Preliminary Establishment Of Efficient Tissue Culture And Genetic Transformation System Of Celery

Celery is an important vegetable crop belongs to Apiaceae family,originating from the Mediterranean region of Europe.Celery prefers cool temperatures,the high temperatures in summer throughout China greatly affect the production of celery and make it difficult to achieve open-air cultivation in summer.Meanwhile,the immaturity tissue culture and genetic transformation system of celery has limited the molecular improvement of celery breeding.Therefore,in this study,the following research was conducted on the heat-sensitive celery cultivar ‘Lvling Huangxinqin’:using ‘Lvling Huangxinqin’ seeds for germination on a culture medium and obtaining sterile seedlings,and exploring the establishment of an efficient and repeatable tissue culture system using cotyledon,hypocotyls,and roots as explants;using callus tissue obtained from tissue culture system to explore the Agrobacterium-mediated method for genetic transformation of ‘Lvling Huangxinqin’ with heat-resistant related transcription factor Ag HSFA1 a,and obtained positive transgenic healing tissues.The main contents of this study are as follows:1.Establishment of an efficient regeneration system for ‘Lvling Huangxinqin’.(1)Selection of culture medium for celery seed germination.Using ‘Lvling Huangxinqin’ seeds,the most suitable germination medium was determined by soaking and disinfecting the seeds and then sowing them on hormone-free 1/2 MS,1/2 B5,MS,and B5 media.The results showed that the most suitable medium for celery seed germination was hormone-free B5 basic medium.(2)Effects of different explants and hormones on celery regeneration.Using leaves,hypocotyls,and roots as explants,callus induction was carried out,and the effects of different types and concentrations of hormones on the induction of callus tissue from ‘Lvling Huangxinqin’ explants were studied.The optimal medium formula for inducing callus and differentiation of celery was then determined to establish an efficient regeneration system for callus induction and shoot regeneration.The results showed that the optimal callus induction medium formula for hypocotyls was B5 basic medium supplemented with 2.0 mg/L IBA and 1.5 mg/L 6-BA,and the optimal differentiation medium formula was supplemented with 1.5 mg/L IBA and 1.0mg/L 6-BA.2.Establishment of genetic transformation for ‘Lvling Huangxinqin’.(1)Establishment of hydomycin concentration and infection time in celery callus transformation system.The results showed that the optimal concentration of hygromycin was 40 mg/L,and the optimal infection time was 30 minutes with an OD600 of 0.5.The callus was then co-cultured for 3 days,resulting in the best experimental effect with a transformation rate of up to 46.67%.(2)Verification of genetic transformation efficiency.Agrobacterium-mediated transformation was used to introduce the transcription factor Ag HSFA1 a as an exogenous marker gene into celery callus tissue.After GUS staining and PCR detection,positive transgenic callus tissue of celery was obtained.