1960) Terrestrial plants may be grown with their roots in the mineral nutrient solution only orin and inert medium, such as perlite, gravel, or mineral wool. Hydroponics is the technique of growing plants in soilless condition with their roots immersed in nutrient solution.(Maharana et.al. 2011). Soil is usually the most available growing medium for plants. It provides anchorage, nutrients, air, water, etc. for successful plant growth. (Ellis et.al., 1974). However, soils do pose serious limitations for plant growth too, at times. Presence of disease causing organisms and nematodes, unsuitable soil reaction, unfavorable soil compaction, poor drainage, degradation due to erosion etc. are some of them. (Beibel, et.al.,1960) In addition, conventional crop growing in soil is somewhat difficult as it involves large space, lot of labour and large volume of water. Moreover, some places like metropolitan areas, soil is not available for crop growing at all, or in some areas, we find scarcity of fertile cultivable arable lands due to their unfavorable geographical or topographical conditions. (Beibel, et.al.,1960). Of late, another serious problem experienced since is the difficulty to hire labour for conventional open field agriculture. (Butler et.al.,2006) Under such circumstances, soil-less culture can be introduced successfully (Butler et.al.,2006). This system helps to face the challenges of climate change and also helps in production system management for efficient utilization of natural resources and mitigating malnutrition. (Butler et.al.,2006). Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and Abiotic stresses.

Nutrient solution Among factors affecting hydroponic production systems, the nutrient solution is considered to be one of the most important determining factors of crop yield and quality. A nutrient solution for hydroponic systems is an aqueous solution containing mainly inorganic ions from soluble salts of essential elements for higher plants. Eventually, some organic compounds such as iron chelates may be present. (Steiner, 1968). An essential element has a clear physiological role and its absence prevents the complete plant life cycle.(Taiz et.al.,1998) Currently 17 elements are considered essential for most plants, these are carbon, hydrogen, oxygen, nitrogen, phosphorus, potassium, calcium, magnesium, sculpture, iron, copper, zinc, manganese, molybdenum, boron, chlorine and nickel.(Salisbury et.al., 1992) With the exception of carbon (C) and oxygen (O), which are supplied from the atmosphere, the essential elements are obtained from the growth medium. Other elements such as sodium, silicon, vanadium, selenium, cobalt, aluminum and iodine among others, are considered beneficial because

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some of them can stimulate the growth, or can compensate the toxic effects of other elements, or may replace essential nutrients in a less specific role.(Trejo-Téllez et. al.,2007) The most basic nutrient solutions consider in its composition only nitrogen, phosphorus, potassium, calcium, magnesium and sculpture; and they are supplemented with micronutrients. The nutrient composition determines electrical conductivity and osmotic potential of the solution. Moreover, there are other parameters pH of the nutrient solution, electrical conductivity of the nutrient solution, composition of the nutrient solution; temperatures of the nutrient solutions are considered.

Composition of the nutrient solution As previously stated, nutrient solutions usually contain six essential nutrients : N, P, S, K, Ca and Mg. Thereby Steiner created the concept of ionic mutual ratio which is based on the mutual ratio of anions : NO3-, H2PO4- and SO42-, and the mutual ratio of captions K+, Ca2+, Mg2+. Such a relationship is not just about the total amount of each ion in the solution, but in the quantitative relationship that keep the ions together; if improper relationship between them take place, plan performance can be negatively affected. (Steiner, 1968,1961). In this way, the ionic balance constraint makes it impossible to supply one ion without introducing a counter ion. A change in the concentration of one ion must be accompanied by either a corresponding change for an icon of the opposite charge, a complementary change for other ions of the same charge, or both. (Hewitt, 1996) When a nutrient solution is applied continuously, plants can uptake ions at very low concentrations. So, it has been reported than a high proportion of the nutrients are not used by plants or their uptake does not impact the production. For example, it was determined that in anthodium, 60% of nutrients are lost in the leachate (Dufour et.al.,2005) but in closed systems, however, the loss of nutrients from the root environment is brought to a minimum. (Voogt, 2002) Also it has been shown that the concentration of nutrient solution can be reduced by 50% without any adverse effect on biomass and quality in gerbera (Zheng et.al 2005) and geranium. (Rouphael et.al., 2009) Accordingly, Siddiqi et. al. (1998). reported no adverse effect on growth, fruit yield and fruit quality in tomato when reduction of macronutrient concentrations to 50% of the control level as well as cessation of replenishment of the feed solution for 16 days after 7 months of growth at control levels were applied. However, it is expected that in particular situations, too low concentrations do not cover the minimum demand of certain nutrients. On the other hand, high concentrated nutrient solutions lead to excessive nutrient uptake and therefore toxic effects may be expected. Conversely, there are evidences of positive effects of high concentrations of nutrient solution. In salvia, the increase of Hoagland concentration at 200% caused that plants flowered 8 days previous to the plants at low concentrations, increasing total dry weight and leaf area. (Kang et.al., 2004) Likewise, high levels of K+ in the nutrient solution (14.2 meq L-1 vs 3.4 meq L-1) increased fruit dry matter, total soluble solids content and lycopene concentration of tomato. (Fanasca et.al., 2006) The explanation of these apparent controversial responses is the existence of optimal concentrations of certain nutrients in a solution for a culture under special

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environmental conditions as well as their relative proportions and not their absolute concentrations as determining factors. (Juárez et.al., 2006). In order to prevent contradictory observations, Dufour & Guérin (2005). recommend : a) to monitor the availability of nutrients through changes in the ionic composition of the substrate by analysis of percolate, and b) to asses plant nutrient uptake by nutrient content analysis in leaves. Moreover, Voogt (2002). indicates that the nutrient solution composition must reflect the uptake ratios of individual elements by the crop and as the demand between species differs, the basic composition of a nutrient solution is specific for each crop. It must also be taken into account that the uptake differs between elements and the system used. For instance, in open-systems with free drainage, much of the nutrient solution is lost by leachate. There are several formulations of nutrient solutions. Advantages of Soil-less culture There are many advantages of growing plants under soil-less culture over soil-based culture. (Savvas, 2002). These gardens produce the healthiest crops with high yields and are consistently reliable; gardening is clean and extremely easy, requiring very little effort. (Silberbush et.al., 2001). Here nutrients are fed directly to the roots, as a result plants grow faster with smaller roots, plants may be grown closer, and only 1/5th of overall space and 1/20th of total water is needed to grow plants under soil-less culture in comparison to soil-based culture, there is no chance of soil-borne insect pest, disease attack or weed infestation too. Overall soil-less culture provides efficient nutrient regulation, higher density planting, and leading to increased yield per acre along with better quality of the produce. It is also effective for the regions of the World having scarcity of Arabic of fertile land for agriculture. (Sonneveld 2000). Due to rapid urbanization and industrialization as well as melting of icebergs (as an obvious impact of global warming), arable land under cultivation is further going to decrease. Again, soil fertility status has attained a saturation level, and productivity is not increasing further with increased level of fertilizer application. Besides, poor soil fertility in some of the cultivable areas, less chance of natural soil fertility buildup by microbes due to continuous cultivation, frequent drought conditions and unpredictability of climate and weather patterns, rise in temperature, river pollution, poor water management and wastage of huge amount of water, decline in ground water level, etc. are threatening food production under conventional soil-based agriculture. Under such circumstances, in near future it will become impossible to feed the entire population using open field system of agricultural production only. Naturally, soilless culture is becoming more relevant in the present scenario, to cope-up with these challenges. In soil-less culture, plants are raised without soil. Improved space and water conserving methods of food production under soil-less culture have shown some promising results all over the World. Future scope of this technology Hydroponics is the fastest growing sector of agriculture, and it could very well dominate food production in the future. (Butler et.al.,2006) As population increases and arable land declines due to poor land management, people will turn to new technologies like hydroponics and aeroponics to create additional channels of crop production.


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(Maharana et.al. 2011). To get a glimpse of the future of hydroponics, we need only to examine some of the early adopters of this science. (Singh et.al., 2012) In Tokyo, land is extremely valuable due to the surging population. To feed the citizens while preserving valuable land mass, the country has turned to hydroponic rice production. (De Kreij, et.al., 1999). The rice is harvested in underground vaults without the use of soil. Because the environment is perfectly controlled, four cycles of harvest can be performed annually, instead of the traditional single harvest. Hydroponics also has been used successfully in Israel which has a dry and arid climate. (Van, et.al., 2002). A company called Organitech has been growing crops in 40-foot (12.19-meter) long shipping containers, using hydroponic systems. They grow large quantities of berries, citrus fruits and bananas, all of which couldn’t normally be grown in Israel’s climate. (Van, et.al., 2002). The hydroponics techniques produce a yield 1,000 times greater than the same sized area of land could produce annually. Best of all, the process is completely automated, controlled by robots using an assembly linetype system, such as those used in manufacturing plants. The shipping containers are then transported throughout the country. (Singh et.al., 2012). There has already been a great deal of buzz throughout the scientific community for the potential to use hydroponics in third world countries, where water supplies are limited. (Butler et.al.,2006; De Kreij, et.al., 1999). Though the upfront capital costs of setting up hydroponics systems is currently a barrier but in the long-run, as with all technology, costs will decline, making this option much more feasible. Hydroponics has the ability to feed millions in areas of Africa and Asia, where both water and crops are scarce. As we learn more and more about the science of hydroponics, it will start to be used more and more because of the improvements of it. NASA, which stands for National Astronautical Space Administration set a goal, which is to fulfill the dream of space exploration and head to Mars. We all know that growing plants is the most efficient and effective way of obtaining food, if you are in an enclosed area. Plants are a good source of food for astronauts; they remove carbon dioxide from the oxygen inside the spaceship. NASA has started experimenting on hydroponics and how the effects of space will change how the growing method functions. They are planning on traveling to faraway places such as Mars or even further. It would be way too expensive to send new food to the astronauts each time they run out or get close to running out. NASA is also trying to fulfill the Vision of Space Exploration, which will be very lengthy, long missions. Scientists have started testing on how lighting, pressure, and gravity affect the growing of the plants. “We want to see how plants are affected if we reduce the pressure inside their environment, to make it more like that of the surface,” Ray Wheeler, plant physiologist at Kennedy Space Center’s Space Life Sciences Lab, explains. “Some benefits of lower pressure would be more leeway in structural material choices, better visibility because you wouldn’t need as thick a cover, and fewer leaks.”

Conclusion Sometime harvesting of crop is tedious due to soil, like harvesting of potato, onion. If we talking about in Hydroponic System, its easy and also good for monitoring of

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maturation of product. Maintaining of minerals in soil is not easy and examination of soil productivity is also tuff, but in Hydroponic System it is easy to handle. The disease control system in Hydroponics is also good. We hope it will be farm for farmer in future.

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Salisbury, F. B. & Ross, C. W. (1992). Plant Physiology. Wadsworth Publishing Company, ISBN 0-534-15162-0, California, U. S. A.

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Hewitt, E. J. (1996). Sand and Water Culture Methods Used in the Study of Plant Nutrition. Technical Communication No. 22. Commonwealth Bureau of Horticulture and Plantation Crops, East Malling, Maidstone, Kent, England.

12. Dufour, L. & Guérin, V. (2005). Nutrient Solution Effects on the Development and Yield of Anthurium andreanum Lind. in Tropical Soilless Conditions. Scientia Horticulturae, Vol.105, No.2, (Jun 2005), pp. 269-282, ISSN 0304-4238 13. Voogt, W. (2002). Potassium management of vegetables under intensive growth conditions, In: Potassium for Sustainable Crop Production. N. S. Pasricha & S. K. Bansal SK (eds.), 347-362, International Potash Institute, Bern, Switzerland. 14. Zheng, Y.; Graham, T. H.; Richard, S. & Dixon, M., (2005). Can Low Nutrient strategies be Used for Pot Gerbera Production in Closed-Loop Subirrigation? Acta Horticulturae, Vol.691, No.1, (October 2005), pp. 365-372. ISSN 0567-7572 15. Rouphael, Y.; Colla, G., (2009). The Influence of Drip Irrigation or Subirrigation on Zucchini Squash Grown in Closed-Loop Substrate Culture with High and Low Nutrient Solution Concentrations. HortScience, Vol.44, No.2, (Apr 2009), pp. 306-311, ISSN 00185345


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16. Siddiqi, M. Y.; Kronzucher, H. J.; Britto, D. T. & Glass, A. D. M. (1998). Growth of a Tomato Crop at Reduced Nutrient Concentrations as a Strategy to Limit Eutrophication. Journal of Plant Nutrition, Vol.21, No.9, (Sep 1998), pp. 1879-1895. ISSN 0190-4167 17. Kang, J. G. & van Iersel, M. W. (2004). Nutrient Solution Concentration Affects Shoot: Root Ratio, Leaf Area Ratio, and Growth of Subirrigated Salvia (Salvia splendens). HortScience, Vol.39, No.1, (Feb 2004), pp. 49-54, ISSN 0018-5345 18. Fanasca, S.; Colla, G.; Maiani, G.; Venneria, E.; Rouphael, Y.; Azzini, E. & Saccardo, F. (2006). Changes in Antioxidant Content of Tomato Fruits in Response to Cultivar and Nutrient Solution Composition. Journal of Agricultural and Food Chemistry, Vol.54, No. 12, (Jun 2006), pp. 4319-4325, ISSN 0021-8561 19. Juárez H., M. J.; Baca C., G. A.; Aceves N., L. A.; Sánchez G., P.; Tirado T., J. L.; Sahagún C., J. & Colinas D. L., M. T. (2006). Propuesta para la Formulación de Soluciones Nutritivas en Estudios de Nutrición Vegetal. Interciencia, Vol.31, No.4, (Apr 2006), ISSN 0378-1844 20. Savvas D (2002). Nutrient solution recycling inhydroponics. In: HydroponicProduc tion of Vegetables andOrnamentals (Savvas D; Passam H C, eds), pp 299 – 343.Embryo Publications, Athens, Greece 21. Silberbush M; Ben-Asher J (2001). Simulation study ofnutrient uptake by plants from soilless cultures as affected bysalinity buildup and transpiration. Plant and Soil, 233, 59 – 69 22. Sonneveld C (2000). Effects of salinity on substrategrown vegetables and ornamentals in greenhouse horticulture.PhD Thesis, University of Wageningen, The Netherlands 23. Singh, S. and Singh, B.S. (2012). Hydroponics – A technique for cultivation of vegetables and medicinal plants‖.In.Proceedings of 4th Global conference on Horticulture for Food, Nutrition and Livelihood Options Bhuvaneshwar, Odisha, India. p.220. 24. De Kreij C; Voogt W; Baas R (1999). Nutrient solutions and water quality for soilless cultures. Research Station for Floriculture and Glasshouse Vegetables (PBG), Naaldwijk,The Netherlands, Brochure 196 25. Van Os E A; Gieling Th H; Ruijs M N A (2002).Equipment for hydroponic installations. In: Hydroponic Production of Vegetables and Ornamentals (Savvas D; Passam H C,eds),pp 103 – 141. Embryo Publications, Athens, Greece

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Single-cell techniques for stress tolerance in breeding plants Niranjan Kumar Chaurasia1*, Rahul Singh1, Renu2 and Praveen Kumar3 Department of Plant Breeding and Genetics, Bihar Agricultural University, Sabour, Bhagalpur-813210 2 Department of MBGE, Bihar Agricultural University, Sabour, Bhagalpur-813210 3 Department of Plant Breeding and Genetics, Assam Agricultural University, Jorhat, Assam-785013 *corresponding author: [email protected] 1

Abstract Due to continuous increase the global population demands the increased food production for feed. At the same time due to global warming and potential climate change hinder the food production. There are several biotic (drought or excess light, flood, temperature, minerals, salinity, soil pH, air pollutionor biotic) and abiotic (bacteria, fungi, viruses and insect pest etc) stresses which directly or indirectly reduces the yielding potential of crop plant. Plant performances are governed by several environments factors, plant genotypes and their physiological factors. Differential physiological response of cell of cell population against the particular stress/s leads the selection misleading if selection against particular stress is on the basis of whole plant selection. Hence, single cell techniques are potential tool for breeding stress tolerance plants where selection against particular stress is exercise on the basis of single cell selection for resistant/ tolerant to stress. Introduction ♦ Due to global warming, and potential climate abnormalities associated with it, crops typically encounter an increased number of abiotic and biotic stress combinations, which severely affect their growth and yield (Ramegowda and Senthil-Kumar, 2015) ♦ Stress refers to the factors of environment that interferes with complete expression of genotypic potential.Stresses can be abiotic, such as drought or excess light, flood, temperature, minerals, salinity, soil pH, air pollutionor biotic, such as pathogens like’s bacteria, fungi, viruses and insect pest etc. Concurrent occurrence of abiotic stresses such as drought and heat has been shown to be more destructive to crop production than these stresses occurring separately at different crop growth stages (Mittler, 2006; Prasad et. al., 2011). ♦ Performance of plant governed by both environments factors and plant genotypes and physiological factors likes cellular metabolisms, photosynthesis, transpiration, nutrients and its transport mechanisms etc.

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♦ Traditional whole plant tissues analysis is difficult due to heterogeneity of physiological response against different types of stresses. The heterogeneity in both eukaryotic and prokaryotic cell populations giving unclear picture of plant response during stress and hence the selection for tolerance/resistance to stress will be misleading. Hence, single cell techniques are potential tool for breeding stress tolerance plants. ♦ Single cell techniques is nothing but the study of genomics, transcriptomic, proteomics and metabolomics at the single cell level instead of cell populations/whole plant tissues. ♦ Analysing a single cell makes it possible to discover mechanisms that not seen when studying a bulk population of cells. ♦ Single-cell studies in plants have the great potential to enhance the resolution of two main areas such as (i.) developmental dynamics of plant tissues to identify non-anatomical markers for important cell populations; and (ii.) plant stress signalling, responses, and adaptation.

Single-cell sampling and analysis (SiCSA) ♦ Different cell types have different solutes as a result the composition of every cell type will respond differently to alternations at the whole plant tissues level. Hence, modification of solutes composition of plant need a detail knowledge of control operating at the level of individuals cell because assumptions based on whole plant tissues analyses may be in the error. ♦ SiCSA technique (Thomoset. al.1994) is an elaboration of pressure probe technique developed originally to measure turgor pressure in plant cells (Husken et. al.1978). ♦ Here micro capillary inserted into a cell, a sap sample forced into capillary tip, volume of sample taken in Pico liter sized and then stored under oil, and subsamples taken for determining the osmotic pressure and the concentrations of inorganic and organic solutes. ♦ Since speed of sap, forcing in capillary very slow hence speed of sampling can increased by some modification of probe so that dilution of sample by water flow can be avoided (Malone et. al.1989). Pros of SiCSA ♦ Has ability to measure a number of parameters simultaneously. It is possible to measure turgor, sap osmotic pressure and major solutes all at the resolution of single cell. Hence, both water and solute relation of individual cells can be determined and compositions of different cell types analyzed in detail in space and in time. ♦ Freezing point depression of cell sap sample ♦ Quantitative measurement of the inorganic elemental composition. ♦ Determine organic solute (sugar) and metabolites (nitrate, sulphate etc).

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♦ It determines single-cell enzymatic activities (acid invertase, acid phosphates, and cysteine protease and nitrate reductase). ♦ Determine both organic and inorganic solutes. Cons of SiCSA ♦ Very difficult techniques. ♦ Especially ideal for sampling of cell from leaf epidermis growing in air. ♦ Sampling of root cell (hydroponic plant) required extreme care to avoid contamination. ♦ Cannot distinguish intracellular components of the cell, the micro samples are a mixture of vacuole and cytoplasm.

X-Ray microanalysis Technique for determining the distribution of elements in various materials. The advent of imaging techniques has advanced the analysis of elemental distributions and the quantification of elements in cells and tissues. Elemental imaging also provides improved spatial information for the analysis of biological materials. Energy-dispersive X-ray microanalysis (EDX) is particularly suited to investigations of stresses imposed by toxic elements or excess salinity. TEM-EDX used to study salt distributions in Populus euphratica Oliver, a salt-tolerant woody species. P. euphratica used as a model plant to address tree-specific mechanisms underlying salt tolerance. Thus, TEM-EDX used to examine elements of interest within cell compartments with high spatial resolution (Chen. et. al. 2014). Principle

Pros

♦ Used to determine elemental composition of plant tissues both inter and intracellular level. ♦ To map distribution of nutrients and ions within the plant tissues. ♦ Used to map accumulation of Ni, Zn, Cd and other heavy metals to know heavy metals tolerance.

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♦ Used for frozen hydrated tissues. ♦ Used for dried micro droplets obtained from SiCSA. Cons ♦ It is nonquantitative. ♦ It is difficult to interpret observation. ♦ Elements cannot be detected if do not produce detectable X-ray signal.

Capillary electophoresis Capillary electrophoresis is an analytical technique, which separates ions based on electrophoretic mobility using an applied voltage. The electrophoretic mobility depends on the charge in molecule, viscosity, and the atom’s radius. The rate at of movement of particles is directly proportional to the applied electric field. Greater the field strength, faster the mobility. ♦ The analysis and simultaneous determination of solutes and metabolites in individual pant vacuoles. ♦ It work more effectively and give accurate result with SiSCA and X-ray microanalysis.

There are three approaches such as: 1. Indirect UV detection, 2. Direct UV detection after dynamic labeling and 3. Fluorescence detection after capillary inlet. Pros

♦ Simultaneous determination of many compounds within even complex plant matrices. ♦ Not require further sample pretreatment like extraction and filtration. ♦ Used to measure the effects of stress events on concentration and distribution of numerous solutes and metabolites within plant tissue.

Cons ♦ Inefficient when used alone

Single-cell gene expression (RT-PCR): The cells have an ability to cooperate and jointly construct complex structures like is tissues, organs, organ systems and whole organisms. During development process, cells undergoes differentiation to form specialized cell types, each with particular functions in the environment in which they reside. In many aspects, individual cell exhibit a high degree of variability and responses to identical stimuli even in a homogeneous cells population. The cells population measurement will not reveal how a particular transcript distributed among the cells. The most powerful technique for single-cell expression profiling is currently quantitative reverse transcription real-time PCR (RTqPCR) whichis suitable to detect even a single molecule.


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♦ It detects the presence of specific mRNA and amplification of single-cell mRNA in micro droplets derived from SiCSA. ♦ Nondestructive analysis of specific genes at single cell level of living plant. There are several sequential steps involved in this technique, which outlined below: CELL COLLECTION

CELL LYSIS

Pros

By Glass capillaries, Flow cytometry, Laser capture.

