Gene Editing In MADS-box B Class Genes Of Hydrangea Macrophylla ’Dooley’

Hydrangea is a type of flower with extremely high ornamental value.Gene editing is an important direction for the development of hydrangea molecular breeding in the future.This research focuses on the research of hydrangea gene editing technology,editing the MADS-box B genes that control the development of flower organs,and hopes to achieve petals Transition to sepals to obtain more ornamental varieties.The main results of the test are as follows:（1）The indirect and direct regeneration system of Hydrangea‘Dooley’has been established.With Hydrangea‘Dooley’leaves as explants,the best callus induction medium is MS+2.25 mg/L6-BA+0.2mg/L NAA,the induction rate can reach 85.34%,and the best bud induction The medium is MS+2.25 mg/L 6-BA+0.1 mg/L IBA,and the adventitious bud differentiation rate can reach 80.32%.The best direct regeneration medium is MS+2.0 mg/L 6-BA+0.1 mg/L IBA,the regeneration frequency can reach 84.42%,and the average number of regeneration buds can reach 5.33.The best rooting medium is 1/2 MS+0.2 mg/L IBA.（2）Three MADS-box class B genes of Hydrangea‘Dooley’were cloned:BWPI,BWTM6,BWAP3.The full length of BWPI gene sequence is 639bp,the full length of BWAP3 gene sequence is 546bp,and the full length of BWTM6 gene sequence is 681bp.None of them contain intron regions.Phylogenetic analysis and identification of conserved domains revealed that all three genes have K-box conserved domains;they belong to the MADS-box class B gene of Hydrangea‘Dooley’.（3）Designing g RNA with the three MADS-box class B genes of Hydrangea‘Dooley’as target genes,of which BWPI and BWTM6 each selected 2 suitable target sites,and BWAP3 selected 3 suitable target sites.Seven single-target CRISPR/Cas9 gene editing vectors were constructed.Using Agrobacterium GV3010 to edit the B gene of Hydrangea‘Dooley’,and experiment with leaves,petioles and callus as explants.The following results were obtained:The leaves and petioles of Hydrangea‘Dooley’were used as the test recipients.The selection pressure of hygromycin for screening culture is 3 mg/L,and the best infection time is 15min.The selection pressure for the screening culture with callus as the test recipient is 2 mg/L,the best infection time is 10 min,and the best bacterial solution OD₆₀₀=0.4,the petiole is obtained,and the callus is directly regenerated from the resistance.15 buds,mixed detection found that resistant seedlings contain Cas9 sequence,which provides a good research foundation for subsequent gene editing of hydrangea..