Recent Molecular Genetics and Biotechnological ...

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PSK (polysaccharide-K). Trametes versicolor,. PSP (polysaccharopeptide), Lingzi. Ganerderma lucidum. Grifron-D. Grifola frondosa lentinan. Lentinula edodes.
2014, Microbial Biodiversity in Sustainable Agriculture Editor: Dr. Ram Chandra Published by: DAYA PUBLISHING HOUSE, NEW DELHI

Pages 323–343

Chapter 16

Recent Molecular Genetics and Biotechnological Approaches in Mushroom Research A. Chattopadhyay1, H. S. Kadappa1* and S. C. Meena2 Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 2 Department of Plant Pathology, RCA, MPUAT, Rajasthan 1

Introduction Mushroom is a macro fungus with a distinctive fruiting body and has been cultivated since ancient times for their nutritional value, texture and flavour. It is a novel source of proteins, vitamins, minerals, crude fibres, carbohydrates, etc. They have low fat content with high fibre and all essential amino acids and contain all most all the important minerals too (Sadler, 2003). Mushrooms also have various therapeutic properties like the production of polysaccharides, triterpenoides, antioxidants (Netravathi et al., 2006), antitumourgenic and antiviral agents and it also produces large amounts of vitamin D, which are essential to ward off hypertension, hypercholesterolemia, cancer, AIDS and numerous other diseases (Beelman et al., 2003; Mallikarjuna et al., 2013). Thus it is considered to be ‘the foods of the Gods’, according to the ancient roman history (Smith et al., 2002). It is an economical crop to cultivate, requiring low resources and area, can be grown throughout the world and all over the year from low initial cost. There is tremendous potential and appeal for growing such type of highly nutritious food –––––––––– * Corresponding Author: E-mail: [email protected]

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with excellent taste from non-expensive substrates (Beetz and Kustudia, 2004). Also, it is very environmental friendly, capable of converting the lignocellulosic waste materials into feed and fertilizers (Hadar et al., 1992; Jaradat, 2010). These also have immense importance in the production of enzymes, in fermentation technology, in paper industry, etc. Although investment in the mushroom industry is not very large, but production and consumption of mushroom is relatively low in comparison to other crops (Chang, 2006).These may be due to various causes related to social, economical, political sectors of our day to day life, but one of major concern is related to the field of scientific research for the development and production good quality of mushroom for various purpose like food, industry, medicine, etc. There is greatest need of application of various molecular concepts and techniques in this field that may lead to the improvement in the biology and physiology of the mushroom.

Common Cultivated Mushroom and their Use The ‘mushroom’ includes a large group of fungi and these can be roughly divided into four different categories (Chang, 2002a): (1) edible mushroom, (2) medicinal mushroom, (3) poisonous mushroom, (4) other miscellaneous group. In this text we will mainly concern about the research and application of recent molecular approaches for the development of edible mushroom, medicinal mushroom and others having major economic importance. There are about 5000 species of edible mushrooms; of which 20-25 species are exploited for commercial cultivation and large scale production all around the world (Change, 2002b). Some of them are listed in Table 16.1.These have diverse importance in various economic aspects like nutritional importance, medicinal importance, industrial importance and other uses.

Nutritional Importance Mushroom is a very good, nutritious and palatable food and a rich source of protein and carbohydrates and also contains almost all essential amino acids. The carbohydrate content of mushrooms is varying from 50 to 65 per cent on dry weight basis. It is a rich source of glucose, sucrose, fructose, raffinose, xylose and mannitol. But starch is absent. The protein content of cultivated species is varying from 1.755.95 per cent of their fresh weight (Flegg and Maw; 1976; Chang, 1980) and easily digestible. The protein found in mushrooms is less than the animal source but much more than vegetables. Thus it can be used to fulfil the protein malnutrition in poor human being. Unlike to carbohydrates and proteins, the fat content in mushrooms is very low (0.6-3.1 per cent) and dominated by unsaturated fatty acids and it is important for patients of obesity as it is cholesterol free. Mushrooms are the rich source of several vitamins, especially vitamin B-complex like thiamine (B1), riboflavin (B2), niacin, biotin etc and also ascorbic acids (C) (Lau et al., 1985) but poor in vitamins A, D and E (Anderson and Fellers, 1942).Among the minerals, mushroom is rich in elements like K, P, Na, Ca, Mg as a major constituents and Cu, Zn, Fe, Mo, Cd as minor constituents (Bano and Rajarathanam, 1982; Bano et al., 1981; Mallikarjuna et al., 2013) and most of them are present in easily available form. Thus mushrooms are quality food for poor population. Due to its quick availability, easy palatability and