Purification, detergents, heat, osmotic and mechanic.

REVERSE TRANSCRIPTION

Reverse transcriptase, priming, temperature profile and pre-amplification.

REAL-TIME PCR

Master Mix, priming, temperature profile, detection chemistry, and preamplification.

DATA ANALYSIS

Normalization, quality assessment and statistics

♦ Widely used and easy to perform. ♦ Cell metabolism, membrane transport and ionic changes at single-cell gene expression level can be know.

Cons ♦ Most challenging step in single-cell expression analysis is to obtain representative individual cells with unperturbed expression profiles. The expression of all genes changes over time, consequently the use of reference genes is not valid to normalize single cell data. In addition, express most actively expressed genes at some time below the detection limit of the RTqPCR assay due to the stochastic behaviour of gene expression.

Fluorescence microscopy techniques ♦ The fluorescence microscope is the microscope, which uses fluorescence to generate an image. ♦ It determines the pattern of metabolite distribution within plant cell.

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♦ Today more widely used and current protocols include methods for quantitative measurements of substrates and their respective enzyme activities. Substrates

Using enzymes

Nitrate

Nitrate reductase

Glucose & Fructose

Glucose-phosphate dehydrogenase

Malate

Malate dehydrogenase

Mannitol

Mannitol dehydrogenase

Glucosinolate

Thioglucosidase

Amino acids

O-phthaldialdehyde

Limitation:♦ Simultaneous determination of analytes is not possible. ♦ Not give accurate ratio of different solutes within cell or cell compartment.

Cell turgor pressure and osmotic pressure ♦ Monitoring of the cell response to osmotic, salt, water stress, heating and ozone pollution. Cell pressure probe: - measure cell turgor pressure Picolitreosmometer: - measure cell osmotic pressure and melting point depression of microdroplets obtained from the cell. ♦ Used to show both the osmotic and turgor gradients within barley apoplasts are modified by ozone fumigation and salt stress.

Fig 1: To measure cell osmotic pressure from the cell.

Electrophysiological techniques This technique commonly used for the study of ionic currents and the ion channels that mediate them. This technique include electroencephalograms (EEGs), electrocardiograms (ECGs), single- and multiunit extracellular recording, multi electrode arrays, impedance measurements, and current-clamp, voltage-clamp, patchclamp, and lipid bilayer recording. It measures the voltage output from ions and in the change in voltage output resulting from the movement of such ions. It analyses


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intracellular ions content and membrane transport mechanisms at a single-cell level under stress condition Pros

♦ High speed ♦ Simplicity ♦ Tremendous sensitivity for recording membrane current

Ion-selective microelectrodes Microelectrode is a glass micropipette which is pulled to a fine tip and filled with an electrically conductive solution. The use of conventional microelectrodes has opened a new possibility for experimental physiology and fundamental parameters like cell membrane potential, membrane resistances and net ion transports became assessable for the first time. Hence many basic properties of electrophysiological processes become clear but fail to identify the specific ions. So to overcome the problem of these ion selective microelectrodes has been invented for answering the questions of cellular ion transport. It offers a unique approach to measurements of electrical parameters and ion activities of single cells. It also measures the membrane potential when inserted into a cell in voltage between inside and outside of the cell. Pros It provides important information of intracellular compartmentation of nutrients. ♦ Measure dynamics of cellular ion activities ♦ Measure the intracellular pH and membrane potential Cons ♦ It report ion activity at a single point within the cell ♦ Incomplete information ♦ Electrodes are sensitive to interference by protein other ions

Ion-invasive microelectrode ion flux measurement (Mife): Here the chemicals in solution move under the influence of chemical forces of diffusion directed towards lower concentration regions. Ions which are charged experienced the electrical forces if an electric field is present as well. The movement of an ion in solution can be described in terms of these chemical and electrical driving forces and other parameters of the ion and solution. It is used to study non-invasive measurement of membrane transport processes in the tissues boundary with resolution of 10 seconds in time and 20 micrometer in position Principle ♦ Measuring of electrochemical potential gradient by a slow square-wave movement of electrodes containing an ion-selective membrane between two positions, close to, and distant from sample surface.

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♦ It record electrode voltage at each position and convert it into concentration using the calibrated Nernst slope of the electrode and give the uptake and extrusion of an ion across cell membrane. Pros

♦ Give the ion concentration at two positions simultaneously. ♦ Estimate the ion uptake or ion extrusion across cell membrane.

Cons ♦ It may change the cellular membrane potential.

Patch-clamp technique The development of the patch-clamp technique in the late 1970s has given electro physiologists new prospects. This technique allows high-resolution currents recordings through ion channels in the cell membrane not only of whole cells, but also of excised cellular patches. It has been the technique of choice for measurements of ion channel activities in the cell with resolution up to a single channel. Principle 1. A glass pipette with blunt end containing electrolyte solution is tightly sealed onto the cell membrane and suction is applied to aid the development of a high resistance seal in the gig ohm range. 2. This tightly seal isolates the membrane patch electrically, which means that all ions fluxing the membrane patch flow into the pipette and are recorded by a chloride silver electrodes connected to a highly sensitive electronic amplifier. A bath electrode is used to set the zero level. 3. To prevent the alterations in the membrane potential, a compensating current that resembles the current that is following through the membrane is generated by the amplifier as a negative feedback mechanism. 4. The membrane potential of the cell is measured and compared to the command potential. If there are difference between the command potential and the measurement, a current will be injected. 5. This compensation current will be recorded and allows conclusion about the membrane conductance. 6. The membrane potential can be manipulated independently of ionic current and this allows investigation of the current voltage relationships of membrane channels. 7. Patch-clamp recordings can also be combined with live-cell imaging approaches such as Ca2+ imaging. In this case a Ca2+-sensitive fluorescent dye is applied to the cell via the patch pipette. The membrane current and changes in fluorescence are recorded simultaneously.


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Advantage ♦ It gives the wide picture about transport mechanisms single cell and even single channel within individual cell. ♦ It identify multiple types of calcium channels ♦ It measures the effect of potassium channels openers ♦ It investigates a wide range of electrophysiological cell properties ♦ Measures the cell membrane conductance. Limitation ♦ Require high level technical and data interpretation skill ♦ Cost of process is expensive ♦ Time consuming ♦ Chance of membrane distortion ♦ Number of samples required is more at times

Fluorescence microscopy The phenomenon of fluorescence was known by the middle of 19th century. The British scientist Sir George G. Stokes first observed that the mineral fluorspar exhibits the fluorescence when illuminated with ultraviolet light and coined the word fluorescence. Fluorescence is the light produced by a substance when it is stimulated by another light. Fluorescence is called cold light because it does not come from a hot source like an incandescent light bulb. Hence fluorescence microscope refers to an optical microscope that uses fluorescence and phosphofluorescence instead of, or in addition to, scattering, rejection and attenuation or absorption to study the properties of organic or inorganic substances. The common fluorophores used are Fluorescein, DAPI, Propidium iodide, Green Fluorescent Protein (GFP), Texas Red etc. Based on fluorescence probes accumulate inside cell and change their fluorescence properties when bound to distinct ions. Inflorescence image obtained by a video camera or photomultiplier. Advantage ♦ Different molecules can be stained with different dye allowing multiple types of molecule to be tracked simultaneously. ♦ It identify the Na and K activity in the cytosol. ♦ Most popular method for studying the dynamic behavior exhibiting in living cell ♦ Easy and quick to perform analysis ♦ Sensitivity is high enough to detect as few molecules per cubic micrometer Limitation ♦ Interaction of ion probe with cell metabolism ♦ difficulty of calibration ♦ photo bleaching effect ♦ poor discrimination of probe

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Conclusion The plant faces several types of stresses during their growth and development. Whole plant tissues analysis is difficult and misleading due to heterogeneity of physiological response against different types of stresses. Hence, single cell technique give fully understanding the physiological processes governing the response of a plant to stress events and efficient selection those able to breed the plant that withstand and tolerate such abiotic condition. References 1.

Chen S, Diekmann H, Janz D and Polle A (2014). Quantitative X-ray Elemental Imaging in Plant Materials at the Subcellular Level with a Transmission Electron Microscope: Applications and Limitations. Materials. 7: 3160-3175.

2.

Mittler, R. (2006). Abiotic stress, the field environment and stress combination. Trends Plant Sci. 11, 15–19.

3.

Prasad, P. V. V., Pisipati, S. R., Momcilovic, I., and Ristic, Z. (2011). Independent and combined effects of high temperature and drought stress during grain filling on plant yield and chloroplast EF-Tu expression in spring wheat. J. Agron. Crop Sci. 197, 430–441.

4.

Ramegowda, V., and Senthil-Kumar, M. (2015). The interactive effects of simultaneous biotic and abiotic stresses on plants: mechanistic understanding from drought and pathogen combination. J. Plant Physiol. 176, 47–54.

5.

Real-time PCR. Methods.50: 282–288.

6.

S Anders and B Martin (2010). Single-cell gene expression profiling using reverse transcription quantitative.

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Marker assisted backcrossing (MAB) an approach for selection by using molecular markers Shahina Perween1*, Anjani Kumar1, N. Swathi Rekha1, Swapnil1 , Priyanka Kumari1, Surya Prakash1 1 Department of Genetics and Plant Breeding, BAU Kanke, Ranchi-834006 *Corresponding Author : [email protected]

Abstract In breeding programs, one of the most anticipated and frequent cited benefits of molecular markers is the Marker assisted backcrossing (MAB) in which the goal is to incorporate a major gene from an agronomically inferior source(donor-parent) into an elite cultivator or breeding line (recurrent parent). It is routinely applied in breeding programs for gene introgression. Rising population requires increased crop production and rate of increase in crop yields is currently declining because of the fact that during cultivation, crops are facing various problems (biotic and abiotic factors), resulting in reduction in crop yield.To overcome these problems molecular breeding is an emerging and promising tool to develop plant varieties against stress condition. The plant varieties developed by marker assisted backcrossing are developed in a significant short span of time as contrary to the conventional backcross breeding and thus this is rapid and time saving beneficial technique. Keywords: Conventional backcrossing, molecular breeding, MAS, molecular markers.

Introduction A rising global population requires increased crop production . Some research suggests that the rate of increase in crop yields is currently declining (Pingali and Heisey 1999) and traits related to yield,stability and sustainability should be of major focus for plant breeding efforts.Many different crops faces lots of problems (boitic & abiotic) during cultivation,which ultimately effects the yields.To overcome these problems molecular breeding is an emerging tool to develop plant varieties against stress condition. Applying marker assisted backcross breeding is an artistic tool to develop varieties within a short span of time which is nearly impossible through conventional backcross breeding as the Conventional backcrossing takes longer time ( 5 to 6 generations ) to get desired trait(s) of the recurrent parent. That’s why we use molecular markers, primarily SNP markers, to assist backcrossing programs, for reducing the number of backcross generations, and reducing the time taken for getting recurrent parent along with desired traits. During MAB process ,not only we can track the gene of interest but we can also visualize and monitor the contribution of recurrent parent genome. MAB

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is the simplest form of MAS, in which the goal is to incorporate a major gene from an agronomically inferior source (the donor parent) into an elite cultivar or breeding line (the recurrent parent). The desired outcome is a line containing only the major gene from the donor parent, with the recurrent parent genotype present everywhere else in the genome.It is routinely applied for gene introgression in plant and animal breeding. Its efﬁciency depends on the experimental design, most notably on the marker density and position,populationsize,andselectionstrategy.Gene introgression programs are commonly designed using guidelines taken from studies focusing on only one of these factors (Hospital et. al. 1992; Visscher 1996; Hospital and Charcosset 1997; Frisch et. al. 1999). But Conventional Backcrossing is the introgression of a target gene from a donor line into the genomic background of a recipient line.

Fig 1: Comparing Conventional and Marker Assisted Backcrossing Source: -IRRI Knowledge bank

Now a days MAB is available for many different crops such as alfalfa, Brassicas, cereals (rice,wheat), corn, cotton, cucurbits, flowers, grasses, leafy vegetables (spinach), legumes, solanaceous (tomato), soyabeans, sunflowers, many green vegetables (brinjal, onion, capsicum, cabbage) etc. MAB may improve the efficiency of backcross breeding in three ways:1. If the phenotype of the desired gene from the donor parent is not easily assayed, BC progeny possessing a marker allele from the donor parent at a locus near the target gene can be selected with good probability of carrying the gene (forground selection). 2. Markers can be used to select against BC progeny with larger amounts of donor parent germplasm in the genome outside of the target region (background selection) 3. Markers can be used to select rare progeny that are the result of recombinations near the target gene, thus minimizing the effects of linkage drag (recombinant selection)

Marker assisted backcrossing (MAB)...

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Foreground selection Marker-assisted foreground selection was proposed by Tanksley (1983) and investigated in the context of introgression of resistance genes by Melchinger (1990), in which the breeder selects plants having the marker allele of the donor parent at the target locus. The main objective of this selection is to maintain the heterozygosity of the target gene (one donor allele and one recurrent parent allele) until backcross is completed.After backcrossing we select the plants which are self pollinated and identify the progeny plants for donor allele that are homozygous. So that the target locus selection is particularly useful for those traits that have laborious or more time taken procedures for phenotypic screening. It can also be used for the selection of those traits which appears on reproductive stage and seedling stage. And also help to identify the best plants for backcrossing. After this the selection of recessive allele is very easy by target locus selection, which is difficult for conventional breeding methods. Background selection In Background selection, the breeder selects for recurrent parent marker alleles in all genomic regions except the target locus,and the target locus is selected based on phenotype. It is important in order to eliminate potentially deleterious genes introduced from the donor.So-called ‘ linkage drag ‘, the inheritance of unwanted donor alleles in the same genomic region as the target locus, it is very difficult to overcome by conventional backcross breeding, but it can be easily done by using molecular markers. Background selection refers to the use of tightly linked flanking markers for recombinant selection and unlinked markers to select for the RP (Hospital & Charcosset 1997; Frisch et. al. 1999). These are those markers that are unlinked to the target locus/QTL on every single other chromosome,in another words, those markers that can be utilized against the donor genome for selection process.It is very useful because the recovery of the recurrent parent can be enormously quickened.But in conventional backcrossing, the recovery of the recurrent parent takes atleast six backcross generations and there may at present be a few donor chromosome fragments unlinked to the target gene or target locus. By using molecular markers,the recovery of recurrent parent can be achieved by two or three or four backcross generations (Visscher et. al.1996; Hospital & Charcosset 1997; Frisch et. al. 1999), so it saves two to four Backcross generations. The utilization of background selection during MAB to quicken the improvement of a Recurrent parent with an extra(or a few)genes has been referred to as‘complete line conversion’ (Ribaut et. al. 2002). Recombinant selection The size of the donor chromosome segment containing the target locus is to be reduced by the recombinant selection (i.e. size of the introgression). This selection process important because the rate of decrease of this donor segment is slower than the unlinked loci and the performance ofcrop are negatively affected by many undesirable genes that might be linked with the target allel from the donor parent.this process is termed as‘linkage drag’(Hospital 2005). Using conventional breeding methods,the donor segment can remain very large even with many BC generations (e.g.more than 10; Ribaut & Hoisington 1998; Salina et. al. 2003). By utilizing these markers that flak

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a target gene (e.g. less than 5 cM on either side), thus it can be minimized the linkage drag. Since double recombination events occurring on both sides of a target locus are very rare,selection is generally performed utilizing at least two Backcross generations (Frisch et. al 1999).

Fig 2: Three levels of Selection during Marker Assisted Backcrossing Source: - IRRI Knowledge bank

The efficiency of marker-assisted backcrossing depends on a number of factors,these factors are size of population in each backcross generation ,distance between target region and markers,total numbers of markers which are used in background selection. Data from Hospital (2003) show faster recovery of the recurrent parent genome with MAS compared to conventional backcrossing when foreground and background selection are combined (Table A).Many difficulty creates in breaking linkage with the target donor allele so the recovery of recurrent parent genome is more slowly on the chromosome carrying target region than the other chromosome .Bonnett et. al. (2005), Frisch and Melchinger (2001), and Frisch et. al. (1999) suggests the methods for enhancing the size of samples strategies for selection in marker assisted selection.

Backcross generation BC1

Number of individuals 70

Homozygosity of recurrent parent alleles at selected markers(%) Chromosome All other chrowith target locus mosomes 38.4 60.6

Recurrent parent genome(%)

BC2

100

73.6

87.4

92.2

87.5

BC3

150

93.0

98.8

98.0

93.7

BC4

300

100.0

100.0

99.0

96.9

Marker-assisted backcross 79.0

Conventional backcross 75.0

Table A. Expected results of a typical marker-assisted backcrossing program, based on simulations of 1000 replicates (Hospital, 2003). In each backcross generation, heterozygotes were selected at the target locus. Recurrent parent alleles were selected at markers flanking the target locus (2 cM on either side) and at three markers on each non-target chromosome.


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The success of MAB depends upon following factors:1. Distance from the target locus (genes)and closest markers, 2. Total number of target genes to be transferred, 3. Genetic bases of the traits or charactors, 4. Number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred, 5. The type of molecular marker(s) used, and available technical facilities (Weeden et. al., 1992; Francia et. al., 2005). Principle of marker assisted backcrossing It is a molecular technique which is apply to overcome all the problem that occurs in conventional plant breeding by changing the selection process from the selection of target genes that controls the trait of interest, either directly or indirectly. Molecular markers are not directly influenced by environment ( not affected by the environmental condition in which plants are grown). These markers are used in all plant growth stages for selection of desirable trait along with target gene(s). In the presence of of molecular markers and genetic maps (Semagn et. al., 2006), MAB is possible for both traits which is governed by single gene and quantitative trait loci (QTLs) (Francia et. al., 2005). MAB is the process which is used for DNA tests to assist in the selection of individuals and after selection these individuals become parents in next generation of a breeding programme. Steps of marker development and implementation can be :1. Identification of the parents which are differ in the traits of interest . 2. Mapping population or develop a plant population which segregates for the traits of interest. 3. Screen the population for the traits of interest in segregating population. 4. Construction of the genetic linkage maps ( Semagn et. al., 2006) of the cross with a proper number of polymorphic markers which have uniformly spaced for accurate location of desired QTLs or major gene(s). 5. Molecular markers linked are identified to the traits of interest. 6. Applicability and reliability tests of the markers for predicting the traits in related groups or families (marker validation or verification is done). 7. For assaying the markers we produce very simple and clear protocols. 8. For enhancing the use of MAB as compare to other selection techniques We should be modifying breeding strategy. 9. Implement into the breeding programs (use the markers directly in the breeding program to follow the introgression of desirable regions of the genome) (Gupta et. al., 1999; Babu et. al., 2004; Francia et. al., 2005).

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The above steps can be divided into three broad categories: genetic mapping, identify the association between markers and the trait of loci, and Marker Assisted Backcrossing. Applications of MAB Successfully integration of MAB into breeding programs are very useful. There are many successful applications of MAB in which markers have real advantages over conventional breeding techniques . MAB offers significant advantages in cases : 1. Phenotypic screening is most important trait for breeding program, MAB is applied in those cases where it is difficult or impossible and expenisive to carry out by conventional backcrossing. 2. Some traits are expressed very late in the plant development (like fruit and flower features or adult characters in species with a juvenile period),then this time selection process takes more time ,so application of MAB is very good for time saving and rapid selection before fruiting,flowering etc. 3. When the trait is of low heritability (incorporating genes that are highly affected by environment). 4. Few genes requires special environmental showing their expression ,in this condition we cannot be easily identified diseases or pests,in this case MAB helps to screen out diseases or pests ( for incorporating genes for resistance to diseases or pests) . 5. In breeding programme we target few genes for selection process,when the expression of this target genes are recessive then it is very difficult to select out those genes by conventional backcrossing. 6. To accumulate multiple genes for one or more traits within the same cultivar, a process called gene pyramiding (Han et. al., 1997; Huang et. al., 1997; Yousef and Juvik, 2001). Few examples where MAB is used ♦ The introgression of transgenes into an adapted variety or commercial genetic backgrounds has been done by using marker assisted backcrossing. (e.g.,Bt insect resistance transgene was introgressed into different maize genetic backgrounds). Often the target gene can be detected phenotypically, and markers are used to select for the recurrent parent genome. Hospital, 2003 reported that this technique is very quickened or rapid for the recovery of recurrent parent ( only 2 backcross generations ) ♦ A marker linked (0.7 cM) to the Yd2 gene for resistance to barley yellow dwarf virus was successfully used to select for resistance in a barley backcross breeding scheme in Australia (Jefferies et. al., 2003). Data recorded after field test showed that BC2F2 lines containing the linked marker had less leaf symptoms and more grain yield when infected by the virus as compare to those lines which are lacking the marker.


167

♦ In soyabean the introgression of yield QTL from a wild accession into commercial varieties by using marker assisted backcrossing,the yields of soyabean were increased more rapidly. (Concibido et. al., 2003). In spite of the fact the yield improvement was seen in just of two of six varieties, the study demonstrates the capability of incorporating wild alleles with the help of molecular markers. Reasons for unexpected results in MAB Marker Assisted Backcrossing is very useful technique in breeding program specially for time saving and help in the rapid development of plant variety.but it is not always necessary that marker assisted introgression is successful. One of the major limitation is the inability of marker based selection to produce the objective of selection at molecular level. Few reasons for unexpected results in MAB are discussed below:♦ After marker assisted interogression ,most of the QTL effects are additive in nature when the additive effect fails to exhibit in target QTL means the QTL have non additive effect (no effect at all i.e it may be a false positive.) ♦ Interaction between the QTL and environment and interaction between epistatis and genes due to linkage (Ribaut et. al.,2002). ♦ One of the major reason for unexpected results of marker assisted introgression is epistatis between QTLs or QTL and genetic background. ♦ QTL contain few genes and recombination between those genes would simply modify the effect of introgressed segments (Eshed and zamir, 1995;Monna et. al.,2002) Cost effectiveness of MAB The developmental cost associated with the development of genetic maps,genes,or QTL identification and verification,DNA extraction,genotyping, and analysis by use of molecular markers in selection process. Molecular markers are costly to develop, and returns from the initial research can take time. Financial aspects, consequently, the key determinant for the use or application of molecular markers in genetic improvement programs.There are many factors ( such as inheritance of trait, methods of phenotypic evaluation, field/glasshouse and labor costs, and the cost of resources) which increases the cost of MAB or MAS. Now and again, phenotypic screening is less expensive contrasted with markerassisted selection (Bohn et. al., 2001; Dreher et. al., 2003). In different circumstances, phenotypic evaluation might be time consuming and additionally troublesome and in this manner utilizing markers might be less expensive and ideal (Young, 1999; Yu et. al., 2000; Dreher et. al., 2003). Recreation examines have demonstrated that in a few conditions the adoption of MAB can enhance selection efficiency over phenotypic selection in breeding programs (Hospital and Charcosset, 1997; Knapp, 1998; Charmet et. al., 1999; Kuchel et. al., 2005). Recent genetic simulation and economic analysis in wheat has shown that MAB may not only provide genetic gain but also reduce cost (Kuchel et. al., 2005).

