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.

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.

Micropropagation of Agave Species.

The genus Agave originates from the American continent and grows in arid and semiarid places, being México the center of origin. Many species of the genus are a source of diverse products for human needs, such as food, medicines, fibers, and beverages, and a good source of biomass for the production of biofuels, among many others. These plants are gaining importance as climate change becomes more evident as heat is reaching temperatures above 40 °C worldwide and rains are scarce. Many species of the genus grow in places where other plant species do not survive under severe field conditions, due to their CAM pathway for fixing CO2 where gas exchange occurs at night when stomata are open, allowing them to avoid excess loss of water. Most of the important species and varieties are usually propagated by offshoots that develop from rhizomes around the mother plant and by bulbils that develop up in the inflorescence, which are produced by the plant mostly when there is a failure in the production of seeds.Areas for commercial plantations are growing worldwide and therefore in the need of big amounts of healthy and good quality plantlets. Although many Agave species produce seeds, it takes longer for the plants to reach appropriate maturity and size for diverse purposes. Micropropagation techniques for the genus Agave offer the opportunity to produce relatively high amounts of plants year around in relatively small spaces in a laboratory. Here, a protocol for micropropagation that has proven good for several Agave species (including species from both subgenera) is presented in detail with two different kinds of explants to initiate the process: rescued zygotic embryos and small offshoots that grow around a mother plant.

Application of in Casa Pollination and Embryo Rescue Techniques for Breeding of Agave Species.

Species of the genus Agave are distributed originally in the tropical and subtropical areas of the American continent with about 200 taxa and 136 species, and its center of origin is probably limited to México. These kind of plants usually grow and live in extreme environmental conditions such as heat and drought where their CAM pathway for fixing CO2 allow them to survive in conditions where other plants cannot survive. Although this kind of plants resist harsh environmental conditions, climate change is imposing stronger kinds of stress that diminish their productive potential and in some cases are cause of death. Because of this, genetic improvement becomes a need of fundamental importance in this kind of species. Despite their economic importance, Agave species have received scarce attention with regard to its genetic improvement, probably due to their unique botanical features such as plant architecture, spines, long life span, and monocarpy, among others, which make hybridization a difficult task for the intra- and interspecific gene transfer and creation of genetic variability among many other breeding techniques.The protocol here presented is a combination of a novel hybridization technique and biotechnological tools, and allows the use of several procedures for the genetic improvement of agaves such as pollen selection, clonal selection, and somatic cell selection, among others, since the rescued embryos can be used for micropropagation, for phenotype/genotype selection or the production of cell lineages for diverse genetic improvement purposes.