Rapid regeneration of stable transformants in cultures of potato by improving factors influencing Agrobacterium-mediated transformation
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Abstract
An efficient and rapid Agrobacterium tumefaciens-mediated transformation protocol was developed to generate activation-tagged mutant lines with the aim of large-scale functional analysis of the potato genome. The explants were inoculated with an Agrobacterium strain harboring the binary plasmid pSKI074 containing four CaMV 35S enhancers in the T-DNA region which activates the downstream genes in the host plant after its integration. Various parameters investigated to increase transformation efficiency were the type and age of explant, cultivar, hormone combinations, preculture of explants, period of co-cultivation with bacteria and concentration of bacterial cultures used for transformation. Stem explants from 5 week old plantlets of cv. Bintje which had undergone phytohormone pretreatment for 4 days, inoculation with diluted bacterial concentration of OD600 = 0.2 containing acetosyringone followed by 2 days of co-cultivation and selection in media with IAA and trans-zeatin all helped in greatly improving the transformation efficiency. The total time required from infection to rooted shoots was 6-7 weeks. Initial evidence for stable integration and expression of the transgenes by PCR analysis showed that over 93% of the regenerated lines were transgenic and this was confirmed by Southern hybridization.
- An, G., Watson, B.D. and Chiang, C.C. (1986) Transformation of tobacco, tomato, potato and Arabidopsis thaliana using a binary Ti vector system. Plant Physiology, 81, 301-305.
- Romano, A., Raemakers, K., Visser, R. and Mooibroek, H. (2001) Transformation of potato (Solanum tuberosum) using particle bombardment. Plant Cell Reports, 20, 198-204.
- Chang, M.M., Culley, D., Choi, J.J. and Hadwiger, L.A. (2002) Agrobacterium-mediated co-transformation of a pea β-1,3-glucanase and chitinase genes in potato (Solanum tuberosum L. cv. Russet Burbank) using a single selectable marker. Plant Science 163, 83-89.
- De Block, M. (1988) Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theoritical and Applied Genetics, 76, 767-774.
- Badr, A., Mabrouk, Y., Rakha, F. and Ghazy, A.H. (2008) Agrobacterium tumefasciens-mediated transformation of potato and analysis of genomic instability by RAPD. Research Journal in Agriculture Biological Science, 4(1), 16-25.
- Wenzler, H., Mignery, G., May, G., Park, W. (1989) A rapid and efficient transformation method for the production of large numbers of transgenic potato plants. Plant Science, 63, 79-85.
- Yee, S., Stevens, B., Coleman, S., Seabrook, J.E.A. and Li, X.-Q. (2001) High efficiency regeneration in vitro from potato petioles from intact leaflets. American Journal of Potato Research, 78, 151-157.
- Banerjee, A.K., Prat, S. and Hannapel, D.J. (2006) Efficient production of transgenic potato (S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation. Plant Science, 170, 732-738.
- Lin, Y.J. and Zhang, Q. (2005) Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Reports, 23, 540-547.
- Sambrook, J. and Russell, D.W. (2001) Molecular cloning: a laboratory manual. Vol. 2, 3rd edn. Cold Spring Harbor Laboratory Press, New York.
- Doyle, J.J. and Doyle, J.L. (1990) Isolation of plant DNA from fresh tissue. Focus, 12, 13-15.
- Soto, N., Enriquez, GA., Ferreira, A., Corrada, M., Fuentes, A., Tiel, K. and Pujol, M. (2007) Efficient transformation of potato stem segments from cv. Desiree using phosphinothricin as selection marker. Biotech Applicada, 24, 139-144.
- Beaujean, A., Sangwan, R.S., Lecardonnel, A. and Sangwan-Norreel, B.S. (1998) Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. Journal of Experimental Botany, 49, 1589-1595.