Agaricus bisporus

Lentinus edodes

Pleurotus ostreatus

Volvariella volvacea

Auricularia auricula

Ganoderma lucidum

Pholiota nameko

Tremella fuciformis

Tuber aestivum, T. melanosporum,T. magnatum

Morchella angusticeps, M. esculenta

Grifola frondosa

Trametes versicolor

Calvatia gigantea

Laetiporus sulphureus

Flammulina velutipes

Coprinus comatus

Craterellus cornucopioides

Boletus edulis

Tricholoma matsutake

Shiitake mushroom

Oyster mushroom

Paddy straw mushroom

Ear fungus

Reishi mushroom

Nameko mushroom

White jelly fungi

Truffles

Morels

Maitake

Turkey Tail

Giant Puffball

Chicken of the Woods

Enokitake

Shaggy Mane

Black Trumpet

Porcini

Matsutake

Scientific Name

Button mushroom

Common Name

Table 16.1: List of Mushroom having Commercial Importance.

Flavour and aroma in cooking

Used in soups and sauces

Best tasting edible mushroom

Antibiotic properties

Used in soups

Taste similar to chicken

Edible when young

Medicinal mushrooms (too tough to be edible)

Edible mushroom with anti-tumour properties

Delicious edible mushroom

These gourmet delights

Food supplements

Food supplements

Medicinal mushroom

Medicinal value

Edible mushroom

Edible mushroom with cholesterol-reducing effect

Edible mushroom with medicinal properties

Edible mushroom with medicinal and industrial use

Importance

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better nutritional importance, the mushrooms can be considered as a complete foodstuff and have significant importance in our diet and to our health also.

Medicinal Importance Medicinal property of mushroom is another most important attribute in mushroom research that have been recognised in China, Korea and Japan a long ago (Thakur, 2005) and applied in various traditional remedies for the treatment of various physiological disorders using bioactive compounds of mushrooms which have recently been isolated and characterized (Rai, 1997).The recent interest in mushroom research is now shifted from the production of healthy edible mushroom to medicinally important healthy edible mushroom using modern biotechnological approaches with aim to supplement medicinally important bio-reactive molecules in dietary food. Such bioreactive molecules are called as ‘mushroom nutriceuticals’ (Chang and Buswell, 1996). These compounds may be polysaccharides, triterpenoids, immunomodulatory proteins, lentinans, adenosine, lingzhi-8, polysaccharide-Krestin etc. Some of these are listed in Table 16.2.

Industrial Importance Most of the mushroom fungi have lignocellulolytic activity and can be able to secrete a wide range of oxidizing enzymes of industrial importance. These enzymes have significant applications not only in the chemical, fuel, food, textile, laundry and pulp and paper industries but also used in agriculture and for animal feed production (Elisashvili et al., 2006). Degradatory enzymes produced by them are of three kinds: polysaccharide degrading enzymes (cellulolytic and hemi-cellulocytic enzymes), proteolytic enzymes and ligninolytic enzymes. Polysaccharide degrading enzymes include xylanase and polygalacturonases etc and can be used as an alternative to boiling with NaOH for removal of non-cellulosic materials in the retting of flax fibres. Ligninoltic enzymes produced by them include phenol oxidases, peroxidases, catalases and laccases are most used for delignification of lignocellulolytic wastes to produce pulp for the cardboard or paper manufacture (Thakur, 2005). On the other hand, proteolytic enzymes can be used in mushroom food processing.

