Protoplast Fusion And Culture Technology Of ’Huashuo’ And ’Wangmo 25’

Camellia oleifera is an important woody edible oil tree species in China,and the seed industry is the strategic commanding height of the development of C.oleifera industry.However,due to the long childhood and highly heterozygous genome,the breeding of C.oleiera has always had problems such as long cycle and low efficiency.Protoplast fusion can break through the barriers of distant hybridization,shorten the breeding period,and improve the efficiency of germplasm creation.In the early stage,the research group established an efficient separation technology for C.oleifera protoplasts,but there was still a lack of systematic research on the fusion and cultivation of C.oleifera protoplasts.Therefore,this study used suspension cell lines and tissue culture seedlings such as C.oleifera ’Huashuo’ and excellent germplasm ’Wangmo 25’as materials to explore the optimal conditions for the fusion and culture of C.oleifera protoplasts,and lay a technical foundation for the hybrid breeding of C.oleifera cells.The main findings are as follows:1.The methods of electrotoplast fusion and PEG fusion of C.oleifera were established.(1)Using ’Huashuo’ and ’Wangmo 25’ mesophyll protoplasts as materials,the effects of PEG species,concentration,treatment time,protoplast density and other factors on the PEG fusion of C.oleifera protoplasts were explored,and the results showed that the fusion rate reached the highest rate of 8.92%when the density of mesophyll protoplasts was 4×105 per ml,PEG species was PEG6000,PEG concentration was 35%,and the PEG treatment time was 20min.The fusion rate of’Huashuo’ and ’Wangmo 25’ suspension protoplasts was carried out by this method,and the fusion rate could reach 8.74%.(2)Using ’Huashuo’ and Wangmo 25’ suspended protoplasts as materials,the effects of alternating electric field strength,alternating electric field action time,DC pulse intensity,DC pulse duration,DC pulse number,DC pulse interval and other factors on the electrofusion of protoplasts were explored,and the results showed that the protoplast density was 1.0×106 pcs/ml,the alternating electric field strength was 150V/cm,the alternating electric field action time was 75s,and the DC pulse intensity was 500V/cm.When the DC pulse width is 50μs,the pulse interval is 0.5s,and the number of pulses is 5,the fusion rate is the highest,reaching 19.28%;The fusion rate of ’Huashuo’ and ’Wangmo 25’ suspension protoplasts was carried out by this method,and the fusion rate could reach 9.37%.In general,the electrofusion method has a higher fusion rate and simple operation,which is more suitable for the fusion of C.oleifera protoplasts.2.The culture conditions of C.oleifera suspension protoplasts and mesophyll protoplasts were explored,and a culture method for C.oleifera suspension protoplasts was established.(1)Using ’Huashuo’ suspension protoplasts as experimental materials,the effects of osmotic pressure regulating substances,basic media types,hormone ratios,culture density and other factors on division were explored.The optimal medium for’Huashuo’ suspension protoplasts was KM8p medium with 0.5 mg/L BAP and 2.0 mg/L 2,4-D with an osmolality of 0.4M and sucrose for osmolality,and the initial density of culture was 2.5×105 cells/ml.The first cell division can be initiated 1～3 days after the culture of the ’Huashuo’ suspension protoplasts,the second division occurs in about 10 days of culture,and small cell clumps can be formed after 30～40 days of culture,and the plate planting rate is as high as 55.58%;Calluses with protoplast.regeneration can be obtained after 60 days of culture.(2)Using C.oleifera ’Huashuo’ and excellent germplasm ’Wangmo 25’ as experimental materials,the culture conditions of C.oleifera leaf meat protoplasts were explored,and the results showed that C.oleifera leaf meat protoplasts survived the longest time in KM8p medium,up to 11 days,while the mesophyll protoplasts in other media were all broken at 5-7 days,but the initiation of cell division was not observed;However,all mesophyll protoplasts gradually broke down and died during the culture process,and the initiation of cell division was not observed,indicating that C.oleifera protoplasts could not be regenerated under the current culture system alone,and further research was needed.