In Vitro Shoot Multiplication of Gloriosa Superba L.

International Conference on Biotechnology, Biological and Biosystems Engineering (ICBBBE'2012) December 18-19, 2012 Phuket (Thailand)

In Vitro Shoot Multiplication of Gloriosa Superba L. – An Important Anticancer Medicinal Herb P. Venkatachalam*, N. Ezhili And M. Thiyagarajan

It is also used in the treatment of gout because it contains colchicine. Paste of the tuber is externally applied for parasitic skin diseases.The plant was under threatened category due to its imprudent harvesting from wild as it is extensively used by medicinal industries for its colchicine content. It also faces a low seed set problem, but due to its industrial demand it is now under cultivation (Sonali Jana and Shekhawat, 2011). Glory lily is an industrial medicinal crop in South India, for its high colchicine content, which is still collected from wild. Due to its over-exploitation and unscientific collection in wild as well as problems faced during field cultivation, it was on the verge of extinction and G. superba is included in the world record of endangered plants i.e. Red Data Book by International Union for Conservation of Nature (IUCN) (Sivakumar and Krishnamurthy 2000; Badola, 2002). In vitro propagation method is urgently needed not only to conserve this taxon but also to meet the tremendous demands of this medicinal plant as a source of colchicine. The main advantage of this technology is the year-round availability of plant material for colchicine production without geographical constraints; the products can be free of pesticide contamination, and production cost and time can be substantially reduced (Sivakumar et al., 2004). Somani et al. (1989) reported in vitro propagation and corm formation in G. superba. Samarjeeva et al. (1993) studied clonal propagation of G. superba from apical bud and node segment of shoot tip, cultured on solidified agar (0.8% w/v) Gamborg’s B5 medium containing BA, IAA, Kinetin, NAA, IBA or 2,4-D. Custers and Bergervoet (1994) reported micropropagation of G. superba by shoot cuttings and explants from node, internode, leaves, flowers, pedicels and tubers. Sivakumar and Krishnamurthy (2004) reported in vitro organogenetic responses of G. superba. The maximum number of multiple shoot (57%) was observed in cormderived calluses. Hassan and Roy (2005) reported 92% of the cultures of apical and axillary buds of young sprout from naturally grown G. superba plants regenerate four shoots per culture in MS basal medium fortified with 1.5 mg/l BA + 0.5 mg/l NAA. In the present study, to develop an efficient protocol for in vitro shoot multiplication of Gloriosa superba using tuber explant.

Abstract--Tuber explants Gloriosa superba were cultured on MS medium supplemented with different concentrations (0.5-3.0 mg/l) of BAP and KIN for shoot bud induction. Newly initiated shoots were excised and subcultured onto MS medium fortified with different concentrations (0.5-3.0 mg/l) of BAP combination with 0.5 mg/l KIN. The shoots were proliferated and elongated upto 7.0 cm with little callus within 3 weeks of culture. Highest frequency (80.2%) of shoot proliferation (6.2 shoots/explant) was observed on MS medium supplemented with 2.0 mg/l BAP combination with 0.5 mg/l KIN. In another experiment, regenerated shoots were subcultured onto MS medium fortified with 2.0 mg/l of 2,4 D where profuse, whitish yellow intact callus produced within 2 to 3 weeks of culture. The callus was cultured onto MS medium supplemented with different concentrations of BAP (1.0-3.0 mg/l) in combination with KIN (0.5 mg/l) for shoot regeneration. The callus was further developed into tuber like structures. The regenerated shoots were cultured on Half Strength MS medium supplemented with different concentrations (0.5 – 2.0 mg/l) IBA and IAA. Of the two auxins used, IBA at 1.0 mg/l was found to be the best concentration for maximum percent of root initiation. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently established in the greenhouse. The present study describes an efficient protocol for in vitro regeneration of Gloriosa superba an important medicinal plant. Keywords-- Gloriosa superba, KIN, BAP, IBA, IAA I. INTRODUCTION

