Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid