how to be a good scientist
AGROBACTERIUM MEDIATED TRANSFORMATION
OF SOYBEAN (GLYCINE MAX L.): SOME
CONDITIONS STANDARDIZATION
Abstract
Present study was aimed to standardize some transformation conditions for soybean cultivar NARC-4 using Agrobacterium tumefaciens strain EHA 101 harboring pGUSintNPTII. Soybean half seed cotyledonary node method was opted. It was found that explant preparation in Agro-suspension culture resulted in highest transformation efficiency (48.3%) than in infection medium and water. One hour infection time was found optimum (55.9% transformation efficiency) in culture OD600 1.0. Co-cultivation of soybean half seed explants with Agrobacterium for five days showed better results as compared with three and four days. Two hr washing in washing medium containing 1g/L cefotaxime controlled prevalence of Agrobacterium in further steps. It was found that varying kanamycin concentration in selection medium resulted in high survival rate of transformed shoots. NARC-4 soybean cultivar showed better transformation efficiency than NARC-7 when genotype dependency was examined using Agrobacterium strain EHA101.
Introduction
Plant genetic engineering mainly depends upon Agrobacterium tumefaciens mediated transformation that accounts so far production of 80% transgenics (Wang & Fang, 1998). Soybean transformation has shown significant improvement and enabled public and private sector for production of commercial cultivars with transgenic traits.
Hinchee et al., (1988) first time reported soybean transformation with Agrobacterium strain. They successfully regenerated plants on media containing kanamycin or glyphosate but transformation efficiency was quite low (Hinchee et al., 1988) but modifications in regeneration protocol may produce high rate of transformants (Parrott et al., 1989; McKenzie & Cress, 1992). Although a number of factors that affect on transformation efficiency has been studied that includes sonication assisted Agrobacterium mediated
OF SOYBEAN (GLYCINE MAX L.): SOME
CONDITIONS STANDARDIZATION
Abstract
Present study was aimed to standardize some transformation conditions for soybean cultivar NARC-4 using Agrobacterium tumefaciens strain EHA 101 harboring pGUSintNPTII. Soybean half seed cotyledonary node method was opted. It was found that explant preparation in Agro-suspension culture resulted in highest transformation efficiency (48.3%) than in infection medium and water. One hour infection time was found optimum (55.9% transformation efficiency) in culture OD600 1.0. Co-cultivation of soybean half seed explants with Agrobacterium for five days showed better results as compared with three and four days. Two hr washing in washing medium containing 1g/L cefotaxime controlled prevalence of Agrobacterium in further steps. It was found that varying kanamycin concentration in selection medium resulted in high survival rate of transformed shoots. NARC-4 soybean cultivar showed better transformation efficiency than NARC-7 when genotype dependency was examined using Agrobacterium strain EHA101.
Introduction
Plant genetic engineering mainly depends upon Agrobacterium tumefaciens mediated transformation that accounts so far production of 80% transgenics (Wang & Fang, 1998). Soybean transformation has shown significant improvement and enabled public and private sector for production of commercial cultivars with transgenic traits.
