Preliminary Study On The Cryopreservation Of Tissue Culture Seedlings Of Vaccinium Uliginosum L.

Because Vaccinium Uliginosum L.fruit has higher nutritional value and better flavor,it is also an important blueberry wild resource.Therefore,in recent years,the research on bilberry is getting more and more attention.However,the current research on the bilberry is mainly focused on domestication and cultivation,nutritional value analysis,and molecular mechanism of cold resistance,but there are few related studies on the protection and preservation of germplasm resources.Tissue culture is an important method for the preservation of germplasm resources.In this experiment,the SL-1 single shoot stem segment of V.uliginousum L.was used as the explant material,and the amplification system of SL-1 was optimized.The seedlings of V.uliginousum L.SL-1 were used as materials.The effects of storage temperature,preservation medium,storage time and storage conditions on the growth,survival rate and rejuvenation rate of tissue culture seedlings was discussed,and MSAP analysis of genomic DNA methylation after reconstitution of cryopreserved tissue culture seedlings.The purpose is to establish a complete cryopreservation system for the seedlings of V.uliginousum L.,it will lay a solid foundation for future genetic engineering and molecular breeding.The main results are as follows:(1)The best induced callus culture medium for the single-bud stem segment of V.uliginousum L.is: WPM+TDZ0.5 mg/L+ZT2.0 mg/L;the best induced differentiation seedling medium for singlebud stem segment is: WPM+ZT3.0 mg/L+IBA0.3 mg/L;the optimum pH value for single-bud stem segment expansion is 5.2;the best illumination time for single-bud stem segment expansion is 14 h/d.(2)The best cryopreservation medium for the tissue culture seedlings of V.uliginousum L.is: WPM+sorbitol20 g/L+ sucrose20 g/L+agar8 g/L;The best storage temperature is 5?;the single day light time is 10 h/d.(3)In the cryopreservation of the bilberry seedlings,malondialdehyde content,chlorophyll a+b content,proline content and soluble protein content were the main factors in the analysis of physiological and biochemical changes during storage,and the cumulative contribution rate was as high as 97.945%.(4)The genomic DNA methylation status of the tissue culture seedlings of V.uliginousum L.was saved for 4 months,one month after rejuvenation and two months after rejuvenation: invariant A-type bands of the genus DNA is the most.The accounting of the total variation strips are 79.61%,67.38% and 80%,respectively.It can be seen that the effect of low temperature preservation is better,and the longer the rejuvenation time,the more the A-type strips are unchanged,and the effect is better;The methylated B-type bands in genomic DNA were 15.15%,26.80%,and 11.92%,respectively.It can be seen that the methylation bands decreased during the rejuvenation process as the rejuvenation time prolonged;The demethylated C-type bands in genomic DNA were 5.24%,5.44%,and 6.54%,respectively.It can be seen that the demethylation band increases during the rejuvenation process as the rejuvenation time increases.In summary,methylate-invariant types of the tissue culture seedlings after cryopreservation and post-rejuvenation were the most,compared with before saving,and the proportion of methylation and demethylation was lower,and the preservation and rejuvenation effects were lower.Good preservation and rejuvenation.(5)The rejuvenation rate of V.uliginousum L.plantlets after storage for 120 days at 5? was as high as 98.42%,and the regeneration rate could reach 100%.