Molecular Approaches in Mushroom Research Keeping the growing importance and increasing public demand of mushroom in day to day life, it is always essential to get a ‘designed mushroom crop’ with desired property of nutritional, medicinal or industrial importance. But it is quite difficult to have a ‘designed crop’ through natural breeding. The early attempts of genetic improvement in the cultivated mushrooms do not have much understanding of genetics and molecular biology of mushroom. There are some biological barriers in the development of desire mushroom strains through traditional breeding programme. “Secondary homothallism” in some mushroom species with a single multiallelic mating type factor is one such type (Elliott and Langton, 1981).The most important impairment to the development of a systematic breeding programme is the absence of any uninucleate propagules during the life cycle of mushroom fungi. Another factor like the compatible matting type nuclei in basidiospore is also important. This

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Table 16.2: Medicinal Importance of some Mushroom Fungi. Property

Bioactive compounds

Source

Anti-tumour

Peptidoglycans (b-glucans, lentinan, schizophyllan, grifolan, and SSG)

Lentinus, Schizophyllum, Grifola, Sclerotinia

Ergosterol, antitumor glucan, Lectin

Agaricus blazei, Agarius bisporus

PSK (polysaccharide-K)

Trametes versicolor,

PSP (polysaccharopeptide), Lingzi

Ganerderma lucidum

Grifron-D

Grifola frondosa

lentinan

Lentinula edodes

Schizophyllan

Schizophyllan commune

Clitocinet

Leucopaxillus giganteus

Agaritine

Agaricus blazei

Irofulven (cytotoxin)

Omphalotus olearius (non edible)

Anti-Hepatitis

Ganopoly

Ganoderma lucidum

Anti-oxidant

Lipid fraction

Grifola sp.

Anti-bacterial

Sesquiterpenoid hydroquinones (ganomycins), Steroids; hypocholesterolemic eritadenine, and cortinellin

Ganoderma pfeifferi, G. applanatum L. edodes

Antimicrobial, Anti-protozoal

Oxalic acid

Lentinula edodes

Epicorazin

Podaxis pistillaris

triterpenes (ganoderiol F, ganodermanontriol,and ganoderic acid B); Farnesyl hydroquinone, ganomycin I, Ganomycin B

Ganoderma lucidum, Ganoderma colossum

Lignins

Inonotus obliquus

Polysaccarides PSK

Trametes versicolor

Anti-cancer

Anti-viral (HIV)

Velutin

Flammulina velutipes

Antiviral (influenza, herpes)

Ganodermadiol, lucidadiol and applanoxidic acid G

Ganoderma sp.

Antiviral (poliomyelitis)

Polysaccharides

Agarius brasiliensis

Anti-allergic

Hispolon, hispidin

Indocalamus hispidin

Ergosterol peroxide

Tricholoma populinum

Illudins, tricyclic sesquiterpenes

Omphalotus olearis, Lampteromyces japonicus

Terpenoid leaianafulvene

Mycena leaiana

Triterpenes (ganoderic acids, lucialdehydes A, B, C and australic acid)

Ganoderma lucidum, G. australe

HIV-induced cytopathic effect

Sulfated lentinan

L. edodes

Apoptosis

triterpenes

Ganoderma concinnum

Asthma

Unspecified bioactive extract

Cordyceps sp.

Cytotoxic to tumour cell

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understanding can be helpful to evaluate the breeding methods previously used and to suggest alternatives methods of strain improvement. There are various methods of new and improved strain selection based on single spores, multispores or tissue culture techniques that may give improvement in the short term but is not as effective as methods like improved molecular breeding with controlled crossing, advanced tissue culture and modern biotechnological methods.

