Effectiveness of R1-nj Anthocyanin Marker in the Identification of In Vivo Induced Maize Haploid Embryos.

Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.

Investigating the Effect of the Interaction of Maize Inducer and Donor Backgrounds on Haploid Induction Rates.

Doubled haploid technology is a feasible, fast, and cost-efficient way of producing completely homozygous lines in maize. Many factors contribute to the success of this system including the haploid induction rate (HIR) of inducer lines, the inducibility of donor background, and environmental conditions. Sixteen inducer lines were tested on eight different genetic backgrounds of five categories in different environments for the HIR to determine possible interaction specificity. The HIR was assessed using the R1-nj phenotype and corrected using the red root marker or using a gold-standard test that uses plant traits. RWS and Mo-17-derived inducers showed higher average induction rates and the commercial dent hybrid background showed higher inducibility. In contrast, sweet corn and flint backgrounds had a relatively lower inducibility, while non-stiff stalk and stiff stalk backgrounds showed intermediate inducibility. For the poor-performing donors (sweet corn and flint), there was no difference in the HIR among the inducers. Anthocyanin inhibitor genes in such donors were assumed to have increased the misclassification rate in the F1 fraction and, hence, result in a lower HIR.

Haploid Induction in Tomato (Solanum lycopersicum L.) via Gynogenesis.

The generation of new hybrid varieties of tomato (Solanum lycopersicum L.) is the most widely used breeding method for this species and requires at least seven self-fertilization cycles to generate stable parent lines. The development of doubled haploids aims at obtaining completely homozygous lines in a single generation, although, to date, routine commercial application has not been possible in this species. In contrast, obtaining doubled haploid lines via gynogenesis has been successfully implemented in recalcitrant crops such as melon, cucumber, pumpkin, loquat and walnut. This review provides an overview of the requirements and advantages of gynogenesis as an inducer of haploidy in different agricultural crops, with the purpose of assessing the potential for its application in tomato breeding. Successful cases of gynogenesis variants involving in vitro culture of unfertilized ovules, use of 60Co-irradiated pollen, in vivo haploid inducers and wide hybridization are presented, suggesting that these methodologies could be implemented in tomato breeding programs to obtain doubled haploids.

Mejoramiento de arveja (Pisum sativum L): avances del programa de mejoramiento genético de la Universidad Nacional de Rosario / Pea (Pisum sativum L.) breeding: advances of the breeding program at Universidad Nacional de Rosario

RESUMEN En 2005 se inició un programa de mejoramiento de arveja para aumentar la producción en cantidad y calidad en la Facultad de Ciencias Agrarias (FCA), Universidad Nacional de Rosario (UNR). Los primeros pasos fueron reunir una colección activa de germoplasma de todo el mundo y analizar la variabilidad genética a través de rasgos morfo-agronómicos y moleculares. En 2014, el Instituto Nacional de Tecnología Agropecuaria (INTA) y la FCAUNR unieron esfuerzos para promover el desarrollo local de genotipos de arveja adaptados a la región. Este programa, utilizando metodologías convencionales, ha obtenido hasta el momento una nueva variedad comercial (Primogénita FCA-INTA) de color de cotiledón verde, semi-áfila, con alta adaptación a las condiciones agroecológicas locales y alto potencial de rendimiento. El mejoramiento genético, sin embargo, es un proceso lento. El desarrollo de nuevas variedades requiere una década o más utilizando metodologías tradicionales, por lo que se propusieron diferentes alternativas para la reducción de este período. Los haploides duplicados y el cultivo in vitro han sido algunas de las metodologías desarrolladas, sin embargo, en legumbres no se han podido implementar de manera eficiente en los programas de mejoramiento. En este contexto, Speed Breeding surge como una tecnología que permite incrementar la eficiencia de los programas, reduciendo los costos y el trabajo requerido.

ABSTRACT A pea breeding program to increase production in quantity and quality was started in 2005 in the College of Agrarian Sciences (FCA), National University of Rosario (UNR). The first steps were to gather an active collection of germplasm from around the world and to analyze genetic variability through morpho-agronomic and molecular traits in order to set objectives. In 2014, the National Institute of Agropecuarian Technology (INTA) and the FCAUNR, joined forces to unite inter-institutional efforts for promoting the local development of pea genotypes adapted to the region. This program, using conventional methodologies, has so far obtained a new commercial line (Primogénita FCA-INTA) of green cotyledons, semileafless, with high adaptation to local agro ecological conditions and high yield potential. Breeding, nevertheless, is a slow process. Developing new pea varieties usually takes a decade or more when using traditional methodologies; thus, different alternatives were proposed for the reduction of this period. Doubled haploids and in vitro culture have been some of the methodologies developed; in pulses, however, they have not been efficiently implemented in breeding programs. In this context, Speed Breeding emerges as a technology that allows increasing the efficiency of the programs, while reducing costs and the required labor.

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.

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.

Production of wheat-doubled haploids resistant to eyespot supported by marker-assisted selection

BACKGROUND: Wheat is one of the most important crops cultivated all over the world. New high-yielding cultivars that are more resistant to fungal diseases have been permanently developed. The present study aimed at the possibility of accelerating the process of breeding new cultivars, resistant to eyespot, by using doubled haploids (DH) system supported by marker-assisted selection. RESULTS: Two highly resistant breeding lines (KBP 0916 and KBH 4942/05) carrying Pch1 gene were crossed with the elite wheat genotypes. Hybrid plants of early generations were analyzed using endopeptidase EpD1 and two SSR markers linked to the Pch1 locus. Selected homozygous and heterozygous genotypes for the Pch1-linked EpD1b allele were used to produce haploid plants. Molecular analyses were performed on haploids to identify plants possessing Pch1 gene. Chromosome doubling was performed only on haploid plants with Pch1 gene. Finally, 65 DH lines carrying eyespot resistance gene Pch1 and 30 lines without this gene were chosen for the eyespot resistance phenotyping in a field experiment. CONCLUSIONS: Results of the experiment confirmed higher resistance to eyespot of the genotypes with Pch1 in comparison to those without this gene. This indicates the efficiency of selection at the haploid level.

Reproductive biology in cassava: stigma receptivity and pollen tube growth.

Knowledge on the reproductive biology of cassava, relevant to breeders and molecular geneticists, is still limited. Therefore, different studies were carried out to determine the duration of stigma receptivity and the rate of pollen tube growth. Inflorescences were covered for up to 3 days after the first opening of the bracts (e.g. anthesis day) to prevent open pollination. Results indicate that fruit and seed set are drastically reduced when flowers were covered for 2 or 3 days. However, fruits and seeds were obtained even from flowers that had been covered for 3 days after anthesis, although at low frequency. The rate of pollen tube growth was assessed in many combinations of female and male progenitors crossed through controlled pollinations and collecting the pistils at varying hours after pollination (HAP). Pollen tube growth is fast during the first 6 HAP reaching the tip of the nucellar beak. The growth slows down thereafter, taking 10 additional hours to reach the end of the beak. The growth of pollen tubes slows down even further until they enter the embryo sac. Only 10% of samples showed pollen tubes entering the embryo sac between 48 and 66 HAP. Although several tubes may reach the nucellar beak, only one was observed entering the embryo sac. Results, across the different experiments, were highly variable suggesting that the timeline of fertilization is influenced both by genotypic and environmental factors as well as the manual manipulation of inflorescences and cyathia.