Micropropagation of Chlorophytum borivilianum: In vitro Clonal Fidelity Test and Antioxidant Enzymatic Study
The regeneration of Chlorophytum borivilianum is generally through tuberous roots, which have become scarce in nature. Seed germination is only 14-16%, thus an in vitro method for conservation and multiplication of this species could be valuable. In vitro clonal propagation of safed musli has been achieved on MS medium augmented with auxins: 2, 4-Dicholorophenoxyacetic acid (2, 4-D), 1-Naphthaleneacetic acid (NAA) and cytokinins: Benzyladenine (BA), Kinetin (Kn). There was a 90% shoot multiplication response obtained at MS + 4.4 µM BA. Isolated shoots were transferred on ½ MS medium supplemented with 2.4 µM IBA (indole-3-acetic acid) which gave 90% rooting response, were further acclimatized and hardened to garden successfully. In vitro regenerated plants have been tested for clonal fidelity using RAPD markers. It is first report on biochemical changes of catalase, peroxidase, super oxide dismutase, protein and chlorophyll content during in vitro regeneration in C. borivilianum.
Chlorophytum borivilianum, RAPD, Catalase, Genetic Fidelity, Superoxide Dismutase
[1]
Aebi, H. 1974. “Catalases.” In Methods of Enzymatic Analysis. Edited by H.U. Bergmeyer, pp. 673-684. New York: Verlag Chemie, Weinheim, Academic Press.
[2]
Agrawal, R., Upadhyay, A., & Nayak, P. S. 2013. Drying characteristics of Safed Musli (Chlorophytum borivilianum) and its effect on colour and saponin content. Journal of Pharmacognosy and Phytotherapy 5(8):142-147.
[3]
Arnon, D.I. 1949. Copper enzymes in isolated chloroplast: polyphenol oxidases in Beta vulgaris. Plant Physiology 24:1-15.
[4]
Beuchamp, C., & Fridovich, I. 1971. Superoxide dismutase: improved assays and an acrylamide gels. Analls of Biochemistry 44: 276-287.
[5]
Bhat, S.R., Chandel, K.P.S., & Malik. S.K. 1995. Plant regeneration from various explants of cultivated Piper species. Plant Cell Reports 14: 398-402.
[6]
Bindiya, K., & Kanwar K. 2003. Random amplified polymorphic DNA (RAPDs) markers for genetic analysis in micropropagated plants of Robinia pseudoacacia L. Euphytica 132: 41-47.
[7]
Cassells, A.C., Croke, J.T., & Doyle, B.M. 1997. Evaluation of image analysis, flow cytometry, and RAPD analysis for the assessment of somaclonal variation and induced mutation in tissue culture-derived Pelargonium plants. Angewandte Botanik 71(3-4): 125-130.
[8]
Dave, A., Bilochi, G., & Purohit, S.D. 2003. Scale–up production and field performance of micropropagated medicinal herb ‘safed musli’ (Chlorophytum borivilianum). InVitro Cellular and Developmental. Biology- Plant 39: 419.
[9]
Doyle, J.J., & Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12:13-15.
[10]
Fernando, A., Barney, V.E., Tohme, J., & Roca, W.M., 1996. Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage. Euphytica 90: 307-313.
[11]
Gaikwad, P.D., John, S.C., & Billore, M. 2003. Callus induction and regeneration studies in safed musli (Chlorophytum borivilianum Sant & Fern). Research on Crops 4: 249–253.
[12]
Gaspar, T. 1995.The concept of cancer in in vitro plant cultures and the implication of habituation to hormones and hyperhydricity. Plant Tissue Culture and Biotechnology 1: 126-136.
[13]
Gupta, S. D., & Datta. S. 2003. Antioxidant enzyme activities during in vitro morphogenesis of gladiolus and the effect of application of antioxidants on plant regeneration. Biologia Plantarum 47: 179-183.
[14]
Hussain, Z., Tyagi, R.K., Sharma, R., & Agrawal, A. 2008. Genetic diversity in in vitro-conserved germplasm of Curcuma L. as revealed by RAPD markers. Biologia Plantarum 52: 627-633.
[15]
Jana, S., & Shekhawat, G.S. 2010. Plant growth regulators, adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of Anethum graveolens. Acta Physiologiae Plantarum 33: 305-311.
[16]
Jana, S., & Shekhawat, G.S. 2011. In vitro regeneration of Anethum graveolens, antioxidative enzymes during organogenesis and RAPD analysis for clonal fidelity. Biologia Plantarum DOI: 10.1007/s10535-011-0223-3.
[17]
Joshi, A., Ganesh, R., Sharma, A., & Sharma, G. S. 1999. Plant regeneration from stem-disc derived callus cultures of Chlorophytum borivilianum. Annual Review of Plant Physiology 13:79–83.
[18]
Joshi, N., & Purohit, S.D. 2012. Optimization of factors influencing shoot multiplication during micropropagation of chlorophytum borivilianum Sant. et Fernand. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 82: 421-429.
[19]
Kemat, N., Kadir, M.A., Abdullah, N.A.P., & Ashraf, F. 2010. Rapid multiplication of safed musli (Chlorophytum borivilianum) through shoot proliferation. African Journal of Biotechnology 9: 4595-4600.
