Organogenesis, Somatic Embryogenesis And Regeneration Of Immature Male Flowers Of Banana Cultivars

This study was carried out to establish a reliable and practical protocol of plant regeneration through organogenesis and somatic embryogenesis of immature male flowers of four cultivars of Musa spp. cvs. Berangan, Rastali, Mas and Raja as well as micromorphological studies and optimization of plant transformation protocol via microprojectile bombardment of immature male flowers of Musa spp. cv. Rastali. Immature male flowers are highly proliferated meristems which are ideal materials for organogenesis and embryogenesis in Musa spp. Immature male flowers of four cultivars of Musa spp. cvs. Berangan, Rastali, Mas and Raja were cultured on a modified MS medium containing 0, 9, 18 and 36 μM BAP. The study showed a reasonably high percentage of adventitious bud induction on MS medium supplemented with 9 μM BAP for cultivars Rastali and Raja and 18 μM BAP for cultivars Berangan and Mas respectively within 4 to 8 weeks from the initiation of culture. Regeneration of shoots from the adventitious buds derived from immature male flowers of cultivars Berangan, Rastali, Mas and Raja were investigated on MS medium supplemented with 4.5, 9, 18 and 36 μM BAP after four weeks of culture with a weekly subculture interval. It was observed that BAP at 4.5 μM produced the highest number of shoots and BAP at 36 μM produced the highest shoot-length for all cultivars tested after four subcultures. Subculturing significantly affected the mean number of shoots produced with the highest mean number of 40.33, 5.66, 23.66 and 19.00 shoots produced in cvs. Rastali, Raja, Berangan and Mas respectively in the third subculture (out of four subcultures). The mean shoot height also increased over subculture cycles in all cultivars. Different coconut water preparations (filter-sterilized and autoclaved) in combination with the best BAP concentration for adventitious bud induction determined earlier for each cultivar were investigated. Highest mean number of adventitious buds after three subcultures (17.30) was attained on medium containing 50 mlL-1 filter-sterilized coconut water combined with BAP at 9 μM for cvs. Rastali and Raja while 18 μM for cvs. Berangan and Mas. Shoots obtained from the immature male flowers of cvs. Rastali, Raja, Berangan and Mas were rooted on half-strength MS medium supplemented with 1.0 μM IBA and the plantlets produced were successfully acclimatized in the growth chamber. Histological and Scanning Electron Microscopy (SEM) studies were carried out on male flowers of Musa spp. cv. Rastali placed on 9 μM BAP treatment at the initial stage of culture and the first, second and fourth subculture. Sequential changes were observed starting from globular mass like structures (bulges) to adventitious buds and finally producing multiple shoots. Somatic embryogenesis of Musa spp. cv. Raja was established using immature male flower hands. Highest percentage of embryogenic callus formation (41.97%) was obtained on 13.5 μM 2,4-D for all flower hand positions assessed. Flower hand position 8 produced the highest percentage (48.25%) of embryogenic callus formation for all levels of 2,4-D tested. The study revealed that the embryogenic cell suspensions initiated from the embryogenic callus/complex had high potential towards somatic embryogenesis. The highest percentage of somatic embryos germination (62.36%) was attained on medium with 0.17 μM BAP after two weeks of culture. Transformation study showed that the target distance of 9 cm along with helium pressure of 1350 and 1550 psi were the most efficient combinations for particle gun bombardment of immature male flower buds of cv. Rastali whereby 58.26% and 57.63% of the bombarded plates showed a high GFP gene expression. Overall, this study indicated that immature male flower buds of Musa spp. cultivars Berangan, Rastali, Mas and Raja can be the appropriate materials for in vitro regeneration via organogenesis and somatic embryogenesis as well as for gene transformation via particle bombardment