| Cucumis melo L.is widely cultivated in all parts of China to meet people’s needs,but the current planting mode is mainly seed propagation,and powdery mildew and downy mildew seriously affect the quality and yield of muskmelon.At present,the conventional breeding methods on the market restrict the quality,yield and variety diversity of muskmelon,and can no longer meet people’s needs.The application of molecular marker assisted breeding,transgenic breeding,mutation breeding and other new breeding technologies cannot be separated from efficient regeneration system.In this study,melon seeds and mature leaves were used as materials to establish a loose embryogenic callus induction system for melon,laying a foundation for melon germplasm resources innovation,plant tissue detoxification,artificial seed development and genetic transformation.The results are as follows:(1)Using melon seeds and mature leaves as materials,adjusting the concentration of sodium hypochlorite solution and disinfection time,it was found that in the process of melon seeds disinfection,Daiyu,Hongyu and Jinyu were at 10%(V/V),while the thin-skinned melon was crisp and dense,and the disinfection effect was the best in 15%(V/V)sodium hypochlorite solution for 10 minutes;The best time for mature melon leaves to be disinfected in 10%(V/V)sodium hypochlorite solution was 12 min,and the survival rate was 72.89%;(2)The cotyledon,hypocotyl,the first true leaf and mature leaf of sweet melon were used as explants to induce callus under different hormone regulation.The results showed that the cotyledon induced callus was in the best condition with 0.25 mg/L 6-BA and 1.0 mg/L2,4-D and 0.5 mg/L NAA,which was ideal soft callus;(3)The loose callus induced by cotyledon was inoculated on MS+0.25 mg/LBA+0.5mg/L2,4-D+30 g/L sucrose medium,which could induce loose callus with loose structure,soft texture and yellow-green color;(4)The loose embryogenic calli of muskmelon were successfully induced by transferring the loose embryogenic calli above to MS+0.1mg/L6-BA+0.5mg/L2,4-D+30g/L sucrose;By adjusting the ratio of hormone and sucrose concentration,it was found that the best medium for maintaining the embryogenic callus of muskmelon was MS+0.1mg/LBA+0.25mg/L2,4-D+45g/L;(5)When the calli from the loose embryogenic callus medium after five subcultures were transferred to the MS medium,the embryoid formation was less and the embryoid development was poor.The addition of hydrolyzed casein,hydrolyzed milk protein and coconut juice can effectively improve the formation ability of embryoids,and hydrolyzed casein is more suitable for inducing embryoids than milk protein,and coconut juice is more helpful for improving embryogenesis ability;Adding drought,starvation,low temperature,high osmotic pressure and other adverse conditions had a significant impact on the induction of melon loose embryogenic callus.Low temperature(10 ℃)treatment for four days could effectively improve the embryogenic degree of callus and the embryoid formation ability.Drought and starvation treatment for 24 hours can improve the growth rate of callus,but the tissue is more serious,but can effectively promote the formation of embryoids.Increasing the concentration of sucrose to 90g/L can effectively improve the embryogenic ability of callus,and the percentage of nucleus can reach 75.98%;(6)The soluble protein content,soluble sugar content,total free amino acid content and polyphenol oxidase activity of different types of muskmelon calli were detected.It was found that the soluble protein content was high at the stage of embryoid formation,and low temperature and high osmotic pressure treatment could improve the soluble protein content in embryogenic calli,while high osmotic pressure could also improve the soluble sugar content of muskmelon calli;The content of amino acid in embryoid formation stage was higher than that in non-embryogenic callus.Low temperature and subculture times had significant effects on the content of amino acid in callus;During embryogenesis,the activity of polyphenol oxidase was significantly decreased.Stress treatment could reduce the enzyme activity to some extent,but the increase of hydrolyzed casein could increase the activity of polyphenol oxidase in callus.Through the combination of adversity,additives,hormone regulation and physiological index detection,the induction of embryogenic callus was explored in multiple directions,and an efficient embryogenic callus induction system of melon was established,which is easier to form an efficient plant regeneration system,laying a foundation for melon germplasm resources innovation,plant tissue detoxification,artificial seed development and genetic transformation. |