G

loriosa superba L., a member of the Liliaceae family, is very important medicinal plant which contains alkaloids, mainly colchicine and colchicoside. The seeds of this taxon are highly priced in the world market as they are the main source of colchicines and colchicoside. In order to provide enough plant material for commercial exploitation, mass multiplication through tissue culture is urgently needed not only to conserve this taxon but also to meet the demands for this medicinal plant as a source of colchicine. Different parts of the plant have a wide variety of uses especially within traditional medicine practiced in tropical Africa and Asia. The tuber is used traditionally for the treatment of bruises and sprains, colic, chronic ulcers, hemorrhoids, cancer, leprosy and also for inducing labour pains. Medicinally, the tuber is used as abortifacient, and in smaller doses it acts as a tonic, stomachic and anthelmintic.

II. P. Venkatachalam*, N. Ezhili And M. Thiyagarajan are with Plant Genetic Engineering and Molecular Biology lab, Department of Biotechnology, Periyar University, Salem-636011, TN, India [* For Correspondence: E-Mail:[email protected]]

MATERIALS AND METHODS

Plant material and surface sterilization Mature and healthy tubers of Gloriosa superba were collected from Kolli Hills. The explants were washed thoroughly under running tap water along with 5.0% (v/v) 246


Tween- 20 for 1hr, followed by soaked with 0.1% (w/v) of Bavistin for 30 min and dipped in 70% (v/v) ethanol for 1.0 min. Then they were surface sterilized with 0.1% (w/v) mercuric chloride for 10 mins, followed by five times rinsed with sterile distilled water. Culture conditions For in vitro experiments, the medium used was MS basal salts supplemented with 3.0 % (w/v) sucrose. The pH of the medium was adjusted to 5.8 before adding 0.7 % (w/v) agar. The cultures were incubated at 24±2◦ C under 16/8 h (light/dark cycle) photoperiod (60 µEm-2s-1) irradiance provided by cool-white fluorescent tubes (Philips, India). Shoot bud induction The surface sterilized tubers were sized to 1.0-1.5cm length and were placed vertically on MS medium supplemented with different concentrations of BAP (1.0-4.0 mg/l) alone and/or 1.0–3.0 mg/l BAP in combination with 0.5 mg/l KIN and 2, 4D (1.0, 2.0 and 3.0 mg/l) for shoot bud induction. Rooting and acclimatization Regenerated shoots with approximately 3-5 cm in length were transferred to root induction medium with half - strength MS salts supplemented with different concentration of (0.52.0 mg/l) IBA and IAA for root induction. Healthy shoots with well developed roots were transferred to plastic cups containing sand and soil in the ratio 1:2. Polyethylene covers were placed over the plantlets for the first 2 weeks for acclimatization and then transferred to green house and finally acclimatized in the field.

Statistical analysis Experiments were set up in a completely randomized block (CRB) design and each experiment had three replicates. The cultures were observed periodically and percent of response for shoot bud regeneration, multiple shoots development and rooting. A total number of shoots as well as roots were also recorded by visual observations. The analysis of variance (ANOVA) was performed using SAS programme. The differences among means were determined by Student– Newman–Keuls Test at 5% significance level. III. RESULTS AND DISCUSSION Tuber explants were cultured on MS medium supplemented with different concentrations (1.0-4.0 mg/l) of BAP for shoot bud induction. After two weeks of culture, the shoots were elongated upto 4.5-5.0 cm long and a green tuber like structure was developed from the base of each aerial shoot. Elongation of the aerial shoots as well as these structure attached to the mother tuber differed on different basal media. Newly initiated shoots were excised and subcultured onto MS medium fortified with different concentrations (1.0-3.0 mg/l) of BAP combination with 0.5 mg/l KIN. The shoots were proliferated and elongated upto 7.0 cm with little callus within 3 weeks of culture. Highest frequency (87.3%) of shoot proliferation (6.2 shoots/explant) was observed on MS medium supplemented with 2.0 mg/l BAP combination with 0.5 mg/l KIN (Table 1). The percentage response and the induction of multiple shoots declined with increase in concentration of BAP beyond the optimal level (2.0 mg/l). Reduction in the number of shoots regenerated per explant at BAP concentration higher than the optimal level was also reported in A. squamosa (Nagori and Purohit, 2004).