Molecular Breeding Strategies Karyotype Analysis Karyotypic analysis of various mushroom species using microscopy and pulse field gel electrophoresis (PFGE) will be helpful to determine the number of chromosome of the species and for the identification of species. Sometimes the importance of karyotypic analysis is not justified as chromosomal polymorphism among the strains. This may be due to chromosomal aberration during growth and development of any fungi. Initially electrophoretic karyotype analysis was used as a potent tool to reveal such type of chromosomal aberration in many fungi like Coprinus cinereus (Pukkila and Lu, 1985). However, the results are sometimes ambiguous, for example, the number of chromosomes in Pleurotus ostreatus varying from 6 to10 (Chiu, 1996; Peberdy et al., 1993; Sagawa and Nagata, 1992).This problem can be solved with the optimization of PFGE separation for fungal chromosomes (Sagawa and Nagata, 1992) that allowed the analysis of molecular karyotype of mushrooms and assigning genes to chromosomes and applied in Agaricus bisporus consisting 13 chromosomes in genome with 31 Mbp size (Sonnenberg et al., 1996) and also in Pleurotus ostreatus containing 11 pairs of chromosomes in genome (Larraya et al., 1999).While, light microscopic observations and electrophoretic karyotype analysis have suggested that this fungus contains at least eight chromosomes in the haploid genome (Tanaka and Koga, 1972; Nakai, 1986; Arima and Morinaga, 1993).There is no report for other mushrooms in this respect and the genomic organization of different species remains poorly understood. Genetic Linkage Map A genetic linkage map is a partial representation of the genome that shows the relative position and distances between markers and genes along a chromosome in terms of genetic distance (in cM). Genetic linkage map can be developed by using a large number of genetic markers distributed within genome and has been analysed for various organisms like plants (Maheswaran et al., 1997; Harushima et al., 1998; Hayashi et al., 2001), animal and fungi (Tzeng et al., 1992; Forche et al., 2000; Larraya et al., 2000) etc. These linkage maps will be a suitable support for whole genome sequencing and for localization of genes of interest or quantitative trait loci (QTL) breeding and map-based cloning of genes of interest (Farman and Leong, 1998). By studying the segregation of genetic markers and the relevant trait in the population in which genetic markers are tightly linked to gene loci associated with that particular trait, we can easily select the population bearing desire trait without any phenotypic testing or phenotypic expression. The generation of a genetic linkage map consists of three basic steps: 1) production of mapping population,

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2) identification of polymorphic molecular markers and their genotyping on mapping population and 3) statistical analysis of segregating data and correlation of genotypic data with phenotypic one for the construction of linkage map. The primary requirement for construction of linkage map is to have a segregating mapping population. Unlike the use of backcrossed and F2 based mapping population in plant breeding, in mushroom breeding one can obtain individual post meiotic products directly from each generation to study the marker segregation. Since the principle of genetic linkage map is based on the analyses of the allelic reshuffling caused by meiosis, population derived from sexual reproduction are more appropriate for mapping (Foulongne-Oriol, 2012c). Therefore, F1 homokaryotic progenies derived from meiotic spore are mainly used in fungal mapping studies (Foulongne-Oriol, 2012c). In mushroom breeding we can easily get these meiotic spores from each generation and single spore isolates are vegetatively propagated for the segregation analysis of genetic markers. Unfortunately, obtaining such progenies (i.e. homo- or heterothallism) is associated with specific constraint. For heterothallic species most of the SSIs are haploid and can directly be used for linkage analysis. Relevant genetic information present in one of the constituent parental lines of the wild isolate is introduced into both parental lines of the commercial strain via two backcrosses (BC1/BC2) and suitable genetic markers are used to select the traits and for commercial genome analysis. For secondarily homothallic species like A. bisporus var biosporus, this is quite problematic. Almost all the basidiospores of A. bisporus are binucleate and self fertile, containing two nuclei of opposite mating types. Only a low percentage of basidiospores are uninucleate that produce homothallic species which can be used as mapping progenies. Although there is no clear cut morphological distinction between homo- and heterokaryons, homokaryotic single spore isolates can be selected based on their slow growth in comparison to heterokaryotic single spore isolates. By a pre-selection for lower growth, usually sufficient homokaryons are obtained (Fritsche, 1986; Kerrigan et al., 1992) and can be screened successful based on multi-locus test (Kerrigan et al., 1992). Other types of meiotic progenies have been occasionally used for linkage map construction in other fungi. For example, a genetic linkage map based on tetrad analysis has been reported for Lentinula edodes (Miyazaki et al., 2008). The second important step for genetic linkage map construction is to identify polymorphic markers that will be used for genotyping the entire mapping population. Previously, isoenzymes based markers are commonly used for genotyping (Bowden and Royse, 1991). The use of these types of markers is, however, not very efficient. With the advancement of molecular technologies, various DNA based molecular markers are now commonly use for genotyping. The construction of the first generation of linkage maps was based on DNA markers like restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) (Kerrigan et al., 1993; Xu and Leslie, 1996; Kwan and Xu, 2002). Second-generation linkage maps are now developed through advanced high-throughput genotyping techniques using AFLP, CAPS, STS, etc based markers. Among these, amplified fragment length polymorphism (AFLP) markers were advantageous since no sequence information was required for their development and a large number of markers could be rapidly generated to provide high resolution mapping (Vos et al., 1995). These markers are

