| Collection and conservation of plant germplasm are a prerequisite for genetic improvement in both classical and genetic engineering breeding programme. Cryopreservation has been for a long time considered an ideal means for long-term conservation of plant germplasm. Potato (Solanum tuberosum) is the fourth largest staple crop in both the whole world and China. Cryopreservation of potato shoot tips has been well documented since the late of 1970s,and cryo-bank of potato germplasm has been set up and came into routine application at the International Potato Center (CIP) in Peru and at Crop Collection Center in Germany. However, studies on cryopreservation of potato shoot tips have been quite limited in China. Potato virus diseases have been a great thread to the sustainable production of potato. Viral diseases can cause losses of potato yield to 30-50%, up to 80% in the worest case. In practice, control of potato virus is through production of virus-free seed tubers. Meristem culture is the most widely used method for virus elimination. However, difficulties of excision of small meristems and low frequency of virus elimination limited its further application. Recently, cryotherapy has been found to efficiently eradicate pathogens including virus and is becoming a novel biotechnology for pathogen eradication. The present study was, therefore, to develop an efficient cryopreservation protocol for long-term storage of china's potato germplasm and to attempt to eliminate potato viruses by cryotherapy of shoot tips.Potato cv. Zihuabai, a major cultivar widely grown in China, was mainly used for establishment of droplet-vitrification cryopreservation. In vitro shoot stock cultures were maintained on Murashige and Skoog medium (MS, Murasgihe and Skoog, 1962). Apical segments about 0.5 cm in length were taken from 3-week-old stock cultures and cold-acclimated for 3 weeks at 4 oC in the dark. Shoot tips (2-3 mm in size) containing 3-4 leaf primordia were excised from the cold-hardened segments and precultured on MS medium supplemented with 0.3 M sucrose for 3 days, followed by loading of the shoot tips with 2M glycerol and 0.4M sucrose contained in MS for 20 min at room temperature. Loaded shoot tips were dehydrated with plant vitirification solution 2 (PVS2) at 0 oC for 40 min and then transferred into droplets of 6μl PVS2 on aluminum foil ( 2 cm x 0.5 cm ), with one shoot tip in each droplet, prior to a direct immersion in liquid nitrogen for 1 h. After then, the aluminum foils with the frozen shoot tips were transferred into Petri dishes (6 cm in diameter) containing 10 ml MS liquid mdium supplemented with 1.2 M sucrose for 20 min. Thawed shoot tips were post-cultured on a survival medium in the dark for 3 days. Survival medium was composed of MS containing 0.5 mg/l indole acetic acid( IAA), 0.5 mg/l zeatine riboside (ZR) and 0.2 mg/l gibberellic acid (GA3). Survived shoot tips were transferred onto a regrowth medium for shoot regeneration under light conditions. Regrowth medium consisted of MS containing 0.0005 mg/l GA3. With this protocol, 100% and approximately 70% of shoot tips survived and regenerated, respectively, following cryopreservation. The optimized cryopreservation procedure was also successfully applied to the other 13 potato cultivars, with an average survival and regrowth reaching 90% and 50%, respectively. Successful establishment of the droplet-vitrification cryopreservation provides a promising means for long-term preservation of China's potato germplasm.The genetic fidelity of plantlets regenerated from cryopreservation was evaluated by the inter-simple sequence repeat (ISSR) and no significant variation was detected.Enzyme-linked immunosorbent assay (ELISA) and reversed transcript polymerase chain reaction (RT-PCR) were developed and employed to detected potato virus Y (PVY) and potato leafroll virus (PLRV). PVY-infected potato cv. Zihuabai and PLRV-infected cv. Desiree were subject to cryotherapy. Plantlets regenerated from cryotherapy have been obtained and their virus status is under investigation. |
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