Agrobacterium-mediated genetic transformation of safflower (Carthamus tinctorius L.)

Abstract

Reproducible and highly efficient protocols for shoot regeneration and genetic transformation mediated by Agrobacterium have been established for safflower (Carthamus tinctorius L.). Agrobacterium tumefaciens strain LBA 4404 with gus reporter gene and hygromycin (hpt gene) as plant selection marker was used as the plant transformation vector. Genetic transformation experiments were carried out to evaluate the efficacy of various parameters such as genotype, seedling age, co-cultivation period, bacterial titer, enzymatic pre-treatment of target tissues, use of compounds that induce vir-gene enhancer, acetosyringone (AS), explant type and explant injury to enhance transformation efficiency. Transformation frequency was high when root and hypocotyl explants of 8-day-old seedlings of safflower cv. HUS-305 were co-cultivated with bacterial cell density of 0.5 OD 600 during a period of 2 days followed by selection regime of 10-15-15 mg/l hygromycin. The frequency of rooting of the primary transformants was low (18.0%) when compared with the regenerated shoots (70.0%), and seven shoots survived on transfer to soil. The putative transformants were confirmed by b-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR), reverse-transcriptase PCR (RT-PCR) and Southern blot analysis. With the optimized transformation protocol, putative transformed shoots were obtained with frequency of 51.0% within 8-10 weeks of culture initiation.

Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

“…Cattleya orchid (Zhang et al 2010), and in safflower (Sri Shilpa et al 2010). None of these publications addressed the possible presence of any GUS enzyme inhibitors in tissues of these different plant species.…”

Agrobacterium-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (Cucumis melo L.)

“…Factors during co-cultivation can enhance transformation efficiency, i.e., temperature (Fullner and Nester 1996;Yasmin and Debener 2010;Sharma et al 2011;Seo et al 2011), lighting conditions (Zambre et al 2003), co-cultivation period (Fang and Grumet 1990;Shilpa et al 2010;Seo et al 2011), addition of acetosyringone (Costa et al 2006;Afroz et al 2010;Sharma et al 2011) and antioxidants (Dan et al 2009;Olhoft and Somers 2001;Ostergaard and Yanofsky 2004;Toldi et al 2002;Zheng et al 2005;Kumar et al 2011;Dutt et al 2011). Co-cultivation periods of 2-6 d were reported to be optimal for melons (Galperin et al 2003;Vallés and Lasa 1994;Akasaka-Kennedy et al 2004;Dong et al 1991).…”

Transformation of HBsAg (hepatitis B surface antigen) gene into tomato mediated by Agrobacterium tumefaciens

“…After tomato leaf tissues were stained with GUS reaction solution and bleached with absolute alcohol, the whole of the transformed tissues appeared nearly deep blue colour while untransformed tissues appeared colourless. GUS histochemical assay is a convenient and fast technique to detect the integration and expression of target gene which was widely used in many plant species (Švábová et al 2005;Sri Shilpa et al 2010). The GUS histochemical assay of the leaves of T 0 plants showed blue colouration, confirming the presence of the transgene ( Figure 3F).…”