Development of Transgenic Rice (Oryza sativa L.) Plant Using Cadmium Tolerance Gene (YCFI) through Agrobacterium Mediated Transformation for Phytoremediation

Abstract

Cadmium has adverse effects on plant development and ultimately reduces production. The investigation was undertaken for successful gene transfer and phytoremediation through an efficient Agrobacterium-mediated genetic transformation method using cadmium tolerance gene (YCF1). Embryogenic calli induced after 20 days of highly regenerating rice cultivar BRRI dhan29 and Agrobacterium strain GV 3101 was transformed with binary vector pCAMBIA 1303-YCF1 which contained the hygromycin phosphotransferase (HPT) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene, were used for genetic transformation in the experiment. The transformed colonies were selected on 15 mg LG 1 hygromycin and 50 mg LG 1 rifampicin to select hygromycin resistant shoots. Hygromycin-resistant shoots were subsequently rooted on root induction medium. Rooted plantlets were transferred to pot-soil, hardened and grown in a greenhouse until maturity and stable integration, expression of YCF1 gene was also confirmed by using PCR analysis. The maximum transformation efficiency of 22% was obtained using 500 mg LG 1 cefotaxime as a bacteriostatic agent to inhibit growth of Agrobacterium and 100 mM acetosyringone in co-cultivation medium. Southern blot analysis was performed to confirm that transgenes (HPT and YCF1) were stably integrated into the plant genome. All transgenic plants showed single-copy of transgene integration in the host genome. This transgenic rice plant will uptake cadmium from soil and will protect rice grain from cadmium and store into cell vacuoles of rice plants. As a result soil will be free from cadmium through phytoremediation process.

Establishment of Agrobacterium-Mediated Genetic Transformation in Popular Rice Cultivar BRRI Dhan-29 through Reporter GUS Gene Expression System in Bangladesh

“…Among the six hormonal concentrations and combination callus formation was highest (85 %) in BRRI dhan-29 when the medium was supplemented with 2.0 mg/ l 2,4-D + NAA 1.0 mg/l NAA and callus formation was lowest (35 %) when the medium was supplemented with 1 mg/l 2,4-D + 0.5 mg/l NAA. A similar result was recorded by Islam and Khalekuzzaman 6 in BRRI Dhan-29 as 92.0 % in MS having 2.0 mg/l 2,4-D + 1.0 mg/l NAA among four combinations of 2, 4-D and NAA. Callus induction in rice greatly affect on the choice of explants, concentration and combination of hormonal and nutritional element 7,8 .…”

“…The optical density of Agrobacterium is an important factor for genetic transformation. There are several studies describing the Agrobacterium mediated transformation using a range of concentrations of Agrobacterium from 0.5 to 1.5 OD 600 6,12,13 . The highest number of GUS expressing calli was observed at OD 0.8 -0.9 (Fig.…”