In-Vitro Propagation of Selected Cassava (Manihot Esculenta Crantz) Cultivars using Multiple Shoot Induction and Somatic Embryogenesis

Abstract

The study showed that farmers cultivate different cassva cultivars based on popularity, duration to maturity and tolerance to African Cassava Mosaic Virus (ACMV) disease. All the five selected local were propagated in vitro using meristem explants on Murashige and Skoog (1962) basal salts and Gamborg B5 vitamins modified with benzylaminopurine (BA) (0.0 - 0.15 mg/I). There was profuse callus formation in all the cultivars. The optimal BA concentration for shoot proliferation in this medium was 0.10 mg/l BA. With reduced NAA and (GA3) concentrations (0.02 and 0.04 mg/l respectively) in the MS medium 0.05 mg/l was optimum and resulted in 100% and 46% shoot regeneration in Bosomnsia ans Santom respectively compared to 37% and 0% in the previous treatment. In M. Col 22 multiple shoots were produced from apical meristems as well as single nodal cutting explants. In the local cultivars multiple shoots were produced from nodal cuttings alone. The number of shoots produced by nodal cuttings in M. Col 22 were comparatively higher than the local cultivars at all the BA levels in the medium. In all, the number of shoots produced was dependent on the BA concentration in the medium. Leaf lobe explants of both greenhouse and in vitro plantlets developed embryogenic calli on MS amended with 0.0-16 mg/l 2,4-D on a step one-induction medium. However, calli formation was depended on the type of explants. Calli formation from young leaf lobe and apical meristem explants was significantly higher than stipule explants. On transfer to a Step two BA amended maturation medium, embryogenic calli derived from in vitro plantlets only formed matured somatic embryos. Embryo formation was depended on the concentration of the auxin in the induction medium and the cassava cultivar. Somatic embryo formation was higher on a medium with 16 mg/l 2,4-D. Santom produced the highest percentage of embryos (25%) among all the cultivars tested. Embryogenic calli which did not form somatic embryos formed foliose structures and/ or roots which also depended on the concentration of 2,4-D in the induction medium. NAA induced somatic embryos required desiccation to stimulate normal germination in all the four cultivars studied. The best desiccation procedure was the petri dish method. The desiccated embryos required a medium supplemented with BA and kinetin for germination . Incubation of cultures in darkness increased frequency of germination and also reduced germination period to 14 days when compared to cultures incubated in light under growth room conditions. The development of the seedling was dependent on the concentration of BA as well as the dark/light photoperiod of the incubation period. Cultures on medium with 0.1 mg/l BA and incubated in the dark produced normal single shoots whereas most cultures on 1 mg/l in the dark produced multiple shoots. Germination of somatic embryos was dependent on the level of moisture loss and the BA concentration in the medium. At optimal moisture loss (40%) higher frequency of germination was achieved on a medium with lower concentration of BA (0.1 mg/l). At lower moisture loss 1 mg/l BA was needed for higher germination in Gading and Adira 4. Wrapping of cultures with aluminium foil to achieve desiccation in complete darkness did not enhance embryo germination. Low light intensity (64 lux) resulted in optimum germination. Light regimes had significant effect on seedling morphology but not on frequency of germination 2,4-D-induced embryos showed a different response from NAA-induced embryos. In both NAA- and 2,4-D - induced somatic embryos desiccation followed by incubation in the dark stimulated root formation than shoot. At a concentration of 1 mg/l, ABA shoot formation only in somatic embryos of Adira 4; while at 40 mg/l, it had an inhibitory effect on the germination of these embryos. Prewashing of somatic embryos with water for 24 hours enhanced whole plant germination on MS medium modified with BA.