Genotype Independent Regeneration and Agrobacterium‐mediated Genetic Transformation of Sweet Potato (Ipomoea batatas L.)
Plant Tissue Culture and Biotechnology
Development of an efficient genotype independent regeneration and genetic transformation system in sweet potato continues to be of great interest. Agrobacterium-mediated genetic transformation protocol was established in two different cultivars of sweet potato using Agrobacterium strain EHA105 harbouring binary plasmid pBI121 containing GUS and nptII genes. The internodal stem segments from 30-day-old micropropogated plants were used as explant with different combinations of media and hormones.
... media and hormones. MS and LS media with various concentrations of growth regulators proved to be non-responsive and the infecundity was severe with the addition of cytokinins. Nonetheless, MS with 2,4-D and TDZ gave a good percentage of callusing but with low differentiation. In different concentrations of NAA, significant amount of callusing was observed but percentage of rooting remained low in both the genotypes. Gamborg's B5 supplemented with NAA proved to be the most suitable media and hormone combination, which yielded shoot formation after 8 -10 weeks with a regeneration efficiency of 40 -70%. Stable integration of transgene was confirmed by PCR analysis. Furthermore, qRT-PCR analysis was performed to assess the transcript accumulation in addition to the GUS enzymatic assay in the transgenic lines. References Aloufa MA (2002) Some factors affecting the callus induction and shoot formation in two cultivars of sweet potato (Ipomoea batatas L. POIS). Cienc. Agrotec. 26: 964-969. Al-Juboory KH and Skirvin RM (1991) In vitro regeneration of Agrobacteriumtransformed sweet potato (Ipomoea batatas L.). PGRSA Quarterly 19: 82-89. Al-Mazrooei S, Bhatti MH and Henshaw GG (1997). Optimisation of somatic embryogenesis in fourteen cultivars of sweet potato [Ipomoea batatas (L.) Lam.]. Plant Cell. Rep. 16: 710-714. Carelli MLD, Skirvin RM and Harry DE (1991) Transformation and regeneration studies of "Jewel" sweet potato. In: Hill WA, Bonsi CK and Loreatan PA (eds.) Sweet Potato Technology for the 21st Century. Tuskegee University, Tuskegee. pp. 52-60. Carswell GK and Locy RD (1984) Root and shoot initiation by leaf, stem, and storage root explants of sweet potato. Plant Cell Tiss. Org. Cult. 3: 229-236. Chen L, Du Z, Nishimura Y, Hamaguchi T, Sugita T, Nagata R, Terao H and Tsuzuki E (2010) Approach to establishment of plant regeneration and transformation system in sweet potato (Ipomoea batatas) by culture of leaf segments. Bull. Minamikyushu University 40 A: 59-63. Dhir SK, Oglesby J and Bhagsari AS (1998) Plant regeneration via embryogenesis and transient gene expression in sweet potato protoplasts. Plant Cell Rep. 17: 665-669. Dellaporta SL, Wood J and Hicks JB (1983). A plant DNA minipreparation: version II Plant Mol. Biol. Rept. 1: 19-21. Dodds JH, Merzdorf C, Zambrano V, Sigüeñas C and Jaynes J (1991) Potential use of Agrobacterium-mediated gene transfer to confer insect resistance in sweet potato In: Jansson RK, Raman KV (Eds.) Sweet potato pest management: a global perspective. West View Press, Oxford, U.K. pp. 203-219. Fior S and Gerola PD (2009) Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS. Plant Methods 5: 19. Gama MIC, Leite JRP, Cordeiro AR and Cantliffe DJ (1996) Transgenic sweetpotato plants obtained by Agrobacterium tumefaciens mediated transformation. Plant Cell Tiss. Org. Cult. 46: 237-244.