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.

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana.

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.

Somatic embryogenesis in sisal ( Agave sisalana Perr. ex. Engelm).

A protocol has been developed for somatic embryogenesis and plant regeneration of sisal (Agave sisalana Perr. ex. Engelm). Embryogenic callus cultures were initiated from young shoots raised in vitro from the stem portion of the bulbil on medium supplemented with 1-2 mg l(-1) kinetin (KN) and 0.2-0.5 mg l(-1) alpha-naphthaleneacetic acid plus KN or 1-1.5 mg l(-1 )benzylaminopurine (BAP) or 0.25-0.5 mg l(-1 )2,4-dichlorophenoxyacetic acid plus BAP or 0.5-1.0 mg l(-1) KN. Embryos at various developmental stages (globular-, heart- or torpedo-shaped) produced mature and germinating embryos on being transferred to a new medium containing 0-0.25 mg l(-1 )KN. After 28 days, a maximum of 76% germinated embryos was obtained on a medium supplemented with 0.1 mg l(-1) KN. The capacity for embryogenesis remained constant in the callus upon subculturing on the same medium for more than 48 months. Histological observations showed a distinct multicellular origin for most of the somatic embryos as they developed from epidermal, sub-epidermal and inside callus cells, while a few of them originated from a superficial callus cell. Plantlets regenerated from embryos were transferred to the field where their survival rate was 100%.