TABLE 1. EFFECT OF DIFFERENT CONCENTRATIONS OF BAP ALONE AND/OR BAP+KIN COMBINATIONS AND 2, 4 D FOR SHOOT BUD INDUCTION OF GLORIOSA SUPERBA

Hormone Conc. ( mg/l) BAP 1.0 2.0 3.0 4.0 BAP+ KIN 1.0+0.5 2.0+0.5 3.0+0.5 2, 4 D 1.0 2.0 3.0

Percent shoot regeneration (Mean ± SE)

No of shoots/explant (Mean ± SE)

63.10±0.89c 84.00±1.35a 60.00±0.57c 46.10±1.49e

2.3±0.32d 3.4±0.45c 2.5±0.23d 1.8±0.19

68.45±2.13c 87.34±1.87a 63.42±1.34c

4.5±1.21b 6.2±1.56a 3.9±0.67c

80.70±0.65b 85.70±0.78a 57.14±1.32d

0.00(callusing) 0.00(callusing) 0.00(callusing)

Mean values within the column followed by the same letter in superscript are not significantly different at P< 0.05 level

In another experiment, regenerated shoots were subcultured onto MS medium fortified with 2.0 mg/l of 2,4 D where profuse, whitish yellow intact callus produced within 2 to 3 weeks of culture. The callus was proliferated and doubled in size after every subculture (21days) onto the fresh medium 247

which was further developed into globular like embryos. 2,4D also improved callus formation from shoot tips of ginger, another member of Zingiberaceae (Guo et al. 2007). The callus was cultured onto MS medium supplemented with different concentrations of BAP (1.0, 1.5, 2.0, 2.5 and 3.0


mg/l) in combination with KIN (0.5 mg/l) for shoot regeneration. The callus was further developed into tuber like structures. Initially the tuberous like structure of Gloriosa

callus culture were whitish in color and later turned into greenish colour.

Fig 1. A), B) and C) Different stages of Multiple shoot bud induction of Gloriosa superba from tuber explants

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TABLE 2. EFFECT OF DIFFERENT CONCENTRATION OF AUXIN FOR ROOT INDUCTION OF GLORIOSA SUPERBA

Hormone Concentration (mg/l) IBA 0.5 1.0 1.5 2.0 NAA 0.5 1.0 1.5 2.0

Percent of root induction

Mean number of roots per shoot

-65 ---

-7.2 ---

-----

---(callusing)

[2]

The regenerated shot buds were cultured on half- strength MS medium supplemented with different concentrations (0.5 – 2.0 mg/l) of IBA and NAA for root induction. Of the different concentrations of auxin tested, the root induction was noticed at 1.0 mg/l IBA. The success of IBA in promoting efficient root induction has been reported for Swaisona formosa (Jusaitis 1997), Hemidesmus indicus (Sreekumar et al., 2000), Cunila galoides (Fracaro and Echeverrigaray 2001), Ceropegia candelabrum (Beena et al., 2003), and Mucuna pruriens (Faisal et al., 2006). The rooted plantlets with expanded leaves were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and covered with polythene bags to ensure high humidity. The plantlets were kept in the controlled environment for two weeks and the polybags were gradually removed in order to acclimatize the plantlets under greenhouse conditions. Subsequently they were transferred to the field conditions and the regenerated plants grew well and phenotypically similar to the parental stock.

[3]

[4]

[5]

[6] [7]

[8]

[9]

[10]

IV. CONCLUSION The present study reveals an efficient protocol for regeneration of Gloriosa superba. from tuber explants. Two different cytokinins and auxins were tested; among these the highest multiple rates were recorded at 2.0 mg/l BAP and 0.5 mg/l KIN found to be best for shoot regeneration and 1.0 mg/l IBA for rooting.

