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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.

This article describes the complete microsporogenesis and pollen formation in cassava (Manihot esculenta Crantz) at the various developmental stages (pollen mother cell, meiosis, tetrads, early free spore, mid uninucleate, late uninucleate, binucleate and mature pollen grain). Light microscopy, transmission electron microscopy and confocal laser scanning microscopy were used for the study. Floral bud size and other inflorescence characteristics were correlated with specific stages of the microspore development. This association allows a rapid selection of floral buds with similar microspore developmental stages, useful when a large number of homogeneous cells are needed for analysis and for in vitro induction of androgenesis. This article also compares methods for digestion the exine wall in microspores

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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 handpollinated 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.

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.

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Rice hoja blanca virus (RHBV) is a major virus disease of economic importance affecting rice in northern South America, Central America and the Caribbean. This is the first report of transgenic resistance to RHBV and the transformation of an indica rice variety from Latin America. Rice transformed with the RHBV nucleocapsid protein ( N) gene had a significant reduction in disease development. Several reactions were observed that ranged from susceptible to completely resistant plants (immunity). The resistant reactions were characterized by the production of local lesions like a hypersensitive reaction or a recovery phenotype with the emergence of symptom-less new leaves. These transgenic RHBV-resistant rice lines expressed the N gene RNA at low levels that were below the detection limit by Northern blots and only resolved by RT-PCR. The nucleocapsid protein could not be detected in any of the transgenic plants either by Western or ELISA tests. These results suggest that the resistance encoded by the N gene in these plants appears to be mediated by RNA. When challenged with RHBV, the resistant transgenic lines showed a significant increased performance for important agronomic traits including the number of tillers, the number of grains per plant and the yield as compared to the susceptible control. Furthermore, upon inoculation some of the most-resistant transgenic lines showed agronomic traits similar to the uninoculated non-transgenic Cica 8 control. Using both agronomic traits and disease severity as criteria, several of the most-resistant lines were followed through the R(4) generation and demonstrated that the N gene and RHBV resistance was inherited in a stable manner. These transgenic rice lines could become a new genetic resource in developing RHBV-resistant cultivars.