Induction Of Loose Embryogenic Callus And Somatic Embryogenesis In Apium Graveolens

Apium graveolens is the main vegetable crop in China,which has a long history of planting and a wide area of cultivation.Because Apium graveolens is rich in minerals,vitamins,flavonoids and other nutrients,it has gradually become one of the most widely consumed vegetables in China.However,its relatively lagged breeding technology of improved varieties still restricts the development of Apium graveolens.In this study,different tissues and organs of Apium graveolens were used as materials to systematically study the tissue culture techniques such as embryogenic callus induction,somatic embryogenesis and plant regeneration,in order to establish an efficient and stable plant regeneration system for Apium graveolens in vitro culture and lay a foundation for further Apium graveolens somatic mutation breeding.The main contents and results of the study are as follows:（1）Establishment of the induction and proliferation system of Apium graveolens loose embryogenic callus:The hypocotyls,cotyledons and true leaves of celery No.2and Parsley were used as explants,and different hormone types and concentration ratios and different culture methods were added to the MS medium to study the key technologies of Apium graveolens loose embryogenic callus induction and embryogenic preservation.The results showed that hypocotyl was an ideal explant for inducing Apium graveolens callus,it was induced on MS+0.5 mg/L KT+0.5 mg/L 2,4-D+30 g/L sucrose medium,once every 15 subcultures,after three subcultures,the loose callus with loose texture and hard structure of Apium graveolens was induced;It was induced on MS+0.1 mg/L KT+0.5 mg/L 2,4-D+30 g/L sucrose,subcultured once every15 days,and through three subcultures,the ideal loosely embryogenic callus of Apium graveolens was induced;It was cultured on MS+0.1 mg/L KT+0.5 mg/L 2,4-D+30 g/L sucrose medium under the conditions of 2000 lx light,24 h light cycle,23℃culture temperature,subculture once every 15 days,and the loose embryogenic callus of Apium graveolens stably proliferated and grew with good embryogenicity.（2）To establish a method to identify the complete embryogenesis of Apium graveolens loose calli:under the microscopic observation,the cell division frequency and nuclear proportion were observed and calculated under the condition of magnification of 200-400 times,on the premise of being able to see the cell nucleus and the basic outline of the cell.The results showed that when the percentage of the cells in the mitotic phase in the counted cells reached more than 1-2%of the total cells observed,and when the percentage of the nucleus reached 40-60%of the total cells,it could be determined that the Apium graveolens callus had reached complete embryogenesis.Moreover,the calli of Apium graveolens with complete embryogenization were induced on MS medium without added hormone,and somatic embryos were produced.The embryogenesis rate and embryoid normal rate were significantly higher than those of calli without complete embryogenization,which could also indirectly indicate that the method for identifying the complete embryogenization of Apium graveolens with loose callus was accurate.（3）The system of somatic embryogenesis and plant regeneration of Apium graveolens was established:the loose embryogenic callus was transferred to MS+0.25mg/L KT+0.25 mg/L 2,4-D+30 g/L sucrose medium,and each subculture time was 15days.After three subcultures,Apium graveolens somatic embryos were induced;The somatic embryos were transferred to MS+2.5 mg/L Ag NO₃+30 g/L sucrose medium,and the culture conditions were adjusted to 2500 lx light,24h light cycle,and culture temperature at 25℃.The time of each subculture is 15 days.After three subcultures,normal celery regenerated plants can be grown.