Somatic Embryo Formation from Co-cultivated Protoplasts of Brassica rapa & B. juncea

Aims: The objectives of this experiment was to establish initial steps of somatic embryogenesis from the fused protoplasts between two Brassica species: Brassica rapa L. (var. Agrani, AA, 2n=20) and Brassica juncea L. (var. BINA Sharisha-7 and var. BINA Sharisha-8, AABB, 2n=36).
Study Design: The experiment was carried out in completely randomized design with three replications for each hormone treatment of each variety combination.
Place and Duration of Study: An experiment was conducted in growth room of tissue culture laboratory at the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh during the period from April, 2013 to May, 2014.
Methodology: Mesophyll protoplasts were isolated from the young leaves of aseptically grown plants. Variety combinations for protoplasts fusion were: Agrani+BINA Sharisha-7 and Agrani+ BINA Sharisha-8. 100% polyethylene glycol-400 was used as a fusogen. Pellieter different compositions of liquid media were used for protoplasts cultured, microcalli, calli and somatic embryo formation.
Results: Fused products were cultured in a modified Pellieter B liquid media with different combinations of auxins and cytokinin for callus induction. A medium having 1 mg L-1 NAA + 0.5 mg L-1 2,4-D produced micro-calli of 518 µm diameter on 15 days after dark treatment. The same hormone combinations produced 1.6 mm visible calli after 24 days of incubation. After 82 days of culture, a medium composition of 0.5 mg L-1 BAP + 0.5 mg L-1 NAA resulted in 76% somatic embryo formation. Agrani+BINA Sarisha-7 produced significantly higher somatic embryos compared to Agrani+BINA Sarisha-8.
Conclusion: Our results might be helpful for developing a complete and efficient protocol of somatic hybridization in Brassica species.