Desarrollo de procedimientos de crioconservación basados en la vitrificación en Mentha sp. para minimizar el crecimiento de bacterias endófitas

Abstract

Cryopreservation is the preservation of living material at very low temperatures (< -150ºC, usually in liquid nitrogen), stopping metabolism so that it can be resumed later, recovering the viability of the material. Therefore, it is a procedure of great interest for the long-term conservation of plant germplasm, especially in the case of species with vegetative propagation or recalcitrant seeds. One of the main problems in the development of cryopreservation protocols is the emergence of endophytes. They are usually bacteria that do not hinder the growth of plants in vitro, but greatly reduces the success of cryopreservation protocols. The main objective of this work was to develop cryopreservation procedures for apices of a Mentha aquatica L. genotype, with endophytic bacteria, kept in in vitro culture for several years. To fine-tune certain procedures, in vitro cultures of two Mentha x piperita L. genotypes, that do not show bacterial contamination, have also been used. The methodology used includes common procedures for in vitro plant culture regarding culture maintenance and monitoring. The apices cryopreservation procedure used was droplet-vitrification, based on that developed by Senula et al. (2007) for mint. This method is based on cold hardening of mint nodes and subsequent treatment of shoot apices with vitrification solutions. Rapid cooling is achieved by immersion in liquid nitrogen of apices arranged in small droplets of the vitrification solution in an aluminum foil inside the cryovial. Procedures to decrease bacterial development during the application of the protocol based on the use of antibiotics and various vitrifying solutions as PVS2 and PVS3 have been considered. The results show that the cryopreservation of Mentha sp. is more efficient using PVS3 as the vitrification solution, improving the recovery of cryopreserved apices compared to the use of PVS2. Besides, the addition of the antibiotic cefotaxime to the apices culture medium after cryopreservation had a beneficial effect on their recovery, by significantly reducing the growth and proliferation of endophytic bacteria.