i. The protocol for callus induction, somatic embryogenesis and whole plant recovery optimized in this study is suitable and therefore recommended for regenerating all the four ecotypes. Using the best performing plant growth regulators and their combinations, it will be easy to regenerate these ecotypes during any improvement program. For instance maturation medium M5 will be suitable for regenerating all cultivars using immature leaf lobe explants while M3 will be suitable for Karembo and Kibanda meno mkubwa using stem explants.
ii. Further studies need be carried out to understand the mechanisms involved in conversion of somatic embryos from stems to cotyledonary embryos and the eventual whole plant development owing to the low frequencies obtained. This will help come up with a robust medium to aid improve the conversion rates of somatic embryos obtained from stem explants and enable their use in cassava regeneration since they are readily available. iii. Further molecular work is needed to ascertain stability of the transgene in
transgenic plants in subsequent generations. Studies such as southern blot (to determine transgene copy numbers integrated into the cassava genome) and reverse transcriptase polymerase chain reaction (RT-PCR) to
determine whether the transgenic plants express the transgene are recommended.
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APPENDICES Appendix I
Composition of culture media and regeneration and transformation protocol of cassava
Media
name/preparation Stage Basic composition
Hormones and antibiotics Conc. Mg/L Treatment period CBM In vitro propagation MS,sucrose pH5.8 N/A N/A 2-6wks CIM Callus induction and somatic embryogenesis MS with B5 vitamins,sucrose pH 5.7 Picloram 2,4-D 4-10 4-10 4 wks CMM Maturation of somatic embryos MS with B5 vitamins,sucrose pH 5.7 BAP NAA GA3 1-3 0.01-0.03 0.5-1.5 3-4wks CEM Germination of cotyledonary embryos MS,sucrose, activated charcoal pH 5.8 BAP NAA GA3 1-3 0.01-0.03 0.5-1.5 2 wks
Bacteria media Bacteria growth LB Kanamycin Spectinomyci n 50 100 24hrs
Pre-infection Infection MS with vitamins, glucose,
galactose,acetosyringon e
pH 5.5
N/A N/A 4-8hrs
Co-cultivation Cocultivation MS,sucrose,acetosyring one
pH5.7
2,4-D 10mg/L 3days
Resting Resting MS with B5
vitamins,sucrose, pH 5.7 Carbenicillin 2,4-D 500 10 7 days
Selection Selection MS with B5vitamins 2,4-D Basta
10 1.5-3