Hinchee et al., (1988) first time reported soybean transformation with Agrobacterium strain. They successfully regenerated plants on media containing kanamycin or glyphosate but transformation efficiency was quite low (Hinchee et al., 1988) but modifications in regeneration protocol may produce high rate of transformants (Parrott et al., 1989; McKenzie & Cress, 1992). Although a number of factors that affect on transformation efficiency has been studied that includes sonication assisted Agrobacterium mediated
References: Cho, H.J., S.K. Farrand., G.R. Noel and J.M. Widholm. 2000. High efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta, 210: 195-204. Donaldson, P.A. and D.H. Simmonds. 2000. Susceptibility to Agrobacterium tumefaciens and coptyledonary node transformation in short-season soybean. Plant Cell Rep., 19: 478-484. Doyle, J.J. and J.L. Doyle. 1990. Isolation of DNA from fresh plant tissue. Focus, 12: 13-15. Gamborg, O.L., R.A. Miller and K. Ojima. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50:150-158. Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep., 5: 387-405. Ko, T.S. and S.S. Korban. 2004. Enhancing the frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons of soybean [Glycine max (L.) Merrill]. In Vitro Cell Dev. Biol. Plant., 40: 552-558. Ko, T.S., S. Lee., S. Krasnyanski and S.S. Korban. 2003. Two critical factors required for efficient transformation of multiple soybean cultivars: Agrobacterium strains and orientation of immature cotyledonary explant. Theor. Appl. Genet., 107: 439-447. Kuta, D.D. and L. Tripathi. 2005. Agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. African J. Biotechnol., 4(8): 752-757. Liu, H.K., C. Yang and Z.M. Wei. 2004. Efficient Agrobacterium tumefaciens-mediated transformation of soybean using an embryonic tip regeneration system. Planta, 219: 1042-1049. Liu, S.J., Z.M. Wei and J.Q. Huang. 2007. The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties. Plant Cell Rep., 27(3): 489-498. McKenzie, M.A. and W.A. Cress. 1992. The evaluation of South African cultivars of soybean for their susceptibility to Agrobacterium tumefaciens and the production of transgenic soybean. South Afric. J. Sci., 88(4): 193-196. Meurer, C.A., R.D. Dinkins and G.B. Collins. 1998. Factors affecting soybean cotyledonary node transformation. Plant Cell Rep., 18: 180-186. Murashige, T. and E. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Olhoft, P.M., L.E Olhoft, P.M., L.E. Flagel and D.A. Somers. 2004. T-DNA locus structure in a large population of soybean plant transformed using the Agrobacterium-mediated cotyledonary-node method. Plant Biotechnol. J., 2(4): 289-300. Parrott, W.A., L.M. Hoffman, D.F. Hildebrand, E.G. Williams and G.B. Collins. 1989. Recovery of primary transformants of soybean. Plant Cell Rep., 7: 615-617. Paz, M.M., J.C. Martinez, A.B. Kalvig, T.M. Fonger and K. Wang. 2006. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep., 25: 206-213. Sharma, R., H.R. Mahla, T. Mohapatra, S.C. Bhargava and M.M. Sharma. 2003. Isolating plant genomic DNA without liquid nitrogen. Plant Mol. Biol., 21(1): 43-50. Sheng, J. and V. Citovsky. 1996. Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell, 8: 1699-1710. Shyamkumar, B., C. Anjaneyulu and C.C. Giri. 2007. Genetic transformation of Terminalia chebula Retz. and detection of tannin in transformed tissue. Curr. Sci., 92(3): 361-368. Stachel, S.E., E.W. Nester and P.C. Zambryski. 1986. A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc. Nat. Acad. Sci. USA, 83: 379-383. Trick, N.H. and J.J. Finer. 1997. SAAT: sonication-assisted Agrobacterium-mediated transformation. Trans. Res., 6: 329-336. Wang, G. and Y. Xu. 2008. Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA Interference. Plant Cell Rep., 27: 1177-1184. Wang, G.L. and H.J. Fang. 1998. Plant genetic engineering: principle and technique, (in Chinese) Beijing: Science Press. Xue, R.G., H.F. Xie and B. Zhang. 2006. A multi-needle-assisted transformation of soybean cotyledonary node cells. Bitechnol. Lett., 28: 1551-1557. Yan, B., M.S.S. Reddy., G.B. Collins and R.D. Dinkins. 2000. Agrobacterium tumefaciens – mediated transformation of soybean [Glycine max (L.) Merrill] using immature zygotic cotyledon explants. Plant Cell Rep., 19: 1090-1097. Yi, X. and D. Yu. 2006. Transformation of multiple soybean cultivars by infecting cotyledonary-node with Agrobacterium tumefaciens. African J. Biotechnol., 5(20): 1989-1993. (Received for publication 29 August 2009)