Cultivo in vitro de anteras como estrategia para el mejoramiento genético de Buffelgrass (Cenchrus ciliaris L)

Abstract

Buffelgrass es una gramínea forrajera que se reproduce principalmente por apomixis. El objetivo del presente trabajo es generar variabilidad genética, mediante cultivo in vitro, en caracteres asociados a tolerancia a sequía a partir de germoplasma introducido de buffelgrass, con la finalidad de incorporarla en un programa de mejoramiento genético de esta especie. El estudio se realizó en una colección activa que se encuentra en el IFRGV, perteneciente al CIAP-INTA. Los resultados del presente trabajo demostraron que el germoplasma apomíctico presentaba diferentes niveles de ploidía. La constitución genética está representada por individuos tetraploides (2n=4x=36) y aneuploides (2n=43,44 y 52). Se encontró una correlación entre el número de cromosomas y el contenido de ADN nuclear. Tres genotipos tuvieron la capacidad de generar callos embriogénicos utilizando anteras como explanto. Las plántulas se originaron a partir de embriones somáticos y el proceso morfogénico se indujo del tejido somático. Se determinó que la eficiencia de inducción y regeneración dependía del genotipo y fue afectada por la fecha de recolección de macollas, los pre-tratamientos o la interacción entre ambos factores. Plantas regeneradas in vitro con menor y mayor contenido de ADN nuclear fueron identificadas mediante análisis de citometría de flujo.

Buffelgrass is a forage grass that reproduces mainly by apomixis. The objetive of this research was to increase the genetic variation, by in vitro culture, in characters associated with drought tolerance from introduced buffelgrass germplasm, in order to incorporate it into a breeding program for this specie. The study was conducted in an active germplasm collection that is located in the IFRGV belonging to CIAP-INTA. The results of the present study showed that the apomictic entries had different ploidy levels. The genetic constitution is represented by tetraploid (2n = 4x = 36) and aneuploid individuals (2n = 43, 44 and 52). A correlation between the number of chromosomes and DNA content was found. Three genotypes had capacity to generate embryogenic callus using anthers as explant. Seedlings were originated from somatic embryos and the morphogenic process was induced from somatic tissue. It was determined that the efficiency of induction and regeneration depended on genotype and was affected by the collection date of the tillers, pre-treatments or interaction between both factors. The flow cytometry analysis detected in vitro regenerated plants with lower and higher nuclear DNA content. The linear regression analysis indicated that in the first case these materials lost a chromosome and cytology studies in one of them confirmed, while in two plants, increased at double the nuclear DNA content indicated an in vitro polyploidization. In six of the regenerated materials, the use of 22 primers ISSRs detected 12% of polymorphism and divergence of 5 to 24% compared to the material used as control. Plants (R 1 ) grown in the field showed different morphological characteristics. Additionally, the progeny of any of these materials (S 1 ) showed differential behavior in an assay of water stress. The changes observed in some in vitro regenerated plants showed somaclonal variation. In one of the entries, mannitol as an osmotic agent incorporated in the process of in vitro selection allowed to obtain tolerant materials with characters associated to water stress.