Optimization of in vitro regeneration protocol for a popular Indica rice (Oryza sativa L. cv Swarna)

Vijaya Naresh Juturu, Gopala Krishna Mekala, Mallikarjuna Garladinne, Puli Chandra Obul Reddy, Akila Chandra Sekhar*


Though regeneration system in rice has been very well established compare to other crop plants, the fact remains that, most of the indica rice varieties are still recalcitrant for regeneration and genetic transformation. Therefore, refinement of tissue culture protocol for generation of embryogenic calli and regeneration of the fertile plants from a single cell should be a pre requisite event for development of transgenic plants. Here, in this study we reported high frequency robust regeneration protocols for a popular Indica cultivar Swarna.Mature seeds were used as initial material as explants. Highest callus induction % was observed in MSCIMP medium containing 2.0 mg-1 2,4, D + 0.5 mg-1 Kn as phytohormonal combinations. In addition, maximum regeneration was observed in 2.0 mg-l KN + 0.5 mg-l NAA. Regenerated plants were shifted to rooting medium followed by polyhouse for hardening. The callus induction and regeneration reported in this study were well suited for transformation agronomical important genes or functional genomics studies.


Oryza sativa; Somatic embryogenesis; Swarna; Variety

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Aananthi N, C. R Anandakumar, R. Ushakumari, P. Shanthi, "Regeneration study of some indica rice cultivars followed by Agrobacterium mediated transformation of highly regenerable cultivar Pusa Basmati", Electronic Journal of Plant Breed (2010): 41249-1256.

Ali S, Q.Z Xue, X.Y Zhang, "Assessment of Various Factors Involved in the Tissue Culture System of Rice", Rice Sci, 11(2004): 345-349.

Biswas A, A.B Mandal, "Plant regeneration in different genotypes of indica rice", Indian J of Biotech, 6 (2007): 532-540

Cooke T.J, R.H Racusen, J.D Cohen, 'The role of auxin in plant embryogenesis", Plant Cell, 5 (2007): 1494-1495

Hiei Y, S. ohta, T. komari, T. kumashiro, "Efficient transformation of rice (Oryza sativus L) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA", Plant J, 6 (1994): 271-282

Hoque K.M.A, Z.A Azdi, S.H Prodhan, "Development of Callus Initiation and Regeneration System of Different Indigenous indica Rice Varieties', J Biol, 1 (2013): 46-51

Huang Li-Chung, C. Kohashi, R. Vangundy, T. Murashige, "Effects of Common Components on Hardness of Culture Media Prepared with Gelrite", In Vitro Cell Dev Biol Plant, 31 (1995): 84-89

Juturu V.N, G.K Mekala, P.B Kirti, "Current status of tissue culture and genetic transformation research in cotton (Gossypium spp)", P lant Cell Tissue Organ Cult, 120 (2015):813-839

Khaleda L, M. Al-Forkan, "Genotypic variability in callus induction and plant regeneration through somatic embryogenesis of five deep water rice (Oryza sativa L) cultivars of Bangladesh", Afr J Biotech, 6 (2006): 1435-1440

Khalequzzaman M, N. Haq, M. Hoque, T. Aditya, "Regeneration efficiency and genotypic effect of 15 indica type Bangladeshi rice (Oryza sativa L) Landraces", Plant Tissue Cult & Biotech, 15 (2005): 33-42

Kumar K.K, S. Maruthasalam, M. Loganathan, D. Sudhakar, P. Balasubramanian, "An improved Agrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars", Plant Mol Biol Rep, 23 (2005): 67-73

Kumria R, M.V Rajam, "Alteration in polyamine titres during Agrobacterium-mediated transformation of indica rice with ornithine decarboxylase gene affects plant regeneration potential", Plant Sci, 162 (2002): 769-777

Kumria R, B. Waie, M.V Rajam, "Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium", Plant Cell Tiss Organ Cult, 67 (2001): 63-71

Merkle S.A, W.A Parrott, B.S Finn, "Morphogenenic aspects of somatic embryogenesis, In: Thrope TA (ed) In vitro embryogenesis in plants", Kulwer Dordrecht, (1995) pp 155-203

