Study On The Method Of Cryopreservation The Potato Stem Tip

Potato germplasm resources preserved in National potato germplasm repository has reachedmore than2100copies at the present stage in China. The quantity of germplasm resources isconstantly increasing.This study on comparing potato in vitro stem tip vitrification method with embeddingvitrification cryopreservation method,conducting experimental analysis of the influence factors ofcryopreservation, and detecting the genetic stability of regenerated plants, building thecryopreservation technique of potato in vitro with high survival and regeneration rate, groping fora set of optimal, common cryopreservation processing technology, establishing a set ofcryopreservation technology system of gene pool, and applying to large-scale cryopreservation ofpotato germplasm resources. Main conclusions are as follows:1.The research on potato stem tip vitrification cryopreservation technology:2wild species、2local variety and2breeding varieties were provided，respectively, researches through the variousfactors showed that the survival and regeneration rate had significant difference when potato stemtip cultured in different time, survival rate and regenerative rate pre cultured successively1days in0.3mol·L-1and0.5mol·L-1sucrose MS liquid medium is highest, between86.7%-95.3%and70.4%-95.3%respectively. With the increase of pre bake time, survival rate aftercryopreservation and regeneration rate presents the trend of first increases and then decreases, andthe survival rate of stem tip without culture is0;the potato stem tip size have significant influenceon survival and regeneration rate. In the length of stem tip1-1.5mm in the survival andregeneration rate is highest, between86.7%-95.3%and70.4%-95.3%respectively, when the stemtip increases, the survival and regeneration rate fell sharply, this may be related to stem tip is toolarge, dehydration is not uniform, causing stem tip local cell moisture content is exorbitant,freezing injury during frozen storage, lower the rate of survival and regeneration; Differentvitrification solution on the survival and regeneration rate of research shows that the influence ofthe use of vitrification solution PVS1and PVS2treatment6copies of material survival andregeneration rate is significantly higher than that of vitrification solution PVS3and PVS4processing, it account for that DMSO entering into stem tip tissue can fast frozen stem tip, makethe cells into glass transition state quickly, and reduce the damage to the organization, improve thesurvival rate and regeneration rate. After vitrification solution PVS2treatment the survival rateand regenerative rate were higher than glass transition liquid PVS1, showed that vitrificationsolution PVS2reagent can achieve the best effect; researches on the influence of processing timeto survival and regeneration rate showed that potato stem tip without PVS2treatment after beingsaved all did not have survival rate and regeneration rate, the survival rate and regenerative rate ofstem tip after PVS2treatment were increased first and then decreased with the increase of culturetime. Processing time for30min, the survival rate and regenerative rate reached to the peak, respectively (86.7%~95.3%) and (70.4%~89.5%). With extended PVS2treatment time, survivalrate and regenerative rate began to fall, With extended PVS2treatment time, survival rate andregenerative rate began to fall, may be the DMSO in potato stem tip caused by excessive poisondamage; studies on influence of recovery media on the survival and regeneration rate showed thatthe survival and regeneration rate had significant difference after thawed potato stem tiptransferred into different recovery medium. Recovery medium B have the highest the survival andregeneration rate. The potato stem tip droplet vitrification cryopreservation system method wasbuilt basically by studying influence factors of preincubate, vitrification solution, PVS2dehydration time and recovery medium, etc. That is stem tip of1.0mm was pre-cultured in orderin0.3mol·L-1and0.5mol·L-1liquid MS medium1d respectively,then treated with PVS2under0℃for30min, then saved in the liquid nitrogen at least1d, thawed with MS culture medium of1.2mol·L-1sucrose at room temperature and washed30min, at last transferred into MS Zeatin+0.10mg·L-1+0.5mg·L-1NAA+1.0mg·L-1GA3restore medium to re-culture.2. The research on embedding vitrification cryopreservation technology: sucroseconcentration in the medium influence on the survival and regeneration rate showed that survivalrate and regeneration rate of stem tip presented the trend of first increases and then decreases withthe increase of sucrose concentration in the pre medium, survival rate and regeneration rate of stemtip reached the peak after being cultured in containing0.5mol·L-1sucrose pre culture medium, andthe rates were all0without sucrose pre culture medium. Based on the research of the sucroseconcentration in the culture medium, the potato stem tip embedding vitrification cryopreservationtechnology system was preliminary built, namely about1mm long stem tip in vitro was suspendedin containing3%(w/v) sodium alginate and0.4mol·L-1sucrose free calcium modified MS medium.With sterile rubber head of straw or1.0ml syringes extracting stem tip, transferred to0.1mol·L-1CaCl2solution containing0.4mol·L-1sucrose, let it sit for30min at room temperature (25℃), the formation of embedding pill for about5mm in diameter, one stem tip in each embeddingpill. Embedding pill waspre-cultured1d in improved MS culture medium containing0.5mol·L-1sucrose, processed by PVS2treatment30min, the embedding pills was transferrd in the tube into1.8ml Microtube, add1ml fresh PVS2, put into liquid nitrogen (LN). Material taken out fromliquid nitrogen, thawed in37℃water bath1-2min, washed30min using improved MS culturemedium contain1.2mol·L-1sucrose, absorbed the washing liquor remaining on the surface of theembedding pill with sterile filter paper, inoculated on restore medium to dark culture7days, andthen glimmer training7days finally transferred to the normal light to culture.3. Adaptive validation of different genetic material under different preservation mode: underpotato stem tip vitrification cryopreservation method, the survival rate of three kinds of geneticmaterial had significant difference, the regeneration rate among the three kinds of genetic materialhad no significant difference, the same genetic material had no significant difference among thevarieties. In different genetic materials, the order of the size of the mean survival rate andregenerative rate is the wild> local varieties> breeding varieties. Under potato stem tip embedding vitrification cryopreservation method, the survival rate and regenerative rate had no significantdifference among three kinds of genetic material, and no significant difference among the samegenetic material varieties, in different genetic material, the average size order of the survival rateand regenerative rate is wild> local varieties> breeding varieties. Two kinds of saving mode, therewere no significant difference between the same genetic material, but the average survival rate andregenerative rate of embedding vitrification method was slightly higher than the vitrificationmethod. It showed that after embedding for cryopreservation of the potato germplasm resourceswas more reliable and safe.