Establishment Of Regeneration System Of Mature Embryo And Genetic Transformation Of EPSPS Gene In Foxtail Millet

Foxtail Millet,which is characterized by its drought tolerance,strong stress resistance,and high nutritional value,has made it one of the important food crops in northern China.With the increase of the world's population,the demand for food is also increasing rapidly.On the other hand,the completion of whole-genome sequencing of millet has played an important role in the genetic engineering of millet.The establishment of a stable and efficient millet regeneration and transformation system is the basis for transgenic technology to cultivate new millet varieties and increase millet yield.In this study,the mature embryos and shoot tips of Jingu 21 Jingu 40,Jingu 45,and Jingu 57 were used as explants to study callus induction,adventitious bud differentiation,clustered shoot induction of mature stems,and rooting culture.The millet varieties with strong regenerative ability were mixed with the hormones in the medium to establish a highly efficient ex vivo regeneration system for mature embryos and stem tips.Preliminary studies were conducted on the germination of millet shoot tips mediated by Agrobacterium-mediated transformation of EPSPS genes.The results show:1.The induction and differentiation of callus induced by mature foxtail embryos as explants showed that the optimum medium for callus induction of mature embryos of Jinu 21 was MS+2.0mg/L2,4-D+0.5mg/LKT+5%sucrose,the callus produced was subcultured once on the medium,the callus became dense,the color was yellowish,and the condition was better,which was conducive to further differentiation;the optimal differentiation medium was MS+1.0mg/L6-BA+0.5mg/LNAA+1.5%sucrose,and the differentiation rate was 75.71%.Sucrose had different effects on callus induction and differentiation of mature embryos.Higher concentrations of sucrose(5%)were beneficial to the induction of high-quality callus,and lower concentrations of sucrose(1.5%)favored the differentiation of buds.There were differences in the in vitro regeneration system of millets,Jingu 21,Jingu 40,Jingu 45,and Jingu 57 all had good yields on the induction medium,and the differences were not significant.In the differentiation rate,the varieties The difference between the two was significant.Jingu 21 had the best differentiation rate.2.Induced cluster buds from germinated millet shoot tip as explants.The results showed that the best germination medium for Jingu 21 was MS+3mg/LTDZ;the best growth medium for bud clusters was MS+3mg/LTDZ+0.5mg/LIBA;the best rooting medium is 1/2MS+1.0 mg/LNAA.There were significant differences in cluster shoot induction between the four varieties of Jingu 21,Jingu 40,Jingu 45 and Jingu 57.Jingu 21 had the highest induction rate of multiple shoots(79.17%)and 4.77 shoots per bundle.3.Agrobacterium tumefaciens-mediated transformation of the EPSPS gene into shoot tip showed that the OD600 value of A.tumefaciens solution was 0.7 and the vacuum infiltration time was 10 min.It was the best condition for Agrobacterium infection.Six genotypes survived through glyphosate screening.The survival rate was 3.33%.The glyphosate resistance of the surviving plants was identified by smearing at the four leaf stage,and the resistance of the isolated leaves to glyphosate was higher than that of untreated millet leaves.