Identification of rice mutants tolerant to cold stress at the germination stage by TILLING.

Cold stress is a common factor affecting rice culture in temperate regions, which impairs seed germination, crop establishment and grain yield. This work aimed to identify, through a TILLING assay, rice mutant families displaying cold tolerance during the germination stage. The mutant analyses were performed in 4000 M3 plants obtained through chemical mutagenesis with ethyl methanesulfonate. We screened for mutations in the Os03g0103300 (qLTG3-1) gene, which is responsible for cold tolerance during germination. The TILLING assay identified a mutant (516 A3) which was tested for germination efficiency in cold stress (13°C). The mutant genotype showed a higher relative performance in germination and germination velocity index, which was more than 50% higher compared with wild-type. The mutation induction was efficient in creating genetic variability for cold stress tolerance during germination. Gene expression analyses demonstrate that Os03g0103300 was downregulated in stage S3 in the mutant and wild-type plants germinated under cold stress. However, downregulation in the Os03g0103300 gene was less severe in the mutant, which suggests that the expression related to germination ability under cold stress may be detected in the previous stages, embryo activation and weakening of the tissues that cover the embryo. Overall, the mutant 516 A3 presents a new genetic variant for cold tolerance during germination.