| Potato (Solanum tuberosum L.) is the fourth largest stample crop in the whole world,behind wheat, corn and rice. China is the largest potato producer country worldwide.Avalaibility of germplasm resources is a basic requirement for breeding of potato novelcultivars. Cryopreservation has been recognized as an ideal method for long-term preservationof germplasm resources. Up to date, there have been a numerous reports on cryopreservationof potato in the world. However, information on cryopreservation of China's potato has beenquite liminted. The objective of the present study was, therefore, to establish two simple andefficient cryopreservation procedures for China's potato and to assess genetic stability of theplants regenerated from three cryopreservation procedures.In this study, cv. Zihuabai, a major potato cultivar, was used to optimize variousparameters affecting survival and regrowth of cryopreserved potato shoot tips. In vitro shootstock cultures were maintained on MS medium (Murasgihe and Skoog1962). Nodal segments(0.5cm in length) in length were taken from3-week-old stock cultures and cultured on basalmedium (BM). After10days of culture, apical shoot tips (1-2mm in length) were excised andprecultured on BM containing0.45M sucrose concentrations for1day. Precultured shoot tipswere stepwise dehydrated (at00C) with60%and80%PVS2, with each step for30min andfinally with100%PVS2for40min. Dehydrated shoot tips were transferred into cryovials,followed by a direct immersion into LN for1h. Frozen cryovials were rapidly thawed in awater bath at38oC for2min. PVS2solution was drained from the cryovials and shoot tipswere unloaded with MS medium containing1.2M sucrose at room temperature for20min.Thawed shoot tips were post-cultured on a survival medium in the dark for3days and thentrasnfered onto shoot regrowth medium under light conditions. With optimized parameters,90.5%survival and41.4%shoot regrowth were obtained by vitrification.For encapsulation-vitrification, nodal segments (0.5cm in length), each containing anaxillary bud, were removed from3-week-old stock cultures and cultured on BM. After7daysof culture, apical shoot tips (2.0mm in length) were excised and precultured on BMcontaining0.3M sucrose concentrations for1day. Precultured shoot tips were suspended inMS medium containing2.5%(w/v) Na-alginate and0.4M sucrose. The mixture, including theshoot tips, was dropped into0.1M calcium chlorite solution containing0.4M sucrose in MS medium, to form beads (about4-5mm in diameter). The beads were treated with a loadingsolution made of MS containing0.6M sucrose with2M glycerol on a rotary shaker for90minat room temperature. Loaded shoot tips were dehydrated with PVS2solution at00C for5h.Following PVS2dehydration, ten beads were transferred into cryovials, followed by a directimmersion into LN for1h. Thawing was performed by immediately placing the cryovail intoa water bath at380C for2min. The beads were then washed with an unloading solutioncontaining MS medium and1.2M sucrose at room temperature for20min. Thawed shoot tipswere post-cultured on a survival medium in the dark for3days and then transfered onto shootregrowth medium. With optimized parameters,90.2%survival and72.5%shoot regrowth inencapsulation-vitrification.Three cryopreservation procedures, i.e, droplet-vitrification, encapsulation andvitrification, were also successfully applied to8major China's potato cultivars, whichrepresent a wide range of China's potato genotypes. The highest survival (92.5%) and shootregrowth (92.5%) were found in cryopreserved shoot tips by droplet-vitrification, while thelowest survival (78.0%) and shoot regrowth (45.0%) by vitrification. Average survival andshoot regrowth from8potato cultivars were93.9%and64.9%for droplet-vitrification,81.5%and44.1%for encapsulation-vitrification, and84.7%and24.0%for vitrification.Two molecular marker techniques (ISSR and AFLP) were employed to assess the genticstability of cv. Zihuabai plants regenerated from droplet-vitrification,encapsulation-vitrification and vitrification. With ISSR,30primers were screend, from which10primers produced reproducible and well resolved bands. Each primer was able to produce4bands, with7440bands in total obtained in30from mother plants and90plants regeneratedfrom cryopreservation. With AFLP,6primer combinations were selected out from20primercombinations tested. Each primer combination produced29amplified bands, with24360bands in total produced in30from mother plants and90plants regenerated fromcryopreservation. Both ISSR and AFLP assay did not reveal any polymorphism beweenmother plants and plants cryopreserved by three different procedures. These data clearlydemonstrated that plants regenerated from cryopreservation were highly stable in terms ofgenetic stability. |
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