[11] [12]

[13]

[14]

ACKNOWLEDGEMENT Dr. P. Venkatachalam gratefully acknowledges financial assistance for this work provided by University Grant Commission under UGC Major Project No. 37-297/2009 (SR), Govt. of India, New Delhi. REFERENCES [1]

Badola, H.K. 2002. Endangered medicinal plant species in Himachal Pradesh. A report on the international workshop on Endangered Medicinal Plant species in Himachal Pradesh organized by G.B. Plant Institute of Himalayan Environment and Development at Himachal Unit. Curr. Sci. 83:797–8.

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Bhakuni, D.S. and Jain, S. 1995. Advances in horticulture, 11. Delhi: Malhotra Publishing House; 1995. p. 98–9. Custers, J.B.M. and Bergervoet, J.H.W. 1994. Micropropagation of Gloriosa: towards a practical protocol. Scientia Horticulturae. 57:323334. Guo, Y.H., Bai, J.H. and Zhang, Z.X. 2007. Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Plant Cell Tissue Org. Cult. 89:151–157. Hassan, S.A. and Roy, S.K. 2005. Micropropagation of Gloriosa superba L. through high frequency shoot proliferation. Plant Tiss. Cult. 15(1):6774. Nadkarni, K.M. 1996. Indian Materia Medica, 3rd ed, vol. 2. Popular Prakashan Mumbai. p. 579–583. Nagori, R. and Purohit, S.D. 2004. Plantlet regeneration in Annona squamosa through direct shoot bud differentiation on hypocotyl segments. Sci. Hort. 99(1), 89–98. Samarajeewa, P.K., Dassanayake, M.D. and Jayawardena, S.D.G. 1993. Clonal propagation of Gloriosa superba. Indian J. Exp. Biol. 31:719720. Sivakumar, G. and Krishnamurthy, K.V. 2002. Gloriosa superba L. — a very useful medicinal plant. Ethnomedicine and Pharmacognosy, Part II. Texas: Series Sci Tech Pub, Texas, USA p. 465–482. Sivakumar, G. and Krishnamurthy, K.V. 2002. Micropropagation of G. superba L. — an overexploited medicinal plant species from India. In: Nandi SK, Palni LMS, Kumar A, editors. Role of Plant Tissue Culture in India in Biodiversity Conservation and Economic Development. Nainital, India: Gyanodaya Prakashan Publications. p. 345–50. Sivakumar, G. and Krishnamurthy, K.V. 2004. In vitro organogenetic responses of Gloriosa superba. Russian J. Plant Physiol. 51:790-798. Sivakumar, G., Krishnamurthy, K.V., Hao, J. and Paek, K.Y. 2004. Colchicine Production In Gloriosa superba Calluses By Feeding Precursors. Chemistry of Natural Compounds. 40:5 Somani, V.J., John, C.K. and Thengane, R.J. 1989. In vitro propagation and corm formation in Gloriosa superba. Indian J. Exp. Biol. 27:578579. Sonali Jana and Shekhawat, G.S. 2011. Critical review on medicinally potent plant species: Gloriosa superba. Fitoterapia 82:293–301


In Vivo Infection of Mycobacterium sp. in Osphronemus gouramy Uun Yanuhar and Sri Rahmaningsih

S12 code, or also in the pncA gene and embB gene that often makes the bacteria character easy to mutate [1] [2]. The nature of this mutation allow the spread of Mtb in waters especially in fish that are consumed by humans as a meal. Infection Mtb can through feces or urine or the consumption of fish meat. Symptoms of infection arising carp after Mtb infection were tested in vivo showed symptoms such as fish become active, experienced bleaching skin color, decreased appetite, inflamation occurs on the skin (integument), and bleeding or haemorage septicemia [3]. The purpose of this study was to identify Mtb infection in vivo in Osphronemus gouramy and infections response emerging in fish by examination by IHC and ICC.

