Cryopreservation Of Shoot Tips Of Grapevine (Vitis Spp.) And Cryotherapy For Eradication Of Grapevine Leafroll-associated Virus 3

Grapevine(Vitis)is one of the economically most important fruits worldwide.Some wild species and cultivars are endangered by urbanization,industrialization,salinization and desertification of soil.Establishment of efficient long-term conservation of plant germplasm resources can effectively protect diversity of genetic resources.Cryopreservation provides an alternative and ideal approach for the long-term conservation of plant germplasm resources.Virus diseases are among the main factors restricting the development of grape and wine industry.Cultivation of virus-free plants is the most effective approach for virus diseases controlling to which production of virus-free plants is the key.Cryotherapy,based on cryogenics process,provides a new means for the elimination of plant viruses.In this study,a droplet-vitrification protocol was established by in vitro shoots of Vitis to provide an efficient and wild-spectrum conservation method for establishment of cryobanks of Vitis germplasm resources.Establishment of cryotherapy for efficient eradication of Grapevine leafroll-associated virus-3(GLRaV-3)is expected to provide technical platform for production of grapevine-free plants.Meanwhile,in vitro biological indexing,Microtissue direct RT-PCR,and immunohistochemical localization methods are used to ensure virus-free regenerants by virus elimination and quarantine.Main results achieved in the present study are as followings:By optimized key factors affecting success of shoo tip cryopreservation of grapevine,a droplet-vitrification cryopreservation protocol was established for diverse geneic resources of Vitis.Axillary shoot tips(1.0 mm in size)containing 5-6 leaf primordia(LPs)were excised from 8-week-old in vitro tock shoots and precultured for 3 days in the dark on a semi-solid 1/2 MS medium containing 0.3 M sucrose,0.16 ?M glutathione and0.14 ?M ascorbic acid(pH=5.7).Precultured shoot tips were treated for 20 min at ambient temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose,followed by exposure at 0°C to half-strength plant vitrification solution 2(PVS2)for 30 min and then full-strength PVS2 for 50 min.Dehydrated shoot tips were transferred onto 2.5 ?L PVS2 droplets dotted on aluminum foil strips and dropped directly in liquid nitrogen(LN).Frozen foil strips containing shoot tips were rewarmed by transfer to a unloading solutioncontaining 1.2 M sucrose for 20 min at room temperature,and post-cultured on a semi-solid1/2 MS medium containing 0.6 M sucrose in the dark for 24 h,and then transfered on a semi-solid 1/2 MS medium containing 0.5 mg L-1 BA for shoot regrowth.Additional seven genotypes,including 5 cultivars of V.vinifera and 2 genotypes of V.pseudoreticulata,were tested by the droplet-vitrification procudure,with an average shoot regrwoth rate of 50.5%achieved.The highest and lowest regrowth rate is 71.7% for V.vinifera ‘Cabernet Sauvignon' and 21.7% for V.pseudoreticulata ‘Baihe 35-1'.Adding glutathione and ascorbic acid to preculture medium helped coping with the no regrowth problem.Testing of effects of axillary bud position and bud types on survival and regrowth rate,we found that axillary shoot tips were superior to apical shoot tips for grapevine cryopreservation.Effects of duration of exposure to PVS2 on survival and regrowth rate were studied by histological observation to reveal survival patterns of cryopreserved shoot tips.No polymorphic bands were detected by inter-simple sequence repeats(ISSR)and random amplification of polymorphic DNA(RAPD)in the regenerants from cryopreserved shoot tips.Vegetative growth of in vitro regenerants recovered from cryopreservation was greater than the control after 7 cycles of subculture.A droplet-vitrification cryotherapy protocol was established to efficiently eradicate GLRaV-3.Nodal segments,each being 1.0 cm in length and containing one axillary bud,were taken from 6-week-old stock shoot that had been cultured on a semi-solid 1/2 MS medium for 2 weeks to promote bud elongation.Apical shoot tips(1.0 mm in size)containing 5-6 leaf primordia(LPs)were excised from 8-week-old in vitro stock shoots and precultured for 3 days in the dark on a semi-solid 1/2 MS medium containing 0.3 M Sucrose,0.16 ?M glutathione and 0.14 ?M ascorbic acid.Precultured shoot tips were treated for 20 min at ambient temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose,followed by exposure at 0°C to 1/2 PVS2 for 30 min and then full-strength PVS2 for 75 min.After hehydration with PVS2,shoot tips were transferred onto 2.5 ?L PVS2 droplets dotted on aluminum foil strips and dropped directly in liquid nitrogen(LN).Frozen foil strips containing shoot tips were rewarmed by transfer to a unloading solution containing 1.2 M sucrose for 20 min at room temperature,and transferred to a semi-solid1/2 MS medium containing 0.6 M sucrose in the dark for 24 h to recovery and post-thaw cultured on a semi-solid 1/2 MS medium containing 0.5 mg L-1 BA for 6 weeks for shoot regrowth.The virus-free frenquency was 100%,regardless of size of shoot tips or duration of exposure to PVS2.GLRaV-3 was not observed in apical dome and the youngest 4 leaf primordia while localized in the fifth leaf primordia and cells that 0.2 mm away from apical dome in apical shoot tips.All cells in the fifth leaf primordia are killed after cryotherapy.These results could explain why cryotherapy can eradicate GLRaV-3.Microtissue direct RT-PCR,inmmunohistochemical localization,in vitro biological indexing and micrografting were established to detect GLRa V-3.In microtissue direct RT-PCR,a syringe needle was used to pierce a leaf or stem,cut and dipped into the RT reaction mixture in a capped tube for 15 min,followed by RT-PCR.Virus-free frequency in regenerants from cryotherapy was tested by microtissue direct RT-PCR was compatable with RT-PCR.With inmmunohistochemical method,GLRaV-3 was localized successfully in the stems and shoot tips of grapevines,confirming GLRaV-3 is a phloem-limited virus,and cannot infect apical dome and the youngest leaf primordia 1-4.In in vitro indexing of virus,shoot segments(about 3 cm with 2-3 leaves)excised from GLRaV-3 infected in vitro stock shoots were cultured on 1/2 MS supplemented with 150 mM NaCl for symptom expression.Typical virus symptoms,including reddish-purple coloration and downward rolling of leaves,were observed with the earliest time in 5 days after stressed culture mentioned above and with 86% proportion of symptom expression after 4 weeks.Additional three genotypes were tested with 150 mM NaCl.Symptom expression occurred the earliest in the hybrid rootstock ‘6-12-2'(V.pseudoreticulata ‘Baihe-35-1' × V.vinifera ‘Carinena')in 4 days with100% proportion after 4 weeks,latest in ‘Kyoho'(V.vinifera)in 8 days with 70% proportion after 4 weeks.