Establishment Of Millet Gene Editing System And Obtaining SiBADH2 Mutant Of Fragrance Gene

Foxtail millet?Seteria italica?is one of the oldest crops in the world,contains a lot of beneficial traits,such as drought resistance,salt tolerance,barren tolerance.Millet is popular among people due to its rich nutrients.With the improvement of people's living standards,the quality of millet has been pursued.However,the millet genetic transformation system is a worldwide problem.Up to now,there has not been an efficient genetic transformation system around the world.In this thesis,the millet Ci846 is used as the research material,and a set of efficient millet transformation system is established by the method mediated by Agrobacterium.It was the first to establish a knockout system using mature CRISPR/Cas9 technology in millet.Taking the homologous gene SiBADH2 of the rice fragrance gene OsBADH2 as an example,the SiBADH2 gene was successfully knocked out in millet.The specific research contents are as follows:1.In view of the problem of low genetic transformation efficiency of millet,this thesis optimized the millet transformation system.After several parallel comparison tests,the best treatment time was screened,sterilized with 70%ethanol for 1 min,and then 20%sodium hypochlorite solution was sterilized for 5 min.Under these conditions,the average bacterial infection rate was 0.52%,and the germination rate was 99.83%;MS medium was selected as the basic medium;CuSO₄ and ZnSO₄ need to be added to the medium,and 2 mg/L 2,4-D was added.Need to add KT,proline and AgNO₃,add 40 mg/L Hyg to the selection medium to increase the resistance callus rate;in the differentiation medium,the hormone could be 2,4-D?1 mg/L?It was used in combination with KT?1 mg/L?;the concentration of Agrobacterium-infected is OD₆₀₀=0.4?0.6,the infection was 40 minutes,and the total culture was 4 days.2.Three CRISPR/Cas9 knockout vectors was constructed for knocking out the SiBADH2 gene:pRLG103-BADH2-1,pRLG103-BADH2-2,and pRLG103-BADH2-3.There are 2 targets in each vector.3.After genetic transformation,only pRLG103-BADH2-3 vector obtained 22 lines,because of the somatic mutation,only 3 lines were obtained.A batch of progeny with vector backbone insertion and without vector backbone were identified by sequencing.Among them,two homozygous knockout lines without vector backbone were obtained.The homozygous mutant#20 was 99 bases deletion from the ATG 97 bp;the homozygous mutant number 18 had a base C inserted 196 bp from ATG.The next step is to evaluate the agronomic traits and metabolite content of these two lines.