Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation.

This work describes the application of clearing on vibratome sections to study the embryo formation in cassava. This procedure provides high-resolution images and reduces significantly the number of sections that need to be analyzed per ovule. This methodology was instrumental for the development of the protocol for embryo rescue in cassava. It has been also applied to monitor the embryo formation response when optimizing seed setting from regular and broad crosses for cassava breeding. Broad crosses between cassava and castor bean (incompatible-euphorbiaceae species) were made aiming to induce doubled haploids through the elimination of the incompatible-male parent genome as done in cereals. Castor bean is widely available and provides continues supply of pollen. Our results suggest that this methodology is easy and effective to assess the response of hundreds of cassava ovules pollinated with castor bean pollen, allowing the identification of multicellular structures in the embryo sac without apparent formation of endosperm. The protocol is also useful when developing and optimizing a methodology to induce doubled haploids in cassava via gynogenesis or from ovules pollinated with irradiated cassava pollen.

Protocol for Rescuing Young Cassava Embryos.

Embryo rescue (ER) in cassava breeding has several relevant applications, from the recovery of broad crosses to the recovery of seeds from the standard pollination program. Cassava fruit setting may drop from 100%, during the 1st week after pollination, to less than 40% during the 2nd week after pollination due to the abscission of fruits depending on genotypes. Therefore, the availability of an ER protocol for early stages of embryo development, in particular during the first 2 weeks after pollination (prior the cotyledonary stage), could have practical implications for cassava breeding. Until now, attempts to recover cassava immature embryos at stages of development earlier than the cotyledonary stage failed. The earliest successful rescue reported in cassava is from embryos excised 32-36 days after anthesis (DAA). However, limited information was available regarding embryo development in cassava. This work studied and documented the stage of embryo development in histological sections of hand-pollinated ovules fixed from 1 to 30 days after anthesis (DAA). At 7 DAA, zygotes were just at the first stages of cell division (pro- embryo stage). At 14 DAA, embryos were at the pre-globular stage. Embryos at the early globular stage were observed in sections fixed at 21 DAA, and at the proper globular stage at 24 DAA. Samples at 30 DAA contained cotyledonary embryos that easily developed after ovule culture into viable plants using existing protocols. A second contribution of this work is the development of a protocol for the recovery of fully developed plants from immature embryos rescued and cultured in vitro as early as 7-14 DAA. Since embryos collected at this age are at the pro-embryo to pre-globular stage, ovary/ovule culture was necessary. A method is described whereby ovules were cultured to allow the development of pro-embryos and pre-globular stage embryos into the cotyledonary stage. Subsequently, these mature embryos were excised from the ovules to induce germination and the recovery of fully developed plants.

Studies on Gynogenesis Induction in Cassava (Manihot esculenta Crantz) Unpollinated Ovule Culture.

Cassava (Manihot esculenta Crantz) is an important crop for subsistence farming in tropical and subtropical regions. There is a need to increase the rate of genetic gain to develop varieties adapted to new environmental conditions affected by climate change, which also influences the patterns of pests and diseases. The rate of cassava genetic improvement is limited by the difficulty in obtaining true-breeding types (inbred/homozygous lines). Cassava inbreeding obtained through conventional sequential self-pollination increases exposure of useful recessive traits and breeding value of progenitors. However, it takes 10-15 years to produce homozygous lines through successive self-pollination. Doubled haploid (DH) technology is a functional alternative to progressive self-pollination, and is already widely used in major crops to accelerate inbreeding. This work aimed at developing a protocol for the culture of isolated ovules and the induction of gynogenesis in cassava. Basic groundbreaking studies on cassava embryo sac development are presented. A protocol using unpollinated ovules collected from ovaries 1 day after anthesis is described. In the unpollinated-cultured ovules, the presence of embryos formed probably from the egg cells and not surrounded by the endosperm, was documented by anatomical analyses. This achievement is an important first step in the development of a reproducible gynogenesis protocol for the generation of doubled haploids in cassava. This protocol can also be useful as a starting point to obtain DHs using alternative methods of induction such as pollination of cassava with pollen of distant species or with cassava pollen irradiated with gamma rays.

Deep Assessment of Genomic Diversity in Cassava for Herbicide Tolerance and Starch Biosynthesis.

Cassava is one of the most important food security crops in tropical countries, and a competitive resource for the starch, food, feed and ethanol industries. However, genomics research in this crop is much less developed compared to other economically important crops such as rice or maize. The International Center for Tropical Agriculture (CIAT) maintains the largest cassava germplasm collection in the world. Unfortunately, the genetic potential of this diversity for breeding programs remains underexploited due to the difficulties in phenotypic screening and lack of deep genomic information about the different accessions. A chromosome-level assembly of the cassava reference genome was released this year and only a handful of studies have been made, mainly to find quantitative trait loci (QTL) on breeding populations with limited variability. This work presents the results of pooled targeted resequencing of more than 1500 cassava accessions from the CIAT germplasm collection to obtain a dataset of more than 2000 variants within genes related to starch functional properties and herbicide tolerance. Results of twelve bioinformatic pipelines for variant detection in pooled samples were compared to ensure the quality of the variant calling process. Predictions of functional impact were performed using two separate methods to prioritize interesting variation for genotyping and cultivar selection. Targeted resequencing, either by pooled samples or by similar approaches such as Ecotilling or capture, emerges as a cost effective alternative to whole genome sequencing to identify interesting alleles of genes related to relevant traits within large germplasm collections.