| Foxtail millet[Setaria italica L. Beauv.], which made contribution to human civilization development as one of the most ancient cereal crops, is a food crop and a forage grass that grown in northern China and many Asian countries.. Foxtail millet was rich in nutritional value and characterized with its drought tolerance, salt tolerance, infertile soil tolerance for human cultivation and other special traits. But because of being a kind of regional-important crop, biotechnology research of foxtail millet was relatively little. At present, foxtail millet genetic transformation is through bombardment or Agrobacterium-mediated co-cultivation.In this study, a high efficiency foxtail millet tissue culture and regeneration system was established, then, the Agrobacterium-mediated genetic transformation system for foxtail millet was optimized and transgenic plants which contain kanamycin-resistant gene were obtained.(1) Through screening of foxtail millet explants and genotypes, an efficient and stable tissue culture regeneration system was developed. It was found that explant of immature inflorencences of foxtail millet, compared to immature embryos and mature embryos, showed an easier way of operation, the highest rate of embryogenic callus iniciation, and higher rate of plantlet regeneration. So, in foxtail millet tissue culture and Agrobacterium transformation, immature inflorencences is the ideal explants. Immature inflorencences at the late branch differentiation stage, which was about 1-1.5cm in length, is the best callus induction stage. By screening 26 foxtail millet varieties, it was found that Jigu 11, Jingu, Zhaogu 12, Gao 39 and Yu 1 were the excellent genotypes for the hard granular embryogenic callus iniciation and plantlet regeneration.(2) Factors that influence foxtail millet transformation mediated by Agrobacterium were studied in this experiment, and the transformation conditions were optimized:A transformation system for foxtail millet was established using Agrobacterium tumefaciens strain LBA4404 (-pBI2300,-pSgt,-pRac,-pRar). Embryogenic callus obtained after about 1 to 2 times of subculture were used as recipient material for genetic transformation, the concentration of Agrobacterium tumefaciens suspension was 0.3-0.6 (OD600) added with AS of 100mg/L, and then infected within 30 min. Callus culture medium were added on filter paper in the Agrobacterium co-culture with the material. After 6-7 weeks, the resistant calli were gained by screening on the selection medium containing 25-60mg/L of kanamycin and 200-500mg/L of carbenicillin. The new resistant calli differentiated into plantlets. Finally, 281 resistant plants were regenerated.(3) Two hundred transformed plants confirmed by PCR assay were obtained, with a transformation frequence of 9.50% and 11.50% respectively by primers designed with 35S promoter and with NPTâ…¡gene sequences. Experiment of preliminary screening of transgenic plants at seedling stage to determine the critical concentration of antibiotic showed that 200mg/L is proper with Kanamycin and50mg/L with G418. So far,90% of T0 generation of transgenic plants can be harvested, and a variety of anomalies in morphological traits were observed and recorded. |
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