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also more reproducible than RAPD markers. Thus, this marker has been used increasingly to develop genetic linkage map of mushroom fungi like Lentinula edodes (Terashima et al., 2002a,b), Pleurotus eryngii (Okuda et al., 2012) etc. However, their conversion into sequence specific PCR markers like AFLP-converted markers (ACM), cleaved amplified polymorphic sequences (CAPS) and sequence characterized amplified regions (SCAR) etc. enhanced their usefulness in linkage mapping (Terashima et al., 2006; Okuda et al., 2009; Foulongne-Oriol et al., 2011a). Microsatellites, also known as simple sequence repeats (SSR), are presently the most popular markers for genetic linkage mapping in mushroom fungi (Okuda et al., 2009). The final step of the construction of a linkage map is the analysis of the genotyping data to the finalized map. Two approaches to linkage analysis were followed. One is statistical analysis of the pair wise segregation of all genetic markers using chisquare (c2) test to analyse for deviation from the expected Mendelian segregation ratio in the mapping population. That can be performed manually for a few markers, but it is not feasible to determine linkages between large numbers of markers that are used to construct maps. The computer programs or software packages like Mapmaker/ EXP (Lander et al., 1987; Lincoln et al., 1993), MapManager QTX (Manly et al., 2001) and JoinMap (Stam, 1993) are required for this purpose. Linkage between markers is usually calculated using odds ratios (i.e. the ratio of linkage versus no linkage). This ratio is more conveniently expressed as the logarithm of the ratio and is called a logarithm of odds (LOD) value or LOD score (Risch, 1992). LOD values of >3 are typically used to construct linkage maps. A LOD value of 3 between two markers indicates that linkage is 1000 times more likely (i.e. 1000: 1) than no linkage (null hypothesis). LOD values may be lowered in order to detect a greater level of linkage or to place additional markers within maps constructed at higher LOD values. Linked markers should be grouped together into ‘linkage groups’ present within a chromosome (Collard et al., 2005). The ordering of the markers in the linkage groups can be carried out using MAPL. Referring to the road map analogy in linkage mapping, linkage groups represent roads and markers represent signs or landmarks. Marker Assisted Selection (MAS) and QTL Breeding Marker-assisted selection (MAS) is a method of identifying the desirable phenotypes in a population based on the genotype of a marker. Today MAS is used as a fast, easy and cheap method for the screening of cultures and selection in mushroom breeding (Kerrigan, 2000). The markers associated with phenotypic traits such as yield, fruit shape, colour, quality (Miyazaki et al., 2010), time of fruiting and disease resistance can be used to identify the homokaryons with desirable traits without waiting for the fruiting stage, making it easier and faster than conventional breeding. Complex (multigenic traits) such as yield, disease resistance and quality characteristics are usually inherited quantitatively (Sonnenberg et al., 2005). These traits are found to be associated with quantitative trait loci (QTL). For quantitative traits, more than one quantitative trait locus (QTL) is normally found. The dissection of these quantitative traits in individualized loci may greatly facilitate their effective manipulation in subsequent breeding program.

