Enhancement of somatic embryogenesis in recalcitrant east African highland bananas

Lack of appropriate in vitro protocols has limited advances in the use of tissue culture in rapid multiplication of planting material and development of reliable genetic transformations for AAA-EA banana. Embryogenic callus induction in AAA-EA banana has been difficult. Conventionally auxins have been used in MS medium with or without purine-based cytokinins for induction of embryogenesis. The low embryogenic response aside, the existing protocols have been cultivar dependent. The main objective of this study was to enhance plant regeneration through somatic embryogenesis in economically important AAA-EA banana cultivars for efficient propagation and genetic improvement. The specific study areas included: i) establishment of proliferation potential of AAA-EA banana cultivars in MS medium, ii) enhancement of embryogenic callus induction from scalps of AAA-EA banana cultivars through application of urea-type cytokinins, N-phenyl-N‟-1,2,3-thidiazol-5-ylurea (TDZ) and N-(2-chloro-4-pyridyl)-N‟-phenylurea (4-CPPU), and iii) establishment of appropriate basal salt mixture in presence of TDZ and 4-CPPU for embryogenic callus induction from immature male flower buds of AAA-EA banana cultivars. Seven AAA-EA banana cultivars namely Mpologoma, Musakala, Mbwazirume, Nfuuka, Nakinyika, Nakabululu and Kabula from different clone sets were used. Yangambi Km5 (AAA) and Sukali Ndizi (AAB) were included for comparison in some of the experiments. Proliferation potential and number of recoverable shoots from one shoot-tip explant of AAA-EA banana cultivars on the MS derived medium for banana multiplication were determined through normal shoot-tip culture. Various combinations of TDZ and 4-CPPU were tested for embryogenic callus induction from scalps and immature male flower buds. By substituting only the MS inorganic salts from the callus induction medium with the best TDZ and 4-CPPU combination, salt formulations: Chu (N6), Eriksson, Gamborg B5, Nitsch, NLN, SH and White were tested for embryogenic callus induction from immature male flower buds of cultivars Mpologoma and Nakinyika. The AAA-EA banana cultivars produced 3-4 new shoots in each subculture cycle and 57-169 recoverable shoots from one starting shoot-tip explant in 18 weeks. Non AAA-EA banana cultivars, Sukali Ndizi and Yangambi Km5 produced 5 and 9 shoots from each subculture cycle and 322 and 352 recoverable shoots, respectively in the same period. Scalps were achieved on MS medium supplemented with TDZ and 4-CPPU in over 50% of the shoot-tip cultures within 4 subculture cycles especially in the medium containing equal proportions of 9 – 13 μM of TDZ and 4-CPPU. Depending on cultivar and, TDZ and 4-CPPU combination, embryogenic callus was formed directly on 2.5 – 20% of the scalp cultures on the auxin-free scalp induction medium. Cultivars Nakinyika, Nakabululu, Mbwazirume and Nfuuka developed regenerable embryogenic callus and recorded plant regeneration except Mpologoma. All the cultivars recorded between 4.6 - 22.2% embryogenic response from immature male flower buds on TDZ and 4-CPPU combinations. The callus induction medium supplemented with 10μM TDZ+10μM 4-CPPU induced more embryogenic response. When male flowers of cultivars Mpologoma and Nakinyika were cultured on the medium containing 10μM TDZ+10μM CPPU with the MS salts substituted by other salt formulations, they recorded 11.4% and 8.3% embryogenic response, respectively, on Gamborg B5, which was almost twice their response on pure MS medium. Though the embryo to plant conversion rate was low, in both methods most of the studied cultivars showed embryogenic responses which in some instances were greater than those reported before. This suggests that a combination of TDZ and CPPU especially at 10 μM each can enhance somatic embryogenesis in a range of AAA-EA banana cultivars and more in interaction with Gamborg B5 salt formulation. Application and optimization of combined TDZ and 4-CPPU especially in Gamborg B5 salt formulation for routine induction of embryogenic callus and development of embryogenic cell suspensions, respectively, is thus worthwhile. This will improve plant regeneration and consequently propagation and genetic improvement of AAA-EA banana cultivars.