| Foxtail millet(Setaria italica L.Beauv)is a species of Setaria of Gramineae.It is not only a significant grain crop and grass,but also a model plant for the emerging C4 cereal research.However,the low genetic transformation rate of foxtail millets greatly limits its application in functional genomics research and genetic improvement of varieties.We screened high-efficiency in vitro regeneration of foxtail millet genotypes and optimized the culture medium,explored direct shoot apex regeneration system preliminarily and established efficient Agrobacterium-mediated genetic transformation system of foxtail millet,which laid a foundation for the functional genomics research and molecular improvement of foxtail millet.1.Establishment of in vitro regeneration system using mature embryos of foxtail milletThe mature embryos of 90 foxtail millet germplasm resources were cultured in vitro and the results showed that the callus formation rate,budding rate and regeneration rate of different genotypes of foxtail millet were significantly different.The callus formation rate ranged from 17.67 to 93.67%,budding rate ranged from 0 to 78.89% and regeneration rate ranged from 0 to 40.00%.Three high-efficiency in vitro germplasm resources Y41,Y176 and Y145 were screened from these resources.The optimum medium for all of the three genotypes was screened and the components were 4.43 g/L MS,30 g/L sucrose,4 g/L Phytagel,2 mg/L 2,4-D and 0.5 mg/L KT.2.Preliminary study on regeneration system of shoot apical meristems of foxtail milletThe resources of xiaomi,Jingu 21 and 110-8 were used as materials for directly regenerating shoot apical meristems to explore another new method of efficient regeneration.A large number of plants were obtained and their shoot apical meristems induction rates were 53.60%,61.60% and 63.20%,respectively.The bud induction rates were 64.91%,55.20% and 63.20%,respectively.The regeneration rates were 29.60%,36.00% and 40.80%,respectively.3.Establishment of Agrobacterium-mediated transformation system of xiaomiThe mature embryos of xiaomi,a model foxtail millet with the ultra-short growth period,were used as the callus-initiated explant,and established an efficient and stable Agrobacterium-mediated transformation system.It takes only 4-5 months for the system from callus induction to obtaining transgenic seeds.NPTII gene of neomycin phosphotransferase and HPT gene of hygromycin phosphotransferase were used as selective marker genes for genetic transformation.A total of 158 transgenic plants were obtained.Among them,115 strains were obtained when NPTII was used as a selection marker,its transformation efficiency ranged from 8.05 to 38.75% with an average of 22.65%.,At the same time,43 transgenic plants were obtained when HPT was used as a selection marker,and the transformation efficiency was significantly decreased,ranging from 3.08 to 16.67% with an average of 8.18%.It showed that the NPTII marker gene is more suitable for the genetic transformation of foxtail millet.4.Agrobacterium-mediated genetic transformation of Si9G328000 geneIn order to analyze the function of Si9G328000 gene and further verify the validity of the established genetic transformation system,we transformed it into the selected high-efficiency foxtail millet varieties Y176 and Y41.A total of 18 transgenic plants were obtained.Among them,8 strains were Y176,6 strains were infected with LBA4404,the transformation efficiency was 2.75%,and 2 strains were obtained by AGL1 infection,the transformation efficiency was 1.77%;The remaining 10 strains were Y41,which were all infected by AGL1,and the transformation efficiency was the highest,4.72%.Therefore,infection of Y41 millet with AGL1 was the best combination and transgenic plants could be used for follow-up studies. |
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