Abstract
The present investigation was undertaken to standardize regeneration and Agrobacterium- mediated genetic transformation protocol with GUS gene and PSCIPKsense salt tolerant gene. Subsequently, these were evaluated for salt tolerant transgenic Brassica varieties through in vitro bio-assay techniques. The study was conducted during 2007 to 2012 at Biotechnology and the Genetic Engineering Laboratory of the Department of Genetics and Plant Breeding, BAU, Mymensingh and the Biotechnology Laboratory of BSRI, Ishurdi, Pabna. In the first experiment, ten Brassica varieties viz. BARI sharisa-15, BARI sharisa-14, Tori-7, Safal, BARI sharisa-13, BARI sharisa-8, BARI sharisa-7, BINA sharisa-4, BARI sharisa-11 and BARI sharisa-10 were used to establish a rapid, efficient and reproducible plant regeneration protocol. Hypocotyl segments of ten Brassica varieties were used as explants with ten different combinations of 2,4-D (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 mgL-1) with constant combinations of BAP and AgNO3 as growth regulators. A wide range of variation in callus induction ability of the used varieties was observed. Among the varieties, Tori-7 produced the highest percentage of callus (97.00%) on MS medium supplemented with 2.0 mgL-1 BAP + 1.0 mgL1 2,4-D + 2.0 mgL-1 AgNO3. Tori-7 also showed the highest shoot regeneration (56.22%) on MS medium supplemented with 2.0 mgL-1 BAP + 2.0 mgL-1 2,4-D + 2.0 mgL-1 AgNO3 followed by BARI sarisha-11 (56.22%) and BARI sharisa- 8 (44.00%) respectively. Safal (15.20%) showed the lowest percentage of shoot regeneration. The highest percentage (53.62%) of root induction was observed in Tori-7 (72.42%) with the hormonal combination of 1⁄2 MS medium supplemented with 1.5mgL-1 NAA. On the successive development of root system, the survival rate of the plantlets in earthen pot was the highest in Tori-7 (82.50%) followed by BARI sharisa-8 (72.73%). In the second experiment, Agrobacterium-mediated genetic transformation was performed by using selected five Brassica varieties viz. Tori-7, BARI sarisha-10, BARI sarisha-11, BARI sarisha-15 and BARI sarisha-8 on the basis of GUS positive performance. Hypocotyl segments were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector PBI21 with GUS (reporter) and nptII (Kanamycin resistant) gene and performed by standardizing two important parameters, pre-culture and co-cultivation periods. Both the pre-culture period of 48 hours and co-cultivation period of 72 hours were most effective for giving the best responses (36.60% and 37.60%) in GUS assay. The maximum transformation was found in Tori-7 (81%) in 48 hours of pre-culture and 72 hours of co-cultivation period. In the third experiment, Tori-7 and BARI sharisa-10 were infected with GUS bacterial strain and confirmed the transformation of GUS gene by using PCR analysis with nptII two sets of primer and displayed expecting 700 bp and 600 bp sized band. In the fourth experiment, the hypocotyl segments were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring Ti plasmid PSCIPKsense salt tolerant gene as well as one reporter gene GUS including selectable marker gene (nptII), resistant of kanamycin. The maximum transformation rate was in Tori-7 (86%) in 48 hours of pre-culture and 72 hours of co-cultivation period. In the fifth experiment, Tori-7 and BARI sharisa-10 were infected with PSCIPKsense salt tolerant bacterial strain and confirmed the transformation of salt tolerant gene by using PCR analysis with nptll two sets of primer and displayed expecting 700 bp and 960 bp sized band. In the sixth experiment, the evaluation was done for salt tolerant transgenic Brassica varieties through in vitro bio-assay techniques. Two transgenic Brassica varieties viz. Tori-7 and BARI sharisa-10 were tested with 0, 3, 6 and 12 dsm-1 (NaCl + KCl solution in a ratio of 1:1) different salt concentration levels in 2 MS media. The better performance was observed in the above two varieties at 6 dsm-1 salt concentration level.