[20]
Lee, S.H., Tewari, R.K., Hahn, E.J., & Paek, K.Y. 2007. Photon flux density and light quality induce changes in growth, stomatal development, photosynthesis and the transpiration of W. somnifera (L.) Dunal. Plantlets. Plant Cell, Tissue and Organ Culture 90:141-151.
[21]
Lowry, O.H., Rosenberg, N.J., Farr, A.L., & Randall, R.J. 1951. Protein measurement with folin phenol reagent. The Journal of Biological Chemistry 393 193: 265-275.
[22]
Maiti, S., & Geetha, K.A. 2005. Plant introduction in medicinal and aromatic plants: achievements and opportunities. Indian Journal of Plant Genetic Resources 18: 22-23.
[23]
Meratan, A.A., Ghaffari, S.M. & Nikam, V. 2009. In vitro organogensis and antioxidant enzymes activity in Acanthophullum sordidum. Biologia Plantarum 53: 5-10.
[24]
Mimaki, Y., Kanmoto, T., Sashida, Y., Nishino, A., Satomi, Y., & Nishino, H. 1996.Steroidal saponins from the underground parts of Chlorophytum comosum and their inhibitory activity on tumour promoter-induced phospholipid metabolism of HeLa cells. Phytochemistry 41:1405-1410.
[25]
Misra, P., Pandey, V., & Kochhar, S. 2010. Study of antioxidant enzymes activity during organogenesis and in vitro propagation of Asiatic hybrid lily. Journal of Plant Biochemistry and Biotechnology 19: 119-122.
[26]
Molassiotis, A.N., Dimassi, K., Diantidis, G., & Therios, I. 2004. Changes in peroxidase and catalase activity during in vitro rooting. Biologia Plantarum 48:1-5.
[27]
Murashige, T., & Skoog, F. 1962. A revised medium for rapid growth and bioassays tobacco tissue cultures. Acta Physiologiae Plantarum 15: 473-497.
[28]
Narasimham, K.R.S.L., & Ravuru, B.K. 2003. A note on some endangered medicinal plants as NTFPs of Eastern Ghats, Andhra Pradesh. EPTRI-ENVIS Newsletter, EPTRI, Hyderabad 9(2):11-12.
[29]
Ostray, M.E., Hackett, W., Michler, C., Serres, R., & McCown, B. 1994. Influence of regeneration method and tissue culture on the frequency of somatic variation in Populus to infection by Septoria musiva. Plant Science 97: 209-215.
[30]
Panigrahi, J., Behera, M., Maharana, S., & Mishra, R.R. 2007. Biomolecular changes during in vitro organogensis of Asteracantha longifolia. Indian Journal of Experimental Biology 45: 911-919.
[31]
Putter, J. 1974. “Peroxidases.” In Methods of enzymatic analysis, Edited by H.U. Bergmeyer, 85–690, 2nd ed. Elzievir, New York.
[32]
Purohit, S.D., Dave, A., & Kukda, G. 1994. Micropropagation of safed musli (Chlorophytum borivilianum), a rare Indian medicinal herb. Plant Cell, Tissue and Organ Culture 39: 93-96.
[33]
Qiu, S.X., Li, X.C., Xiong, Y.S., Dong, Y.M., Chai, H.B., Fransworth, N.R., Pezzuto, J.M., & Fong, H.H.S. 2000. Isolation and characterization of cytotoxic saponin chloromaloside - A from Chlorophytum malayense. Planta Medica 66: 587-590.
[34]
Rani, V., & Raina, S.N., 2000. Genetic fidelity of organized meristem derived micropropagated plants: a critical reappraisal. In Vitro Cellular and Developmental Biology-plant 36: 319-330.
[35]
Samantaray, S., & Maiti, S. 2010. An assessment of genetic fidelity of micropropagated plants of Chlorophytum borivilianum using RAPD markers. Biologia Plantarum 54: 334-338.
[36]
Samantaray, S., Patel, T.K., Kumar, T., Geetha, K.A., & Maiti, S. 2011. Identification and assessment of genetic relationships in three Chlorophytum species and two high yielding genotypes of C. borivilianum through RAPD markers. Biologia 66(2): 244-250.
[37]
Sharifi, G., & Ebrahimzadeh, H. 2010. Changes of antioxidant enzyme activities and isoenzyme profiles during in vitro shoot formation in saffron (Crocus sativus L.). Acta Biologica Hungarica 61: 73-89.
[38]
Shi, X.L., Han, H.P., Shi, W.L., & Li, Y.X. 2006. NaCl and TDZ are two key factors for the improvement of in vitro regeneration rate of Salicornia europaea L. Journal of Integrative Plant Biology 48: 1185-1189.
[39]
Suri, S.S., Jain, S.K., Arora, D.K., & Ramawat, K.G. 1999. In vitro high frequency regenaration of plantlets and tuberous root formation in Chlorophytum borivilianum. Gartenbauwissenschaft 64:106–110.
[40]
Van Breusegem, F.M., Vernova, E., Dat, J.F., & Inze, D. 2001. The role of oxygen in plant signal transduction. Plant Science 161: 405-414.