168


One situation where MAB is often being implemented is in F1 hybrid breeding.When parents of a successful hybrid A x B have been identified, the maternal A frequently should be endorsed with cytoplasmic male sterility and the male B with appropriate restorer genes.and these parents are very valuable. As to prior stages in a breeding program, the experimental results have been published at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico.It is related to the MAB for various maize breeding applications and cost-effectiveness of conventional selection (Morris et. al., 2003; Dreher et. al., 2003). According Morris et. al. (2003) MAB is the transfer of an elite allele at a single dominant gene from a donor line to a recipient line. Neither conventional backcrossing nor MAB showed clear prevalence as far as both expense and speed at CIMMYT. Conventional breeding was less expensive but MAB was quicker and more expensive than conventional breeding. Many researchers suggested that the cost-effectiveness of utilizing MAB relies on some parameters which are given as below:i. The equivalent cost of phenotypic versus marker screening. ii. Less time cosuming by MAB as compare to conventional backcrossing. iii. The size and transient circulation of benefits associated with quickened release of improved germplasm. iv. The availability to the breeding program of working capital. Above parameters can vary significant within breeding projects. Researchers suggested that the detailed economic analysis might be required to predict the proper selection method for a given breeding project. Koebner and Summers (2003) suggested that the current costs of MAB should be significantly reduced before it would be widely used in breeding program. Future prospects of MAB Researchers are continuously working on finding new ways in order to apply MAB for developing variety of crops. Thus MAB bring great expectation, in some cases it has led to exceed expected resulted while in some cases it has led to disappointment as the desired expectation have not been achieved. In coming years, breeders and researchers much work together in order to make research into a reality. Proper implementation of MAB is limited by cost of utilizing markers. Willingness of society in investment in research has made it possible for the usage of DNA markers in studies. In animal breeding, the investment in breeding sire is high as compare to plant breeding wherein the gain from MAB must be greater than gain made from traditional breeding or in other words the time saved by the application of MAB compensates the involved cost. It is expected that with advancement in technology will make it possible to adapt markers in plant breeding as a result of decreased cost of markers. In one research, Young (1999) urged that reliable markers for MAB can be developed if the scientists are able to realize the importance using larger population sizes, accurate phenotypic data, different genetic backgrounds and independent research work. In the coming years, with development in marker technology, the efficiency and effectiveness of


169

QTL mapping and MAB will be greatly affected by the availability of high-density maps and the integration of functional genomics with QTL mapping. Incorporating single nucleotide polymorphisms (SNPs) and expressed sequence tags (EST) to develop high-density maps will surely help the researchers for new finding in QTL mapping and MAB application. Also the growing database (genome sequencing projects) will help data mining task for new market and the discovery of SNPs (Gupta et. al., 2001; Kantety et. al., 2002). It is anticipated that the high-resolution map will help to isolate the actual genes (insteadof markers) via ‘map based cloning’ (Tanksley et. al., 1995). The plant scientist can predict gene function, isolate homologues and conduct transgenic experiments with possibility of identification of gene that controls important traits. Researchers have reported that efficiency of MAB can be enhanced by gaining knowledge of DNA sequence (Ogbonnaya et. al., 2001; Ellis et. al., 2002). Although there is much to be done in future work for example the DNA sequence for the majority of genes that controls the agronomical important traits are still known completely, for the time being the scientist can continue to explore QTL maps and markers that isolate genes. In developing country like Africa where the potential impact of MAB are quite high, several factors such as equipment, skilled manpower, infrastructure, etc. obstruct it’s usage. Also there is lack of genetic improvement with development priorities and the agricultural research work accounts for moderate research in the developing nations. The different stages of MAB development and application processes incurs significant cost. The identification of linkage between genes or QTLs and important traits are one of the most significant cost which act as hurdle for the developing countries. Even after identifying the marker-trait association and the availability of linkage map, the acquisition of consumables for molecular laboratories and timely purchase of equipment is quite challenging for in many African nations. While applying MAB in developing nations, return of investment must be rightly evaluated such that it exceeds that of the developed nations. The future could be very bright for these nations given the fact that both developed and developing nations work together and also the collaboration between public-private sector, assistance from research institutes (Consultative Group on International Agricultural Research (CGIAR)) and organizations (FAO) would also lead to reduction on MAB costs and would lead to high benefits. Overall, the traditional breeding program have a past success record along with some limitations it will be interesting to see the rise and advancement of MAB in future term.

Conclusion It can be concluded that application of MAB in breeding can benefit in carrying out our research work in early stages, by which a lot of time can be saved when compared to the conventional breeding method and thus compensating the cost involved in MAB process. With advancement of technology and research works, cost of MAB will go down which will help in widespread implementation of MAB globally especially in the developing nations. Also an investment in research is very important to explore full benefit of MAB. The future of breeding using MAB is very bright.A proficient costeffective MAB must be developed that will enable breeders to evaluate the genotype over the full genome and incorporating the qualities standard trait to the parent plant.

170


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Chapter -

14

Characteristics features of diara and tal land of some different blocks of Bhagalpur district Bihar Sumitap Ranjan*1and Rajkishore Kumar 2 Department of Soil Science & Agricultural Chemistry, BAU Sabour, Bhagalpur – 813210 2 Department of Soil Science & Agricultural Chemistry, BAU Sabour, Bhagalpur – 813210 *Corresponding Author: [email protected]

1

Abstract In this study the soils of the study area all the physical parameters were estimated that, the soils of ‘Tal’ which are highly clayey throughout their depth, grey in colour. whereas, the ‘Diara’ land consists light texture soil. Taxonomical classification of land along with suggested technological interventions for higher crop production and income and Some of major soil physical constraints relating crop production and income are appropriate soil conservation measures. Keywords: Tal and Diara land, physical status

Introduction Bihar with a geographical area of about 94.2 thousand square km is an important state in India, Diara land, found in between the natural levees of the river and formed due to periodical erosion and deposition of sediments (alluvium) under the influence of meandering and course changing behavior of the rivers. Such a land form is recognized as one of the most valuable natural resources (ICAR, 2005). The Diara land soils are distributed over an area of more than 11 lakh hectares on both sides of river Ganga, Gandak, Kosi, Sone and subsidiary rivers. The topography of Diara land is mostly undulating and intersected with numerous dead and alive streams forming complex type of physiography Sharma et. al. In continuation of available natural resources, the soils of ‘Tal’ which are highly clayey throughout their depths, grey to dark grey in colour, neutral to slightly alkaline in reaction found in adjoining of river Ganges with spatial geomorphic characteristics. In order to understand the soils of Tal and Diara properly, all 8 profiles were excavated in different locations of Bhagalpur district. Based on the horizon wise physical and chemical properties of the soils, taxonomic classification was done by adopting the standard procedure as described in revised version of soil taxonomy (Soil Survey Staff, 2014).

174


Features of Tal land Tal is recognised as a saucer shaped physiography inundated with water for 2 to 4 months in which soils are developed on back-water deposition of the rivers. The soils of tal land in the old alluwial region are characterized by neutral to slightly alkaline reaction, dark brown to very dark greyish brown; colour, clayey texture, low organic matter, high CEC, relatively lower silica and higher sesquioxide content, somewhat showing typical characteristics of Vertisols. They have developed on alluvium of different sources in a saucer shaped physiography under basic environment and impeded drainage condition. The narrow variation in organic carbon, clay and CEC within the pedons are indicative of homogenization due to argillipedoturbation. The specific feature of interseciing slickensides forming parallel epipeds with cyclicped on designated these tal land soils as Vertisols. Features of Diara land The term Diara has been derived from Diya meaning earthen lamp. Keeping in conformity with the shape of the Diya the bowl like systems on the surface (depressions) situated between the natural levees on either side of the river appear like small Diyas when rain water gets accumulated in them during the rainy session. Diara lands are extensively found in Uttar Pradesh, Bihar, Assam, and Orissa. Usually these lands are available only for a short period, and landless, small and marginal farmers cultivate on these lands. Classification of Diara land on the basis of distance from the main stream: a) Lower diara lands: are located in the main river beds that have fine sand to courses and deposits on the surface and are available for cultivation of different crops and vegetables during non-monsoon seasons (November/ December to May/June). b) Middle diara lands: are located on the banks of the rivers. The width of such lands varies considerably as the areas are frequently inundated during rainy seasons by swelling flood waters. The depth of flooding however varies considerably at different locations. c) Upper diara lands: are those lands which, during the course of continuous depositions get elevated and are less frequently flooded, in comparison to the middle diara lands.

Taxonomically classification of the soils of Tal and Diara land in Bhagalpur district Sr. No

Pedon

Location

Soil series

Pedon 1:

Sultanganj

Clayey, mixed, Hyperthermic Typic Haplusterts

Tal land 1. 2.

Pedon 2:

Bihpur

Clayey, mixed, Hyperthermic Dystric Eutruderts

3.

Pedon 3:

Kahalgon

Clayey, mixed, Hyperthermic Typic Hapluderts

4.

Pedon 4:

Sabour

Clayey, mixed, Hyperthermic Typic Ustifluverts

Characteristics features of diara and tal land...

175

Diara Land 5.

Pedon 5:

Sultanganj

Fine-loamy,mixed,hyperthermic Typic Hapludents

6.

Pedon 6:

Bihpur

7.

Pedon 7:

Kahalgon

8.

Pedon 8:

Sabour

Fine-loamy,mixed,hyperthermic Dystric Eutrudepts Fine-loamy,mixed, Hyperthermic Typic Hapludents Fine-loamy,mixed, hyperthermic Typic Ustifluvents

Physical characteristics of profile Soil separates of all the soil-horizons of pedons determined as different size fraction along with morphological characteristics. The result revealed that soil of different pedons viz., pedon 1, 2, 3, 4, 5, 6, 7 and 8 (Sultanganj, Bihapur, Kahalgaon and Sabour block). The result of sand status was observed that the maximum sand content (59.00 %.) was found in Pedon 8 of Sabour block in Diara land areas and minimum was found in Pedon 3 (Kahalgaon block) which was 1.8% in Tal land. Physical properties of different soil profile in selected location Soil depth

Horizon

Sand

Silt

Clay

Texture

(cm)

B.D

CaCO3

(Mgm-3)

(%)

Tal land Pedon 1: Sultanganj: Clayey, mixed, Hyperthermic Typic Haplusterts 0-15

Ap

17.5

41.5

41

sic

1.31

1.75

15-35

Bw 1

7.8

34.8

57.4

sic

1.35

1.92

35-59

Bw 2

6.8

31.8

61.4

c

1.37

2.01

59-90

Bw 3

6.4

25.1

68.5

c

1.43

1.98

90-149

Bw 4

7.9

25.4

66.7

c

1.47

2.26

Max

17.5

41.5

68.5

1.47

2.26

Min

6.4

25.1

41

1.31

1.75

Mean

9.28

31.72

59

1.39

1.98

Pedon 2: Bihpur: Clayey, mixed, Hyperthermic Dystric Eutruderts 0-15

Ap

3.7

27.2

69.1

c

1.51

2.1

15-35

Bw1

3.9

26.1

70

c

1.29

2.34

35-60

Bw2

5.2

25.5

69.3

c

1.32

2.45

60>

Bw3

4.3

27.6

68.1

c

1.39

2.41

Max

5.2

27.6

70

1.51

2.45

Min

3.7

25.5

68.1

1.29

2.1

Mean

4.28

26.60

69.13

1.38

2.33

Pedon 3: Kahalgaon: Clayey, mixed, Hyperthermic Typic Hapluderts 0-20

Ap

2.9

23.3

73.8

c

1.45

3.21

20-41

Bt1

2.1

23.5

74.4

c

1.26

3.45

41-68

Bt2

1.8

22.4

75.8

c

1.27

3.56

176


68--97

Bt3

3.1

20.5

76.4

c

1.32

3.42

97-140

Bt4

2.5

21.5

76

c

1.35

3.45

Max

3.1

23.5

76.4

1.45

3.56

Min

1.8

20.5

73.8

1.26

3.21

Mean

2.48

22.24

75.28

1.33

3.42

Pedon 4: Sabour: Clayey, mixed, Hyperthermic Typic Ustifluverts 0-20

Ap

2.2

25.2

72.6

c

1.37

0.5

20-62

Bw1

2.6

26.1

71.3

c

1.38

0.8

62-96

Bw2

2.5

23.9

73.6

c

1.45

1.1

96-137

Bw3

2.8

23.8

73.4

c

1.47

1.21

137-151

Bw4

2.3

23.6

74.1

c

1.48

1.24

Max

2.8

26.1

74.1

1.48

1.24

Min

2.2

23.6

71.3

1.37

0.5

Mean

2.48

24.52

73

1.43

0.97

Horizon

Sand

Diara Land Silt

Clay

Texture

B.D

CaCO3

(Mgm-3)

(%)

Pedon 5: Fine-loamy,mixed,hyperthermic Typic Hapludents 0 –40

Ap

50.8

21.75

27.45

scl

1.36

3.11

40-59

Bw1

41.7

27.85

30.45

cl

1.41

3.09

50- 91

Bw2

44.2

20.85

34.95

cl

1.43

3.24

91-180

Bw3

45.6

19.35

35.05

cl

1.49

3.15

Max

50.8

27.85

35.05

1.49

3.24

Min

41.7

19.35

27.45

1.36

3.09

Mean

45.58

22.45

31.98

1.42

3.15

Pedon 6: fine-loamy,mixed,hyperthermic Dystric Eutrudepts 0-15

Ap

53.3

20.55

26.15

scl

1.34

1.1

15-35

Bw1

48.8

22.85

28.35

scl

1.38

1.14

35-65

Bw2

45.4

24.85

29.75

scl

1.45

1.05

65-145

Bw3

46.5

25.05

28.45

scl

1.48

1.06

Max

53.3

25.05

29.75

1.48

1.14

Min

45.4

20.55

26.15

1.34

1.05

Mean

48.5

23.33

28.18

1.41

1.09

Pedon 7: fine-loamy,mixed, Hyperthermic Typic Hapludents 0–10

Ap

49.1

28.15

22.75

l

1.31

1.61

25–50

Bw1

46

24.95

29.05

scl

1.32

1.59

50-115

Bw2

47.1

23.25

29.65

scl

1.37

1.41

115–180

Bw3

44.5

28.05

27.45

l

1.41

1.46

Characteristics features of diara and tal land... Max

49.1

28.15

29.65

1.41

1.61

Min

44.5

23.25

22.75

1.31

1.41

Mean

46.68

26.10

27.23

1.35

1.52

177

Pedon 8: fine-loamy,mixed, hyperthermic Typic Ustifluvents 0–30

Ap

58

27.85

14.15

sl

1.44

1.89

30–60

Bw1

59

24.95

16.05

sl

1.51

1.94

60-180

Bw2

58.9

27.55

13.55

sl

1.53

2.04

Max

59

27.85

16.05

1.53

2.04

Min

58

24.95

13.55

1.44

1.89

Mean

58.63

26.78

14.58

1.49

1.96

Max

59

41.5

76.40

1.53

3.56

Min

1.8

19.35

13.55

1.26

0.5

Mean

24.33

25.49

50.18

1.4

2.05

Overall

Conclusion Soil profiles, soil taxonomic classification and physical properties of analysed soil samples were characterized. Information towards land use and natural vegetation under Tal land Diara lands were also incorporated. In continuation of soil analysis, all the soil properties were determined as per the standard procedure outlined by various scientists/ researchers. The study area Bhagalpur district, located at near river Ganga that flows west to east direction in middle portion of the entire district. There are sixteen blocks in which only Sultanganj, Bihpur, Kahalgaon and Sabour blocks were studied towards Tal and Diara included soil series due to high encroachment of such natural phenomena. The drainage of the area plays a vital role in respect of the texture of Tal land clay content varied from 37.7 to 76.00 per cent. Whereas, Diara land was observed light in texture having low clay content that varied from 13.55 to 35.05 per cent. The bulk density of pedon 1, 2, 3, 4 from Tal land varied from 1.31-1.47, 1.29-1.51, 1.26-1.45 and 1.37-1.48 Mg cm-3. However, bulk density of pedon 5, 6, 7 and 8 varied from 1.36-1.49, 1.34-1.48, 1.31-1.41 and 1.44-1.53 Mg cm-3, respectively in Diara land. Bulk density of all soil profiles was between 1.26 to 1.53 Mg m-3. However, soils of Diara land showed higher mean BD (1.53 Mg m-3) as compared to Tal land (1.26 Mg m-3). In all the pedon of Tal and Diara land, sand varied from 1.8-17.50% , 41.7-59.00% , silt varied from 20.5-41.5%, 19.35-28.15% and clay varied from 41.60-76.40%, 13.5535.05% respectively. The overall mean values of sand, silt and clay were 24.33, 25.49 and 50.18 respectively in Tal and Diara. Soil pH of entire pedon viz.1, 2, 3, 4, 5, 6, 7 and 8 (Sultanganj, Bihpur, Kahalgaon and Sabour block) varied from 7.25 to 9.46. The surface and sub-surface samples of Tal land in respect of soil pH varies from 7.25 – 8.69 and 7.55-9.27, respectively. Whereas, pH in case of Diara land, surface and sub-surface samples were varies from 7.80 -8.48

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and 7.71-9.46, respectively. Similarly, Soil pH of pedon 1-4 varies 7.42 -8.75 and pedon 5-8 varies from 7.62 - 8.10, respectively. Soil EC of entire pedons viz. 1, 2, 3, 4, 5, 6, 7 and 8 (Sultanganj, Bihpur, Kahalgaon and Sabour block) varied from 0.08 to 0.86 dSm-1. The surface and sub-surface samples of Tal land in respect of soil EC varies from 0.08-0.46 dSm-1 and 0.13-0.72 dSm1 , respectively. Whereas, EC in case of Diara land, surface and sub-surface samples were varies from 0.08-0.86 dSm-1 and 0.21-0.75 dSm-1, respectively. Similarly, Soil EC of pedon 1-4 varies 0.11 - 0.53 dSm-1 and pedon 5-8 varies from 0.09-0.44 dSm-1, respectively. As far as ESP was concerned, soils of pedon viz. 1, 2, 3, 4, 5, 6, 7 and 8 (Sultanganj, Bihpur, Kahalgaon and Sabour block) were non-sodic to slightly sodic in nature. The ESP value of Tal land of Pedon 1-4 varied from 5.05 to 6.15 per cent and Diara land varied from 1.83 to 3.72 (%). Similarly, the CaCO3 percentage of Tal land varied from 0.50 to 3.56 and Diara land 1.05 to 3.24 per cent, respectively. The CEC of pedon viz. 1, 2, 3, 4, 5, 6, 7 and 8 (Sultanganj, Bihpur, Kahalgaon and Sabour block). The CEC of Tal land pedon 1 to pedon 4 varied from 40.73 to 49.53 (meq/100g) and Diara land pedon 5 to pedon 8 from 14.31 to 29.18 (meq/100g). Soil Organic carbon (SOC) of entire pedon viz. 1, 2, 3, 4, 5, 6, 7 and 8 (Sultanganj, Bihpur, Kahalgaon and Sabour block) varied from 0.12 to 0.77 %. The surface and sub-surface soil of Tal and Diara land was low to medium. The organic carbon of surface and sub-surface samples of Tal land varied from 0.14 to 0.48 % and 0.12 to 0.44 %, respectively. Whereas, in case of Diara land, it varied from 0.08-0.42 % and 0.08-0.39 % in surface and sub-surface, respectively. Similarly, SOC percent of pedon 1-4 varies 0.17 to 0.73 % and pedon 5-8 varies from 0.17 to 0.77 %, respectively. Based on gathered information and generated data ‘Soil characterization, classification, production related constraints and soil-site suitability the following Conclusions are emerged out. Heavy and light s textured soils, surface soil and high bulk density in certain areas of Tal and Diara are some of the major soil physical constraints relating crop production and for which suggested technological interventions for higher crop production and income are appropriate soil conservation measures like mulching, community based small check dam, adoption of agri-silvi, agri- horti and silvi-pasture system, boundary planting of trees and horticultural crops.

References 1.

ICAR, (2005). Ad-hoc research project on Preparation of research inventory for the improvement of in the Punjab region,India. Geoderma, 81: 357-368.

2.

Sharma, B.D., Mukhopadhaya, S. And Sidhu, P.S. (1999). Microtopographic control on soil formation Tal and Ganga Diaraland soils, Rajendra Agricultural University, Bihar.

3.

Soil Survey Staff (2014). Keys to Soil Taxonomy 12th edition, National Resource Conservation Centre, USDA, Blacksburg, Virginia.

Chapter -

15

Allelopathy: a natural and an environment-friendly unique alternative tool and their effect on fruit crops Kanchan Bhamini1* and Anjani Kumar2 Department of Horticulture (Fruit & Fruit tech), BAU Sabour, Bhagalpur – 813210 2 Department of Genetics and Plant Breeding, BAU Kanke, Ranchi – 834006 * Corresponding Author : [email protected]

1

Abstract Term allelopathy elucidated as the adverse effect of chemical discharged by one plant or microorganism on another plant or microorganism. The above discharged chemicals obtained through the interaction among different species of plants or micro-organism is recognized as allelochemicals. These allelochemicals are released by different processes such as volatilization, root exudation, leaching, and decomposition of plant residues. In fruit crops, the allelopathic effect detected in two forms like alloinhibition and auto-inhibition. Several allelochemicals was analyzed in fruit crops like mangiferin in a mango, epicatechin, procyanidin A2, kaempferol-3-0-galactose and 4-hydroxybenzaldehyde in a litchi, juglone in apple, zizynummin, dammarane and saponin in ber, hydrojuglone in black walnut, kevin, yangonin, dihydromethysticin and coumarin in passion fruit. If fruit crops are closely planted, the allelopathic compounds results both stimulatory and inhibitory effect in agriculture ecosystem such as weed control, inhibition of seed germination and seedling growth and increase in soil microbial population around the rhizosphere. The allelopathic effect of different fruit crops needs further studies for their beneficial effect in agriculture ecosystem. Key words: Allelopathy, Allelopathic chemicals, Effect, Fruit crops.

