Development Of Novel Cryopreservation Protocols,Cryotheraoy For Virus Eradication And Virus Detection In Lilium

Lily,a perennial bulbous plant belonging to the family Liliaccae and the genus Lilium,is widely grown throughout the world as cut flowersand also vegetables.Germplasm resources provide valuable genes,which can be used for breeding of novel lily cultivars in both classic breeding and genetic transformation programs.Viral diseases constitute a major constraint in commercial production of lily.Cultivation of virus-free plants is an effective means to control viral diseases in lily production.Availability of efficient methods for virus detection is necessary in virus disease control,production of virus-free plant and quarantine for exchanges of plant materials between countries.The objectives of the present study were 1),to attempt to develop a novel technique for cryopreservation of lily shoot tips;2),to test droplet-vitrification cryotherapy for production of virus-free lilies;3),to develop effective methods for detection of lily virus.The main results obtained in this study are as followings:1.A new cryopreservation method for Lilium Oriental hybrid‘Siberia’was established.Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium（MS）supplemented with 1 mg L^-1α-naphthalene acetic acid（NAA）and 0.5 mg L^-11 thidiazuron（TDZ）.Small leaf squares（SLSs,3×4 mm）,each bearing at least one adventitious bud,were cut from leaf segments,precultured on medium with 0.5 M sucrose for 1 day,and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol,followed by exposure to plant vitrification solution 2 for 7 h at 0°C.Dehydrated SLSs were directly immersed in liquid nitrogen（LN）for 1 h.Cryopreserved SLSs were warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature,followed by post-thaw culture for recovery.With this procedure,85%survival and 72%shoot regrowth were achieved following cryopreservation.2.Droplet-vitrification cryotherapy was applied to liliesinfected with Cucumber mosaic virus（CMV）.They were L.Orientalhybrid‘Siberia’,L.Asiatic hybrids‘Elite’and L.longiflorum Oriental‘Triumphator’.With this method,survival levels of 95%,73%and 60%,and shoot regrowth levels of 85%,72%and 43%were obtained in these three lilies.Preliminary tests of virus sanitary found 85%,80%and 73%virus free frequencies in the plantlets recovered after cryotherapy,and final confirmation of their virus sanitary status is in progress.3.TAS-ELISA,RT-PCR,microtissue direct RT-PCR（MDRT-PCR）and immunohistological localization were applied to detect CMV in Lilium Orientalhybrid‘Siberia’.Comparative results were obtained using MD RT-PCR and RT-PCR.Results obtained in the present study would provide a technical platform for setting-up of cryo-banks of Lilium genetic resources,open a new avenue for virus eradication and provide efficient virus detection for lily virus.