Protoplast Culture Of Hong Yang Kiwifruit And Analysis On Physiological And Biochemical Factors Of Effecting Protoplast Growth And Division

Kiwifruit is one of the most successful wild fruit trees cultivated in the last century.The kiwifruit industry has made great contributions to the development of the world fruit industry.However,with the expansion of kiwifruit cultivation area,the occurrence and harm of kiwifruit diseases becomes more and more serious,which poses a threat to the efficient and safe production of kiwifruit.Bacterial canker disease is a devastating disease for kiwifruit cultivation diseases.So far,there is no effective treatment to cure it,and it has become a bottleneck problem affecting the development of kiwifruit industry.Therefore,breeding new varieties of resistant canker disease has been the focus of breeder research.Studies have shown that A.eriantha and A.arguta are more resistant to canker disease in the wild kiwifruit resources,which provide a good parental material for the cultivation resistant to canker disease and even new varieties.Protoplast fusion is an effective way to introduce the excellent traits of wild species into cultivars.Therefore,the establishment of efficient kiwifruit protoplast culture and fusion technology system is of great significance for the subsequent cultivation of kiwifruit anti-canker disease resources innovation and breeding new varieties.Hongyang kiwifruit is favored by consumers because of its unique and excellent quality.However,this variety has high canker disease and has caused great losses to many producing areas.This study used Hongyang kiwifruit and A.eriantha as materials to screen the protoplasts and culture conditions of callus induced by different explants.The technical system lays the foundation for the subsequent division of hybrid cells and the acquisition of regenerated plants.However,it was found that the protoplast percentage of cell division of two kiwifruits was extremely low,and only the third cell division was observed after continuous subculture under various cultutre conditions.This study used tobacco protoplasts with high cell division rate as a control to observe and analyze the callus structure of protoplasts and the physiological and biochemical indexes of protoplasts in Hongyang kiwifruit and tobacco.To find out the physicochemical factors affecting the protoplast division of Hongyang kiwifruit,and adjust the culture scheme to observe the cleavage effect,and provide a theoretical reference for the subsequent protoplast fusion of kiwifruit and the acquisition of anti-ulcer body cell hybrids.The main results of this study are as follows:1.Callus induction of Hongyang kiwifruit and A.eriantha.The cotyledons,hypocotyls,shoot tips,stem segments,petioles and leaf-induced callus of the sterile seedlings of Hongyang kiwifruit and A.eriantha germination were harvested,and suitable callus induction and proliferation medium were screened.The results showed that the best way to disinfect the seeds of Hongyang kiwifruit and A.eriantha was:20%sodium hypochlorite sterilization for 30 minutes,the pollution rate was zero,and the germination rate of Hongyang kiwifruit seeds was 70.8%.The disinfection method was applied to the A.eriantha,and the disinfection effect was also good.The pollution rate was 0,and the germination rate of the A.eriantha seeds was 75%.The explants of Hongyang kiwifruit and A.eriantha had the best induction effect on MS+1.0mg/L ZT+1.0mg/L 2,4-D callus,resulting in faster callus and 100%recovery rate;The color of the callus is yellowish white and the texture is loose,making it easy to prepare for protoplasts.The callus of different parts of A.eriantha was transferred to the No.6 medium,and it was found that the callus induction rate of different parts of the kiwifruit was 100%on the No.6 medium.The induction speed is fast,and the yellowish white callus is produced and the amount is large.The optimal proliferation medium for callus of stem segments,petioles,cotyledons and hypocotyls of Hongyang kiwifruit was:N6+1.0 mg/L 2,4-D+1.0mg/L 6-BA+1.0 mg/L NAA,The multiplication ratios were 7.83,6.27,5.75,and 8.17;the optimal proliferation medium for leaf callus was:N6+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+0.5 mg/L NAA,The multiplication ratio was 6.75;the shoot tip was N6+1.0 mg/L 2,4-D+1.0 mg/L NAA,and the multiplication ratio was 8.00.The suitable proliferation medium for callus of stem,stem,cotyledon,hypocotyl and petiole of A.eriantha is N6+1.0 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L NAA,a suitable proliferation medium for leaf callus is N6+1.0 mg/L2,4-D+0.5 mg/L 6-BA+0.5 mg/L NAA.2.Isolation and purification of protoplasts from Hongyang kiwi and A.eriantha.Protoplast isolation and purification of callus obtained from different explants showed that the callus from different explants was combined with 2%cellulase R-10+0.5%Macerozyme R-10+1 mmol/L CaCl₂ in enzyme solution.The enzymatic hydrolysis of 2H₂O+0.7mmol/L mannitol and the purification of CPW13 and CPW25 were the best.The protoplast yield of each callus was1.70-11.2×10⁵/g FW,and the viability of protoplasts was also high.81.7%-92.3%.For Hongyang kiwifruit,the highest protoplast yield was stem callus,the yield was 11.2×10⁵/g FW,and the protoplast vigor was 85.0%,and the highest vigor