Cryopeservation Of Potato Shoot Tips And Viruses, And Assessments Of Cryotherapy-derived Virus-free In Vitro Plantlets Under Salt Stress

Purple potato provides valuable potato germplasm.Cryopreservation is considered an ideal method for long-term conservation of plant germplasm.No reports have been published on cryopreservation of purple potato.Cultivation of potato virus-free seed tubers has been for a long time used for efficient control of virus diseases.Cryotherapy has been established as a novel biotechnology for plant pathogen eradication.Evaluations of vegetative growth and mictotuber productions,particularly under abiotic stress,of cryotherapy-derived virus-free potato plants are necessary before the virus-free plants come into practical cultivation.All virus-and viroid-related researches require pathogen-infected material.Establishment of safe and efficient methods for long-term preservation of the pathogen can ensure easy access to the pathogen.（1）Cryopreservation and evaluations of vegetative growth,microtuber production and genetic stability in regenerants of purple-fleshed potato:Buds of in vitro-grown shoots of two purple-fleshed potato genotypes were successfully cryopreserved by encapsulation-vitrification and droplet-vitrification.Optimal time durations of exposure to PVS2 for shoot regrowth of cryopreserved buds were 5–7 h and 6 h for‘E03-2677’and for‘Blue Congo’,respectively,in encapsulation-vitrification,and 30–50 min and 40 min for the former and the latter,respectively,in droplet-vitrification.Higher rates of shoot regrowth were obtained in 1.5–2.0 mm-buds than in 1.0–1.4 mm-ones in encapsulation-vitrification for‘E03-2677’and‘Blue Congo’,while opposite results were found in droplet-vitrification.In‘Blue Congo’,only apical shoot tips survived and developed into shoots,with one shoot produced in one cryopreserved bud in encapsulation-vitrification and droplet-vitrification.In‘E03-2677’,survival and shoot regrowth patterns were similar to those of‘Blue Congo’in encapsulation-vitrification.However,both apical and axillary buds survived and developed into two shoots in one bud in droplet-vitrification.In‘E03-2677’,histological observations revealed only apical shoot tips survived following encapsulation-vitrification,while both apical and axillary shoot tips survived following droplet-vitrification.Vegetative growth in shoots regenerated from encapsulation-vitrification and droplet-vitrification after 3 weeks of post-thaw culture was significantly lower than that from control,but markedly increased after 6 months of post-thaw culture.In both‘E03-2677’and‘Blue Congo’,number of microtubers per shoot,per vessel and≥3 mm in diameter were significantly greater in shoots regenerated from cryopreservation than in those from the control.Assessments by ISSR and RAPD of genetic stability did not find any polymorphic bands in regenerants recovered from both methods.（2）Growth,microtuber production and physiological metabolism in virus-free and virus-infected potato in vitro plantlets grown under NaCl-induced salt stress:virus-free plantlets were obtained from the infected in vitro plantlets of potato‘Zihuabai’by the method of droplet-vitrification,and virus-carrying samples were set as control.Using in vitro cultures,we demonstrated that virus infection（singly and in combination）or salt,and co-stress by virus infection（singly and in combination）and salt significantly reduced growth and microtuber production,and caused severely oxidative cell damage determined by levels of O₂·^-and methane dicarboxylic aldehyde,and H₂O₂ localization in situ.Alterations in physiological metabolism by increasing total soluble sugar and free proline,and by decreasing chlorophyll content are responses of potato plantlets to virus infection（singly and in combination）or salt stress and co-stress by virus infection（singly and in combination）and salt.Oxidative cell damage and reduced chlorophyll content caused by virus and/or salt are believed to be responsible for the reduced growth,eventually resulting in decreased tuber yield.（3）Long-term preservation of potato leafroll virus,potato virus S and potato spindle tuber viroid in cryopreserved shoot tips:A protocol was described for long-term preservation of potato leafroll virus（PLRV）,potato virus S（PVS）,and potato spindle tuber viroid（PSTVd）in cryopreserved shoot tips of potato cv.‘Zihuabai’.Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips（58–60%）than in0.5-mm-ones（30–38%）.All shoots recovered from 0.5-mm-shoot tips were PVS-and PSTVd-preserved,but none of them were PLRV-preserved.Cryopreservation of1.5-mm-shoot tips resulted in 35%and 100%of PLRV-and PVS-and PSTVd-preserved shoots.Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips.Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation,similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation,compared with the diseased in vitro stock shoots（the control）.Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots.The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants.Successful cryopreservation of purple potato shoot tips provide technical supports for setting-up of cryobanks of purple potatoes.Results of evaluations of cryotherapy-derived virus-free plants plantlets under salt stress provide theoretical evidence for application of cryotherapy-derived virus-free plants to the field production.Shoot tip cryopreservation of PLRV,PVS and PSTVd opens a new avenue for safe and long-term preservation of obligate pathogens in living tissues of plants.