Establishment and Maintenance of Friable Embryogenic Callus Lines in Cassava (Manihot esculenta)
A.B. Ibrahim; C.P. Barbosa; T.F. Machado; F.A.P. Campos
Depto. Bioquímica e Biologia Molecular, Univ. Fed. Ceará, Fortaleza, CE
An important prerequisite for the development of a genetic transformation system is the availability of morphogenic culture which allows for the production of tissues that can be used as targets for gene transfer techniques, and subsequently regenerated into complete plants, with little or no risk of somaclonal variation. In cassava (Manihot esculenta), the best system for the production of these tissues is somatic embryogenesis. Despite the relative ease with which somatic embryos can be produced from several genotypes, regeneration of plants from these organized structures has been difficult especially after the explants have been subjected to genetic transformation. In order to overcome these difficulties, a regeneration system based on the production of friable embryogenic callus (FEC) was developed and successfully applied to model genotypes. Here, we report the establishment and maintenance of ten (10) independent lines of FEC from an economically important genotype (Bujà Preta) of cassava from Northeast of Brazil. Following the establishment and production of somatic embryos via a cyclic system of somatic embryogenesis through inoculation of isolated shoot apexes on MS based media supplemented with 8mg/l picloram and 2uM CuSO4, FEC was induced by transferring somatic embryos to Gresshoff and Doy (GD) medium supplemented with MS vitamins and 12mg/l picloram. These tissues were continuously selected and subcultured every three weeks, on the same media. The effect of photoperiod on the induction of the FEC lines was evaluated as well as the percentage of explants capable of generating homogenous FEC over time. Our findings have shown that this genotype produces FEC when maintained in a photoperiod of 16 hrs. Over 33% of ten (10) explants of embryo clusters per Petri dish produce FEC over a period of 6 weeks, giving rise to homogenous and highly friable FEC culture. When transferred to an MS based media supplemented with 5uM NAA, these tissues differentiated into embryos at a rate of 400 cotyledon-stage embryos per 1 gram of FEC. The recovery of complete plants from these cotyledon-stage embryos can be achieved either by embryo maturation and germination or via organogenesis using cotyledon pieces of green mature somatic embryos as explants. Supported by CAPES, CNPq, FUNCAP and Cassava Biotechnology Network
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