Optimization Of Regeneration And Agrobacterium-Mediated Transformation Protocols For Selected Kenyan Cassava (Manihot Esculenta Crantz) Genotypes

ABSTRACT

Cassava (Manihot esculenta Crantz) is a tropical root crop that serves as a staple food and a vital source of income to small holder farmers in the tropics. Despite its contribution to food security, cassava production and utilization is faced by several challenges that include post-harvest physiological deterioration, insect and disease susceptibilities and accumulation of cyanogenic glycosides. Cassava crop improvement by conventional breeding has failed to address these constraints because of unsynchronized flowering, lack of resistance genes, high heterozygosity, allopolyploidy and poor seed set. Genetic transformation which begins with the establishment of embryogenic callus cultures can be used as one of the ways to complement these challenges faced by cassava breeders. This study investigated the effects of explant source (immature leaf lobes and meristematic stem segments), auxins (2, 4-dichlorophenoxyacetic acid (2, 4-D) and picloram), and photoperiod (0/24 and 16/8 light/ dark) on callogenesis and embryogenesis in five African cassava genotypes (KME 1, 08/080, 08/354, 08/274 and TMS 60444). Callus formation and embryogenesis were successfully achieved in both explant sources. The leaf explants recorded significantly higher frequencies (p < 0.05) of somatic embryogenesis compared to the stem explants in all the five genotypes. This study showed that the 0/24 light/ dark photoperiod was superior to the 16/8 light/ dark cycle for both callogenesis and embryogenesis. Although statistically insignificant, 8 mg/l 2, 4-D was the best concentration for the induction of embryogenesis in 08/354, TMS 60444, 08/274 and 08/080 while 10 mg/l gave the best results for genotype KME 1. For picloram, 10 mg/l showed the best results for embryogenesis across all genotypes. This study also determined the effects of varying formulations of BAP (6- Benzylaminopurine), NAA (α-Naphthalene acetic acid) and GA3 (Gibberellic acid) on somatic embryo maturation and plant recovery of the selected cassava genotypes Embryos in the cotyledonary stage were incubated in maturation medium supplemented with five different combinations of plant growth regulators: BAP, NAA, and GA3. Significant differences (p < 0.05) were recorded in shoot formation frequencies with combination 2 mg/l BAP, 0.01 mg/l NAA, 1.5 mg/l GA3 and combination 1 mg/l BAP, 0.02 mg/l NAA, 1.5 mg/l GA3 giving the highest rates. Transformability was determined by carrying out a histological GUS (β-glucuronidase) assay on callus transformed using Agrobacterium tumefaciens strain EHA 101 harbouring plasmid pTF 102 with a GUS visual marker gene and a bialaphos selectable marker gene. All the genotypes were found amenable to Agrobacterium mediated transformation with TMS 60444 and 08/274 recording the highest transformabilities of 73.33 % and 68.33 % respectively. A positive polymerase chain reaction (PCR) amplification targeting the GUS gene confirmed the transfer of the transgenes into cassava cells. The validated regeneration and transformation protocols reported here can be used for the integration of desired traits in African cassava genotypes.