Research On Agrobacterium Rhizogenes-mediated Genetic Transformation In Cotton

Cotton is one of the largest cash crops in China, the production and security of which suffers limits of national arable land and water resource, and negative shocks aroused by plant disease and insect pest. With the booming development of biotechnology, utilization of genetic engineering to transfer beneficial genes into cotton is becoming increasingly attractive and currently, Agrobacterium tumefaciens-mediated transformation is widely used in cotton. However, there are several unfavorable factors, including cotton genotype limitation, low transformation efficiency and time-consuming process, which set barriers for the development and application of transgenic cotton.Here, the study innovatively applied Agrobacterium rhizogenes to transform cotton. Through induction of hair root and its cultivation, we successfully obtained regenerated cotton and transgenic hairy roots harboring three different MYB transcriptional factors.1. Under sterile condition, cotyledon stabbing was applied to establish a system of hairy root generation in cotton. The resulting hairy roots appeared spontaneous growth without adding any phytohormone into the medium and partly lose gravitropism, both of which are classic characteristics of hairy root.2. To investigate into the effect of bacterial strain and cotton cultivar on generation of hairy root in cotton, three different Agrobacterium rhizogenes strains A4, K599 and ATCC15834, were used to induce hairy root of three cotton cultivars Shiyuan 321, YZ-1 and Coker 312, respectively. After PCR screening of rolAB gene in hairy root, the result indicates that the combination of wide type Agrobacterium rhizogenes A4 and Coker 312 produces the maximal induction efficiency of hairy root.3. Discussed the effect of various explants and seedling age on hairy root induction. The result shows cotton cotyledon of 10 to 15 days old seedling most benefits induction of hairy root in cotton.4. Discussed the optimal combination of carbon resources, inorganic and organic compounds in solid culture for hairy root induction. The result reveals that Murashige-skoog with 30g/L glucose as carbon resource is the best.5. Initially established regeneration system of cotton hairy root and got five regenerated lines.6. Agrobacterium rhizogenes carrying three different MYB transcriptional factors GmMYBZD2, GmMYBJL6 and GmMYBJL7, were applied to get transgenic hairy roots of cotton proved by genomic PCR and Southern-blot. Parts of these transgenic hairy roots were subjected to determine its growth index. In our results, we found that transgenic lines grew more vigorously than non-transgene ones. And combined with previous studies, we hold that cotton hair root has a great potential in producing gossypol, and suppose that gossypol productivity is positively related to growth ability of hairy root in cotton.