Abstract
Lacking of an efficient regeneration protocol for the recalcitrant crop chickpea is a limiting factor for adapting genetic engineering approaches for its improvement. The present study describes a rapid and efficient method for multiple shoot regeneration for three Indian cultivars, B115, C235, ICCV89314, using single cotyledons with half embryos as explant. Modified MS medium with 1.5 mg l−1 6-benzyladenine (BA) and 0.04 mg l−1 α-naphthaleneacetic acid (NAA) induced a maximum of 26 shoots from a single explant after 20 days of culture. When cultured in modified MS medium containing 0.2 mg l−1 indole-3-acetic acid (IAA), 80% of the shoots from each regenerating explant elongated in another 20–25 days. Following a root-grafting protocol, 90–95% of the elongated shoots survived in soil which subsequently produced seeds. The regeneration process from explant preparation to complete plants took 55–60 days. The presently optimized rapid regeneration method holds promise for facilitating the deployment of agronomically important components through genetic transformation for betterment of this important food crop.
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References
Al-Khayri JM, Al-Bahrany AM (2001) In vitro micropropagation of Citrus aurantifolia (lime) Curr. Sci. 81:1242–1246
Barna KS, Wakhlu AK (1994). Whole plant regeneration of Cicer arietinum from callus cultures via organogenesis Plant Cell Rep. 13:510–513
Brandt EB, Hess D (1994). In vitro regeneration and propagation of chickpea (Cicer arietinum L.) from meristem tips and cotyledonary nodes In Vitro Cell Dev. Biol. Plant 30P:75–80
Chakrabarty R, Singh DT, Garg GK (2000) Transformation studies on chickpea embryo axis J. Plant Biochem. Biotechnol. 9:107–110
Chandra A, Pental D (2003) Regeneration and genetic transformation of grain legumes: An overview Curr. Sci. 84:381–387
Gamborg OL, Miller RA, Ojima K (1968). Nutrient requirements of suspension cultures of soybean root cells Exp. Cell Res. 50:151–158
Hoagland DR, Arnon DI (1950). The Water-Culture Method for Growing Plants Without Soil. Circ. 347 California Agricultural Experimental Station, College of Agriculture, University of California Berkley
Jayanand B, Sudarsanam G, Sharma KK (2003). An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from In vitro – germinated seedlings In Vitro Cell Dev. Biol. Plant 39:171–179
Kar S, Johnson TM, Nayak P, Sen SK (1996). Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of Chickpea (Cicer arietinum L.) Plant Cell Rep. 16: 32–37
Kar S, Basu D, Das S, Ramkrishnan NA, Mukherjee P, Nayak P & Sen SK (1997) Expression of Cry1A(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod-borer (Heliothis armigera) larvae. Transgenic Res. 6: 177–185
Murashige T, Skoog F (1962). A revised method for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant 15:473–497
Murthy BNS, Victor J, Singh RP, Fletcher RA, Saxena PK (1996). In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron Plant Growth Regul. 19:233–240
Polisetty R, Patil P, Deveshwar JJ, Khetarpal S, Chandra R (1996). Rooting and establishment of in vitro grown shoot tip explants of chickpea (Cicer arietinum L.) Ind. J. Exp. Biol. 34:806–809
Polisetty R, Paul V, Deveshwar JJ, Khetarpal S, Suresh K, Chandra R (1997). Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.) Plant Cell Rep. 16:565–571
Sanyal I, Singh AK, Kaushik M, Amla DV (2005) Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cry1Ac gene for resistance against pod borer insect Helicoverpa armigera Plant Sci. 168: 1135–1146
Sarmah BK, Moore A, Tate W, Molvig L, Morton RL, Rees DP, Chiaiese P, Chrispeels MJ, Tabe LM, Higgins TJV (2004) Transgenic chickpea seeds expressing high levels of a bean α-amylase inhibitor Mol. Breeding 14:73–82
Sharma L, Amla DV (1998). Direct shoot regeneration in chickpea (Cicer arietinum L.) Ind. J. Exp. Biol. 36:605–609
Shri PV, Davis TM (1992) Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons Plant Cell Tiss. Organ. Cult. 28:45–51
Singh R, Jat RS, Sahoo PD, Srinivasan (2002a). Thidiazuron induced multiple shoot formation in chickpea (Cicer arietinum L) J. Plant Biochem. Biotechnol. 11:129–131
Singh R, Srivastava K, Jaiswal HK, Amla DV, Singh BD (2002b). High frequency multiple shoot regeneration from decapitated embryo axes of chickpea and establishment of plantlets in the open environment Biol. Plantarum 45:503–508
Sonia, Singh RP, Sharma KK, Jaiwal PK (2003). In vitro regeneration and genetic transformation of chickpea In: Jaiwal PK, Singh RP (eds). Applied Genetics of Leguminosae Biotechnology Kluwer Academic Publisers Great Britain pp 69–87
Srivastava K, Tiwari KN, Singh R, Singh BD, Jaiswal HK (2001). Shoot regeneration from immature cotyledons of Cicer arietinum Biol. Plantarum 44:333–337
StatSoft I (1995). Statistica for Windows (Computer program manual) Statsoft Inc. Tulsa, OK
Subhadra, Vashishat RK, Chowdhury JB, Singh M, Sareen PK (1998). Multiple shoots from cotyledonary node explants of non-nodulating genotype (ICC435M) of chickpea, Cicer arietinum L Ind. J. Exp. Biol. 36:1276–1279
Acknowledgement
The authors are thankful to Swiss Agency for Development and Cooperation, Government of Switzerland and the Department of Biotechnology, Government of India under the Indo-Swiss Collaboration in Biotechnology for financial contributions. The help of Dr. K.K.Sharma, ICRISAT, Dr. B.K.Sarmah, AAU, Prof. Thomas Hohn and Prof. Barbara Hohn, Basel, Switzerland are sincerely acknowledged.
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Chakraborti, D., Sarkar, A. & Das, S. Efficient and rapid in vitro plant regeneration system for Indian cultivars of chickpea (Cicer arietinum L.). Plant Cell Tiss Organ Cult 86, 117–123 (2006). https://doi.org/10.1007/s11240-005-9072-0
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DOI: https://doi.org/10.1007/s11240-005-9072-0