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Many QTL loci that are responsible for traits have been located. Larraya et al. (2002) found 1 to 4 QTL controlling growth rate and several production traits in the oyster mushroom (P. ostreatus). In the button mushroom, the genetics of yield-related traits and cap colour have also been investigated through QTL mapping (FoulongneOriol et al., 2012a, b). QTLs for disease resistance as well as sensitivity have also been detected. Sonnenberg et al. (2005) found two significant QTL in one homokaryon of the wild line and five QTL in the other wild homokaryon of A. bisporus resistant to Verticillium fungicola. QTLs for resistance in A. bisporus to three major diseases like bacterial brown blotch (caused by Pseudomonas tolaasii), dry bubble (caused by Lecanicillium fungicola) and green mold (caused by the fungal competitor Trichoderma aggressivum) have been found (Moquet et al., 1999; Foulongne-Oriol et al., 2011b; 2012a, b). Moquet et al. (1999) found only one QTL for sensitivity to bacterial blotch in A. bisporus. Hybridization Breeding The hybridization breeding in mushroom research was introduced in the 1980s with the development of first cross breeding in 1983, generating two Agaricus bisporus hybrids Horst U1 and Horst U3 (Fritsche, 1983) and open up a new way to develop hybrid mushroom strains with desire traits and choice able production characteristics like tolerance to environmental and cultural stresses etc (Chakravarty, 2011). These techniques gradually become popularised and well received. Since then, cross breeding has been carried out in Lentinula sp. (Zhang and Molina, 1995), Pleurotus sp. and Agaricus sp. (Fritsche, 1983) and in many other mushroom species (Ma et al., 2004) with the production of new hybrids for resistance to diseases and pests but also reduced the dependence and risks of environmental and cultural stresses. The major aim of hybridization (cross breeding) is to combine desirable characteristics from different strains and create variability in the existing germplasm. That can be obtained only by pairing monosporic cultures. Hybridization for strain improvement of two oyster mushrooms (Lanka Oyster and American Oyster) was achieved by duel culture technique of monospore cultures (Wasantha Kumara and Edirimanna, 2009). But in Agaricus bisporus due to secondarily homothallism that could limit outcrossing and recombination among homokaryons in natural populations and also creates difficulties in mushroom breeding, an alternative source of recombinant genotypes is from somatic pairings of heterokaryons (Xu et al., 1996). The cross breeding of strains has traditionally been accomplished by trial and error, and large numbers of hybrids generated by pairing monosporic cultures (Chakravarty, 2011); need to be cultivated to evaluate the production characteristics. An enormous increase in the use of DNA markers like RAPD or RFLPs methods during the last decade also help in the selection process (Moore et al., 2001; Fukuda et al., 1995; Mary et al., 2001). The use of genetic makers is straightforward for monogenic traits that segregate in distinct phenotypes such as cap colour in mushrooms (Kerrigan, 2004; Baars et al., 2004). However, complex traits such as yield, resistance to disease and quality characteristics that are usually inherited quantitatively will be selected with QTLs. A first marker-assisted backcrossing program for a polygenic trait introgression has been mentioned in Sonnenberg et al. (2005).

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Mutation Breeding There are various breeding strategies to improve the strains of mushroom for better quality and more yield. Among them mutation breeding is potent one. In this breeding method, selection of the desirable strains from a diverse population generated by natural mutation or induced mutation by ionizing radiation (X-rays, neutrons, g-rays, laser beams, ultraviolet light) and chemical mutagens was done (Fan et al., 2006). Mycelia plugs of the actively growing parent strains were subjected to various doses of mutagens and mutated mycelia were assessed in laboratory experiments for desire selection and were used to prepare mother flasks, spawn and fruiting bags for production experiments. The principal aim of irradiation is to improve selected strains mushroom by creating genetic variation. Two types of irradiation, namely irradiation using Gammaray and UV radiation were mostly used in order to attempt to improve parental strains. Gamma radiation induced mutants have been reported in several works on Pleurotus sp to reduced mycelia spawn run time and stimulated growth, yield without any effect on protein and carbohydrate content (Roy et al., 2000), to recycle biowaste (Lee et al., 2000), to enhance enzyme production (Chang et al., 2003) and the use of UVray irradiation has been reported in others.

