Establishment And Optimization Of Protoplast Isolation And Culture System In Sugarcane

In order to obtain high-yield and high quality sugarcane protoplasts,and lay the foundation for the regeneration of sugarcane protoplasts.The influencing factors of protoplast isolation and protoplast culture program and the related technology were studied,isolation and purification conditions of sugarcane protoplasts were optimized by using the ROC22 and GT28 leaves.Results indicate that:1.Before the enzymolysis,the wall separation time of 0.5 h was compared with that of Oh:The yield and activity of GT28 increased by 65.62%and 2.08%,respectively,and the yield and activity of ROC22 increased by 105.94%and 1.84%.2.Under the condition of enzyme combinations 0.5%pectinase + 2%cellulase + 0.3%hemicellulase + 0.1%macerozyme,the results showed that the yield and activity of ROC22 protoplasts were 2.55×107·g-1 and 98.54%,respectively.The yield and vigor of GT28 protoplasts were 2.05×107·g-1,and 99.83%,respectively.3.Enzymolysis time had great effect on protoplast yield.When enzymatic hydrolysis 10 h,the yields of ROC22 and GT28 protoplasts were 35.4%and 89.81%,respectively higher than that of 5 hours.10 h is the most suitable for the separation of sugarcane protoplasts;4.When the centrifugal rate was 800 rpm,the yield of protoplasts of these two varieties were 2.1×107·g-1 and 2.02×107·g-1,they were 70.73%and 82.23%higher than that of 500 rpm.The activity of ROC22 and GT28 protoplasts decreased by 10.13%and 11.12%,respectively,when the centrifugal rate increased to 1200 rpm.As a result,the optimum centrifugal rate of sugarcane protoplasts was 800 rpm.5.The most suitable medium for the purification of sugarcane protoplasts is KM8P.In the KM8P medium,the division frequency of ROC22 protoplasts 6.14%and 13.17%higher than that of MS and N6 respectively,and the plating efficiency increased by 5.21%and 8.91%,respectively.Similarly,when the protoplasts of GT28 were cultured in KM8P medium the rate of cell division was increased by 9.84%and 18.1%,respectively,compared with MS and N6 medium,and the plating efficiency increased by 8.56%and 10.26%,respectively.6.The optimum concentration of 2,4-D was 3 mg · L-1 in the two protoplast cultures.At this concentration,the division frequency and plating efficiency of GT28 protoplast regeneration cells was the highest,which was 19.24%and 14.18%.Compared with no addition of 2,4-D,the division frequency and plating rate increased by 6.88%and 5.74%respectively.For ROC22,they reached up to 19.31%and 13.95%at this concentration,when compared with no addition,these data were increased by 8.07%and 7.31%.7.The microdroplet culture was the most suitable for ROC22 and GT28 protoplast culture.During the culture,GT28 cell division rate and the plating efficiency reached the highest,respectively,which were 8.89%and 5.36%higher than that of solid-liquid culture,respectively,which were 13.58%and 6.52%higher than that of the liquid culture.The frequency and efficiency of ROC22 were highest,which were 5.27%and 7.07%higher than that in the solid culture,respectively,when compared with the liquid culture,they were increased by 10.5%and 7.81%.8.The combination of 1 mg · L-1 6-BA + 0.5 mg · L-1 KT was the most suitable for the differentiation of sugarcane protoplasts.When the hormone combination was 1 mg · L-1 6-BA+ 0.5 mg · L-1 KT,the frequency of division of sugarcane protoplasts was the highest,it was 20.77%in GT28 and 18.88%in ROC22,respectively.After 21 days of culture in differential medium,The length of GT28 and ROC22 protoplast regenerative cells,compared with the hormone combination B and C,respectively,increased by 9.57 μm,10.57 μm and 11.98 μm,17.38 μm.the width changed to 12.59 μm and 14.38 μm,then the cells become long strip,the area also increased to 473.59 μm2 and 564.33 μm2.In summary,it is necessary for 0.5 h of wall separation,and then,with the 10 h enzymolysis of the best enzyme combination of pectinase + 2%cellulase + 0.3%hemicellulase + 0.1%segregation enzyme,In additon,in order to achieve the highest yield and viability of the sugarcane protoplasts,you need control the centrifugal rate at 800 rpm and the microdroplet culture is carried out in the high efficient medium of KM8P medium,and during the culture,you had better add to 2,4-D of 3 mg · L-1,the above results provide a technical support for the quality of protoplast cells in the process of protoplast fusion breeding,and lay the foundation for sugarcane somatic cell fusion breeding technology,which also provides a scientific basis for the single cell separation and culture of sugarcane somatic cell fusion breeding.