Study On Agrobacterium-mediated Transformation Of Hulless Barley

Hulless barley,namely naked barley,is one of important crops in Qinghai-Tibetan plateau, is also the main food for the Tibetan. Traditional method for breeding is always the main way for hulless barley. With the development of molecular biology, transformation is used as one of the ways to improve the genetic resources and quality. Agrobacterium-mediated transformation is thought as the base for genes transferring. In this study, we focus on the effects of the genotypes, cutting ways, culture medium and hormones on rates of induction and differentiation of hulless barley. Based on this, it is the foundation for the Agrobacterium-mediated transformation. The results are showed below:1. In order to establish a highly efficient plant regeneration system suitable for plant transformation, we employed the immature embryos from Dulihuang, one of the main cultivars of hulless barley in Qinghai Province, as the explants material, to study the effect of the time of candle and sampling, and to study the effect of 6-BA to the differentiation. The results show that the callus initiation rate of Dulihuang reaches 91.2%, and the differentiation rate reaches 35.4%. Therefore, Dulihuang is identified as the genetic transformation recipient materiel due to higher performance both in induction of callus and plant regeneration.2. In order to establish a highly efficient plant regeneration system by using mature embryos, we employed the mature embryos from Dulihuang, one of the main cultivars of hulless barley in Qinghai Province, as the explants material, to study the effects of different ways of cutting mature embryos, base medium, carbons, 2, 4-D, and hormone combination on callus induction and plant regeneration. The results indicated that the modified MS medium, which includes 30g/l maltose and 6mg/l 2,4-D is the optimal medium for mature embryo totally broken. The results showed that the callus initiation rate of Dulihuang in above medium reaches 96.7%, and the differentiation rate reaches 44.5%. Therefore, Dulihuang is identified as the genetic transformation recipient materiel due to higher performance both in induction of callus and plant regeneration.3. In order to widen the genotypes for transformation, the rate of induction and differentiation of 14 cultivars were compared. The results showed that four genotypes(Kunlun 12, Beiqing 3, Kunlun 14, Zangqing 320), except for Dulihuang, were observed, which had the higher rate of induction and plant regeneration. Therefore, they are the suitable genotypes for transformation by using mature embryos.4. Meanwhile, some factors that effecting expression including Agrobacterium concentrations, infection time and culture time were investigated by detecting transient expression of GUS gene and PCR to increasing the frequency of transient expression mediated by Agrobacterium-mediated transformation and to optimize the transformation conditions. The results showed that the higher transient expression frequency of GUS gene could be resulted from Agrobacterium-mediated transformation bacterial concentration of OD600 being 0.8, 10 min. And the transformation frequency is 0.2% by PCR detection in GM hulless barley.