Tissue Culture Techniques-Protoplast Fusion The protoplast fusion is a new approach for genetic manipulation in fungi. This method of hybridization has been successfully used to produce heterokaryons between strains that are incompatible by conventional breeding methods. This protoplast fusion technology is advantageous for introducing one or more polygenic traits or traits with unknown genetic mechanism or to avoid the cross breeding barriers of two biological species. This technique brings two distinct genomes in a common cytoplasm. It involves two steps: (1) isolation of protoplast, (2) fusion of protoplast. The isolation of protoplast is depending on three major factors: the lytic enzymes used for cell wall degradation, osmotic stabilizers to prevent the lysis and the physiological status of the organisms. The fusion of isolated protoplast was efficiently done by chemical fusogens (PEG) or by electrofusion methods followed by the reversion of the protoplast by plating on osmotically stabilized agar media for regeneration of cell wall. In recent years several studies have been reported to show that this technique has been applied successfully to produce the somatic hybrids in various mushroom fungi intraspecifically (Kiguchi and Yanagi, 1985; Toyomatsu and Mori, 1987), interspecifically (Takehara et al., 1993; Matsumoto et al., 1997), intergenerically (Eguchi et al., 1993; Zhao and Chang, 1996) and even interheterogenerically (Eguchi and Higaki, 1995; Toyomatsu and Mori, 1987). A protoplast fusion experiment between white oyster mushroom (Pleurotus floridae) and brown oyster mushrooms brown oyster mushroom (Pleurotus cystidiosus) was conducted to obtain an oyster mushroom strain showing high productivity and long storage life (Djajanegaraa and Masduki, 2010) and between Pleurotus eous and Pleurotus flabellatus for strain improvement (Parani and Eyini, 2010). Interspecific hybridization between Ganodarma licidum and G. tsugae enhance the high and low temperature tolerance and production of bioactive

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compounds (Luk and Chiu, 2005) and intergeneric protoplast fusion between Calocybe indica (milky mushroom) and Pleurotus florida aids in the qualitative and quantitative improvement of sporophore of the milky mushroom (Chakraborty and Sikdar, 2010).

Biotechnological Approaches Recombinant DNA Technology and Transgenic Breeding The application of recombinant DNA technique in mushroom research has created numerous possibilities and opportunities for the genetic enhancement of mushroom. For creating transgenic mushroom, the desirable transgene from unrelated sources was introduced into the commercial strains. The first such transformation was made in Agaricus bisporus in 1993 (Mooibroek et al., 1993) and later on applied in other case also. There are two approaches in transgenic breeding either site directed integration of transgenes or antisense RNA technology. An efficient homologous site-directed integration of the transformation plasmid was done by isolating the tyrosinase genes responsible for mushroom browning from Agaricus bispous and introducing it in antisense orientation (Van de Rhee et al., 1996b). Another gene isolated and identified in mushrooms was the mannitol-dehydrogenase (MtDH) gene and its 3- dimensional structure has now become available (Sassoon et al., 2001). Isolation of this gene can allow the production of mushrooms with altered mannitol profiles and ultimately yield strains with higher dry matter content or better pathogen resistance (Stoop and Mooibroek, 1998). There is various transformation techniques adapted for transgenic breeding such as Agrobacterium mediated transformation and direct transformation like polyethylene glycol/CaCl2 (Li et al., 2006), electroporation and particle bombardment have been used to incorporate DNA into protoplasts, mycelium or basidiospores etc. Among these, PEG/CaCl2 was effectively used in transforming different species of Coprinus (Binninger et al., 1987; Challen et al., 2000), Pleurotus ostreatus and Volvariella volvacea (Jia et al., 1998) but remain experimental in Agaricus bisporus. While electroporation is effectively applied for transforming Agaricus bisporus by Van de Rhee et al. (1996a). The direct gene delivery by particle bombardment has also been experimented as an alternative method for genetic transformation in mushrooms (Li and Horgan, 1993). It has the advantage of being labour intensive and avoids problems associated with production and regeneration of protoplasts. In many laboratories, attempts have been undertaken to introduce hygromycin-B resistance and other selectable markers by particle bombardment. Successful transformation of Coprinus cinereus was reported by Moore et al. (1995). However, this technique has not yet resulted in the selection of stable transformants or an applicable system. As these previous transformation techniques are not very reliable or stable, The Agrobacterium mediated transformations become popularized. This system allows transformation of both homokaryons and heterokaryons, simultaneously (Mikosch et al., 2001). The use of A. tumefasciens for efficient transformation was first carried out in Agaricus bisporus by De Groot et al. (1998). But the problems associated with this method were non-reproducible, low level of integration and DNA modification after