Introduction Allelopathy is a biological aspect in which an organism produces one or more biochemical that effects the growth, survival and reproduction of other organisms. Etymologically, the term allelopathy was coined by Hans Molish in 1937. It is the combination of two Greek words allelon-meaning mutual and pathos-meaning harm. It means that it is the mutual influences, which may be stimulatory or inhibitory of one plant or microorganism to another plant or microorganism. According to Whittaker and Feeny (1971) allelopathic substances not only play an important role in plan communities but also plan as relationship builder among other major group of organisms. This constituted both detrimental and beneficial interactions between the plants that mediated through chemicals released by the donor. Allelopathic interactions specify the inter-organism relationship of allelopathic substances i.e.,

180


choline is an inhibitor or toxin produced by a higher plant, which affect another higher plant, phytoncide is in Gummer’s sense a substance produced by a higher plant which suppresses microorganisms and macro fungi and marasmin substance produced by microorganisms which suppress the growth of higher plant. Antibiotic releases by microorganism which suppress the growth of another microorganism. They compared both plant form and function, particularly in relation to nutrition. The root exudation may play a role in plant system as suggested by Dutchman Boerhoove. The concept of allelopathy is based on theory of root excretions. The plant interaction theory via root excretions was developed by Swiss botanist Auguste Candolle being influence of available information on phytochemistry. Molisch (1937) pointed out that ethylene influences the growth in length and thickness of seedlings, hastens the ripening of fruit in amazing ways, promotes proliferation of lenticels, hastens callus formation, hastens leaf fall, prevents the negative geotropic curvature of hypocotyls, and cancels epinastic curvatures. Tukey (1969) also discussed several important allelopathic responses in horticulture resulting from grafting or budding. Among these were dwarfing, resistance to diseases, change in time of maturation of fruits, change in size and color and quality of fruits. However, in practice, allelopathy is generally used for detrimental plant-plant interactions. Occurrence It is a natural phenomenon, representing the release in the environment of plants substances (named allelochemicals) that can exert beneficial or detrimental effect on neighboring plants (Soltys et. al., 2012). This influence is mediated by the production of chemical compounds that can lead to death or slow of the growth of other plants. Production of these substances is one of the mechanisms for the protection of plants against pests, pathogens, as well as a means of improving the competitive position of a plant species in relation to other plants (Rice, 1984). The discharge of allelopathic substances is considered a major factor influencing the distribution of species and their density and multiplication within plant communities, especially related to the success of invasive plants in both natural and agro ecosystem (Inderjit et. al., 2011; Zheng et. al., 2015). The integration of allelopathic principles into current agricultural practices can considerably attenuate the extensive utilization of pesticides (Shennan, 2008) and synthetic fertilizers, some of these resulting in devastating changes for the plants biology (Lingorski and Churkova, 2011) inducing environmental (air, soil and water) degradation. Allelopathic interactions and allelopathic compounds Allelopathy is also considered a multi-dimensional phenomenon occurring constantly in natural and anthropogenic ecosystems (Gniazdowska & Bogatek, 2005). It is a biological process where the chemical inhibition of one species by another species. The “inhibitory” chemical is released into the soil environment where it affects the growth and development of neighboring plants. This chemical is present in any part of the

Allelopathy: a natural and an environment-friendly...

181

plant. They can be found in leaves, stems, roots, flowers, fruits, seeds, pollen, rhizomes and stem bark or sometimes found in just one or two of such organs (Zeng et. al., 2008). They can also be present in the surrounding soil. These toxins affect target species in several various ways. The shoot or root may be suppressed by toxic chemicals. The interaction between plants and microorganisms by a variety of compounds usually referred to as allelopathins, allelochemicals or allelopathic compounds. Allelopathins are products of the secondary metabolism and are non-nutritional primary metabolites (Weir et. al., 2004; and Iqbal & Fry, 2012). It is isolated from plant tissue, collected from exudates or even synthetic compounds identical to natural ones. These compounds belong to numerous chemical groups including : triketones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, strigolactones, phenolic acids, fatty acids, tannins, lignin, and non-protein amino acids. Allelochemicals can be classified into 10 categories (Li et. al., 2010) according to their different structures and properties such as water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes and ketones; simple lactones; long-chain fatty acids and polyacetylenes; quinines (benzoquinone, anthraquinone and complex quinines); phenolics; cinnamic acid and its derivatives; coumarins; flavonoids; tannins and steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids). There are mainly two types of allelopathy which are direct and indirect allelopathyIf one plant or microorganism inhibit the growth of another plant or microorganism without help of another mediator called direct allelopathy or primary allelopathy but in case of indirect allelopathy an intermediate organism is involved. The mode of action of a chemical can be grouped into a direct and an indirect action (Rizvi et. al., 1992). Indirect action represent the effects through the alternation of soil properties, nutritional status and an altered population or activity of microorganisms and nematodes. The direct action of allelopathic compound involves the biochemical, physiological effects of allelochemicals on various types of important processes of plant growth and metabolism. The various processes affected by allelochemicals are mineral uptake i.e., these chemicals can change the rate at which ions are absorbed by plants. This is also reported that the reduction in both macro and micronutrients are encountered in the presence of phenolic acids (Rice, 1974). Phytohormone and plant growth hormones for example, indole acetic acid (IAA) and gibberellins (GA) control cell enlargement in plants. IAA is present in both active and inactive forms, which is inactivated through IAA- oxidase. IAA oxidase is also encountered by various allelochemicals (Rice, 1974). Other inhibitors block GA-induced extension growth. Exudation of compounds from roots on root slices have been used as an index of permeability because plant membranes are very difficult to study (Harper and Balke, 1981). Photosynthesis photosynthetic inhibitors may be electron inhibitors or uncouplers, energy transfer inhibitors electron acceptors or a combination of the above (Einhellig and Rasmussen, 1979; Patterson, 1981). Respiration allelochemicals can stimulate or inhibit respiration, both of which can be harmful to the energy producing process (Rice, 1974). Under natural conditions the action of allelochemicals seems to be organised by a fine tuned regulatory process where various compounds may act together on one or more of the above processes (Rizvi et. al., 1992).

182


Allelopathic compound of fruit crop and their roles The allelopathic effect in fruit crop is observed in two ways allo-inhibition and autoinhibition. Many allelochemicals was identified in different fruit crops. Mangiferin compounds are extracted from mango leaves, are used for weed management in rose basins. These compounds were identified as mangiferin which is basically 1, 3, 6, 7-tetra hydroxyl 2-C-B-glucopyranosylxanthone. Old mango leaves extract was found most effective in wheat germination and growth than that of young leaves. Young leaf extract reduced shoot length and grain weight of wheat. This indicated that total phenolic contents were more in new mango leaves as compared to old ones (Venkateshwarlu et. al., 2001). Phenolic compounds such as 4-hydroxybenzaldehyde, m-coumaric, pcoumaric, 4-hydroxy benzoic, vanillic, caffeic, gallic and protocatechuic acids were identified through Mass Spectrometry and High Performance Liquid Chromatography in mango leaves. It was also observed that leaves extract of old mango leaf could be used as herbicide to suppress the canary grass and enhance the germination of wheat (Saleem et. al., 2013). The allelopathic potential of Litchi chinensis utilises as natural herbicide in the field. Leaf extracts of litchi tree applied on test weeds, which slow down the radical growth of it (Islam et. al., 2013). Chromatography, mass analysis and NMR spectroscopic analysis identified epicatechin, procyanidin A2, Kaempferol-3-0-galactose and 4-hydroxybenzaldehyde as an allelopathic compound in litchi. Litchi leaf powder reduced the growth of weeds including Bidens pilosa, Eleusine indica, and Portuiaca oleracea, which further reduce their competition with main crop. It shows that beneficial usage of litchi leaf as natural herbicides. In case of Ber fruit crop, allelochemicals such as Zizynummin, Dammarane, Saponin are found in Ber fruit. These allelochemicals shows different allelopathic effect in Ber fruit like root exudates, leaf leachates, litter decomposition, volatile toxicants and sick soil toxicants. Black walnut is considered as the most notorious of allelopathic trees. It has long been recognized that the principal chemical responsible for walnut allelopathy is a phenolic compound called juglone (5-hydroxy-1, 4- naphthoquinone). However, installation of polyethylene root barriers proved to be efficient in preventing juglone from getting into the alley where associated crop species are normally planted. This implies that management practices such as root pruning, fertilizer injection, or root disking can be used to limit the impacts of juglone. A colorless nontoxic reduced form called harmless hydrojuglone is abundant, especially in leaves, fruit hulls, stem and roots of walnut. When exposed to air or oxidizing substances, hydrojuglone is oxidized to its toxic form, juglone. Rain washes juglone from the leaves and carries it into the soil. Thus, neighboring plants of the walnut are affected by absorbing juglone through their roots. This allelochemicals is found in leaf extracts of Black walnut (Juglans regia L.), which shows allelopathic influence on yield, growth, chemical and nutritional composition of fruit.


183

In another study are bioassay and greenhouse studies of allelopathic effect of passion fruit on germination and growth of indicator plants of paddy [barnyard grass (Echinochloa crusgall), monochoria-(Monochoria vaginallis), radish (Raphanus sativus) and lettuce (Lactuca sativa)] and major paddy weeds. Various chemical substances, which belonging to alkaloids, phenolics, flavonoids and volatiles were found in passion fruit; these are kavin, yagonin, dihydromethysticin and coumarin. These allelochemicals completely suppressed the barnyard grass growth so it may be used as a natural herbicide in place of synthetic herbicides and other agrochemicals, which is injurious to human health and environment (Khanh et. al., 2008). In another study Terminalia catappa (tropical almond) fruit or leaf extracts were applied to the weeds E.heterophylla and C. bengalensis. Allelopatic compound was identified as dichloromethane and ethyl-acetate. Allelochemical potential of T. catalpa is highest in fruit extract compared to leaf extract. It suppresses the growth of weeds E. heterophylla and C. bengalensis (Baratelli et. al., 2012). These allelochemicals are used to treat venous insufficiency and haemorrhoidal symptomatology. Allelochemicals, juglone having a maximum potential as a specific bactericidal effects on E. amylovora, which is causal organism of fire blight in apple. It shows both inhibitory and stimulatory effect on other living organism in different fruit crops. This allelopathic compound also affects the agriculture ecosystem. Table.1: Allelopathic compounds of some fruit crop Fruit crop

Allelochemicals

References

Mango

Mangiferin (1,3,6,7-tetra hydroxyl 2-C-B glucopyranosylxanthone)

Venkateshwarlu et. al., (2001)

4-hydroxybenzaldehyde, m-coumaric, p-coumaric,4-hydroxy benzoic, vanillic, caffeic, gallic and protocatechuic acids Epicatechin, ProcyanidinA2, Kaempferol-3-0-galactose and 4-Hydroxybenzaldehyde

Saleem et. al., (2013)

Ber

Zizynummin, Dammarane, Saponin

Black walnut

Hydrojuglone

Passion fruit

Kavin, Yagonin, Dihydromethysticin and Coumarin Dichloromethane and Ethyl-acetate

Saroj et. al., (2002) Cui et. al., (2012) Khanh et. al., (2008) Baratelli et. al.,(2012)

Litchi

Tropical almond

Islam et. al., (2013)

Growth and yield potential of fruit crops through allelopathic chemicals Allelochemicals influence plant growth and development which further decide the final yield of tree. Roots of the fruit tree release various allelochemicals in its rhizosphere which shows its effect on the adjoining crops. Juglone is allelochemicals found in leaf extracts of Persian walnut (Juglans regia L.), which shows allelopathic influence on yield, growth, chemical and nutritional composition of the strawberry

184


cultivar Fern. Juglone and undiluted walnut leaf extracts both inhibited vegetative and reproductive plant growth. It reduced fruit yield per plant, number of fruit per plant, average fruit weight, crowns per plant, number of leaves, leaf area, fresh root weight, total soluble solid (TSS), vitamin C and acidity in comparison to control (Ercisli et. al., 2005). Ercisli et. al. (2005) conducted an experiment in a heated greenhouse to investigate the effects of juglone (5-hydroxy-1,4-napthoquinone) on yield, growth, chemical and plant nutrient element composition of short-day strawberry cultivars “Camarosa” and “Sweet Charlie”. Result shows that inhibition of strawberry plants growth under walnut-based intercropping systems. Murligopal et. al., (2006) reported that allelopathic effects of root and leaf leachates of coconut on selected beneficial microorganisms around coconut rhizosphere. Many agriculturally beneficial microbes, phosphate solublizers and plant growth promoting rhizobacteria provide the minimal nutrition to palms. However their population and activities are affected by chemicals (tannins and phenols) released from biomass, leachates and root exudates. The leachates affected the rhizosphere micro flora in coconut based cropping system. Role of allelochemicals as bioherbicides Some allelochemicals behave like a synthetic herbicides that means both are same mode of action. These features have permitted them to be considered for use in weed management as bioherbicides. These allelochemicals are highly attractive as new classes of herbicides due to a variety of advantages. Though, in the view of bioherbicides based on allelopathins, effects caused by these compounds on target plants are also classified as “phytotoxic”. In mango fruit, Allelopathic compounds i.e. Mangiferin which are extracted from mango (Mangifera indica L.) leaves and these are used for weed management in rose basins. Saleem et. al. (2013) observed that leaves extract of old mango leaf could be used as herbicide to suppress the canary grass and enhance the germination of wheat. In another study, the allelopathic potential of Litchi chinensis utilises as natural herbicide in the field. The leaf extracts of litchi tree applied on test weeds, which slow down the radical growth of it (Islam et. al., 2013). As well as the allelochemical potential of Terminalia catappa (tropical almond) is found highest in fruit extract compared to leaf extract. It suppresses the growth of weeds E. heterophylla and C. bengalensis (Baratelli et. al., 2012). Fungicidal effect on fruit crop through the allelochemicals Allelochemicals are reported to have antifungal activity. They have inhibitory effect on fungal growth. It also delay or complete inhibition of germination on the fruit plant species. It may be required for 50% growth inhibition on the hypocotyls/coleoptiles and roots of the fruit plants. Lovett et. al. (1989) and Liu et. al. (2011) reported that allelopathic substances can stimulate the seedlings growth at lower doses but inhibited the growth at higher doses. On the other hand, the higher sensitivity of root growth to the extracts as compared to their hypocotyls/coleoptiles might be due to the more intensive contact of roots to the extracts than their hypocotyls/coleoptiles (Qasem, 1995; Islam and Kato-Noguchi, 2013). The inhibitory effect of allelochemicals on the bacterial fire blight pathogen Erwinia amylovora found in apple. Allelopathic influence


185

of juglone from walnut fruit was restricted to browning of petals; later fruit rusting was not observed. Juglone is a most promising allelochemicals to control the growth of fire blight bacteria in a new environment to replace the bactericide antibiotic streptomycin for fire blight control (Fischer et. al., 2012).

Conclusions Allelopathy must be recognized as a dynamic process that influences the growth and development of agricultural and biological systems with positive and negative effects. Allelochemicals, as a group of substances also called bio-communicators, seem to be a productive challenge for combining traditional agricultural practices and innovative approaches for crop improvement. References 1.

Gniazdowska, A, & Bogatek, R. (2005). Alleopathic interaction between plants. Multi side action of allelochemicals. Acta Physiologiae Plantarum, Pp: 395-407.

2.

Soltys, D, Rudzinska-langwald, A, Gniazdowska, A, Wisniewska, A, & Bogatek, R. (2012). Inhibition of tomato (Solanum lycopersicum L.) root growth by cyanamide is due to altered cell division, phytohormone balance and expansin gene expression. Planta, 236(5):1629-1638.

3.

Weir, T. L, Park, S-W, & Vivanco, J. M. (2004). Biochemical and physiological mechanisms mediated by allelochemicals. Current Opinion in Plant Biology, 7(4):472-479.

4.

Iqbal, A. & Fry, S. C. (2012). Potent endogenous allelopathic compounds in Lepidium sativum seed exudate: effects on epidermal cell growth in Amaranthus caudatus seedlings. Journal of Experimental Botany, 63(7):2595-2604.

5.

Li, Z. H., Wang, Q, Ruan, X, Pan, C. D, & Jiang, D. A. (2010). Phenolics and Plant Allelopathy Molecules. doi:10.3390/molecules15128933, 15(12):8933-8952.

6.

Molisch, H. 1937. Der Einjlus einer Pfruiize aufdie aitdei-e-Allelopathie. Jena: Gustave Fishcher Verlag.

7.

Inderjit, Wardle, D.A., Karban, R., Callaway, R.M. (2011). The ecosystem and evolutionary contexts of allelopathy. Trends Ecol. Evolut. 26, 12: 655-662. doi:10.1016/j. tree.2011.08.003.

8.

Rice, E.L. (1984). Allelopathy. Academic Press, New York.

9.

Zheng, Y.-L., Feng, Y.-L., Zhang, L.-K., Callaway, R. M., Valiente-Banuet, A., Luo, D.Q., Liao, Z.-Y., Lei, Y.- B., Barclay, G. F., Silva-Pereyra, C., (2015). Integrating novel chemical weapons and evolutionarily increased competitive ability in success of a tropical invader. New Phytol. 205, 3: 1350-1359.

10.

Lingorski, V. and Churkova, B. (2011). Correlative dependences between forage chemical composition and crude protein productivity of grass – legume mixture under variable mineral fertilizing. Banat J. of Biotechnology. 2, 4: 9-13.

11.

Zeng, R.S., Malik, A.V., Luo, S.M. (2008). Allelopathy in Sustainable Agriculture and Forestry. Springer Verlag, Germany. 412p.

12.

Whittaker, R. and Feeny, P. (1971). Allelochemics: chemical interactions between species. Science. 171: 757-770.

13.

Rice E. L. (1974). Allelopathy. Academic Press.

14.

Ercisli, S., Esitken, A., Turkkal, C. and Orhan, E. (2005). Allelopathic Effects of Juglone and Walnut Leaf Extracts on Growth, Fruit Yield and Plant Tissue Composition in

186

Recent advances in Chemical Sciences and Biotechnology strawberry Cvs. ‘Camarosa’ and ‘Sweet Charlie’. Journal of Horticultural Science and Biotechnology. 80(1): 39-40.

15. Ercisli, S., Esitken, A., Turkkal, C. and Orhan, E. (2005). The allelopathic effects of juglone and walnut leaf extracts on yield, growth, chemical and PNE compositions of strawberry cv. Fern. Plant Soil Environ. 51 (6): 283–287. 16.

Cui Cui, Jingcai, Zaimin, Jiang and Shuoxin Zhang. (2012). Isolation and identification of allelochemicals in rhizosphere and adjacent soil under walnut (Juglans regia) trees. Allelopathy Journal. 29(1): 25-36.

17.

Khanh, T. D. Xuan, Linh, L. H. and Chung, I. M. (2008). Allelopathic plants: Passion fruit (Passiflora spp). Allelopathy journal. 21(2):199-206.

18.

Venkateshwarlu, Ravindra, G. V. and Challa, Prabha. (2001). Mangiferin: anallelopathin from mango (Mangifera indica L.) leaves. Allelopathy Journal. 8(2): 221-224.

19.

Patterson, D.T. (1981). Effects of allelochemicals on growth and physiological responses of soybeans (Glycine max). Weed Science. 29- 53.

20.

Rizvi, S.J.H., Haque, H., Singh, V.K. and Rizvi, V. (1992). A discipline called allelopathy. In: S.J.H. Rizvi, and V. Rizvi (eds.). Allelopathy: Basic and applied aspects. Chapmann and Hall Publishers. 1-8.

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Khanh, T. D. Xuan, Linh, L. H. and Chung, I. M. 2008. Allelopathic plants: Passion fruit (Passiflora spp). Allelopathy Journal. 21(2):199-206.

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Einhellig, F. A. and Rasmussen, J. A. (1979). Effects of three phenolic acids on chlorophyll content and growth of soybean and grain sorghum seedlings. Journal of Chemical Ecology. 5: 815.

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Harper, J.R. and Balke, N.E. (1981). Characterization of the inhibition of K+ absorption in oat roots by salicylic acid. Plant Physiology. 68:1349.

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Saroj, P. L., Sharma, B. D., Bhargava, R. and Purohit, C. K. (2002). Allelopathic influences of aqueous leaf extracts of ber (Ziziphus mauritiana) on germination, seedling growth and phytomass of ground storey crops, Allelopathy Journal. 4(1): 57-61.

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Tukey, H.B. (1969). Implications of allelopathy in agricultural plant science. Botanical Review, 35: 1-16.

26. Shennan, C. (2008). Biotic interactions, ecological knowledge and agriculture. Philosophical Transactions of the Royal Society B: Biological Sciences. 363: 717-729. 27.

Kamran saleem, Shagufta, Perveen, Nighat sarwar, Farooq, latif, Khalid, Pervaiz Akhtar; Hafiz, Muhammad and Imran, arshadpak. (2013). Identification of phenolics in mango leaves extract and their allelopathic effect on canary grass and wheat. J. bot. 45(5): 15271535.

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Gopal, Murali, Gupta, Alka, Sunil, E. and Thomas, G. V. (2006). Allelopathic effects of roots and leaf leachates of coconut on selected beneficial microorganism. Allelopathic Journal. 18(2): 363-368.

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Islam, A.K.M.M., Khan, M.S.I. and Kato-Noguchi, H. (2013). Allelopathic activity of Litchi chinensis Sonn. Acta Agriculturae Scandinavica, Section B - Soil & Plant Science, 63(8):669–675, http://dx.doi.org/10.1080/09064710.2013.850531.

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Tatiana de Gouveia Baratelli, Anne Caroline Candido Gomes, Ludger A. Wessjohann, Ricardo Machado Kuster, Naomi Kato Simas, (2012). Phytochemical and allelopathic studies of Terminalia catappa L. (Combretaceae). Biochemical Systematics and Ecology. 41: 119–125.

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Lovett, J.V., M.Y. Ryuntyu and D.L. Liu. (1989). Allelopathy, chemical communication and plant defense. J. Chem. Ecol., 15: 1193-1202.


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Liu, Y., X. Chen, S. Duan, Y. Feng and M. An.(2011). Mathematical modeling of plant allelopathic hormesis based on ecological-limiting-factor models. Dose-Response, 9: 117129.