was leaf callus?92.3%?.For A.eriantha,the highest protoplast yield was hypocotyl callus,the yield was 9.5×10⁵/g FW,and the protoplast vigor was 81.7%.The highest vigor was the callus of shoot tip?89.7%?.3.Protoplast culture methods and division.Different culture methods and culture conditions of protoplasts derived from different explants were analyzed.It was found that the first division of protoplasts from the beginning of the 7th-12th day,and the protoplasts derived from petioles and cotyledons were derived from Hongyang kiwifruit and A.eriantha.However,the percentage of cell division is lower under different conditions,and percentage of cell division of different protoplasts from different explants for 20 days is 4.9-15.7%.Among them,the callus of Hongyang kiwifruit cotyledon protoplasts was cultured in a shallow liquid culture method at N6+1.0 mg/L 2,4-D+0.030mg/L ZT+1.0%sucrose+0.2 mol/L glucose+200 mg/L CH+0.45mol/L mannitol,the culture density was 5×10⁴/ml,the culture condition was light intensity 1500lx,the illumination duration was16h,the culture temperature was 24°C,and the culture was started 7-8d.The percentage of cell division was 15.7%after 20 days,and only the second division of one protoplast was observed under this condition.Under the conditions of each culture condition,after continuous subculture for 6months,no continuous division was found.The callus protoplasts of kiwifruit leaves were cultured in a shallow liquid layer using N6+1.0 mg/L 2,4-D+0.025 mg/L ZT+1.0%sucrose+0.2 mol/L glucose+200 mg/L CH+0.45mol/L mannitol medium,the culture density was 5×10⁴/ml,dark,and the culture temperature was 26°C,and the division began at 7d.The statistical division frequency was 13.4%at 20d,but only two divisions were observed in the continuous culture.4.Difference analysis of physicochemical factors between protoplasts of Hongyang kiwifruit and tobacco.The protoplasts derived from tobacco leaf callus had higher cleavage ability than Hongyang kiwifruit.The frequency of division was 67.9%at 14 days,the plate-planting rate was26.3%,and the small cell cluster callus was formed by the naked eye for 30d.The callus structure derived from tobacco leaf callus and different explants of Hongyang kiwifruit was found to be paraffin-embedded.The cells of tobacco callus,red kiwi vine petiole and cotyledon callus were loosely arranged,stem tip and hypocotyl.The cells of the callus of the leaves are arranged closely.Combined with the results of protoplast culture,it was found that the growth division of protoplasts is closely related to the state of callus,and the loosely arranged callus is beneficial to the culture of protoplasts.The protoplasts isolated from Hongyang kiwifruit and tobacco callus were cultured for 14 days,and hormone content?IAA,ABA,ZR?,enzyme activity?POX,SOD?,soluble protein content,amino acid content,phenolic acid,diamine(Physical and chemical indicators such as putrescine and cadaverine and polyamines?spermine and spermidine?showed that the contents of endogenous hormones ZR,IAA and ABA were found in the content of endogenous hormones ZR,IAA and ABA.Stem tip>petiole>cotyledon>stem segment>leaf>tobacco>hypocotyl tendency;and the ABA content of all materials was the highest,while the mass ratio of ZR to IAA was maintained between2.64 and 2.85,there was no significant difference.The SOD,POX activity,soluble protein content and total amino acid content of tobacco were significantly higher than those of kiwifruit,which were more than 1.3-16,2.0-4.5,1.7-3.0,1.6-2.4 times of kiwifruit content.In addition to the high phenolic acid content of the hypocotyls of Hongyang kiwifruit,the other materials are 1.1-2.9 times lower than tobacco.The total amines of Hongyang kiwifruit were significantly higher than that of tobacco,indicating that the amines used to increase the activity of POX enzymes were more expensive.To this end,the authors speculate that the SOD activity,soluble protein content and amino acid content of tobacco protoplasts are high,indicating that its ability to scavenge free radicals is strong,and the protein synthesis ability required for osmotic adjustment and growth division is better than that of Hongyang kiwifruit.Tobacco has high POX activity and high phenolic acid content,indicating that tobacco splits are vigorous and produce more phenolic acids,while POX has strong activity and can quickly scavenge free radicals and phenolic acids,thereby harming protoplasts.These may be important reasons for the differences in protoplast division between tobacco and Hongyang kiwifruit.5.In order to improve the free radical scavenging and anti-oxidation ability of protoplast culture of Hongyang kiwifruit,under the optimized culture scheme obtained above,an additional20mg/L coconut milk was added,as well as different concentrations of PVP,glycine,Vc,GSH and other free radical scavenging and antioxidant media.The results showed that the treatment with glycine 0.05mol/L was the best,the protoplast splitting frequency was 20.1%,which was 1.3 times higher than the value without antioxidant?15.7?,and the planting rate was increased from 2.3 to 2.9.The division began at 7 days,and small cell clusters were observed after 60 days of culture.However,the regenerated plants have not been obtained,and the regeneration conditions need to be further optimized.