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integration. A successful Agrobacterium-mediated transformation was done by infecting the fruiting gill tissue with Agrobacterium strains carrying the gene construct of interest and use of a vector with homologous promoter (Chen et al., 2000). There are many areas for transgenic breeding possibilities that include improvement in yield, size, colour, self life and nutrient composition; tolerance to temperature, heat and water stress; resistance for insects, pathogens, pesticides; and many others like strain stability, fruiting cycle regulation and substrate utilization etc. For example introduction of cry genes from Bacillus thuringiensis for insect resistance and synthetase resistance from Agrobacterium for glyphosphate herbicide resistance, pathogen derived resistance for viral disease resistance, homology depended silencing of tyrosinase gene and genes involved in agaritine biosynthetic pathway, etc. Mushroom Genome Sequencing The whole genome sequence of any organism is the basic requirement in this “Omics” era. The genome sequence of various mushroom is already available in public domain, e.g. Schizophyllum commune (Ohm et al., 2010), Ganoderma lucidum (Chen et al., 2012) and Agaricus bisporus (Morin et al., 2012) where as that of others is underway. The sequencing the mushroom genome is very important in various aspects like environmental safety, commercial application etc (Chakravarty, 2011). This will be helpful keeping in view their role in degradation of plant material into less harmful substances, removal of heavy metals from waste flows and in production of biofuels (Thwaites et al., 2007) etc. After sequencing, investigations will continue to find out the organization of functionally related genes, their distribution, regulation and expression at transcriptomic and proteomic level. That will also be helpful in MAS breeding and in transgenic approach. ‘Omics’ Approaches in Mushroom Research In this post-genome era, various “omics” approaches like genomics, transcriptomics, proteomics and metabonomics alone with bioinformatics can play an important role in assessment of the genotoxicity, teratogenicity and nephrotoxicity of mushroom. Global analysis of genome expression in the form of mRNA, protein profile and metabolite patterns will be helpful to predict the toxic chemicals having the same type of transcriptomics, proteomics and metabonomics profile in a biological system, i.e. toxic fingerprinting; to discover the genes or proteins as effective biomarkers for a given toxicity by in silico genome analysis or through comparative genomics and also to elucidate the inhibitory effects of toxic compounds singly or in mixture (Stierum et al., 2005; Xu et al., 2009; Villeneuve and Garcia-Reyero, 2011). Detail studies on transcriptomics, proteomics and/or metabonomics profiles may helpful to explore potential toxicity of any undesired compounds in mushroom and their toxic outcomes (Aardema and MacGregor, 2002).Similarly, toxicogenomics can also help to predict the possible differential response of different organisms (having different genome

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composition) to various toxic and allergic compounds of various edible and nonedible mushroom via in vitro and in vivo toxicity assessment.

Future Direction in Mushroom Research Mushroom research itself is not a new field. It started about thousands year back with their artificial cultivation in the tropical and subtropical region of the world. But this is restricted to only their use as good healthy foods. The real commercial ventures started mainly in developed countries with better utilization from agri-based industries to pharmaceutical and industrial purpose. The growing research on mushroom provides an immense opportunity for molecular pharming. The main aim of mushroom pharming is de novo synthesis of novel compounds through the introduction of genes from other organisms using transformation technologies. For mushroom pharming to become reality, the detail information about the genetics and molecular biology of different mushroom species along with suitable scientific technologies, adequate biosafety and regulatory measures are essential. This may open up a new way of research in the field of mushroom technology.

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