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Qasem, J.R., (1995). The allelopathic effect of three Amaranthus spp. (Pigweed) on wheat (Triticum estivum). Weed Res., 35: 41-49.

34. Islam, A.K.M.M. and H. Kato-Noguchi, (2013). Plant growth inhibitory activity of medicinal plant Hyptis suaveolens: Could allelopathy be a cause? Emir. J. Food Agric., 25: 692-701. 35.

Thilo Christopher Fischer, Christian Gosch, Beate Mirbeth, Markus Gselmann, Veronika Thallmair, and Karl Stich. (2012). Potent and Specific Bactericidal Effect of Juglone (5-Hydroxy-1,4-naphthoquinone) on the Fire Blight Pathogen Erwinia amylovora. Journal of Agricultural and Food Chemistry. 60 (49): 12074-12081.

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Marker assisted selection a new tools of plant breeding Swapnil1*, Priyanka Kumari1, Jenny Priya Ekka1, Anjani Kumar1, Shahina Perween1, Krishna Prasad1 and Ekhlaque Ahmad2 1 Department of Genetics and Plant Breeding, Birsa Agricultural University, Ranchi 2 Zonal Research Station, Birsa Agricultural University, Chianki *Corresponding Author : [email protected]

Abstract With the increasing integration of biotechnology with the conventional breeding process, genetic mapping of major genes and quantitative traits loci (QTLs) for many important agricultural traits has increased. Marker-Assisted Breeding (MAB) is defined as the application of molecular biotechnologies, in combination with linkage maps and genomics, to alter and improve plant or animal traits on the basis of genotypic assays. This procedure have shown that the success of MAS depends upon various factors like the genetic base of the trait, the degree of the association between the molecular marker and the desired gene, the number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred. DNA markers have huge potential to improve the efficiency of conventional plant breeding via markerassisted selection (MAS). Traits related to disease resistance to pathogens and to the quality of some crop products are offering some important examples of a possible routinary application of MAS. For more complex traits, like yield, abiotic stress tolerance, disease and pest resistance ,a number of constraints have determined severe limitations on an efficient utilization of MAS in plant breeding, then also there are a few successful and useful applications in improving quantitative traits. MAS has only a small impact on plant breeding till date and there are some ways in which the potential of MAS can be realized. Achieving a substantial impact on crop improvement by MAS represents a great challenge for agricultural scientists in the coming few decades. Key words: Molecular markers, Trait, QTL, Selection, Resistance, Pyramiding.

Introduction Marker Assisted Selection or Marker Aided Selection (MAS) is an indirect selection process where any trait of interest is selected which is based on a marker linked to a trait of interest rather than on the trait itself. Marker may be morphological, biochemical, DNA or RNA variation and trait of interest may be productivity, disease resistance, abiotic stress tolerance, and quality. It is the application of molecular biotechnologies, generally molecular markers combined with linkage maps and genomics for the improvement of plant and animal traits that are based on genotypic assays.

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For example, MAS can be used to select individuals with disease or pest resistance and identifying a marker allele that is linked with disease resistance rather than the level of disease or pest resistance. These markers associate at high frequency with the gene or quantitative trait locus (QTL) of interest, because of genetic linkage between the marker locus and the disease resistance locus. Important properties of ideal markers for MAS An ideal marker is abundant in number, polymorphic, Co-dominant, insensitive to environment, multiallelic, recognizes all possible phenotypes (homo- and heterozygotes) from all different alleles, demonstrates differences in expression between trait or gene of interest alleles, early in the organism’s development. Prerequisites for an efficient marker-assisted breeding program It needs complicated equipment and facilities compared to conventional breeding. These are the pre-requisites that are essential for marker-assisted breeding (MAB) especially in plants. a. Good markers and marker system: Reliable markers and suitable marker system are very important for a marker-assisted breeding program. As discussed above, suitable markers should have attributes like easy to use and have low analysis cost, needs small quantity of DNA, co-dominant, reproducible, have high levels of polymorphism and genome wide distribution and closely associated with the target gene(s). If the markers are located in close proximity to the target gene or present within the gene, the marker will increase the success rate in selection of the gene. DNA markers are the predominant types of genetic markers which are used for MAB. These markers can be detected at any stage of plant growth, but the detection of classical markers is generally limited to particular growth stages. Compared to other markers, SSRs have most of the desirable features and are therefore the current marker of choice for most of the crops. SNPs require more detailed knowledge of the single nucleotide DNA changes responsible for genetic variation among individuals. They are also considered as an important type of marker for MAB as more and more SNPs have become available in many crop species. b. Quick DNA extraction and high throughput marker detection: Screening of hundreds or thousands of plants or individuals is required for desired marker patterns and the breeders need the results instantly to make selections in time. Thus a quick DNA extraction technique and a high throughput marker detection system are essentially required to handle a large number of samples for large-scale screening of multiple markers in breeding programs. PAGE and AGE systems are generally used for marker detection. Some labs also provide marker detection service using automated detection systems, e.g. SNP chips based on thousands to ten thousands of markers. c. Genetic maps: Linkage maps are a framework for detection of marker-trait associations and for choosing markers in marker-assisted breeding. Therefore, a genetic linkage map, particularly high-density linkage map is very important for MAB.

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d. Knowledge of marker-trait association: The knowledge of the associations between markers and the traits of interest are very important. Only those markers that are closely associated with the target traits or tightly linked to the genes can provide sufficient guarantee for the success in practical breeding. The more closely the markers are linked with the traits, higher is the possibility of success will be there. e. Efficient and fast data processing and management: In MAB for handling of large number of samples, multiple markers for each sample are also screened at the same time which requires an efficient data processing and management .With the development of bioinformatics and statistical software packages this purpose is solved. Activities of marker-assisted breeding Marker-assisted breeding involves the following activities: a. Breeding populations are planted which has potential segregation for the trait of interest or polymorphic for the markers which is used. b. Generally, at early stages of growth like emergence to young seedling stage, the plant tissues are sampled. c. DNA is extracted from tissue sample of each individual in the population and further, the DNA samples can be used for PCR and marker screening. d. PCR is run for the molecular markers that are linked to the trait of interest. e. PCR/amplified products are separated and scored with the help of appropriate separation and detection techniques like PAGE or AGE. f. Individuals carrying the desired marker alleles are identified. g. The best individuals with both desired marker alleles for target traits and desirable phenotypes of other traits are selected by jointly using marker results with other selection criteria. h. The above activities are repeated for several generations. The number of generations depends upon the linkage between the markers and the trait of interest as well as the status of marker alleles (homozygous or heterozygous) and the individuals are advanced until stable elite lines with improved traits are developed.

MAS procedure General procedure of a single cross is described below1. Selection of parents and making cross, one or both possessing DNA marker allele(s) for the trait of interest. 2. Planting of F1 population and detecting the presence of the marker alleles to remove false hybrids. 3. Planting of segregating F2 population and screening individuals for the marker(s), and harvesting the individuals carrying the desired marker allele(s).

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4. Planting F2:3 plant rows, and screening of individual plants with the marker(s). Bulking of F3 individuals within a plant row can be used for the marker screening for confirmation if the preceding F2 plant is homozygous for the markers. Selecting and harvesting the individuals which have desired marker alleles and other traits of interest. In the subsequent generations (F4 and F5), conducting marker screening and making selection similar as done in F2:3s, but focusing on superior individuals within homozygous lines 5. In F5:6 or F4:5 generations, bulking of best lines according to the phenotypic evaluation of desired trait and the performance of other traits. 6. Planting yield trials and evaluating the selected lines for yield, quality, resistance and other traits of interest. Number of markers in MAS Increasing the number of markers associated with a QTL, increases the rate of success .For more effective and accurate work- markers to be used should be close enough to the gene/QTL of interest (80% are favourable conditions for karnal bunt spread. Control measures: Seed health test and diagnosis is important step for control this disease. Sodium hydroxide soak method is use for seed health test and visual examination are use for detection the karnal bunt seed .another control measures are –always use the resistant varieties. And avoid the excessive irrigation at the time of flowering. Treat seeds Vitavax @ 2-2.5 g/kg seed for eliminating seed borne infection. Black Point Disease of wheat Black point caused mainly by Bipolaris sorokiniana, Alternaria alternata, Cladosporium cladosporioides, Curvularia lunata and Fusarium spp. is one of them (Fakir, 1998). The most commonly isolated fungus from discolored kernels is A. alternata, followed by C. sativus 10 (Fernandez et. al., 1994b). The disease is characterized by brown to black discolouration usually restricted to the embryonic end of the grain, but in case of severe infection, the whole grain may be discoloured and shrivelled (Hanson and Christensen, 1953; Adlakha and Joshi, 1974). The disease occurs almost all over the world wherever wheat is grown (Mathur and Cunfer, 1993). Black point infection becomes severe when prolonged wet weather prevails during grain filling period of the crop. Black point has an adverse effect on seed weight, germination, and seedling emergence (Khanum et. al., 1987; Rahman and Islam, 1998). Black point is an insidious disease. It first appears when the grain begins to lose moisture. Symptoms are not readily observed until the plants are harvested and the grain is threshed from the head (Southwell et. al. 1980b). Symptoms The symptoms of Black Point the embryo tip shows a black to brown discoloration that may extend into the crease of the kernel. Symptoms are not readily observed until the plants are harvested and the grain is threshed from the head (Southwell et. al. 1980b). Black point is an insidious disease. It first appears when the grain begins to lose moisture. Control measures The effectiveness in controlling BP diseases is limited by the multiplicity of its causes. The development of resistant cultivars is generally considered the most practical way to control BP, but no variety is fully resistant (Conner and Thomas 1985; Conner and Davidson 1988). Fungicide application is used for control the Black point disease.

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References 1.

Adlakha KL, Joshi LM (1974) Black point of wheat. Indian Phytopathology 27, 41-44.

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Ahmed, S.M. and C.A. Meisner. 1996. Wheat Research and Development in Bangladesh. Bangladesh-Australia Wheat Improvement Project and CIMMYT-Bangladesh. 201 pp.

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Brown, J. K. M. and Hovmoller, M. S. (2002). Aerial dispersal of pathogens on the global and continental scales and its impact on plant disease. Science 297: 537-41.

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Conner RL, Thomas JB (1985) Genetic variation and screening techniques for resistance to black point in soft white spring wheat. Canadian Journal of Plant Pathology 7, 402-407.

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Conner RL, Davidson JGN (1988) Resistance in wheat to black point caused by Alternaria alternata and Cochliobolus sativus. Canadian Journal of Plant Science 68, 351-359.

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Crous PW, Van Jaarsveld, AB Castelebury, LA Carris, LM Frederick R.D.and Pretorious, ZA (2001). Karnal bunt of wheat newly reported from the African Continent. Plant Disease 85:561.

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Duran (1972).Further aspects of teliospore germination in North American smut fungi. Canadian Journal of Botany 50:2569-2573.

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E.J. Warham, (1984) “A comparison of inoculation methods for Karnal bunt Neovossiaindica”, vol. 74, Phytopathol,, pp. 856–7.

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Fakir, G.A. (1998). Black point disease of wheat in Bangladesh. 2nd Edition. Seed Pathology Laboratory, Bangladesh Agricultural University, Mymensingh. 81 pp.

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Fernandez, M.R., J.M. Clarke, R.M. DePauw, R.B. Irvine, and R.E. Knox. (1990-1992). 1994b. Black point and red smudge in irrigated durum wheat in southern Saskatchewan in Can. J. Plant Pathol. 16: 221-227.

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Hanson EW, Christensen JJ (1953) The black point disease of wheat in theUnited States. Minnesota Agricultural Experiment Station Technical Bulletin 206, 30.

12. Konzak, C. F., Line, R. F., Allan, R. E. and Schafer,J. F. (1977). Guidelines for the production, evaluation and use of induced resistance to stripe rust in wheat. Proc. Induced Mutation to Plant Diseases, International Atomic Energy, Vienna. pp. 437-60. 13.

Line, R. F. (1972). Recording and processing data on foliar diseases of cereals. Proc. European and Mediterranean Cereal Rusts Conference, Prague. pp. 175-78.

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Line, R. F. and Chen, X. M. (1995). Successes in breeding for and managing durable resistance to wheat rusts. Plant Dis. 79 :1254-55.

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Mathur SB, Cunfer B (1993) Black point. In: Mathur SB, Cunfer B (Eds)Seed-borne Diseases and Seed Health Testing of Wheat, Danish GovernmentInstitute of Seed Pathology for Developing Countries, Copenhagen, Denmark,pp 13-21.

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Mitra M (1931).A New Bunt of Wheat in India Annals of Applied Biology 18:178-179.

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Munjal RL (1974).Technique for keeping the cultures of Neovossia indica in sporulating condition. Indian Phytopathology 27:248-249.

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Robbelen, G. and Sharp, E. L. (1978). Mode of inheritance, interaction and application of genes conditioning resistance to yellow rust.Fortschr Pflanzenzucht 9 : 88.

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Singh DV, Aggarwal R, Shreshtha JK, Thapa BR and Dubin HJ (1989). First report of Neovossia indica on Wheat in Nepal. Plant Disease 73:277.

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Southwell RJ, Wong PTW, Brown JF (1980b) Resistance of durum wheat cultivars to black point caused by Alternaria alternata. Australian Journal of Agricultural Research 31, 1097-1101.

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Wan, A. M., Chen, X. M. and He, Z. H. (2007). Wheat stripe rust in China. Aust. J. Agric. Res. 58: 605-19.

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Ykema RE, Floyd JP, Palm ME and Peterson GL (1996).First report of Karnal Bunt of wheat in the United.

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Genetically modified food a revolutionary change Priyanka Kumari, Swapnil, Jenny Priya Ekka, Anjani Kumar, Shahina Perween and S.K. Tirkey Department of Genetics and Plant Breeding, BAU Kanke, Ranchi – 834006 *Corresponding Author : [email protected]

Abstract Throughout the ages humans have used selective breeding techniques to create plant and animals with desirable traits. With the emergence of transgenic technologies, new ways to improve the agronomic performance of crops for food, feed, and processing applications have been achieved. The role of genetically modified (GM) crops for food security is the subject of community controversy. GM crops greatly contribute to increasesd food production and higher food availability. The food quality and nutrient composition have also been improved. Moreover, growing GM crops may also influence farmers’ income, thereby providing economic access to food. Even large quantities of commercially important industrial or pharmaceutical products in plants may be produced due to the expression of foreign genes. One of the reasonable steps after creating a transgenic plants to evaluate its potential benefits and risks to the environment and these should be compared to those generated by traditional agricultural practices. To monitor the release of GM organisms or frame guidelines for the appropriate application in agriculture based industries to reassure the safe use of recombinant organisms and for overall development, several agencies are available in different countries. This chapter basically deals with the genetically modified crops which brought gene revolution in food and agriculture such as herbicide tolerant, insect tolerant, drought tolerant, biofortified crops, etc.. and also the methods of gene transfer, its benefits, outcoming risks related to GM foods on the environment, human health, non target pest, creation of superweeds and impact on biodiversity. Keywords: Transgene, Resistance , Tolerance, GMO, Transformation,

Introduction History Foremost efforts to genetically modify crops were undertaken in the 1980s and were done in the tobacco plant, not to food plants. The first GM product to be released for human consumption was the Flavr Savr tomato, which was characterized by prolonged shelf-life in 1994. But unfortunately this tomato was not a commercial success, possibly due to lack of consumer acceptance, and was ultimately withdrawn from the market. There are about 200 different GM crop varieties which were accepted for

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use as food or livestock feed in the United States by 2012. Most of these GM crops (almost 90 percent) are grown in five countries—the United States, Brazil, Argentina, Canada, and India. These include varieties of soybeans, corn, sugar beets, cotton, canola, papaya, and squash. However, herbicide tolerant or insect resistant are most popular planted varieties. What Are GM Foods? GM foods are basically evolved from genetically modified organisms (GMOs), particularly plants and animals of agricultural importance. GMOs are defined as those organisms whose genomes have been altered in ways that do not occur naturally by mating or natural recombination according to World Health Organization. These foods are also known as genetically engineered foods, or bioengineered foods. The other genetically engineered agronomic traits are also being developed apart from herbicide-tolerant and insect resistant GM crops such as fungal resistance, drought tolerance, salt tolerance , and nematode resistance . Several agriculturally important animals such as goats and sheep have also been genetically modified to produce pharmaceutical products in their milk. Another exciting field of modern plant biotechnology is represented by the enhancement of crop nutritional properties through genetic modification (Rein and Herbers, 2006). For the last decade and half, conventional crops have been genetically modified for a variety of reasons including longer shelf life, improved nutritional value, enhanced agronomic traits such as herbicide tolerance, microbial/ insect resistance, and tolerance to various severe environmental perturbances (Engel et. al. 2002; Konig et. al. 2004). Basically GM crops were first introduced in 1996 into the commercial market in the United States and were promptly accepted by farmers. During 20th century prodigious success was achieved in increasing agricultural productivity to fulfill human needs due to the Introduction of GM crops (Yan and Kerr 2002; Rommens et. al. 2004). However, 90% of GM crops produced worldwide consist of just four plants : soybean, maize, rape and cotton. Moreover in Europe only two GM plants have been approved for cultivation : MON 810 maize (Monsanto) and Amflora potato (BASF). The potential benefits of agricultural biotechnology, such as improving nutritional quality or reducing pesticide use, are too great to be withheld from the majority of the world’s population due to regulatory issues. Dr. Norman Borlaug as recipient of Nobel Peace Prize noted (Borlaug, 1997), “this issue goes far beyond economics and regulatory issues since it is a matter for bottomless ethical consideration.

Methods of gene transfer The organisms are modified through genetic engineering or recombinant DNA technologies. In this technology one or more genes are to be cloned and transferred from one organism to another. The organism is called transgenic when genes are transferred between unrelated species and cisgenic when gene transfer takes place within a species.

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The prerequisites for genetic transformation in plants are construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the essential controlling sequences i.e. promoter and terminator, thereby introducing the genes into the host plant. The gene transfer methods in plants are mainly categorized into two :

Vector-mediated or indirect gene transfer The foreign genes are basically transferred by a particular vector into the host plant. The Ti plasmid of Agrobacterium tumefaciens has been extensively used amongst the various vectors used in plant transformation. They are also known as “natural genetic engineer” of plants because they have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and an unorganized growth of a cell occurs known as crown gall. Ti plasmids are specifically used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The region in which desired foreign gene is cloned is the T-DNA region of Ti-plasmid . Procedure for Transformation The leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected to transform plants . The collected parts of plants are then infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissues are cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant

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regeneration medium which is supplemented with lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are kept in root-inducing medium, and complete plants are transferred after another 3-4 weeks, to soil thereby hardening (acclimatization) of regenerated plants. To discover the presence of foreign genes in the transgenic plants, molecular techniques like PCR and southern hybridization are used .

Vectorless or direct gene transfer In these, the foreign gene of interest is delivered into the host plant cell without the aid of a vector which includes various methods listed below. Chemically mediated gene transfer: To induce DNA uptake into plant protoplasts certain chemicals are used such as polyethylene glycol (PEG) and dextran sulphate. Sometimes Calcium phosphate may also be used to transfer DNA into cultured cells. Microinjection: In this technique the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometer diameter) glass needle or micropipette. It is basically used to incorporate DNA into large cells such as oocytes, eggs, and the cells of early embryo. Electroporation: This process requires high voltage which are applied to protoplasts/ cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA. For this the cells are placed in a solution containing DNA and are subjected to electrical shocks to cause holes in the membranes through which the foreign DNA enter into the cytoplasm and then to nucleus. Particle gun/Particle bombardment: This method was initially named as biolistics by its inventor Sanford (1988). Here , the foreign DNA containing the genes which is to be transferred is coated on top of the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded against the target tissue or cells with the help of a particle gun also called as gene gun, shot gun or microprojectile gun. Transformation: This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The uptake of plasmid DNA by E. coli takes place which is carried out in ice cold CaCl2 (0-50C) supervised by heat shock treatment at 37-450C for about 90 sec. The CaCl2 breaks the cell wall at certain regions and binds the DNA to the surface of the cell. Liposome mediated gene transfer or Lipofection: The circular lipid molecules along with an aqueous interior containing nucleic acids are known as liposomes. The DNA fragments are encapsulated with liposomes which adheres to the cell membranes to transfer DNA fragments. Consequently, the DNA incorporates into the cell and then to the nucleus. Lipofection is a very efficiently used to transfer genes in bacterial, animal and plant cells. Some of the genetically modified crops have been listed below which may be of major concern relating to its yield, resistance against biotic and abiotic stress, improvement in its quality and nutrient enriched food.


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Golden rice: All the GM crops presently available in the market are traits that benefit the farmers (‘input traits’), but none of them are benefiting the consumer directly (‘output traits’). ‘Golden Rice’is one of the classic example of ‘output trait’ . Its development started in 1992 and is estimated to be commercially released in 2011 in the Philippines and India (Mayer, 2007). From the humanitarian prospective, this 20year wait for the release of ‘Golden Rice’ was however found harmful if we consider that 124 million children who are deficient in vitamin A (Humphrey et. al., 1992), and fortunately improved nutrition in vitamin A and could prevent 1 to 2 million deaths annually (West et. al., 1989). Paine et. al. (2005) hypothesized that the daffodil gene encoding phytoene synthase (psy) was the limiting step in β-carotene accumulation. Through systematic testing of other plant psy genes, they identified a psy from corn that considerably increased carotenoid accumulation in a model plant system. When expressed in rice, this ‘Golden Rice 2’ accumulated total carotenoids of up to 37 µg·g-1, or a 23-fold increase compared to the original Golden Rice. This translates to delivering 50% of the children’s Recommended Daily Allowance (RDA) in 72 g of dry new ‘Golden Rice 2’ (Paine et. al., 2005). Golden potatoes: Recently, the accumulation of carotenoids was further increased and engineered in GM potatoes (‘Golden potatoes’). Assuming a β-carotene to retinol conversion of 6:1 , this newly developed GM crop is sufficient to provide 50% of the RDA of vitamin A with 250 g (fresh weight) of ‘golden potatoes’ according to Diretto (Diretto et. al., 2007). Vitamin-Rich Tomato: It contains carrot Gene which are transferred naturally through the bacterium Agrobacterium that infects plants. It carries some genes on a circular piece of DNA called a plasmid and inserts those genes into plant cells. MON 810 maize: It contains a Cry gene from the bacterium Bacillus thuringiensis, which expresses a Bt protein, a toxin of insecticidal properties. Amflora potato: It was modified in such way as to avoid production of amylose starch, and to produce only amylopectin starch, which is desirable in many industrial applications (papermaking, fibre and adhesive sectors). Amflora is therefore an industrial potato, but one cannot exclude that it can, unintentionally, be introduced into the human and farm animal food chain. Many European countries have banned the cultivation of GM maize, and some have banned the GM potato. Potato Vaccines: Researchers tried making a cholera vaccine using plants. The bacterial disease Cholera causes deadly diarrhea that spreads rapidly where people don’t have clean water and kills two to three million children each year. Researchers pinpointed part of the cholera bacterium that the human immune system can recognize, so it could be used as a vaccine. Scientists found the genes that make that bacterial part. After some trial and error, they put those genes into potatoes to turn potatoes into a handy vaccine.

