Detection And Further Screening Of Cold-resistant Cell Germplasm Of Pineapple

Pineapple(Ananas comosus)is an important tropical fruit,it is vulnerable to cold damage.Chilling injury will appear when the environment temperature is around 7℃.Cold stress was one of the important factors limiting the development of pineapple industry in China.Researches about the pineapple cold-resistance mechanism and the breeding of improved cold-resistant pineapple varieties are problems to be solved.The cold-resistance cell germplasm used in this study were selected as the primary cold-resistant cell germplasm of pineapp le(J-5-S、K-6-S、K-7-S)after three consecutive low-temperature screening(0℃ + 72 h,0℃ + 72 h,0℃ + 96 h).We make identification from the physiological,morphological,cell and molecular in order to have further low temperature screening.The main results are as follows:Stomatal density observation showed that the stomatal density of primary cold-resistant cell germplasm was lower than that of ordinary ’Shenwan’ germplasm.Analysised of the primary cold-resistant cell germplasm and ordinary ’Shenwan’ germp lasm by flow cytometry.The results showed that J-5-S doubled the number of cells(tetraploid)while J-5-S,K-6-S and control germplasm were still diploid.ISSR molecular markers were used to analyze the genetic diversity of the three primary cold-resistant cell germplasm which were selected after screening.The results showed that polymorphic bands were found compared to the ordinary ’Shenwan’ germplasm.26 ISSR primers were used to screen out 5 primers that could amplify clear bands and had polymorphism.Among the three primers,the polymorphic bands appeared in the three primary cold-resistant cell germplasm.Two primers showed polymorphic bands only in primary cold-resistant cell germplasm(K-6-S).Genetic variation analysis showed that the primary cold-resistant cell germplasm had different degrees of somaclonal variation,and the genetic variation of J-5-S and K-6-S was more abundant.The results of 24 differential gene expression were analysised by task group according to the pre-transcriptome screening.They were analyzed by fluorescence quantitative PCR.After 0h,4h,12 h,24h,72 h cold treatment,7 genes were up-regulated and the expression level of primary cold-resistant cell germplasm.5 genes which the regulation of the trend and the amount of expression in the two materials is basically the same.1 gene was down-regulated and expressed the same in two materials.ZAT10 and MYB73 played an important role in the cold response network,multiple verification results showed that the two genes expressed obviously compared to the ordinary ’Shenwan’ germplasm.Selecting the primary cold-resistant cell germplasm(J-5-S,K-6-S)which has the same size with the ordinary ’Shenwan’ germplasm as the material.The semi-lethal temperatures of the two primary cold-resistant cell germplasm were 0.42℃ and 2.51℃ lower than the control.While the plants at the low temperature(-4℃ + 12 h)all died.The survival rates of J-5-S and K-6-S were 11.1%,16.7% at low temperature(-2℃ + 12 h),it showed that cold resistance of primary cold-resistant cell germplasm were better than the control.The difference between the results of the two treatment methods is known that directly using of low temperature treatment than the semi-lethal temperature measurement of the cold temperature is higher.While the low temperature treatment method is closer to the actual cold temperature of pineapple under natural conditions.In the base of the established the primary cold-resistant cell germplasm in vitro culture system to have further low temperature screening(-2℃ + 72 h)used in direct cooling refrigerator.The result show that the survival rate of somatic embryogenesis(K-6-S,J-5-S and K-7-S)germplasm were 1.38%,3.85% and 5.27% respectively.After having further low temperature screening(-2℃ + 48 h)used in air-cooled refrigerator,the average survival rate of regenerated plants rates were: 4.23 %,10.29 %,34 % and the survival rate of the control germplasm was 0%.Both indicators indicated that K-7-S had stronger cold resistance than the o ther two cold-resistant cell germplasm.