Transgenic sweet potato expressing mammalian cytochrome P450

Abstract

Sweet potato [Ipomoea batatas (L.) Lam] is considered to be recalcitrant to transformation and regeneration because of its genotype-dependent in vitro responses. The lack of a genotype-independent transformation and regeneration system limits biotechnological applications in this plant species. To establish a transformation system for a diverse group of sweet potato genotypes, we examined sweet potato regeneration after transformation in five cultivars. An Agrobacterium tumefaciens transformation system was used for the introduction of mammalian cytochrome P450 genes, which are capable of conferring herbicide tolerance. Among the different factors studied, including explant type, plasmid vectors, and auxin type in the initiation media, auxin type had the greatest effect on the regeneration response. Of the auxins tested, only 4-fluorophenoxyacetic acid (4FA) induced regeneration from all cultivars. In terms of the quality of calli, 4FA promoted the induction of type I calli, which were capable of somatic embryo formation, whereas type II calli fail to produce somatic embryos. The frequency of somatic embryo formation was also affected by the composition of the embryo-induction media. Transgenic plants were regenerated from all cultivars. The stable integration and expression of transgenes was confirmed by several approaches. This Agrobacterium-mediated transformation system should be applicable to a wide range of sweet potato cultivars.

Agrobacterium-Mediated Transformation of Cry8db Gene in Vietnam Sweet Potato Cultivar

“…However majority of the most efficient and robust Agrobacterium transformation systems succeed in transformation of marker genes such as gfp, gusA, nptII, bar [5,10,[34][35][36][37]. Therefore, genetic transformation of sweet potato remains cumbersome and labor-intensive due to the difficulty in plant regeneration of several recalcitrant cultivars, and the reproducibility of the used protocol [15,17,34,[36][37][38][39] .…”

“…Since the Agrobacterium mediated transformation system does not involve sophisticated equipment and frequently produces cleaner events (intact integrations and single copy) than other methods [13], it remains to be the more favorable approach. Previous studies on sweet potato transformation via Agrobacterium tumefaciens transformation have been reported including herbicide resistance [14,15], viral resistance [16], insect resistances related to the CryIIIA delta-endotoxin, cowpea trypsin inhibitor and snowdrop lectin [17,18] and enhancing starch accumulation in tubers [19,20]. To date, effective transformation protocols have been developed for only few of sweet potato cultivars, indicating that it is a very recalcitrant species and transformation methods of sweet potato are still genotype dependent.…”

Induction of somatic embryogenesis in recalcitrant sweetpotato (Ipomoea batatas L.) cultivars

“…The three different auxins tested in this study were 1 mg/L 4-fluorophenoxyacetic acid (4FA), 1.3 mg/L 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). These concentrations were derived from previous studies on sweetpotato somatic embryogenesis (Anwar et al, 2010;Cipriani et al, 2001;Song et al, 2004;Triqui et al, 2007). All auxins were filter-sterilized and added to autoclaved media that was cooled to about 60°C.…”

“…Efforts to extend this into a wide range of genotypes have shown that most of them are recalcitrant or respond at low frequencies (Al-Mazrooei et al, 1997;Anwar et al, 2010;Luo et al, 2006). Moreover regeneration protocols reported to work in some laboratories have proved difficult to reproduce in other laboratories (Moran et al, 1998).…”

“…Importantly, it has been observed that African cultivars are particularly difficult to regenerate in vitro (Luo et al, 2006). Apart from genotype, the medium composition and type of plant organ providing the explants have been shown to influence regeneration in vitro (Anwar et al, 2010;Song et al, 2004). Thus, there is need to develop an efficient system of somatic embryogenesis and plant regeneration for recalcitrant African cultivars.…”