Optimize Of Sugarcane Protoplast Fusion And Optimizing The Miscellaneous Nuclear Cell Regeneration

Sucrose is the main source of sugar in our country.More than half of the sugar production from guangxi,guangxi sugarcane planting area of 1.0815 million hectares,accounting for 60%of the country Natural disasters have important effects on sugarcane production.Chilling injury is one of the disasters of sugar cane.Sugar cane after cold,a massive sugar cane leaf dry,growing point died a death,sugarcane stalk is boiled,lateral bud serious damage,sucrose conversion,purity of sugarcane juice drops,sugar rate drop,quality become worse,affect the output of sucrose,caused great economic losses.ROC22 as guangxi sugar cane take charge of varieties,high germination rate,high tillering rate,high yield and high sugar,drought and other good agronomic characters.But cold resistance is poor,the breed and GT28 has the characteristics such as high sugar,cold resistance,can be in breeding as a good gene pool to exploit and use ROC22 and GT28 for cell fusion breeding,restructuring their good genes and can be concluded that the excellent agronomic traits and cold hardiness more hybrids.Cell fusion has its incomparable advantage than other breeding way.Is not subject to the limitations of species,overcome sexual cell fusion,can transfer a large number of genes,still can obtain the expression of multiple population genetic traits,but at the same time the cytoplasm and nucleus gene.etc.These results for cold resistance varieties of high yield produced to provide the basis.To do this,try the following research:(1)the sugarcane protoplast of cryopreserved recovery research,in order to get the right frozen storage conditions,the protoplast can save lots,solve the problem of experimental materials affected by seasonal(2)to complement inhibitors IOA and the concentration of R-6G is optimized(3)the PEG fusion and fusion process is optimized(4)to the cultivation of the protoplast fusion followed and cultivate combination of design optimization.The results show that:1.The cryopreserved materials of liquid,frozen storage temperature and different parts,after protoplast cryopreserved vitality significant difference.Three combination of freeze-stored liquid,serum + 10%DMSO + 20%in medium,the cryopreserved strongest vitality after 30 d,up to 72%.Cryopreserved recovery,within 90 d-196 ℃ of liquid nitrogen and-80 ℃ refrigerator cryopreserved,sugar cane protoplast energy difference was not significant,vigor were above 75%.But recovery after frozen storage,after 90 d-196 ℃ after liquid nitrogen frozen storage-80 ℃ refrigerator cryopreserved than after plasma strong vitality.Comparing different based site,young leaves cryopreserved protoplast energy recovery after 30 d income is higher,79.2%,after 30 d stem tip cryopreserved income protoplast energy recovery is only 42.7%.2.Freeze-stored liquid temperature is different,and cryopreserved cells divide the first time you start and form of the time difference was not significant.About 5-6 d general training,basic form a complete cell walls,training after 6 d,cell division,training after 15 d form cell mass.Compared different material parts,protoplast regeneration stem tip enzyme solution was the strongest,the young leaf protoplast enzyme solution,the formation of cell wall time as early as 3 d,2 d division for the first time the earlier.3.IOA(0-10 mmol/L)and R-6G(0-70 g/mL)passivation of two sugarcane varieties,can make the protoplast vitality and value board rate dropped significantly.Training after 30 d,the highest concentrations,all protoplast loses its ability to form callus.IOA on sugar cane GT28 inhibitory effect is more apparent,R-6G of sugar cane ROC22 inhibitory effect is more apparent,and can make R-6G of sugarcane protoplast glow red fluorescence microscope,do use fluorescent dye.Considering two kinds of passivator on sugar cane protoplast vitality and value rate and the influence of passivation effect,it is concluded that appropriate for sugarcane protoplast the concentration of passivator is:sugarcane ROC22 with 50 mu g R-6G/mL;Sugarcane GT28 with 6 tendency for R-6G/L.4.PEG fusion research,to a concentration of 50%PEG-6000 sugar cane young leaf protoplast fusion fluid processing,the volume of PEG and cell suspension is for 4:5,Ca2 +concentration is 0.3 mol/L,25 and 30 ℃ in the fusion of about 30 min,the highest rate.5.Electrical integration study,after mixing volume 2 parents protoplast and sugar cane,adjustable density to 5×105/mL,and electrical integration.To the best conditions for the electric field,electric field intensity of 200 V/cm,dc pulse 2 kv/cm field intensity,pulse wide 40 μs,pulse number 2,convergence rate is highest,lowest breakage.6.The cultivation of chimeras,the optimal combination is to cultivate density of 5 X 105/mL,cultivating mode for solid liquid double cultivation,2,4-D concentration for 2 mg/L,6-BA concentration for 3 mg/L.In this combination,the train after the cell walls of protoplast formation around 5 d,6 d about start of cell division,form small cell mass around 15 d,30 d around to form small callus.