Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx. (muscadine grape)

Abstract

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l(-1) in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l(-1), which permitted the proliferation of more non-transformed cells. Transgenic plants of "Alachua" and "Carlos" were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.

Green fluorescent protein as a visual selection marker for coffee transformation

“…In the mature cotyledon leaves and young leaves which developed subsequently, the intensity of green fluorescence was maintained (Figs 2f-h). Similar green fluorescence pattern was also demonstrated in transgenic grape (Dhekney et al 2008), tobacco (Hraska et al 2008) and other plant species transformed with gfp gene (Hraska et al 2006) during various developmental stages.…”

“…In the present study, Agrobacterium strain EHA105 harbouring pBECKS 2000.7 was chosen for co-cultivation. The Agrobacterium EHA105 is an agropine type of bacterial strains and found to be hypervirulent in many woody plant species, such as peach (Padilla et al 2006), pear (Yancheva et al 2006) and muscadine grape (Dhekney et al 2008). In coffee, Agrobacterium EHA105 was found to be more hypervirulent than other A. tumefaciens strains (Mishra et al 2009).…”

The Family Rhizobiaceae

“…Vectors carrying the border sequence and the genes of interest can be transferred via triparental mating by Escherichia coli into the disarmed A. tumefaciens or A. rhizogenes, which will be used for infection and transformation of the plants of interest (Zambryski et al 1983;Matthysse 2006;Kiyokawa et al 2009). Plant explants used for transformation are selected using the appropriate antibiotic, and the transformed plant is regenerated using tissue culture, whereas the Agrobacterium is eliminated with the application of cefotaxime (Valvekens et al 1988;Dhekney et al 2008;Marcondes and Hansen 2008).…”

An efficient method for transgenic callus induction from Vitis amurensis petiole

“…Kanamycin is a widely used antibiotic in grape post-transformation selection, and the tolerance of different tissues to kanamycin is quite variable [10, 16, 36, 37]. In order to find a suitable concentration of kanamycin for transgenic calli selection, petiole segments were cultured on Petri dishes with medium containing 5, 10, 15 or 20 mg L -1 kanamycin for 30 days in the dark at 26°C.…”