Transformation of green fluorescent protein reporter gene into rice Oryza sativa cv. KDML 105 by co-cultivation with agrobacterium

Abstract

Agrobacterium-mediated transformation was used to transfer green fluorescent protein (GFP) reporter 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 EHA105 carrying pCAMBIA1301 (hygromycin resistance gene (hpt) as a selectable marker and GUS (uidA gene) as the reporter gene), pCAMBIA5305 (hpt as a selectable marker and GFP as the reporter gene) and pCAMBIA1306IC (hpt as a selectable marker and GFP and GUS as the reporter gene). Each was driven under the CaMV35S promoter. Mature embryos were cultured on 2MS and 2NB medium for callus induction. 2NB medium gave 79.2+-3.4% embryogenic calli induction, higher than 2MS medium (54.6+-2.5%). The embryogenic callus was transferred to four regeneration medium (MS1-RE, NB1-RE, NB2-RE and NB4-RE. High percentage (68.2+-13.6%) of plantlet regeneration was obtained from the BN4-RE medium. Embryogenic calli were co-cultivated with Agrobacterium. Primarily, GUS expression from pCAMBIA1301 in the callus stage was examined immediately after 3 days of co-cultivation revealed 15.8% transformation efficiency. Selection was made on selective medium containing 50 mg/l hygromycin. Hygromycin resistant calli were obtained after 4 weeks selection. The uninoculated calli did not show continuous growth and died. After 8 weeks, the frequency of transformed calli, based upon hygromycin resistance and GUS activity was 19.4%. Use of A.tumefaciens strain EHA105 (pCAMBIA5305) and EHA105 (pCAMBIA1306IC), GFP transformed calli were obtained and regenerated. The presence of GFP gene was confirmed by PCR analysis and detection of GFP fluorescence. The transformation frequency obtained in this work determined by plants that stably expressed GFP for pCAMBIA5305 and expressed both GFP fluorescence and GUS activity for pCAMBIA1306IC was 9.8% and 8.2%, respectively.