Optimization of Agrobacterium tumefaciens-mediated transformation and shoot regeneration after co-cultivation of cabbage (Brassica oleracea subsp. capitata) cv. KY Cross with AtHSP101 gene

Abstract

A number of parameters which have been reported to influence genetic transformation via Agrobacterium-mediated transformation method were evaluated to increase the frequency of transformation of cabbage (Brassica oleracea subsp. capitata) cv. KY Cross with AtHSP101 gene. The binary vector pCAMHSP was designed and mobilized into two Agrobacterium tumefaciens strains C58 and GV2260. The study was carried out on hypocotyl and shoot tip explants of cabbage cv. KY Cross. Transformation parameters optimized were pre-culture medium, acetosyringone application, bacterial density and inoculation time. The polymerase chain reaction (PCR) assay and production of mRNA of AtHSP101 gene were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The expression of LacZ gene in the transgenic plants also showed that it could be applied as a plant transformation reporter gene in genetic transformation studies. Multiple shoot regeneration of hypocotyl and shoot tip explants of cabbage after co-cultivation with Agrobacterium was optimized and medium containing 2 mg/L BAP was observed to be the best for shoot regeneration after cocultivation. In this study, 45% and 32.5% transformation efficiencies were achieved for hypocotyl and shoot tip explants, respectively using the optimized procedure.

Influence of phenyl-urea and adenine-type cytokinins on direct adventitious shoot regeneration of cabbage (Brassica oleracea subsp. capitata) ^|^#8220;KY Cross^|^#8221;

“…capitata or commonly known as cabbage is among members of the Cruciferous family commonly associated with numerous health benefits such as containing high levels of antioxidants, anticancer compounds and vitamins A, B1, B2 and C (Christey and Braun 2001;Rafat et al 2010;Singh et al 2006). In Malaysia, the production of cabbage was estimated around 30 metric ton per hectare.…”

“…The control treatment was PGR-free MS medium. In addition, TDZ, BAP and 2ip in combination with IBA (Table 2) were also tested for cotyledonary explants since earlier reports (Rafat et al 2010;Ravanfar et al 2009) indicated poor shoot regeneration in terms of percentage and mean shoot number when cytokinins were applied alone. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121°C and 1.03 kPa for 20 min.…”

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel

“…For acetosyringone treatment, the A. tumefaciens strain LBA 4404 harboring pGFHSP binary vector was cultured in LB broth without acetosyringone (W1) and with 200 µM acetosyringone (W2). In this experiment, an Agrobacterium (OD 600 = 1.6) dilution (1:5) in liquid hormone-free MS medium and cocultivation time (10 min) were applied to the hypocotyl explants (Metz et al 1995;Rafat et al 2010).…”

“…Rapifera. In addition, other important factors that could affect the efficiency of Agrobacterium-mediated transformation need to be optimized, which include the preculture and acetosyringone requirements (Rafat et al 2010), bacterial concentration (Zhao et al 2001) and immersion time (Xing et al 2007), co-cultivation condition and period of cocultivation (Vasudevan et al 2007), and temperature conditions (Salas et al 2001). Transformation systems have been developed in almost all economically important Brassica species, and Agrobacterium tumefaciens-mediated transformation is the most commonly used and generally quite efficient and practical for most species in the genus (Chakrabarty et al 2002).…”

“…Transformation systems have been developed in almost all economically important Brassica species, and Agrobacterium tumefaciens-mediated transformation is the most commonly used and generally quite efficient and practical for most species in the genus (Chakrabarty et al 2002). Available reports on Agrobacteriummediated transformation of the Brassicas indicated a high efficiency of transformation (Suri et al 2005;Rafat et al 2010;Metwali et al 2012). Reports have shown increased transformation efficiencies in broccoli (Cao and Earle 2003) and canola (Cardoza and Stewart 2003, 2006, the two most important Brassica crops.…”