Transgenic plants from shoot apical meristems of Vitis vinifera L. “Thompson Seedless” via Agrobacterium-mediated transformation

Abstract

Shoot apical meristem explants of Vitis vinifera "Thompson Seedless" were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L(-1) in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L(-1) kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.

Agrobacterium-mediated transformation of the winter jujube (Zizyphus jujuba Mill.)

“…This lack of a system by which to produce appropriate explants currently limits the transformation of winter jujube. Shoot apical meristems obtained through in vitro micropropagation may be a represent alternative as the target for transformation (Dutt et al 2007). Similar beneficial results have been reported in sugar beet , maize , and cotton (Lv et al 2004).…”

“…In an earlier publication, we reported the establishment of an efficient in vitro culture system of winter jujube for regenerating shoot tips . Shoot tips were capable of regenerating cells of the shoot apical meristem that could serve as targets for genetic transformation (Dutt et al 2007). The currently preferred method for plant transformation is an Agrobacterium-mediated transformation system due to single-or low-copy integration and a high conversion efficiency-with up to 85% transgenic plants being obtained in some systems (Dai et al 2001;Shou et al 2004).…”

Recent Advances in Fruit Species Transformation

“…Other interesting approach was the genetic transformation of shoot apical meristems, previously subjected to a dark growth stage after wounding for transformation. Authors reported that 1% of shoot tips produced stable transgenic lines after weeks (Dutt et al, 2007). Ismail and co-workers (2011) transformed successfully banana apical meristems via microprojectile bombardment and regenerated 80.3% percent of the transformed meristematic tissues.…”

“…Regeneration from transformed embryos can be achieved via direct germination or shoot organogenesis and the method is useful for large-scale and rapid propagation of transformants. In grapevine most of the approaches are being performed by using embryogenic cultures from different tissues such as zygotic embryos, leaves, ovaries and anther filaments to provide cells amenable to gene insertion and regeneration (Mezzetti et al, 2002;Dutt et al, 2007;López-Noguera et al, 2009). However, these techniques are highly genotype dependent and for many cultivars they have been difficult to obtain successful results (Dutt et al, 2007).…”

“…In grapevine most of the approaches are being performed by using embryogenic cultures from different tissues such as zygotic embryos, leaves, ovaries and anther filaments to provide cells amenable to gene insertion and regeneration (Mezzetti et al, 2002;Dutt et al, 2007;López-Noguera et al, 2009). However, these techniques are highly genotype dependent and for many cultivars they have been difficult to obtain successful results (Dutt et al, 2007). Moreover, it is considered that anther filaments, as commonly employed in grapevine for embryogenic calli, are laborious, cultivar-dependent, depend on availability of immature flowers and may affect strongly the phenotype of the regenerated plantlets (Mezzetti et al, 2002).…”