Regenerability And Transformability Of Selected Kenyan Farmer Preffered Cassava (Manihot Esculenta Crantz) Ecotypes

Cassava (Manihot esculanta Crantz) is a perennial shrub grown mainly for its tuberous roots and provides food for over 500 million people in developing countries. The crop is affected by various biotic and abiotic stresses such as diseases, low protein content and presence of cyanogenic glycosides that result in low production. Management strategies against these stresses rely upon conventional improvement programs, but these are not effective owing to vast limitations inherent to such breeding programs. Genetic engineering may allow rapid development of stress-resistance in cassava, but efficient and robust transformation and regeneration protocols for farmer-preferred cultivars need to be optimized for ease of transfer of novel genes since existing regeneration and transformation protocols are not compatible with all the cultivars/ecotypes. In this study, the ability of coastal cassava ecotypes Karembo, Tajirika, Kibanda meno mkubwa and Ex-Mariakani to regenerate via somatic embryogenesis was investigated. Two types of explants (immature leaf lobes and stems) were cultured on Murashige and Skoog (MS) medium supplemented with varying concentrations of auxins; 2,4-dichloro phenoxyacetic acid (2,4-D), picloram and α-naphthalene acetic acid (NAA), a cytokinin; 6-Benzylaminopurine (BAP) and a gibberellin (GA3) under 16 hour light/8 hour darkness and total darkness photoperiod regimes.

Transformability of the regenerable ecotypes was also investigated through Agrobacterium-mediated transformation with the β-glucuronidase (gus) reporter gene. The results show that 2,4-D performed better than picloram in inducing callus across the ecotypes under both photoperiod regimes. There were significant differences (P≤0.05) in frequencies of somatic embryogenesis among the cassava ecotypes. Generally, 2,4-D produced the highest somatic embryogenesis frequencies of >92% (Kibanda meno mkubwa), >83% (Ex-Mariakani), >82% (Karembo) and >85% (Tajirika) under total darkness. All the four ecotypes recorded successful maturation of somatic embryos induced using immature leaf lobes on media with different phytohormone combination ratios. On the other hand, only stem explants of ecotypes Karembo and Kibanda meno mkubwa matured into cotyledons and germinated into whole plants. Molecular analysis by PCR confirmed transformation of the putative transormants from the four ecotypes. From this study, an optimized and reproducible transformation and regeneration protocol for four Kenyan cassava ecotypes has been developed.