Efficient Preparation And Fusion Of Apple Rootstock Protoplasts

In this experimentbegonia tissue culture was carried out with begonia mature embryo as the material,and the effects of different hormones and ratios on the expansion,rooting and induction and proliferation of isolated leaf healing tissues of begonia group culture seedlings were mainly investigated,and a sterile system for efficient regeneration of begonia embryos was established to provide technical support for industrial production and to provide materials for the preparation of protoplasts.Protoplasts have proven to be a powerful medium for modern plant biology.However,the successful preparation of large amounts of viable protoplasts remains a challenge for the genus apple.In this study,we established the protoplast isolation and purification system by optimizing the enzymatic conditions and performed the study of protoplast fusion by using the haploid begonia histoculture seedlings and M9-T337 field seedlings as the materials,and screened the most suitable conditions and methods for protoplast isolation and purification and fusion of the two rootstocks,respectively.It provided a theoretical basis for the research of somatic cell hybridization and germplasm resource innovation of apple plants.The main research results are as follows.1.Establishment of tissue culture system of Begonia bajra（1）The optimal sterilization treatment for Begonia balsamina explants was:before stripping the seed coat:75%ethanol/30 s+10%Na Cl O/10 min,and after stripping the seed coat:10%Na Cl O/1 min,which could reduce the contamination rate of seeds to 0%and the highest germination rate was 93%;the most suitable seedling of Begonia balsamina group culture The most suitable rooting medium for begonia seedlings was:MS+2.0 mg/L6-BA+0.1 mg/LNAA,with a proliferation coefficient of 4.11 and seedling height of 2.57 cm;the most suitable rooting medium for histoponic seedlings was:1/2MS+0.7mg/LIBA+0.5 mg/LNAA,with 100%rooting rate,average root length of 7.97 cm,number of primary roots of 8.33,root surface area of The optimum induction medium for leaf healing tissue was MS+0.1 mg/L TDZ+1.0 mg/L NAA and MS+2.5 mg/L 2,4-D,and the induction rates were 94.70%and 91.33%,respectively,which could induce different states of healing tissue;the optimum medium for healing tissue proliferation was The optimal medium for the proliferation of healing tissues was B5+0.5 mg/L 6-BA+0.2mg/L IBA,and the optimal succession time was 28±2d.2.Establishment of protoplast isolation system（1）The material was submerged in the enzyme solution combination of 1.0%cellulose R-10（Cellulase R-10）+0.2%pectolyase Y-23（Pectolyase Y-23）+0.65 mol/L mannitol,and the p H was adjusted to 5.6,under dark conditions at 35 rpm,25°C After enzymatic digestion for 10h,the protoplasts were then purified by density gradient centrifugation using 25%sucrose-13%mannitol,and protoplasts with low impurities,high yield and high activity could be obtained.（2）The enzymatic material was treated with 13%mannitol CPW salt solution for 1 h before enzymatic digestion,which could significantly improve the yield and viability of protoplasts.The optimal enzyme solution combination was 1.0%cellulose R-10（Cellulase R-10）+0.3%pectinase Y-23（Pectolyase Y-23）+0.7 mol/L mannitol,p H adjusted to 5.6,and enzymatic digestion at 35 rpm and 25°C for 10 h under dark conditions to obtain high-quality protoplasts.3、Establishment of protoplast fusion systemThe purified protoplasts of Begonia octocarpa and M9-T337 were used as materials,and the PEG-high Ca²⁺-high p H binding fusion method was selected for the fusion test.The optimal PEG-6000 concentration was 35%and the optimal fusion time was 25 min to obtain fusion bodies with the highest fusion rate of4.57%.