Moura D.S, F.J ZapataArias, A. Ando, A.T Neto, "Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars", Euphytica, 94 (1997): 01-05

Pasqualetto P.L, R.H Zimmerman, I. Fordham, "Gelling agent and growth regulator effects on shoot vitrification of phytagel", Apple in vitro J Am Soc Hort Sci, 111(1986): 976-980

Pawar B, P. Kale, J. Bahurupe, A. Jadhav, A. Kale, S. Pawar, "Proline and glutamine improve in vitro callus induction and subsequent shooting in rice", Rice Sci, 22 (2015): 283-289

Rao M.V.R, K.S Behra, N. Baisakh, S.K Datta, G.J.N Rao, "Transgenic indica rice cultivar ‘Swarna’ expressing a potato chymotrypsin inhibitor pin2 gene show enhanced levels of resistance to yellow stem borer", Plant Cell Tissue Organ Cult, 99 (2009): 277-285

Rattana K, Threerakulpisut, S. Bunnag, "The effect of plant growth regulators and organic suppliments on callus induction and plant regeneration in rice (Oryza sativa L)", Asian J Plant Sci, 11 (2012): 182-189

Rueb S, M. Leneman, R.A Schilperoort, L.A.M Hensgens, "Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L)", Plant Cell Tiss Org Cult, 36 (1994): 259-264

Saharan V, R.C Yadav, N.R Yadav, B.P Chapagain, "High frequency plant regeneration from desiccated calli of indica rice (Oryza sativa L)", Afr J Biotechnol, 3 (2004): 256-259

Sahoo K. K, A.K Tripathi, A. Pareek, S.K Sopory, S.L SinglaPareek, "An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars" Plant Methods, 7 (2011): 49

Sankepally S.S.R, B. Singh, "Optimization of regeneration using differential growth regulators in indica rice cultivars", 3 Biotech, 6 (2016): 19, DOI 101007/s13205-015-0343-0

Shobbar M.S, V. Niknam, Z.S Shobbar, H. Ebrahimzadeh, "Effect of salt and drought stresses on some physiological traits of three rice genotypes differing in salt tolerance", JUST, 36 (2010): 1-9

Sivakumar P, P. Sharmila, P. Pardhasaradhi, "Proline alleviates salt-stress-induced enhancement in ribulose-1 5-bisphosphate oxygenase activity", Biochem Biophys Res Commun, 279 (2000): 512-515

Sridevi G, M. Dhandapani, K. Veluthambi, "Agrobacterium mediated transformation of white ponni a non-basmati variety of indica rice (Oryza sativa L)", Curr Sci, 88 (2005): 128-132

Tariq M, G. Ali, F. Hadi, S. Ahmad, N. Ali, A.A Shah, "Callus induction and in vitro plant regeneration of rice (Oryza sativa L) under various conditions", Pak J Biol Sci, 11(2008): 255-259

Toki S, "Rapid and efficient Agrobacterium-mediated transformation in rice", Plant Mol Biol Rep, 15 (1997): 16-21

Toki S, N. Hara, K. Ono, H. Onodera, A. Tagiri, S. Oka, H. Tanaka, "Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice", The Plant J, 47 (2006): 969–976

Tyagi H, S. Rajasubramanium, I. Dasgupta, "Regeneration and Agrobacterium mediated transformation of popular indica rice variety ADT39", Curr Sci, 93 (2007): 678-683

Zaidi M.A, M. Narayanan, R. Sardana, I. Taga, S. Postel, R. Johns, M. McNulty, Y. Mottiar, J. Mao, E. Loit, I. Altosaar, "Optimizing tissue culture media for efficient transformation of different indica rice genotypes", Agronomy Res, 4 (2006): 563-575

Zhou J, X. Wang, Y. Jiao, Y. Qin, X. Liu, K. He, C.H Chen, L. Ma, J. Wang, L. Xiong, Q. Zhang, L. Fan, X.W Deng, "Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot flag leaf and panicle", Plant Mol Biol, 63 (2007): 591-608.

DOI: https://doi.org/10.21746/aps.2016.08.002

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