| Cryopreservation has been considering the most ideal method for conservation of plantgermplasm. However, cryo-associated injuries can cause sever damages to explants whichreduce considerably the recovery rates after LN exposure. Cryotherapy of shoot tips, based oncryopreservation and its associated injuries, has shown its great potential for plant pathogenseradication than traditional methods. To improve the efficiency of cryotherapy is a criticalissue for develop and improvement this biotechnology.Potato is model plant for researches in cryopreservation. Potato viruses consist one of themost important restrictive factors in potato production. Control of potato viruses by usage ofvirus-free seed potato is most effective worldwide. Thus taking potato as experimentalmaterial, the aim of the present study was to develop two cryopreservation procedures, toanylysis the cell injuries in histology, ultrastructure, ice crystal and oxditive during theprocesses of cryopreservation, to identify possible genegtic changes in plants recovered fromcryoprservation, to evaluate the PLRV and PVY elimination from potato by three vitrificationbased cryotherapy methods, to investagate the mechanism of cryotherapy. The main resultswere as follows.1. Droplet vitrification and vitrification cryopreservation of potato ‘Zihuabai’ shoot tipswere developed through optimized various critical factors. In droplet vitrification,2~3mmshoot tips were precultured on0.3M sucrose medium for2~3days followed by loading with2M glycerol and1M sucrose for30min, loaded shoot tips were dehydrated thereafter withPVS2on ice for40min and then transfered onto aluminum foil and plunged into liquidnitrogen. In vitrification,2~3mm shoot tips were precultured on0.45M sucrose medium for1~2days followed by loading with60%PVS2and80%PVS2for30min, respectively.Loaded shoot tips were dehydrated thereafter with PVS2on ice for40min and then transferedinto cryotubes and plunged into liquid nitrogen.75and48%shoot regeneration rates wereobtained for the two cryogenic methods, respectively. When application of the two cryogenicmethods on20potato cultivars mianly cultivated in China,62and36%shoot regrowth inaverage were obtained. The DMSO droplet freezing method, which has applied in Germanyfor more than one thousand accessions, was compared with our established dropletvitrification method. More survival parts in potato shoot tips and rapid regrowth were found in droplet vitrification than DMSO droplet freezing by Even blue staining and morphologicalobservation. Moreover, mass callus were produced in DMSO droplet freezing, Thecomparison of two cryogenic methods were conducted in6potato cultivars, and71and30%shoot regrowth in average were obtained for droplet vitrification and DMSO droplet freezingmethod, respectively.2. The histological injuries, ultrastructural changes, thermal analysis, oxditive injuriesand possible changes in DNA and chromosome ploidy were evaluated. The percentages ofsurviving cells and histological injuries in shoot tips of potato following critical steps inthree-vitrification based cryopreservation methods were studied. As results showed, sucrosepreculture did not cause obvious changes in cell viability and histological, PVS2dehydrationwas the major step causing histological injuries, LN exposure killed much more cells but didnot change surviving patterns. Shoot tips by vitrification were most damaged, only about50%cells in this region were found survive. ISSR and AFLP makers did not detect any DNAvariations in plants recovered from three cryogenic methods. The ploidy in chromosome wasalso confirmed stability in these plants. The ice crystal formation and ultrastructure changes inshoot tips subjected to droplet vitrification and DMSO droplet freezing were also compared.As different scanning calorimeter (DSC) results showed, in the freezing-thawing cycle ofDMSO droplet freezing, mass ice crystals were formed and DSC detected no vitrificationtransition. While in droplet vitrification, only small recrystallization was detected in thethawing process, the amount of ice is about one sevenths of DMSO droplet freezing. Mostimportant, vitrification transition occurred during freezing. Transmission electron microscopewas used to observe the ultrastructural changes in shoo tips following the two cryogenicmethods. Vacuoles were divided into many small ones in cells located in apical dome afterPVS2dehydration, many large starch grains in the plastids and electrical-dense polyphenolsubstances in the small vacuole were observed in the cells located in apical dome and youngerleaf primodium. These changes were not found in shoot tips treated, however, by DMSOdroplet freezing. There was no obvious injuries in meristematic cells following dropletvitrification, while DMSO droplet freezing always killed the apical dome, only some cells inleaf primordium were found survive LN. The oxidative injuries were also studied usingwild-type and transgenic potatoes which enriched the content of exogenous and endogenousantioxidants of ascorbic acid (AsA) and reduced glutathione (GSH). As a result, endogenousenriched AsA and reduced GSH and exogenous application of GSH significantly improvedshoot recovery after droplet vitrification cryopreservation. While exogenous application ofAsA was found not affect the shoot recovery. 3. The effect of cryopreservation on potato virus elimination (PLRV and PVY) and itsmechanism were proved. The effect of PLRV and PVY eradication of three cryogenicmethods from potato was investigated. As a result, PLRV and PVY can be eradicated in ahigh frequency of about80%. To reveal the mechanism this virus eradication by cryotherapy,surviving cells and PLRV were localized, PLRV were demonstrated phloem limited andcould infect the fourth and older leaf primordium by immunohistochemical staning, whilecells in the first3leaf primordium and apical dome less than0.4mm were virus-free. Onlycells in the first ten layers were found survival following three cryogenic methods, thusvirus-free cells regrew into virus-free plants. When removing the forth and older leafprimordium, only PVS2dehydration could enough eradicate PLRV totally, irrespective ofsize of shoot tips (0.5~4mm). PVS2duration can regulate the two types of injuries fromcryoprotectant and ice crystals. However, no obvious effect of regulating the two injuries onPVY eradication was observed. |
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