Utilizing heated pollen and Androgenesis pathways for the production of haploids in cassava

Abstract

It takes no less than eight years to develop a new variety because the cassava breeding cycle is long, and its multiplication rate slow. Cassava breeding is based on the use of heterozygous progenitors, which has important drawbacks. One of them is the impossibility to implement the conventional back-crossing. The doubled haploid (DH) technology offers a shortcut for one of the most time-consuming processes in plant breeding; arriving at homozygous inbred lines that in turn can be used for hybrid production. DH systems have been adopted, and utilized in commercial production, and viewed as an invaluable tool for crops that have it available like maize. Spontaneous production of haploids is a rare phenomenon and is not efficient enough to rely on for commercial production. Developing a reliable method of haploid production, which is not something many crops have, is essential for cassava. This study was therefore undertaken to; i. evaluate the effect of heated pollen on fruit set, seed and haploid embryo development in NASE3 and NASE14 genotypes for production of haploid cassava, ii. induce haploidy in cultured cassava anthers of NASE3 and NASE14 genotypes and iii. induce haploidy in cultured cassava microspores of NASE14 and NASE3 genotypes. To evaluate the effect of heat-treated pollen on fruit set and seed and haploid embryo development, NASE14 and NASE3 genotypes were used as mother plants, and as pollen donors for the different temperature treatments and durations before fertilisation. The pollen was pretreated at 40oC, 50oC, and 60oC for 0.5, 1.0, and 2.0 h. The pre-treated pollen was then tested using four rounds of self-pollinations. Pollen tube penetration capacity into the embryo sac was then monitored and followed by early embryo rescue and ovule culture. Results showed that for 2, 269 and 2,571 self-pollinated flowers from NASE14 and NASE3, respectively pollen germinated on the stigma, and grew within the style through the nucellar beak, but could not reach the embryo sac to achieve fertilization. Furthermore, in vitro and in vivo pollen germination rates, as well as pollen tube penetration into the embryo sac, decreased with increasing severity of temperature treatment. Heated-treated pollen stimulated division of the egg cell and induced development of parthenocarpic fruits. Plant regeneration from ovules pollinated with fresh pollen 14 days after pollination (DAP) was achieved. However, with heat-treated pollen, green-like structures and rooting developed. A high rate of polyembryony was observed with a maximum of 6 embryoids per ovule. Flow cytometry and zygosity analyses of 22,618 nucleotide polymorphism (SNPs) showed that all regenerated plantlets were diploid with up to 93% increased homozygosity. To induce haploidy in NASE3 and NASE14 genotypes using microspore embryogenesis, microspores were heat stressed at 40˚C for 0, 6, 12, 18, and 24 hours. The microspores were then cultured on Murashige and Skoog (MS) medium supplemented with 2-9% sucrose and 2,4-dichloro phenoxy acetic acid. Heating NASE3 anthers at 40˚C for 6 h resulted in a significantly higher percentage of callus induction on MS medium supplemented with 2% sucrose. Callus emerged from the inside of the anthers with production influenced by genotype, sucrose concentration, anther density, and duration in culture (P≤0.001). Limited in vitro callus differentiation was observed on auxin-and cytokinin-supplemented media. In both genotypes, embryogenic callus was obtained in liquid medium, while green callus was achieved on a solid medium. This study provided an analysis of the effect of self-pollination with heat-treated pollen on parthenocarpy and homozygosity in cassava. Furthermore, the study explored induction of haploidy in cultured cassava anthers using anthers sourced from genotypes NASE3 and NASE14. Through these studies, it was found that successful induction of embryogenic callus by heat treatment and inoculation of liquid and solid cultures in two elite Ugandan cassava genotypes is possible. Results also indicated a successful fruit set, induced by heated cassava pollen, and increased homozygosity in derived embryos, following pollination of female flowers with heat-treated pollen. For the first time, cassava proembryos were regenerated from ovules rescued 14 days after pollination (DAP). Until now, this was possible only earliest 28 DAP or later, indicating improved efficiency of our embryo rescue protocol. Finally, the unit of success‘ reported in this study contributes to efforts devoted towards development of cassava DH. This is also a significant step upstream of the double haploid plant production pathway in cassava. Rigorous optimization of protocols downstream of callus induction is needed for regeneration of microspore-derived embryos and haploid plants.