REGENERATION OF BANANA (Musa spp. AAA Group) cv. WILLIAMS via SECONDARY SOMATIC EMBRYOGENESIS AND CELL SUSPENSION FROM IMMATURE MALE FLOWER BUDS

Abstract

Development of embryogenic cultures having high regeneration efficiency from important, commercial varieties of banana is a prerequisite for genetic manipulation and for in vitro propagation. In the present study, we have studied the induction of somatic embryogenesis from young immature male inflorescences of the banana cultivar "Williams" (Musa spp. AAA group) via secondary somatic embryogenesis and cell suspension. Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l alfa naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline (MM1). Secondary somatic embryogenesis (SE2) was developed when primary embryos were subculture on SK4 medium (MS medium supplemented with 10 ml of coconut milk). Embryos differentiated when sub-cultured on SK8 medium (MS medium supplemented with 5 mg/l BA and the embryos germinated when subculture on MS free hormone medium. Suspension cultures were initiated from SE2 embryogenic tissues from when placed in liquid medium supplemented with 2,4-D (1mg/l), biotin (1 mg/l), L-glutamate (100 mg/l), malt extract (100 mg/l), and sucrose (45 g/l), pH of the medium was adjusted to 5.3. The packed cell volume (PCV) of the suspension increased 2-5 fold with each monthly cycle. The somatic embryos were developed when suspension culture were aspirated on MS medium supplemented with biotin (1 mg/l), malt extract (100 mg/l), Glutamine (100mg/l), NAA (1mg/l), Kinetin (0.5 mg/l) Zeatin (0.2 mg/l), sucrose (45 g/l), and phytagel (2.6 g/l) (SK13). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l 6-benzylaminopurine (BA) for development of mature somatic embryos, which were isolated and cultured on hormone-free MS medium for germination and development into plantlets. Approximately 55% of the somatic embryos germinated and developed into plantlets. Somatic embryogenesis via SE2 and cell suspension might be an excellent technique for mass propagation, developing a breeding strategy and genetic transformation of banana.