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The use of genetically modified crops in developing countries Herbicide tolerance: A transgene confers tolerance to a specific herbicide. This trait allows farmers to apply a herbicide which acts on a wide range of weeds while not affecting the modified crop. Currently, the most commonly used GM trait worldwide is Herbicide tolerance, for example in soybean, maize, cotton and oil seed rape. Herbicide tolerant crops are chiefly grown in developed countries with the primary aim of reducing applications of herbicides. Insect/pest resistance: A transgene produces toxins against the specific insects feeding on the crop. Such genes have led to significant reductions in the use of pesticides and insecticides. Insect-resistant cotton, maize and potato varieties are being grown in both developed and developing countries. Overall, Bt crops reduced insecticide usage in the USA by 3,700 tons in 2005, providing benefits for human health and the environment (Sankula, 2006). The risk to non-target insects from Bt pollen is much less than that from insecticides that would be used if the crop did not contain the Bt trait (Pimentel and Raven, 2000). Bacterial, fungal and viral resistance: These transgene provide resistance against biotic stresses such as plant pathogens which often reduce yields to a large extent. Examples of crops in which these traits are being introduced include coffee, bananas, cassava, potato, sweet potato, beans, wheat, papaya, squash and melon. Abel et. al. (1986) reported that transgenic tobacco expressing the coat protein (CP) gene of Tobacco mosaic virus (TMv) was resistant to TMv, which spurred the development of virus resistant transgenic crops. Since then, the initial hope that pathogen-derived resistance (Sanford and Johnson, 1985) might be a practical way to control plant viruses has been firmly established and applied to many viruses and crops (Beachy, 1997). Abiotic stress resistance: Some plants are able to survive in harsh climatic or soil conditions due to the presence of resistant gene. Therefore it can be isolated and introduced into commercial crops. Such applications are particularly valuable for those countries, where abiotic stresses such as drought, heat, frost and acidic or salty soils are common. Research on crops such as cotton, coffee, rice, wheat, potato, Brassica, tomato and barley varieties is currently in different stages of completion. Micronutrient enrichment: Transgenes could play a crucial role in the provision of vitamins or minerals which may eventually prevent malnutrition. GM crops could help to provide people with essential micronutrients through consumption of their main staple crop. Research in this area is currently being undertaken in rice, cassava, millet and potato. Risks emanating from the use of genetically modified organisms Toxins and Poisons: Genetically engineered products may have the potential to be toxic and a threat to human health. However, Harrison et. al. (1996) assessed CP4 EPSPS enzyme that was introduced into soybean to produce a herbicide tolerant crop rapidly degrades during digestion and are found nontoxic.


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Antibiotic resistance: When gene engineers splice a foreign gene into a plant or microbe, they often link it to another gene, called an antibiotic resistance marker gene (ARM). These ARM genes might unexpectedly recombine with disease-causing bacteria or microbes in the environment or in the guts of animals or humans and cause antibiotic resistance. Food Allergies: Genetic engineering is capable of introducing allergens into recipient plants (Nordlee et. al. 1996). Imported genes spliced into food could induce potentially fatal allergies due to novel proteins. Contamination: The non-genetically engineered products are contaminated with genetically engineered products (horizontal or collateral gene transfer) or of endproducts with by-products of GMO-based processes. Agriculture: As transgenic traits tend to be unstable, they could break down and revert to flower development, spreading transgenes to native trees, or creating pollen that poisons bees and other pollinators as well as causing potential harm to human beings. Detrimental effects on nontarget species and the environment: Transgenic crops that express insecticidal transgenes to control agricultural pests may also affect nontarget organisms (Hilbeck et. al. 1998; Saxena et. al. 1999). The ecological risks of releasing transgenic Bt plants could be its toxic effects on organisms that are not pests of the crop itself but are predators and parasites of pests and, therefore, of benefit to agriculture. Lacewing larvae fed on caterpillars reared on one specific variety of transgenic corn (Cry1Ab) showed an increased mortality rate compared with those fed on caterpillars reared on non-transgenic corn (Hilbeck et. al. 1998). However, these problems may be directly related to the application of Bt toxin and not due to cultivation of crops carrying Bt gene as obvious from the studies on the effects of spraying of Bttoxin on nontarget organisms or pests (Janmaat and Myers 2003; Schoenly et. al. 2003; Tabashnik et. al. 1997). Impact on Biodiversity: It was predicted that GM crops could be a threat to the crop diversity or outgrow a local flora to the detriment of native species (Rissler and Mellon 1993) in Mexican maize landraces. The study also addresses socioeconomic and ethical implications of use of GM crops thereon (Garcia et. al. 2005). Creation of new viruses and bacteria due to pest resistance: Genetically altering plants to resist viruses can cause viruses to mutate into new, more virulent forms. The release and wide spread cultivation of GM crops with pest or disease resistance has raised concerns that this will impose intense selection pressure on pest and pathogen populations to adopt to the resistance mechanism. Resistance to transgenic proteins by insect pests may possibly limit the duration that an insecticidal transgenic variety can be feasibly grown. For example, the diamond black moth, an important pest to Brassica crop worldwide, was the first documented pest to develop resistance to Bt toxins applied as microbial formulations in open field populations (Tabashnik 1994). Creation of “Superweeds” and “Superpests”: The significance of assessing weedy characteristics when considering the invasiveness of GM crops has been the subject

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of much debate (Fitter etal. 1990). This would be the genetic engineering of crops to be herbicide-resistant or to produce their own pesticide. Under constant pressure from genetically engineered crops common plant pests such as cotton boll worm may evolve into “superpests” which would be completely immune to pesticides such as BT-sprays and several other ecologically sustainable biopesticides. Some weed-like characteristics are observed in some crops such as Medicago sativa, Brassica napus, and Brassica rapa, Helianthus annuus and Oryzae sativa . Thus their transgenic and novel traits possibly will allow the crop itself to become weedier and invasive (Raybould and Gray 1993; Regal 1994).

Conclusion The only conclusion to be drawn by considering both benefits and concerns raised by GM food is that neither full-scale adoption nor full-scale rejection is a viable option. The technology may be more appropriate for farmers that have difficulty in spraying pesticides and herbicides. The uncertainty in surrounding due to consumers acceptance of GM products, particularly in foreign markets, is a risk that may simply be unacceptable to some farmers. Certainly, GM products are a revolutionary technology in the agricultural industry. Genetically-modified foods have the potential to solve many difficulties such as the world’s hunger and malnutrition problems. Moreover it helps , protects and preserves the environment by enhancing yield and reducing reliance upon chemical pesticides and herbicides. But still, numerous challenges are confronted by governments, especially in the areas of safety testing, regulation, international policy and food labeling. Due to such enormous potential benefits scientists senses that genetic engineering is the unavoidable wave of the future which cannt . However, scientist are proceeding with caution to prevent unintended harm to human health and the environment as a result of our enthusiasm for this powerful technology. References 1.

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Halpin C.(2005). Genes stacking in transgenic plants—the challenge for 21st century plant biotechnology. Plant Biotechnol J 3:141–155.

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Harrison L.A, Bailey MR, Naylor MW, Ream JE, Hammond BG, Nida DL, Burnette BL, Nickson TE, Mitsky TA, Taylor TA, Fuchs RL, Padgette SR. (1996). The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikinate-3-phosphase synthase from Agrobacterium sp. strain CP4 is rapidly digested in vitro and is not toxic to acutely gavaged mice. J Nutr 126:728–740.

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Hilbeck A, Baumgartner M, Fried PM, Bigler F. (1998). Effects of transgenic Bacillus thuringiensis corn-fed prey on mortality and development time of immature Chrysoperla carnea (Neuroptera: Chrysopidae). Environ Entomol 27:480–487.

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Huang, G., Allen, R. Davis, E.L. Baum, T.J. and Hussey, R.S. (2006). engineering broad rootknot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc. Natl. Acad. Sci. USA 103:14302-14306.

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19. Konig, A., Cockburn, A, Crevel, R.W.R, Debruyne, E, Grafstroem, R, Hammerling, U, Kimber, I, Knudsen I, Kuiper, H.A, Peijnenburg AACM, Penninks AH, Poulsen M, Schauzu M, Wal JM .(2004). Assessment of the safety of foods derived from genetically modified (GM) crops. Food Chem Toxicol 42:1047–1088. 20.

Mayer, J. (2007). Golden Rice and nutritional enhancement of seeds. lecture presented at the University of California, Davis, United States. September 19, 2007. www.goldenrice. org

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Rein, D., and Herbers. K. (2006). Enhanced nutritional value of food crops. Pages 91117. In: N.G. Halford (ed.). Plant Biotechnology: Current and Future Applications of Genetically Modified Crops. J. Wiley, Chichester, England.

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A Beam on nanoparticles features and future Prerna Kumari1, Rohit kumar2 and Vikash Kumar Jha3* Deptt. of Plant Breeding & Genetics, Bihar Agricultural University Bhagalpur. 2 Deptt. of Mol. Biology & Genetic Eng., Bihar Agricultural University Bhagalpur. 3 P.G. Deptt. of Biotechnology, T.M Bhagalpur University Bhagalpur. *Correspondence author: - [email protected] 1

Abstract The Nanoparticle (NPs) is the most fundamental component in the fabrication of a nanostructure, and is far smaller than the world of everyday objects that are described by Newton ’ s laws of motion, but bigger than an atom or a simple molecule that are governed by quantum mechanics. In general, the size of a Nanoparticle spans the range between 1 and 100 nm. In this review article, we provide a general overview on the different types, characterizations, properties and applications of NPs. The last section is also provided with the future aspects. Keywords: Nanotechnology , Nanoparticles Types , Applications , Fabrication

Introduction Metallic nanoparticles have different physical and chemical properties from bulk metals (e.g., lower melting points, higher specific surface areas, specific optical properties, mechanical strengths, and specific magnetizations), properties that might prove attractive in various industrial and medicinal applications. the optical property is one of the fundamental attractions and a characteristic of a nanoparticle. For example, a 20 nm gold nanoparticle has a characteristic wine red color. A silver nanoparticle is yellowish gray. Nanotechnology refers to the creation and utilization of materials whose constituents exist at the nano-scale and by convention, be up to 100 nm in size. Types It’s broadly divided into various categories depending on their morphology, size and chemical properties. Based on physical and chemical characteristics, some of the well known classes of NPs are given as below. 1. Carbon-based NPs Fullerenes and carbon nanotubes (CNTs) represent two major classes of carbon-based NPs. Fullerenes contain nanomaterial that are made of globular hollow cage such as allotropic forms of carbon. They have created noteworthy commercial interest also in nanocomposites for many commercial applications such as fillers (Saeed and Khan, 2016, 2014), efficient gas adsorbents for environmental remediation (Ngoy et. al.,

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2014), and as support medium for different inorganic and organic catalysts (Mabena et. al., 2011). 2. Metal NPs Metal NPs are purely made of the metals precursors. Due to well-known localized surface plasmon resonance (LSPR) characteristics, these NPs possess unique optoelectrical properties. NPs of the alkali and noble metals i.e. Cu, Ag and Au have a broad absorption band in the visible zone of the electromagnetic solar spectrum. The facet, size and shape controlled synthesis of metal NPs is important in present day cutting-edge materials (Dreaden et. al., 2012). Due to their advanced optical properties, metal NPs find applications in many research areas. Gold NPs coating is widely used for the sampling of SEM, to enhance the electronic stream, which helps in obtaining high quality SEM images. Ceramics NPs Ceramics NPs are inorganic nonmetallic solids, synthesized via heat and successive cooling. They can be found in amorphous, polycrystalline, dense, porous or hollow forms (Sigmund et. al. 2006). Therefore, these NPs are getting great attention of researchers due to their use in applications such as catalysis, photocatalysis, photodegradation of dyes, and imaging applications. (Thomas et. al., 2015). 3. Semiconductor NPs Semiconductor materials possess properties between metals and nonmetals and therefore they found various applications in the literature due to this property. Semiconductor NPs possess wide bandgaps and therefore showed significant alteration in their properties with bandgap tuning. Therefore, they are very important materials in photo catalysis, photo optics and electronic devices. As an example, variety of semiconductor NPs are found exceptionally efficient in water splitting applications, due to their suitable band gap and band edge positions. 4. Polymeric NPs These are normally organic based NPs and in the literature a special term polymer nanoparticle (PNP) collective used for it. They are mostly nanospheres or nanocapsular shaped (Mansha et. al., 2017). The former are matrix particles whose overall mass is generally solid and the other molecules are adsorbed at the outer boundary of the spherical surface. In the latter case the solid mass is encapsulated within the particle completely (Rao and Geckeler, 2011). 5. Lipid-based NPs These NPs contain lipid moieties and effectively using in many biomedical applications. Generally, a lipid NP is characteristically spherical with diameter ranging from 10 to 1000 nm. Like polymeric NPs, lipid NPs possess a solid core made of lipid and a matrix contains soluble lipophilic molecules. Surfactants or emulsifiers stabilized the external core of these NPs (Rawat et. al., 2011). Lipid nanotechnology is a special

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field, which focus the designing and synthesis of lipid NPs for various applications such as drug carriers and delivery and RNA release in cancer therapy.

Property and features 1. Electronic and optical properties The optical and electronic properties of NPs are Inter dependent to greater extent. For instance, noble metals NPs have size dependent optical properties and exhibit a strong UV–visible extinction band that is not present in the spectrum of the bulk metal. This excitation band results when the incident photon frequency is constant with the collective excitation of the conduction electrons and is known as the localized surface plasma resonance (LSPR). LSPR excitation results in the wavelength selection absorption with extremely large molar excitation coefficient resonance Ray light scattering with efficiency equivalent to that of ten fluorophores and enhanced local electromagnetic fields near the surface of NPs that enhanced spectroscopies. It is well established that the peak wavelength of the LSPR spectrum is dependent upon the size, shape and inter particle spacing of the NPs as well as its own dielectric properties and those of its local environment including the substrate, solvents and adsorbents (Eustis and El- Sayed, 2006). Gold colloidal NPs are accountable for the rusty colors seen in blemished glass door/windows, while Ag NPs are typically yellow. Actually, the free electrons on the surface in these NPs (d electrons in Ag and gold) are freely transportable through the nanomaterial. 2. Magnetic properties Magnetic NPs are of great curiosity for investigators from an eclectic range of disciplines, which include heterogenous and homogenous catalysis, biomedicine, magnetic fluids, data storage magnetic resonance imaging (MRI), and environmental remediation such as water decontamination. The literature revealed that NPs perform best when the size is less than critical value i.e. 10–20 nm (Reiss and Hu¨ tten, 2005). The uneven electronic distribution in NPs leads to magnetic property. These properties are also dependent on the synthetic protocol and various synthetic methods such as solvothermal, co-precipitation, micro-emulsion, thermal decomposition, and flame spray synthesis can be used for their preparation (Wuet. al., 2008). 3. Mechanical properties The distinct mechanical properties of NPs allow researchers to look for novel applications in many important fields such as tribology, surface engineering, nanofabrication and nano manufacturing. Different mechanical parameters such as elastic modulus, hardness, stress and strain, adhesion and friction can be surveyed to know the exact mechanical nature of NPs. Beside these parameters surface coating, coagulation, and lubrication also aid to mechanical properties of NPs. NPs show dissimilar mechanical properties as compared to microparticles and their bulk materials. Fruitful outcomes in these fields generally need a deep insight into the basics of the mechanical properties of NPs, such as elastic modulus and hardness, movement law, friction and interfacial adhesion and their size dependent characteristics (Guo et. al., 2014).

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4. Thermal properties The oxides and metals NPs have thermal conductivities higher than those of fluids in solid form. For example, the thermal conductivity of copper at room temperature is about 700 times greater than that of water and about 3000 times greater than that of engine oil. It is desirable to use the particles with large total surface area. The large total surface area also increases the stability suspension (Lee et. al., 1999). Recently it has been demonstrated that the nanofluids consisting of CuO or Al2O3 NPs in water or ethylene exhibit advance thermal conductivity (Cao, 2002).

Future aspect 1. Drugs and medications The use nanoparticle in therapy is very similar to use of Swarn Bhasma in Ayurveda. NPs have drawn increasing interest from every branch of medicine for their ability to deliver drugs in the optimum dosage range often resulting in increased therapeutic efficiency of the drugs, weakened side effects and improved patient compliance. The development of hydrophilic NPs as drug carrier has represented over the last few years an important challenge. Super paramagnetic iron oxide NPs with appropriate surface chemistry can be used for numerous in vivo applications such as MRI contrast enhancement, tissue repair, and immunoassay, detoxification of biological fluids hyperthermia, drugs delivery and cell separation. TiO2, ZnO, BiVO4, Cu- and Ni-based NPs have been utilized due to their suitable antibacterial efficacies. 2. Manufacturing and materials The Nanocrystalline materials provide very interesting substances for material science since their properties deviate from respective bulk material in a size dependent manner. Manufacture NPs display physicochemical characteristics that induce unique electrical, mechanical, optical and imaging properties that are extremely looked-for in certain applications within the medical, commercial, and ecological sectors (Todescato et. al., 2016). 3. In Environment The removal of heavy metals such as mercury, lead, thallium, cadmium and arsenic from natural water has attracted considerable attention because of their adverse effects on environmental and human health. Superparamagnetic iron oxide NPs are an effective sorbent material for this toxic soft material. So, for no measurements of engineered NPs in the environment have been available due to the absence of analytical methods, able to quantify trace concentration of NPs (Mueller and Nowack, 2008). Photodegradation by NPs is also very common practice and many nanomaterials are utilized for this purpose. Rogozea et. al. used NiO/ZnO NPs modified silica in the tandem fashion for photodegradation purpose. The interaction of contaminants with NPs is dependent on the NPs characteristics, such as size, composition, morphology, porosity, aggregation/disaggregation and aggregate structure.

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4. In Electronics The good example of the synergism between scientific discovery and technological development is the electronic industry, where discoveries of new semiconducting materials resulted in the revolution from vacuumed tubes to diodes and transistors, and eventually to miniature chips (Cushing et. al., 2004). 5. Energy harvestingThe Recent studies warned us about the limitations and scarcity of fossil fuels in coming years due to their non renewable nature. Therefore, scientists shifting their research strategies to generate renewable energies from easily available resources at cheap cost. They found that NPs are the best candidate for this purpose due to their, large surface area, optical behavior and catalytic nature. Especially in photocatalytic applications, NPs are widely used to generate energy from photoelectron-chemical (PEC) and electrochemical water splitting. 6. Mechanical industries The NPs can offer many applications in mechanical industries especially in coating, lubricants and adhesive applications. Besides, this property can be useful to achieve mechanically stronger Nano devices for various purposes. Alumina, Titania and carbon based NPs successfully demonstrated to get the desirable mechanical properties in coatings.

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Molecular techniques for improvement of agricultural yield Anil Kumar1*, N. K. Sharma1, Komal Shekhawat1, Rekha Kumari2 and Swarnlata Kumawat1 1 Department of Genetics and Plant Breeding, Swami Keshwanand Rajasthan Agriculture University, Bikaner- 334 006 2 Department of Plant Breeding and Genetics, Bihar Agricultural University, Sabour, Bhagalpur-813 210 *Corresponding author: [email protected]

Introduction Man discovered agriculture almost 10,000 years ago. Agriculture is domestication of wild plants for personal use of humans. Food is the necessity for sustainability of human life on this planet. Various crops have been harvested since thousands of years. However, it was not possible to cope with the demand of food by using conventional methods of cultivation. There was need to develop new approaches to improve the quality and quantity of yield. Past five decades global food grain production is growing as with increasing population but still 1 billion persons of the world are malnourished because of food insecurity (Hazell and Wood, 2008). It has been estimated that worldwide food production must be increased by 70 % by the year 2050 to fulfill the need of expanding population and growing consumption of food (Godfray et. al., 2010). Plant breeding is based around the identification and utilisation of genetic variation. The breeder makes decisions at several key points in the process. First in deciding on the most appropriate parents to use for the initial cross or crosses and then in the selection strategy used in identifying the most desirable individuals amongst the progeny of the cross. The efficiency of the breeding and selection process can be assessed in many different ways including the ultimate success of the varieties released and the frequency with which new varieties are produced. A major cost and logistical issue in plant breeding are the actual number of lines that need to be carried through the evaluation and selection phases of a program. Large breeding programs for annual crops may carry hundreds of thousands of lines to produce a new variety only once every few years. Field trials can be expensive and evaluation of some traits, such as quality and yield stability can be expensive to assess. Most of the plant breeding programs aim to increase yield, disease and insect resistance, abiotic stress tolerance and to improve quality characteristics. The value of new plant breeding products and varieties in increasing food production has been demonstrated time and again. To meet growing need of ever increasing human population, we need to enhance food production for sustaining food supply. Furthermore, several biotic and abiotic stresses continue to threaten crop productivity. Moreover with urbanization,

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land for cultivation is shrinking and several environment concerns involving excessive use of fertilizers and agro-chemicals, soil and water pollution including water scarcity are key issues in increasing crop productivity and food sustainability. Plant breeders therefore, has the major challenge how to increase crop productivity with limited land, limited water, limited chemicals and limited labour particularly in the context of global climate changes. In the genomics era, advances in molecular biology have opened new opportunities to accelerate plant breeding processes and in overcoming some of the above constraints limiting crop productivity. Molecular markers have become important tools in the hands of plant breeders in marker assisted breeding and for enhancing the selection efficiency for various agronomic traits in precision ‘plant’ breeding. The isolation, cloning and moving of genes from diverse biological sources into plant genomes holds promise to broaden the gene pool of crops and develop new plant varieties for specific traits that determine yield, quality, and resistance to biotic and abiotic stresses. New genomics tools will be of great value to support conventional breeding for sustainable food production especially under the climate change and meet demand of ever growing human population. Since then, major advances have been made in molecular tagging of genes/QTLs governing complex agronomic traits, identification of candidate genes and in applying marker assisted breeding for tolerance to biotic and abiotic stresses and quality traits. Recent advances in transgenic technologies, genome sequencing and functional genomics offer tremendous opportunities to support plant breeding programs. Molecular Techniques: Different type of Molecular Techniques are used for agriculture yield improvement, they are discus below.

