Study On Regeneration And Transformation Of Homozygous Genotypes Of Apple And Construction Of MYB10 Expression Vector

Homozygous genotype plantlets obtained from anther culture of apple are new important material for apple breeding that expanded apple germplasm resources.Homozygous plantlets combined with genetic engineering techniques can accelerate the breeding process,and the useage of homozygous genotypes as host plant in transformation systems for genetic studies has obvious advantages.In this study,the ploidy analysis was performed on 40 homozygous genotypes with different microspore-derived genotypes obtained from the cultivation of 'Gala' anther culture.Plantlets were regenerated,and conditions including hormone concentration,leaf age,dark culture,and seeding density were explored to establish an efficient regeneration system for leaf in vitro culture.Based on the regeneration system,high-frequency regenerative genotypes were used as host materials to conduct antibiotic susceptibility tests on leaves and to select appropriate concentrations of antibiotics for the materials.Exploring and optimizing the conditions of transformation's cultivation by concentration and time of inoculation of the broth,and the number of days of co-cultivation to establish an agrobacterium-mediated homozygous genotypic genetic transformation system.In addition,the MYB10 gene was cloned from the red leaves and pericarp of Malus sieversii ‘ Wumei '.The expression vector was constructed to prepare for the subsequent study of the apple MYB10 gene.The leaf regeneration test results showed that the genotype was the most critical factor for the regeneration of homozygous genotype plantlets,and the regeneration ability of different plantlets was different.Among them,the double haploid plantlets DH2-10,DH2-24,DH2-7,and tetraploid homozygote DH2-15 showed strong regeneration ability,especially DH2-10.Hormone has a great influence on the regeneration.40 days old leaves have better regeneration ability.Dark culture can promote the regeneration of homozygous genotypes.However,seeding density has no effect on regeneration.A high-frequency regeneration system for double haploid DH2-10 was established on the medium MS + 0.5mg/L IBA + 5 mg/L 6-BA to obtain a 100% regeneration rate.The tetraploid homozygous line had a regeneration rate of 93.33% on medium MS + 0.5mg/L IBA + 6mg/L 6-BA.The doublehaploid Plantlets DH2-7 and DH2-24 obtained regeneration rates of 88.88% and94.44%,respectively,in medium MS + 0.5mg/L IBA +2mg/L TDZ medium.Leaf antibiotic susceptibility tests showed that the high-frequency regeneration genotype strain DH2-10 was insensitive to kanamycin and 100mg/L Kan completely inhibited leaf regeneration.Cephalosporinhad little effect on leaf regeneration of DH2-10,but it affected the elongation and growth of shoots.Timentinwas not toxic to DH2-10 leaves..In addition,the coordinated use of cefotaxime and timentin has a better inhibitory effect on Agrobacterium LAB4404,and it is preferable to use the cefotaxime 600 mg/L+Ténitin 200 mg/L for the transformation and culture.Agrobacterium-mediated homozygous genotypic transformation experiments showed that the appropriate concentration of Agrobacterium tumefaciensOD600 was 0.3~0.7,and the appropriate immersion time was 5~10min.Two resistant buds were obtained from the induced bud differentiation on selected medium with Kan 100 mg/L.The RNA was extracted from the red leaves and pericarp of 'Wumei' and the RNA was reverse transcription.The specific primers for MYB10 gene were designed.The gene was amplified by PCR and recombined into the pMD-18 T cloning vector.Vector construction was carried out and the apple MYB10 gene expression vector pEZR(k)-LNY-MYB10 was obtained.