Abstract— This study aimed to identify the infection of Mycobacterium sp. in Osphronemus gouramy. This study used pure isolates of M. tuberculosis (Mtb), and to identify and determine protein molecular weight of Mtb by SDS-PAGE and electro elusion. Mtb infection in fish was observed based on the results of in vivo test for 4 days on pisciculture in the aquarium. Mtb infection was confirmed by molecular weight immunogenic protein using SDSPage and hemagglutination assay (HA), as well as western blotting (WB) and immunohistochemistry (IHC) and or immunocytochemistry (ICC). Studies of fish infection in vivo based on the symptoms the fish that comes up, then an immunogenic protein profiles in fish and specificity cellular response to MTB infection using antibodies labeling against the immunogenic protein of Mtb in fish and the cellular response of immune cells (lymphocytes) that is formed against Mycobacterium tuberculosis by ICC. Result show that analysis of proteins formed in the fish was protein 23 kDa. It is an immunogenic protein expressed in the muscle meat of fish and going haemorrhage septicemia. Early symptoms of infection are darkening the color, the haemorrage septicemia, ulcers and inflammation of fish tissue in vivo as evidenced by examination of IHC and ICC

Keywords— Osphronemus Infection, IHC, ICC.

gouramy,

Mycobacterium

II. PROCEDURE Mycobacterium sp Adhesin Protein Isolation Mtb with isolates code 1070 ST cultured on Middlebrook. Cultures were used for inoculation of 100 Middlebrook 7H11 agar plate with modifications OADC and incubated for 2 weeks. Bacteria harvested using sterile glass Spreader and resuspended in 200 ml PBS. The suspension was divided into 25 ml, and the conducted adhesin isolation mechanically. Results shave bacterial supernatant and pellet were separated. Pellets were washed with PBS to obtain adhesin protein and debris were eliminated by centrifugation at 3000xg 2 times and 18000xg for 10 minutes. Lipid contamination was eliminated after divortex mixed with chloroform / methanol 2: 1 for 1 hour with the same volume. After it was centrifuged at 18 000 xg for 30 minutes until the phase 35 ml, a watery liquid and interphase fraction carefully collected and extracted 2 times and dissolved materials (lipids) removed. Last watery liquid fraction and centrifuged at 120,000 xg for 3 h at 4 °C in the Ti-56 angle rotor (Beckman) and analyzed by SDS-Page 16%. The protein concentration was determined by spectrophotometer.

sp,

I. INTRODUCTION

O

ne of the host or vector of the spread of a disease that is

transferred from one host to another host is a fish, either directly or indirectly. One group of fish diseases can be caused by the bacteria of the type of Mycobacterium sp. This bacterium is one of the causes of Tuberculosis disease or Mycobacteriosis. The disease is caused by bacteria belonging to the Mycobacterium sp which is a type of bacteria Mycobacterium marinum, where bacteria are found in many types of fish. The bacteria can also be found in cold and hot water, in fresh water or sea water. At the genus level, the bacteria is composed of more than 80 species, invade also in cattle, birds and even humans [1]. Mycobacterium (Mtb) easily mutate, because Mtb bacteria in the 16S rRNA gene or the gene rpLs with ribosomal protein

Identification Crude Protein by SDS-Page Isolation of protein performed by electrophoresis using SDSPage, with a 12.5% gel concentration [4]. After electrophoresis, gel electrophoresis resulted was performed staining for 30 to 60 minutes. Then do the destaining overnight to fade staining over the shaker. In this condition we can determine the presence and measuring protein mobility is then determined for molecular weight. .

Uun Yanuhar is with the Faculty of Fisheries and Marine Science, University of Brawijaya, East Java, Indonesia (corresponding author to provide phone: +6281931892262; e-mail: [email protected]). Sri Rahmaningsih, was with University of Ronggolawe, East Java, Indonesia. She is now with the Department of Fisheries and Marine Science, University of of Ronggolawe, East Java, Indonesia [email protected]). .

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