Transformation and expression of barley stress-induced embryogenic hva1 gene in Oryza sativa cv. KDML 105

Abstract

Agrobacterium-mediated transformation was used to transfer green fluorescent protein (GFP) and hva1 gene into indica rice variety (Oryza sativa cv. KDML 105). Embryogenic calli derived from scutellum of mature embryo were co-cultivated with Agrobacterium tumefaciens strain EHA 105 carrying pCAMBIA5305hpt-vector (GFP as the reporter gene), pCAMBIA5305 (hpt as a selectabe marker gene and GFP as the reporter gene) and pCAMBIA5305hpt (bar as a selectable market gene, GFP as reporter gene and hva1). In first study, embryogenic calli were infected with A. tumefaciens carrying pCAMBIA5305hpt-vector. The embryogenic calli were co-cultivated for 3 days and transferred to induction medium without selective agent. After 1 day and 10 days of co-cultivation, the efficiency of transformation calli was 8 and 15% respectively. Four weeks of selection, the infected embryogenic calli failed to detect. Total of GFP positive calli were cut into small pieces and transferred to regenerated medium. The section calli did not show continuous growth. In second study, A. tumefaciens strain EHA 105 habouring pCAMBIA5305 was used for co-cultivation of embryogenic calli. Condition of infection was varied to 10 minutes infected/ 2 days co-cultivated and 15 minutes infected / 3 days co-cultivated. After 8 weeks on selected with hygromycin, the efficiency of transformation of calli upon hygromycin resistance and GFP expression was 10 and 16% to observe from 2 and 3 days co-cultivated calli, respectively. Transgenic rice plants were regenerated from visually selected GFP-positive calli. No difference were observed in the morphology between transformed and non-transformed plants. The frequency of transformation, based upon GFP fluorescence and PCR analysis, was ranged from 4 to 5.5%. Expression of GFP and its inheritance was stable in T1 progeny. In third study, the embryogenic calli were co-cultivated for 3 days. Concentrations of selection medium were varied to 6. 8 and 10 mg/l glufosinate. The efficiency of transformed calli upon resistance to glufosinate and GFP expression were 1.8, 2.7 and 2.2%, respectively. Of the 12 callus lines produced from three concentrations, one line from each 8 and 10 mg/l glufosinate selection gave rise to plants. One line from 8 mg/l glufosinate had normal morphology. Another line from 10 mg/l glufosinate gave rise to albino plants. The frequency transformation of plant upon GFP expression and hva1 (level mRNA), which selected on 8 mg/l glufosinate, was 0.54%