Molecular markers Molecular markers are important genetic tools for plant breeders to detect the genetic variation available in the germplasm collection. In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers are used in molecular biology and biotechnology to identify a particular sequence of DNA in a pool of unknown DNA. Molecular markers are specific fragments of DNA that can be identified within the whole genome. Molecular markers are found at specific locations of the genome. They are used to ‘flag’ the position of a particular gene or the inheritance of a particular character. Molecular markers are phenotypically neutral. DNA markers Non-PCR Based, RFLP- Restriction fragment length polymorphism. PCR Based RAPD- Random amplification of polymorphic DNA. AFLP-Amplified fragment length polymorphism. SCAR -Sequence characterize amplified region. STS-Sequence tagged sites. EST-Express sequence tags. SNP-Single nucleotide polymorphism. SSR -Simple sequence repeats CAPS -Cleaved amplified polymorphic sequences. These markers have been utilized extensively for the preparation of saturated molecular maps (genetic and physical) and their association with genes/QTLs controlling the traits of economic importance has been utilized in several cases for marker assisted selection (MAS) (Varshney et. al. 2005b, 2006). The molecular markers can be

Molecular techniques for improvement of agricultural yield

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grouped in three main categories (Gupta et. al. 2002): (1) hybridization-based markers : restriction fragment length polymorphism (RFLP), (2) PCR based markers : random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellite or simple sequence repeat (SSR), and (3) sequence or chipbased markers : single nucleotide polymorphism (SNP), diversity array technology (DArTs) and single feature polymorphism (SFP). 3. Applications of molecular markers : 1.Measure Of Genetic Diversity 2.Finger Printing 3.Genotypic Selection 4.Genotypic Pyramidying And Introgression 5.Indirect Selection Using Quantitative Traits Loci (Qtls) 6.Marker-Assisted Selection 7.Identification Of Genotype Random amplified polymorphic DNA (RAPD) RAPD is a PCR based method, which employs single primers of arbitrary nucleotide sequence with 10 nucleotides to amplify anonymous PCR fragments from genomic template DNA. In RAPD analysis, the target sequence(s) (to be amplified) are unknown. In RAPD, PCR is generally carried out with arbitrary primers. The amplifications are visualized through agarose gel electrophoresis. For amplification to occur it is essential that primers anneal in a particular orientation (such that they point towards each other) and the primers must anneal within a reasonable distance to one another.

Advantages ♦ No prior knowledge of DNA sequences is required. ♦ Random distribution throughout the genome. ♦ The requirement for small amount of DNA (5-20 g). ♦ Easy and quick to assay. ♦ The efficiency to generate a large number of markers.

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♦ Commercially available decamer primers are applicable to any species. ♦ The potential automation of the technique. ♦ RAPD bands can often be cloned and sequenced to make SCAR (sequencecharacterized amplified region) markers. ♦ Cost effectiveness as compared to other markers. Limitations ♦ Dominant nature (heterozygous individuals cannot be separated from dominant homozygous). ♦ Sensitivity to changes in reaction conditions, which affects the reproducibility of banding patterns. ♦ Co-migrating bands can represent non-homologous loci. ♦ The scoring of RAPD bands is open to interpretation. ♦ The results are not easily reproducible between laboratories. Applications of RAPD ♦ Measurements of genetic diversity. ♦ Genetic structure of populations. ♦ Germplasm characterization. ♦ Verification of genetic identity. ♦ Genetic mapping. ♦ Development of markers linked to a trait of interest. ♦ Cultivar identification. ♦ Identification of clones (in case of soma-clonal variation). ♦ Interspecific hybridization. ♦ Verification of cultivar and hybrid purity. ♦ Clarification of parentage. Restriction fragment length polymorphism (RFLP) RFLP is a molecular marker based on the differential hybridization of cloned DNA to DNA fragments in a sample of restriction enzyme digested DNAs. RFLPs involve digestion of genomic DNA with restriction enzymes (bacterial enzymes that cut DNA at specific sequences known as restriction sites). The resulting DNA fragments are size fractionated on gel electrophoresis, transfer of fractionated DNA fragments on Nylon membranes (a process known as Southern blotting) and finally hybridization with labeled probe to visualize DNA polymorphisms. The first step in RFLP analysis is to derive a set of clones that can be used to identify RFLPs. The two primary sources of these clones for RFLP mapping of plants are cDNA clones and PstI-derived genomic clones. RFLP markers are defined by a specific enzyme-probe combination. This technique is highly reproducible, and the markers are co-dominant in their inheritance therefore, allows the differentiation of heterozygotes from homozygotes. RFLP procedure is time consuming and expensive but they have been used to generate


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saturated genetic map. RFLPs behave like any other Mendelian trait. Each band seen in a Southern blot indicates the presence of one or more restriction sites in a sequence. The sequence containing a restriction site is one allele, while the corresponding sequence missing the restriction site is the other allele. The “phenotypes” of these alleles are the differences in banding patterns, due to presence or absence of bands. RFLP loci are co-dominant (twice as much informative in a genetic cross as compared to dominant markers like RAPDs).

Amplified fragment length polymorphism (AFLP) Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for rapid screening of genetic diversity. AFLPs are DNA fragments with different nucleotide sequence of which large number of copies have been amplified via PCR. This technique is a combination of the RFLP and PCR techniques. Like RFLP, the AFLPs are highly heritable and polymorphic. The technique involves restriction digestion of DNA with two different enzymes and ligation of two adopters, selective amplification of sets of restriction fragments and gel analysis of amplified fragments. The amplified products are generally separated on a denaturing polyacrylamide gel and visualized using autoradiography. The technique is more skill demanding than RAPD and also requires more amount of DNA. The reproducibility of AFLP is ensured by using site specific adopters. AFLP method rapidly generates hundreds of highly replicable markers from DNA of any organism, and thus, they allow high resolution genotyping of fingerprinting quality. The time and cost efficiency, replicability and resolution of AFLPs are superior or equal to those of other markers [allozymes, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), microsatellites], except that AFLP methods primarily generate dominant rather than codominant markers. Because of their high replicability and ease of use, AFLP markers have emerged as a major new type of genetic marker with broad

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application in systematics, pathotyping, population genetics, DNA fingerprinting and quantitative trait loci (QTL) mapping.

Weaknesses of AFLP ♦ Proprietary technology is needed to score heterozygotes and homozygotes. Otherwise, AFLP must be dominantly scored. ♦ Developing locus-specific markers from individual fragments can be difficult. ♦ Need to use different kits adapted to the size of the genome being analyzed. Cleavage amplification polymorphisms (CAPs) The scoring of this type of marker is dependent on the variation of size of fragments following the digestion of the PCR product by a restriction enzyme. A completely new set of CAPs markers would be generated from a different restriction enzyme. polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers. The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to display RFLP. from two related individuals (for example, from two different inbred ecotypes), A/A and B/B, and from the heterozygote A/C. The amplified fragments from A/A and B/B contain two and three RE recognition sites, respectively. In the case of the heterozygote A/C, two different PCR products will be obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or


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acrylamide gel electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as doublets.

Advantages of CAPS ♦ Most CAPS markers are co-dominant and locus-specific. ♦ Most CAPS genotypes are easily scored and interpreted. ♦ CAPS markers are easily shared between laboratories. ♦ CAPS assay does not require the use of radioactive isotopes, and it is more amenable, therefore, to analyses in clinical settings. Developing CAPS markers ♦ Sequence the RFLP probe. ♦ Design primers to amplify 800–2,000-bp DNA fragments. Targeting introns or 3’ untranslated regions should increase the chance of finding polymorphisms ♦ The PCR product is cloned and sequenced. ♦ PCR amplify DNA fragments from target genotypes, separately digest the amplicons with one or more restriction enzymes. ♦ Screen the digested amplicons for polymorphism on gels stained with ethidium bromide. Simple sequence repeat (SSR) marker Microsatellite or Simple sequence repeats (SSRs) provide fairly comprehensive genomic coverage. They are amenable to automation, they have locus identity and they are multi-allelic. Many agronomic and quality traits show quantitative inheritance and the genes determining these traits have been quantified using Quantitative trait locus (QTL) tools. SSR markers have wide applicability for genetic analysis in crop improvement strategies. They are widely used in plants because of their abundance, hyper-variability, and suitability for high throughput analysis.

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Inter-simple sequence repeat (ISSR) marker Inter-simple sequence repeat (ISSR) are semi-arbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Amplification in presence of non anchored primers also has been called microsatellite-primed PCR, or MP-PCR. Such amplification does not require genome sequence information and leads to multi-locus and highly polymorphic patterns. Each band corresponds to a DNA sequence delimited by two inverted microsatellites. Like RAPDs, ISSRs markers are quick and easy to handle, but they seem to have the reproducibility problem because of the longer length of their primers. STS and SCAR markers Random amplified polymorphic DNA (RAPD) is an application of PCR where arbitrarily chosen 10 base primers are used to search for variation in DNA. RAPD data can contain artifacts and are not fully reproducible. However, RAPDs have been used to generate large number of genetic markers useful for linkage mapping quickly and cheaply. RAPD fragments can be separated on agarose gels. The excised bands from the gel can be re-amplified in to individual bands from gel slices using the original RAPDs primer. The fragments can be cloned and sequenced. The sequence data can be used to design PCR primers specific to RAPDs fragments, and use PCR to produce specific RAPD’s fragments from genomic DNA, which then can function as sequence tagged sites (STSs) or Sequenced characterized amplified region (SCAR). This method allows for rapid generation of STSs derived from RAPD fragments and eliminates the problems associated with reproducibility. Factors influencing efficiency of a marker Efficiency of markers depends on their closeness to the linked trait; how the phenotype of marker is affected by environment; consistency in phenotypic expression; how easy is to score the phenotype; and level of polymorphism. Ideally, a marker should be polymorphic, tightly linked with the trait of interest, highly heritable, co-dominant, easily scoreable and it should not affect the fitness of the individual. DNA markers have many advantages over the morphological markers. DNA markers are phenotypically neutral which is a significant advantage compared to traditional phenotypic markers, highly polymorphic, abundant, usually randomly distributed throughout the genome, easily scoreable and as such DNA markers are not affected by environment but the gene of interest may be sensitive to GxE and hence its (DNA marker) association with phenotype of the gene may vary with change of environment.

Uses of DNA markers a. DNA fingerprinting To establish distinctness among biological entities ♦ Genetic diversity studies ♦ Evolutionary studies


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b. Molecular mapping ♦ To prepare saturated genetic map ♦ Chromosome identification c. Map based cloning of genes d. Marker Assisted Selection ♦ QTLs ♦ Disease resistance e. Construction of genetic maps

Application of DNA markers in crop improvement QTL mapping Some of the most difficult tasks of plant breeders relate to the improvement of traits that show a continuous range of values. Genetic factors that are responsible for a part of the observed phenotypic variation for a quantitative trait are called quantitative trait loci (QTLs). The term QTL was coined by Gelderman. Conceptually it can be a single gene or may be a cluster of linked genes of the trait. Although similar to a gene, a QTL merely indicates a region on the genome comprised of one or more functional genes. Among such quantitative traits like yield, plant length and days to flowering etc., are important ones. Selection for quantitative traits is difficult, because the relation between observed trait values in the field (the phenotype) and the underlying genetic constitution (the genotype) is not straight forward. Quantitative traits are typically controlled by many genes, each contributes only a small part to the observed variation. The environmental variations resulting from differences in growing conditions, further create the problem to understand the relation between phenotype and genotype. In practice, this problem is typically dealt with by evaluating large and replicated trials, which allow identification of genotypic differences through statistical analysis. Plant breeders would like to utilize the quantitative traits for genetic factors that are responsible for the observed variability in quantitative traits. In a process called QTL mapping, association between observed trait values and presence/absence of alleles of markers, that have been mapped onto a linkage map is analysed. When it is significantly clear that the correlation that is observed did not result from some random process, it is proclaimed that a QTL is detected. Also the size of the allelic effect of the detected QTL can be estimated. Identification of molecular makers associated with QTL. QTLs involves three basic steps namely, scoring individuals of a random segregating population for a QTL trait; determination of the molecular genotype of each member of the population and determination of association between any of the markers and the quantitative trait. The first step is to make cross and generate marker data. In the next step generate linkage maps of molecular markers. Subsequently collect phenotypic measurements of QTL trait across the environments in replicated trials. Finally, mapping of QTL is done. The most common method of determining the association between marker and QTL is done by analyzing phenotypic observation of trait and scoring of molecular data by one-way analysis of variance and regression analysis.

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For each marker, presence of a specific fragment of DNA is considered a marker class, and all individuals (in a segregating population) possessing that marker class are considered to be positive for that class. If the variance due to a particular class is significant, then the molecular marker used to define, that class is considered to be associated with a QTL. Regression values are calculated for all the markers which have shown association with the quantitative trait which reflect the amount of total genetic variation that is explained by the specific molecular marker. There are very few examples of QTL mapping in pulse crops. In chickpea, two RIL populations were used to construct a composite linkage map with the help of RAPD, ISSR, RGA, SSR and ASAP markers (Radhika et. al. 2007). Marker trait association was observed among three yield related traits : double podding, seeds per pod and seed weight. The double podding gene was tagged by the markers NCPGR 33 and UBC 249z. Seeds per pod was tagged with TA 2x and UBC 465 markers. Eight QTLs were identified for seed weight. Duran et. al. ( 2002) developed a QTL map for plant height, pod dehiscence, number of shoots and seed diameter from inter-subspecific population of Lens culinaris ssp. culinaris x Lensculinaris ssp. orientalis. Tullu et. al., (2008) identified QTLs in lentil for earliness and plant height with RAPD, SSR and AFLP markers. Kahraman et. al. (2004) identified five independent QTLs for winter hardiness in a population of RILs derived from a cross between lentil accessions WA 8649090 x Precoz. One IISR marker Ubc 808-12 was found to be useful in MAS for predicting winter survival in segregating populations. Rubeena et. al. (2003) identified eight QTLs for ascochyta blight resistance gene in lentil through composite interval mapping. Five QTLs were identified in F2 population of ILL 5588/ILL 7537 whereas three QTLs were detected in F2 of the cross ILL 7537/ILL 6002. Young et. al. (1993) identified three QTL associated with powdery mildew resistance in mungbean, while Chaitieng et. al. (2002) and Humphry et. al. (2003) found one QTL responsible for powdery mildew resistance in Vigna species. Sholihin and Hautea (2002) identified six AFLP derived putative QTLs associated with two traits (leaf relative water content and leaf stress rating) used to measure draught tolerance. Tagging of disease resistance genes DNA based markers have shown great promise in expediting plant breeding methods. The identification of molecular markers closely linked with resistance genes can facilitate expeditious pyramiding of major genes into elite background, making it more cost effective. Once the resistance genes are tagged with molecular marker the selection of resistant plant in the segregating generations becomes easy. A chickpea linkage map was established with help of 354 molecular markers (118 STMSs, 96 DAFs, 70 AFLPs, 37 ISSRs, 17 RAPDs, eight isozymes, three cDNAs, two SCARs and three markers linked to fusarium wilt resistance) surveyed among 130 recombinant inbred lines derived from a C. arietinum × C. reticulatum (Winter et. al.2000). The fusarium wilt resistant genes for race 4 and 5 were placed on the linkage group that also contained STMS and a SCAR marker previously shown to be linked to fusarium wilt race 1. This is an indication of clustering of several fusarium


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wilt resistance genes. These markers will pave the way for MAS and searching other useful genes. DNA markers associated with two closely linked genes for resistance to fusarium wilt race 4 and 5 in chickpea were identified from a population of 131 recombinant inbred lines derived from a wide cross between Cicer arietinum and Cicer reticulatum (Benko-Iseppon et. al. 2003). With the aid of bulk segregant analysis nineteen new markers were identified in the vicinity (4.1-9 cM) of fusarium wilt resistance genes on linkage group 2, R-2609-1 showed closest linkage (2cM) with race 4 resistance locus. Gowda et. al. (2009) identified flanking markers for chickpea fusarium wilt resistance genes in a recombinant inbred line population. H3A12 and TA101 SSR flanked the Foc 1 resistance gene whereas Foc 2 was mapped between TA96 and H3A12. The H1B06y and TA194 markers flanked the Foc3 locus. Reddy et. al., (2009) performed bulk segregant analysis on a segregating population of ICPL 7035 x ICPL 8863 for identification of RAPD markers associated with pigeonpea sterility mosaic disease (PPSMD) resistance. The primer OAP18 revealed polymorphism between the parents and the resistant and the susceptible bulks. The OAP18 marker was converted into SCAR marker for identification of PPSMD resistant plants in the segregating population. Dhanasekhar et. al. (2010) identified two RAPD markers OPF04700 and OPA091375 were linked with the open and tall plant type gene in pigeonpea F2 population of the cross between TT44-4 and TDI2004-1through bulk segregant analyses . These markers were validated in 15 genotypes with open-tall plant type. Kotresh et. al. (2006) used bulk segregant analysis with 39 RAPD primers which led to identification of two markers (OPM03704 and OPAC11500) that were associated with Fusarium wilt susceptibility allele in a pigeonpea F2 population derived from GS1 x ICPL87119. Taran et. al. (2003) identified two molecular markers associated with Ascochyta blight resistance in lentil viz., UBC 2271290 linked with ral1 gene and RB18680 linked with AbR1 and a marker (OPO61250) linked with Anthracnose resistance gene were utilized for identifying lines that possessed pyramided genes in a population of 156 recombinant inbred lines (RILs) developed from a cross between ‘CDC Robin’ and a breeding line ‘964a-46’. These markers can be converted into more robust SCAR markers for routine use in marker assisted selection. Tullu et. al. (2003) tagged anthracnose resistance gene LCt-2 of lentil cultivar PI 320937 with RAPD and AFLP markers. Basak et. al. (2004) developed molecular marker linked to yellow mosaic virus (YMV) resistance gene in Vigna sp. from a population segregating for YMV disease resistance. Maiti et. al. (2010) identified molecular markers CYR1and YR4 in a F2 population for screening of MYMIV resistance genes. CYR1 co-segregated with MYMV resistance

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gene in F2 plants and F3 progenies. These two markers can be used simultaneously with the help of a multiplex PCR reaction. Katoch et. al. (2009) identified a powdery mildew resistance gene er2 in pea that was associated with a RAPD marker OPX-17_1400, exhibiting cis phase linkage (2.6 cM) in a F2 population derived from Lincoln/JI2480. The reproducibility of RAPD marker was enhanced by converting it to a sequence characterized amplified region (SCAR) marker. Ek et. al. (2005) used bulk segregant analysis on a F2 population derived from the cross 955180 x Majoret for screening of SSR markers linked with powdery mildew resistance gene in pea. Out of 315 markers, only five showed linkage with the PM resistance gene. It was noted that none of single marker was tightly linked with the gene that can be considered optimal for inclusion in a MAS program. Therefore, a combination of two markers can be utilized for selecting PM resistant plants which would result in only 1.6% false positives. Nguyen et. al. (2001) converted a RAPD marker into a SCAR(SCARW19) for selecting ascochyta blight resistance gene of lentil accession ILL5588. Rubeena et. al. (2003) identified QTLs for ascochyta blight resistance in lentil. Further validation is required to use these markers for MAS. Hamwieh et. al. (2005) mapped microsatellite markers identified from a genomic library of lentil. The linkage spanning about 751cM, consisting of 283 marker loci was derived from 86 recombinant inbred lines derived from the cross ILL 5588 × L 692-161(s) using 41 microsatellite and 45 amplified fragment length polymorphism markers. The average marker distance was 2.6 cM. Two flanking markers (SSR marker SSR592B at 8.0 cM and AFLP marker p17m30710 at 3.5 cM) were linked with fusarium resistance. Saxena (2010a) assessed the DNA polymorphism in a set of 32 pigeonpea lines screened with 30 SSR markers. Based on polymorphism of marker alleles, higher genetic dissimilarity coefficient and phenotypic diversity for Fusarium wilt and sterility mosaic disease resistance data, five parental combinations were identified for developing genetically diverse mapping populations suitable for the development tightly linked markers for Fusarium wilt and sterility mosaic disease resistance.

Tagging of male sterility genes A cytoplasmic male sterile system is desirable for use in hybrid seed production, as it eliminates the need for hand emasculation. CMS is a maternally inheritable trait characterized by the inability to produce viable pollen but without affecting the female fertility, and it is often associated with mitochondrial DNA rearrangements, mutations and editing. Several restorer locus have been identified using RAPD and STS in different crop and DNA markers linked to these locus enable the molecular study of the CMS system. These co-dominant markers are useful in identifying the homozygous restorer genotypes after the backcrossing for production of restorer lines. In this way, the restorer lines could be produced in a shorter period than by conventional methods.


