The increasing demand for cassava (Manihot esculenta Crantz) for food and non-food uses in the tropics necessitates that its breeding for increased root productivity be made faster. The characteristic long breeding cycle and heterozygous nature of this crop, pose a major obstacle to its rapid genetic improvement. This study aimed at inter-pollinating cassava with castor bean (Ricinus communis), with a purpose of inducing and regenerating cassava doubled haploids (DHs). A total of 3,349 flowers from twelve elite cassava varieties were inter-pollinated with caster bean. A total of 803 fruits were harvested for early embryo rescue and/or ovule culture. Of these, three were dissected to obtain seven unique embryos, while 800 were dissected to obtain 1312 young ovules, all of which were cultured in vitro. Overall, 82 (6.25%) of the cultured ovules formed callus that originated from the embryosac region, which is haploid. Four out of seven rescued embryos (57.1%) regenerated into plantlets. Ploidy analyses of 24 samples using flow cytometry revealed that 23 of the analysed samples were diploid. However, one callus sample was anueploid. Only one sample had an exceptionally high level of homozygosity (84.2%). These findings lay a foundation for future research aimed at induction of haploids in cassava.

Production and use of double haploids is important for attainment of systematic genetic gains, and indeed several plant breeding programmes have benefited from it. Gynogenesis, the in vitro culture of unfertilized ovules and/or embryos has specifically been exploited in several economically important crops but not cassava. In this study, we examined possibilities of generating doubled haploids (DH) in cassava through gynogenesis, by bagging female flowers of selected varieties to prevent pollination. A total of 2,466 flowers across 32 elite cassava varieties were bagged for a period of one-to-three days. Early embryo rescue and ovule culture were done at 7 to 42 days after anthesis. Consequently, 517 fruits (21%) were harvested and dissected to obtain 97 seeds from which 47 unique embryos and 18 callus lines were obtained in vitro. Only six of the rescued embryos (12.8%) regenerated into plantlets. Upon undertaking ploidy analysis and single nucleotide polymorphism (SNP) genotyping, it was established that all samples analyzed (regenerated plants and calli) were diploid. SNP genotyping revealed increased homozygosity (up to 85.7%), but no doubled haploids were noticed. The knowledge generated is a significant contribution towards understanding cassava flowering biology and thus a foundation to on-going efforts towards developing protocols for generation of cassava DH.

Cassava’s (Manihot esculenta Crantz) high heterozygosity complicates its genetic improvement via selective breeding. Double haploid (DH) technology can be used to improve the crop’s heterozygosity, thereby improving the capacity for genetic improvement. The objective of this study was to evaluate the effect of self-pollination using heated pollen on pollen tube penetration, fruit set, seed and haploid embryo development in cassava genotypes for the production of haploid cassava. Pollen from two cassava genotypes, NASE3 and NASE14, was heated at 40, 50 and 60 oC for 0.5, 1.0 and 2.0 hr each. The heated pollen was used in six rounds of self-pollinations. Pollen tube penetration was monitored by fluorescent microscopy, followed by early embryo rescue and ovule culture. Ploidy and zygosity were assessed using flow cytometry and single-nucleotide polymorphism analysis, respectively. Pollen germinated on the stigma, grew within the style through the nucellar beak, but did not reach the embryo sac, thus achieving no fertilisation in all the 5756 self-pollinated flowers. There was a reduction in pollen germination (in vitro and in vivo), pollen tube penetration and fruit set with increasing temperature. Heat-treated pollen stimulated division of the egg cell and induced development of parthenocarpic fruits. Up to 6 embryoids per ovule were observed and all regenerated plantlets were diploid, with up to 93.0% increased homozygosity. For the first time, plant regeneration from ovules, pollinated with fresh pollen at 14 days after pollination, was achieved indicating improved speed in plant regeneration. The data generated are important for the development of protocols for cassava DH plant production.

Cassava (Manihot esculenta Crantz) is an important staple and food security crop for millions of people in Africa. However, its nutritional value is limited; yet its productivity is constrained by several pests and diseases. Induced mutagenesis is one approach with the potential to overcome such biotic stresses. The objective of this study was to assess the variability in morphometric traits and prevalence of common viral diseases and whiteflies in cassava to different doses and concentrations of gamma irradiation and ethyl methane sulphonate (ems) treatments. The effects were assessed on seed germination and growth of stakes, as well as foliar viral disease symptoms and whitefly counts. Radio-sensitivity tests revealed LD50 for sprouting as 37.6Gy for g-irradiation and 0.08% for EMS treatment. There was notable decrease in sprout, epicotyl length, shoot height, petiole length and number of leaf lobes, with increasing g-ray doses and EMS concentrations. Contrastingly, total chlorophyll content increased with increasing doses of g-rays and EMS concentrations. Basing on foliar whitefly counts and disease symptoms, there was a general increase in susceptibility to whitefly infestation and cassava mosaic disease (CMD) incidence. Significantly varying levels of resistance or tolerance to whiteflies and CMD were observed among plantlets derived from irradiated and EMS treated stems, compared to the controls. These findings lay a foundation for more future research on breeding for various traits, including disease resistance in cassava using induced mutagenesis approach.