K. Miah
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh
B. Hossen
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh
M.S. Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh
S.N. Begum
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh
M. M. Hasan Sohel
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh
Rice, Agrobacterium tumefaciens, Genetic transformation, Salinity
Tissue Culture Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh
Variety and Species
Transformation, Rice, Climate change
The experiments were carried out at the Tissue Culture Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh in 2010 by using Binadhan-5 and Binadhan-6. Agrobacterium tumefaciens strain LBA4404 was used for infecting the embryogenic calli in the transformation process. This strain contains plasmid pBI121, udiA gene encoding GUS gene GUS (β-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. The nptll gene encoding neomycin phosphotransferase Il (nptll) conferring Kanamycin resistance. The hpt gene conferring hygromycin resistence driven by NOS promoter and NOS terminator and T-DNA border sequences (RB, LB).
2.1 Media used: For callus induction, callus proliferation, shoot regeneration and rooting MS medium was used. MS media was used as a basal medium for both callus induction and regeneration of explant.
2.2 Sterilization: To ensure aseptic condition in in vitro, all instruments, glasswares and culture media were sterilized properly by autoclaving at 121oC for 30 minutes at 1.16 kg cm-2 pressure.
2.3 Agrobacterium culture and inoculation: Two different types of culture media, namely, YMB (Yeast extract Mannitol Broth) medium and LB (Luria Broth) medium were used with kanamycin as antibiotic to grow the strain of genetically engineered Agrobacteriurm tumefaciens. Here two sorts of media were used, such as, Agrobacterium maintenance medium and Agrobacterium working culture medium for transformation.
2.4 Transplantation of plantlets from growth chamber’s to earthen pot: Soil containing 25% garden soil + 50% sand + 25% cowdung in pots placed in growth chamber having 4500 Lux light at 25±1°C for seven days.
2.5 Culture techniques: The following culture techniques were employed in the experiment.
2.5.7 Transfer of the transgenic shoots for root initiations: The green shoots (2-3 cm) were separated from each other and again were cultured on petridishes with freshly prepared rooting medium (RTM) to induce root. The vials containing plantlets were incubated at 22±2°C with 16 hrs photoperiod. 2.5.8 Transplantation: The plantlets were transplanted to pots. Pots were kept in growth chamber for 7 to 15 days under controlled environment, also nourished with Hoagland's solution. After 15 to 20 days they were transferred to the field condition. 2.5.9 Data recording and analysis: The effect of different treatments data were collected on following parameters: Number of explants positive for GUS, transformed plantlet regeneration, days to shoot initiation, no. transgenic plantlets and average number of shoots with root. The Completely Randomized Design (CRD) was used for this experiment. The analyses of variances for different parameters were performed and means were compared by the Duncan’s Multiple Range Test (DMRT) in MSTATC program.
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) e-ISSN: 2319-2380, p-ISSN: 2319-2372. Volume 8, Issue 3 Ver. I (Mar. 2015), PP 28-35 www.iosrjournals.org
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