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Souframanien et. al. (2003) identified RAPD marker linked with male sterility gene. Primer OPC-11 produced a unique amplicon of 600bp in male sterile (A) lines 288A (derived from C. scarabaeoides) and 67A (derived from C. sericeus), which was absent in their respective, maintainers and putative R lines (TRR 5 and TRR 6). Genetic distance based on similiraty index revealed considerable genetic variation between male sterile lines, two putative R lines and donors of male sterility genes. Diversity evaluation Stability and identity of crop variety has assumed great importance for predicting plant breeder’s right/farmers right. Traditionally, evaluation and conservation of bio-diversity/genetic variability is based on comparative anatomy, morphology, embryology, physiology, etc., which provide informative data but of low genetic resolution. Recent advances in molecular biology have provided powerful genetic tools, which can provide rapid and detailed genetic resolution. Molecular marker based genotyping involves the development of marker profile unique to an individual. This unambiguous pattern of crop varieties obtained using DNA a marker is termed as “DNA Fingerprinting”. The technique was developed by Alec Jeffery in 1985 in human and was used first time in crop (rice) in 1988 by Dallas for cultivar identification. The choice of molecular marker to be used for DNA fingerprinting usually depends on technical expertise, available funds as well as the requirements of the experiment. At the same time the most important considerations in the use of molecular techniques are discrimination power and reproducibility. RAPD markers have shown to be of low discrimination power as compared to SSR and AFLP. Now days, microsatellites are the method of choice for varietal identification due to their abundance, high polymorphism, and simple protocol etc. Odeny et. al. (2007) deduced DNA polymorphism in pigeonpea by using 113 primers (designed from genomic SSR) and 220 soybean primers. Sivaramakrishnan et. al. (2002) showed that RFLP of mtDNA can be used for the diversity analysis of pigeonpea. They assayed restriction enzyme digested fragments of 28 accessions representing 12 species of the genus, Cajanus arranged in 6 sections including 5 accessions of the cultivated species C. cajan and 4 species of the genus Rhyncosia with maize mtDNA probes. In addition to inter-specific variability, intra-specific diversity was observed between the accessions of wild species (C. scarabaeoides, C. platycarpus, C. acutifolius) and cultivated species of C. cajan. Saxena et. al. (2010b) designed 23 primer pairs from 36 SSR enriched genomic library of pigeonpea. Sixteen primer pairs produced expected amplification fragments, of which 13 were polymorphic amongst 32 cultivars and 8 wild accessions representing six species. The average polymorphic information content was 0.32 per marker which varied from 0.05 to 0.55 for these 13 primer pairs. Ratnaparkhe et. al. (1995) developed DNA fingerprints for cultivated and wild pigeonpea accessions with the help of RAPD markers. The polymorphism among the cultivated species was low whereas high level of polymorphism was observed among the wild species. All pigeonpea accessions including cultivars under study were

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distinguishable from each other which demonstrated utility of RAPD in the genetic fingerprinting of pigeonpea. Ganapathy et. al. (2011) generated 561 amplified fragment length polymorphism (AFLP) loci for clustering cultivated and wild pigeonpea accessions. Jaccard’s similarity index indicated greater diversity within wild species which clustered into several groups. Most of the cultivated accesions were grouped into one major cluster. Among the cultivated lines, BRG 3, ICP 7035, TTB 7 and ICP 8863 were selected on the basis of morphological and molecular diversity for generating mapping population for identification of markers linked to sterility mosaic disease. Hamwieh et. al. (2009) developed new set of microsatellite markers in lentil for delineating the molecular diversity. Souframanien and Gopalkrishna (2004) used RAPD and IISR markers for deducing the genetic diversity among 18 blackgram cultivars.

Bulk segregant analysis Often a geneticist is not interested in developing a molecular map, but would rather find a few markers that are closely linked to a specific trait. Identification of these markers is often achieved by a procedure called bulk segregant analysis. The essence of this procedure is the creation of a bulk sample of DNA for analysis by pooling DNA from individuals with similar phenotypes. For example, you may be interested in finding a molecular locus linked to a disease resistance locus. You would create two bulk DNA samples, one containing DNA from plants or lines that are resistant to the disease and a second bulk containing DNA from plants or lines that are susceptible to the disease. Each of these bulk DNA samples will contain a random sample of all the loci in the genome, except for those that are in the region of the gene upon which the bulking occurred. Therefore, any difference in RFLP or RAPD pattern between these two bulks should be linked to the locus upon which the bulk was developed. 2. QTLs analysis Scoring individuals of a random segregating population for a QTL trait is done by growing the segregating population in replicated multi-location trials. Determination of the molecular genotype/DNA marker data of each member of the segregating population is done by analyzing the DNA with specific markers. Specific programmes are prepared (with certain theoretical assumptions) for determining association between any of the markers and the quantitative trait. Therefore, experimental procedure must comply with these assumptions. Methods of determining association of QTL and trait ♦ Single marker test ♦ Interval mapping ♦ Composite interval mapping ♦ Multiple interval mapping


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3. Marker assisted selection (MAS) MAS is most useful for traits that are difficult to select e.g., disease resistance, salt tolerance, drought tolerance, heat tolerance, quality traits (aroma of basmati rice, flavour of vegetables). The approach involves selecting plants at early generation with a fixed, favourable genetic background at specific loci, conducting a single large scale marker assisted selection while maintaining as much as possible the allelic segregation in the population and the screening of large populations to achieve the objectives of the scheme. No selection is applied outside the target genomic regions, to maintain as much as possible the Mendelian allelic segregation among the selected genotypes. Aftermselection with DNA markers, the genetic diversity at un-selected loci may allow breeders to generate new varieties and hybrids through conventional breeding in response to targets set in breeding programme. Material required for MAS Molecular markers, a set of authentic lines carrying trait of interest and a population to validate the markers to be used e.g., F2 or BCF2 for each of the individual traits/genes. Following are the basic pre-requisites for MAS : ♦ Search of molecular markers that are linked to the trait of interest ♦ Validate the available markers in parents and breeding population ♦ If markers are not available, it has to be designed and validated before use (if mapping ♦ populations are not available in hand it may take 2-4 years to generate and validate markers) ♦ Design a selection scheme and breeding strategy ♦ Fix the minimum population to be assayed to capture all beneficial alleles ♦ Meticulous record keeping ♦ Progeny testing for fixation of traits. Steps involved in MAS 1. Validation of molecular markers. Extract the DNA from test individuals and find out whether there is one to one relationship with marker and the trait. 2. Extract the DNA of breeding population at the seedling stage and apply MAS. Select the individuals on the basis of presence of desired molecular markers for the concerned trait. For other traits, selection is based on classical breeding methods. Minimum individuals to be assayed should be as per the defined strategy and statistical considerations. Limitations of MAS ♦ Cost factor ♦ Requirement of technical skill ♦ Automated techniques for maximum benefit

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♦ Per se, DNA markers are not affected by environment but traits may be affected by the environment and show G x E interactions. Therefore, while developing markers, phenotyping should be carried out in multiple environments and implications of G x E should be understood and markers should be used judiciously. ♦ DNA marker has to be validated for each of the breeding population. Any apriori assumption regarding the validity of markers may be disastrous. Marker assisted backcross breeding A backcross breeding programme is aimed at gene introgression from a “donor” line into the genomic background of a “recipient” line. The potential utilization of molecular markers in such programmes has received considerable attention in the recent past. Markers can be used to assess the presence of the introgressed gene (“foreground selection”) when direct phenotypic evaluation is not possible, or too expensive, or only possible late in the development. Markers can also be used to accelerate the return to the recipient parent genotype at other loci (“background selection”). It is assumed that the introgressed gene can be detected without ambiguity, and the theoretical study was restricted to background selection only. The use of molecular markers for background selection in backcross programmes has been tested experimentally and proved to be very efficient. Introgressing the favourable allele of QTL by recurrent backcrossing can be a powerful mean to improve the economic value of a line, provided the expression of the gene is not reduced in the recipient genomic background. Yet, recent results show that for many traits of economic importance. QTLs have rather small effects. In this case, the economic improvement resulting from the introgression of the favourable allele at a single QTL may not be competitive when compared with the improvement resulting from conventional breeding methods over the same duration. Marker assisted introgression of superior QTL alleles can then compete with classical phenotypic selection only if several QTLs could be manipulated. Selection scheme for MAS breeding The approach involves selecting plants at early generation with a fixed, favourable genetic background at specific loci, conducting a single large scale marker assisted selection while maintaining as much as possible the allelic segregation in the rest of the genome. First, the identification of elite lines presenting high allelic complementarity and being outstanding for traits of interest is required to capture favourable alleles from different parental lines. Second, after identification of the most favourable genomic regions for each selected parental line, those lines are intercrossed to develop segregating populations from which plants homozygous for favourable alleles at target loci are selected. One objective of the scheme is to conduct the marker assisted selection only once, and it requires the selection of a minimum number of plants to maintain sufficient allelic variability at the unselected loci. Therefore, the selection pressure exerted on the segregating population is quite high and the screening of large populations is required to achieve the objectives of the scheme. No selection is applied


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outside the target genomic regions, to maintain as much as possible the Mendelian allelic segregation among the selected genotypes.

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Chapter -

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Potential approach of bacterial biosorption of arsenic and chromium contamination from soil and water resources Ujjwal Kumar1, Ankita Sinha2, Ghanshyam K. Satyapal3, Sailesh Kumar4 and Ashok K Jha4* 1 P.G. Department of Biotechnology, T. M. Bhagalpur University, Bhagalpur – 812007 2 Department of Plant Breeding & Genetics, Bihar Agriculture University, Sabour, Bhagalpur 3 Centre for Biological Sciences (Biotechnology), Central University of South Bihar, Gaya – 824236 4 University Department of Chemistry, T. M. Bhagalpur University, Bhagalpur – 812007 * Correspondence author: [email protected]

Abstract Arsenic and chromium contamination in soil and water resources become an emerging challenges. Bacteria have capacity to mitigate and transform toxicity of these metals. Some specific microbes permit its recovery and trap metallic ions, its generally less energy intensive and less polluting then other non biological procedures. Bacteria may influence the concentration of arsenic and chromium in environment. Anthropogenic and geogenic activity had increase the risk of metallic pollution and this has forced to the future research area involve bacterial – metal toxicity to achieve bioremediation. The present chapter deals with the role of bacteria and incorporated them into remediation design. Keyword: Arsenic, Chromium, Bioremediation.

Introduction Metallic contamination in soil and ground water resources has been reported as serious hazardous threat globally (Duarte, 2009). Arsenic and Chromium are widely distributed due to natural and anthropogenic activity in the environment, Arsenic is referred to the first class of hazard while Chromium comes under class (Swaine et. al., 1995). Arsenic mostly found naturally and release less amount by anthropogenic sources despite them Chromium is highly toxic and non essential metal for living organism including microbes and its occurrence is rare in nature. Abundance if both these element then permissible limit can cause a series of health hazard via Arsenic and Chromium containing drinking water or agricultural products irrigated by contaminated

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water(Jha et.al., 2015). Chromium causes many acute and chronic effect that is in short term and intermediate exposure it create respiratory irritation, nosebleeds, asthama, sensitization, skin burns and dermatitis, while long term effect of hexavalent chromium in group 1 (carcinogenic to human). In contrast, trivalent chromium compounds are group 3 (not classifiable with regard to carcinogenicity to human) (Kumar, et.al. 2018). Arsenic is a poisonous due to its effect on sulphydral group of cells enzyme (SS) causes arsenicals dermatosis, andarsenicosis (Smith,1998,2003). The widely affected habitat is Gangetic basin of Bihar (Jha et.al.2017). There are two stable oxidation stages of Chromium found in the environment that is chromium(III) and Chromium(VI) which have contrasting toxicity, mobility and bioavalability. Chromium compound of oxidation stage Cr(VI) are powerful oxidents. Cr(VI) compounds are used as pigments for photography and in pyrotechniques, dyes, paints, inks, and plastics, it is also used in stainless steel production, textile dyes, wood preservation, leather tanning and corrosion coating, while Cr(III) is relatively innocuous and immobile (Kumar et.al., 2018). The presence of Arsenic and Chromium above critical concentration in soil and groundwater are great concern of pollution. Its has harmful effect on animals, plants, micro organism as well as on environment also. Apart from existing strategies to remediate these metallic contaminants in soil and water. Arsenic and Chromium occurrence in soil and groundwater Arsenic and chromium is naturally found in soil and groundwater is due to geogenic process and anthropogenic activities such as melting operations, fossil fuel combustion arsenic chromium based agrochemicals, textile dyes, fertilizers and direct disposal of municipal and industrial waste. Arsenic widely distributed in world wide through geogenic distribution as well as in northern, eastern and central India. Arsenic spread in soil and groundwater due to Holocan aquifer originated from Himalayan base and distributed in Ganga – Meghna – Brahamputra basin plains. Although Major source of hexavalent Chromium in soil and groundwater is due to anthropogenic and industrial activities, However some finding of naturally occurring Cr (VI) contamination in soil and groundwater in gangetic basin have been reported. It released by weathering of Ultramafic rocks that contain Cr(VI). These Ultramafic rocks and derived serpentine soil and sediments are encountered in populated area around the world that present hogh Cr(VI) concentration.(Jha et.al., 2014) , with an average of 2000 and 2650 mg/kg for rock and soils respectively. WHO decleared 0.05 ppm concentration as permissible limit in water for arsenic and chromium concentration. Cr release in groundwater occur through the dissolution of trivalent chromium from its mineral hosts, followed by the sorption of cr(III) onto high – valancy Mn oxide and oxidation of Cr(VI), which desorbs its mobile at alkaline pH. Arsenic and Chromium cycle in soil Arsenic naturally exist in more then 200 different minerals forms of which approroximately 60% arsenates, 20% sulphides and sulphosalts forms of arsenic, despite of this Gangetic basin of Bihar more then 87% tested samples were found to trivalent

Potential approach of bacterial biosorption of arsenic...

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arsenic As(III) contamination in groundwater. Most abondent arsenic ore minerals in Ganga – Brahmputra – Meghna basin is arsenopyrite Fe(SAs)2 . Arsenic dissolve in aquifers through a range of pH condition which may lead significant dissolution where high concentration are present in the rocks. Arsenic also exist in various concentration in other environmental media e.g. air, rain, river, lakes, soil, groundwater, sea water etc. Several cycles have been proposed by different experimentalist, Langdonet et.al. proposed a cycle that covers all areas of environment that are arsenic affected.

Environmental chromium cycles, states that Cr(VI) is the most oxidized, mobile, reactive and toxic from oxidation states of Chromium. Cr(VI) only exist thermodynamically in equilibrium with the atmosphere(Hawley, 2004). It is found in small concentration in soil and groundwater, may be due to the result of oxidation of natural Cr(VI), but higher concentration, generally, is the result of industrial discharge of cr(VI) (Chromium in Drinking Water, 2017). Atmoshpheric oxygen that contain both oxidized manganese and reduced carbon exist in partially equilibrium with soil and sediments (Smedley 2002). The oxidation of chromium (III) to Cr(VI) by manganese oxide and reduction of Cr(VI) to Cr(III) by soil carbon compounds are both thermodynamically spontaneous reaction (Hrudayanath, 2014). It is interesting that oxidation and reduction of chromium takes at same time in chromium cycle (Dhal,2013).

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Fig. 2: Environmental Chromium Cycle

Bacterial structure and efficiency responsible for biosorption capacity The presence of arsenic and chromium in soil and groundwater above critical concentration is effects great concern to environment. Soil is a great natural habitat for bacteria, different type of microorganism play a pivotal role in cycling, controlling heavy metals concentration in soil. Bacterial bioremediation is a novel techniques which is ecologically sound alternative to chemical technology. All bacteria have simple cell structure i.e. with nuclei and cellular organelles, but possible cell walls. The bacterial cell wall provides structural integrity due to the presence of peptidoglycan layer to other organism. The bacterial cell wall varies from species to species, either it is gram positive or gram negative. Cell wall is the first component in the bacterial cell that comes in contact with metal ions, solute can be deposited on the surface or within the cell wall structure. The chemical functional groups including carboxyl, phosphate, amino acid and hydroxyl group play a vital roles in biosorption of arsenic and chromium. Negatively charged and abundantly available carboxyl group actively. participate in binding metal cations. Amino groups are also very effective in removing metal ions, as it not only chelates cationic metal ion but also adsorb anionic metal species via electrostatic interactions or hydrogen bonds. Biosorption mechanism during interaction of bacterial biomass and metallic ion such as arsenic(III) and chromium can be use different analytical techniques, including potentiometric titration, Fourier transformed infrared spectroscopy. X – ray diffraction. X – ray photoelectron spectroscopy. Scanning electron microscopy. Atomic force microscopy. Transmission electron microscopy. Energy dispersive X – ray microanalysis. Potentiometric titrations have been used by several researchers for the determination of the nature and number of binding site of metal in bacterial surface.The nature of the binding sites and their involvement during biosorption can be approximately


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determined using FT – IR. XPS are widely used to distinguish different forms of the same element and indentify the existence of particular element in a material. EDX can provide information regarding the chemical and elemental characteristics of a biomass after and before biosorption. It confermed the involvement of an ion exchange mechanism during their biosorption. while, to analyze the morphology of the bacterial cell surface before biosorption, SEM microscopy are generally used. AFM was also used and polyallyamine as a ideal tool for determining change in surface morphology. Arsenic and Chromium resistance mechanism in bacteria The uptake of arsenic by bacteria occure via the glycerol and phosphate transport system. Therefore, the primary mechanism of arsenic resistance uptake by increasing the specificity of phosphate uptake [As(III)] and the glycerol transport (aqualyceroporin) [As(III)]. Once arsenic has entered the cell other defence mechanisms are activated. the most wide spread mechanism of arsenic resistance in microorganism is based on energy dependent efflux of arsenite from the cell . The mechanism of bacterial reduction for Cr(VI) varies from strain to strain depending upon their bio-geochemical activities and nutrient utilization patterns which directly affect the resistance/tolerance to chromate. All the microbial reme-diation of Cr(VI) follows either or a combination of the three reduction mechanisms. These are : (i) in aerobic conditions, it is associated with soluble chromate reductases that use NADH or NADPH as cofactors, (ii) under anaerobiosis, some bacteria, like Desulfotomaculum reducens, can use Cr(VI) as an electron acceptor in the electron transport chain, and (iii) reduction of Cr(VI) may also take place by chemical reactions associated with compounds present in intra/extra cellular such as amino acids, nucleotides, sugars, vitamins, organic acids or glutathione. Cr(VI)- reduction may also follow plasmid resistance mechanism which represents a potentially useful detoxification process for several bacteria . Hyper metal resistance and biosorptive bacteria for Arsenic and Chromium detoxification Microbial biosorption of arsenic and chromium is the passive sequestration. biosorption involves accumulation of metals on the surface of the cells or cell fractions by adsorption or ion exchange. All microbes, which expose negatively charged groups on their cell surface, have the capacity to bind matel ions. Various compounds of bacterial walls sorb different metals, which later get precipitated. Metal cations may bind on the microbial cell surface (biosorption) or with in the cell wall (bioaccumulation) and in tern, metal uptake is augmented through microprecipitation. metal ion may be actively translocated inside the cell through metal binding proteins. Arsenic and chromium resistance bacteria generally exist in arsenic and chromium contaminated areas naturally, such as arsenic or chromium containg irrigated soil feild, coal mines, arsenic 0r chromium contaminated groundwater, industrial and waste water sewage and other high concentrated site of arsenic and chromium exposure. Many bacterial species have reported as arsenic and chromium resistance or biosorptive capacity

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Bacterial surface display for arsenic and chromium Binding sites Biosorption of metal ions is a metabolism-independent metal uptake event at the cell walls, polysaccharides, associated molecules, and functional groups are involves mainly the ion-exchange, chemisorption, adsorption. And, in some cases, also the inorganic microprecipitation of certain heavy metal species. In the search for strategies allowing for enhancements of the biosorption capacity for a specific metal ion, display of particular amino acid sequences with the capacity to form coordination centers for the metal ions at the microbial cell wall has proved to be a promising approach. Biosorption for gram negative bacteria: The cell wall of Gram-negative bacteria is a two component system composed of outer membrane (OM) and periplasmic space, which extends between inner plasmatic and OM. Located in the 13–25 nm thick periplasm is peptidoglycan cell wall structure with a thickness ranging from 5 to 8 nm. The periplasm can be viewed as a true compartment that fulfills important functions and contains: catabolic enzymes that degrade complex molecules for nutrient acquisition; solute-binding components of ABC transporters or chemotaxis receptors; periplasmic components of serial transport system involved in transport of large substrates which are proteins spanning the periplasm as shuttles connecting outer and inner membranes; detoxifying enzymes; enzymes and chaperones involved in biogenesis of cell envelope structures, including redox enzymes of Dsb family responsible for disulphide bond formation; branched oligoglucans derivatized with phosphoglycerol, phosphoethanolamine and succinic acid, which are induced under hypoosmotic conditions; structural lipoproteins integrating outer membrane with peptidoglycan layer Biosorption for gram positive bacteria: The major component of the cell wall of the Gram-positive bacteria is a peptidoglycan (15–80 nm thick), intimately layered just above the plasma membrane and leaving very limited periplasmic space. Unlike in Gram-negative bacteria, the short peptides crosslinking the polysaccharide component of peptidoglycan in Gram-positive species are covalently linked to teichoic acid (polyol phosphate polymers). Besides these components, the rigid cell wall contains secondary cell wall nonproteinaceous polymers (teichuronic acid, lipoteichoic acid, and other neutral or acidic polysaccharides) in various proportions. Proteins displayed within or on the cell wall fall into four different groups based on the anchoring mechanism : proteins anchored in periplasmic membrane by hydrophobic transmembrane domain(s); lipoproteins posttranslationally attached to the membrane diacylglycerols by a covalent bond; proteins covalently attached to peptidoglycan; proteins anchored noncovalently via cell wall-binding domains. Natural metal binding peptides in bacteria To enhance metal-binding properties of specific proteins we can either use the natural metal-binding peptides or attempt to design metal-binding peptide sequencies de novo. The former approach is described in this section whereas the rational molecular design is mentioned in the next section. The intracellular peptides involved in sequestration of transition heavy metal ions in mammals, yeasts and plants had become popularly used


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for surface display to improve the metallosorption properties of bacteria and yeasts. These involve metallothioneins (MTs) and phytochelatins (PCs), which combine small size beneficial for efficient surface display and high number of metal-binding centers. Ubiquitous in eukaryotes, MTs are cysteine-rich peptides capable of high affinity coordination of heavy metal ions via cysteine residues shared along the peptide sequence in characteristic Cys-X-Cys or Cys-Cys motifs. Besides the role in detoxification of excess heavy metal ions, MTs are involved namely in Zn and Cu homeostasis, protection against oxidative damage and redox control. In mammalian MTs of 61 or 62 amino acid residues form their 20 cysteine residues 7 and 12 coordination centers for divalent and monovalent heavy metal ions, respectively. The mammalian MT molecule is composed of two distinct domains, denoted α- and β-domain. The α-domain consists of first 30 amino acids, contains nine cysteine residues and binds three bivalent metal ions. The β-domain (amino acids 31–61 or 62) contains 11 cysteine residues that bind four bivalent metal ions and the cadmium-binding stability of α-domain is known to be much stronger than that of β-domain and intact wild-type MT.

Conclusion Toxicity of arsenic and chromium become hazardous pollutant for groundwater and soil due to the natural and anthropogenic discharge. Microorganisms have been measured not only for use in bioremediation of metals but also as accumulators of arsenic and chromium from dilute medium. A great number of bacteria are accomplished of accumulating these metal and metalloid in their cells to concentrations some orders of degree higher than the background concentrations of these metals. Many laboratory experiments have been developed regarding bacterial biosorption of arsenic and chromium, but problem still threatening due to the lack of implementations. This information is useful to prepare remediation strategies with indigenous microbial communities from contaminated site. References 1.

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