| The availability of genetic resources is a prerequisite for breeding of novel cultivars byboth classic and genetic engineering programs. Cryopreservation has been widely recognizedas an ideal means for long-term conservation of plant genetic resources. Apple is globally oneof the most economically important fruit crops. China is a major apple-producing country inthe world, and an original centre for genetic resources of Malus. Virus-induced diseases havelong threatened sustainable production of apple. Apple stem pitting virus (ASPV) and Applestem grooving virus (ASGV) are the two most damaging diseases for apple production in theworld. Practically, use of virus-free plants is an effective method to combat virus diseases.Recently, cryopreservation of shoot tips has been found to efficiently eradicate plantpathogens including virus. The objective of the present study was, therefore, to establishsimple and efficient cryogenic techniques for germplasm and attempt to eradicate ASPV andASGV using cryopreservation.1．A simple and efficient cryopreservation protocol using encapsulation-dehydration wasestablished by using in vitro grown shoot-tips of apple ‘Gala’(Malus×domestica Borkh.‘Gala’). Shoot tips (2mm) containing5-6leaf primordium (LP) were excised from4-weekold in vitro stock shoots. They were encapsulated into beads (5mm in diameter) using2.5%Na-alginate in0.1M CaCl2solution, each containing one shoot tip. The beads wereprecultured on a preculture medium composed of MS medium containing0.5M sucrose for7days. The precultured beads were dehydrated by air drying in a laminar flow hood. Afterdehydration for6h, the beads were transferred into cryotubes (1.8mL), each containing10beads, followed by a direct immersion in liquid nitrogen (LN) for storage. For thawing, frozenbeads were removed out from LN and immerdiately placed in a water bath set up at38oC for2min. Shoot recovery were conducted by culturing the thawed shoot tips on a post-culturemedium composed of MS,30g L-1sucrose,0.25mg L-1BA,0.01mg L-1IBA and8g L-1agar. Shoots (≥5mm in length) were produced after8weeks of culture. This protocol wassuccessfully applied to7genotypes of Malus (four Malus species and one hybrid). Thehighest and lowest shoot regeneration rates were found in ‘Gala’(75%) and‘Wangshanhong’(Malus×domestica,36%). Histological studies revealed that shoots could be regenerated in cryopreserved shoottips only when many cells most of the cells in the apical dome and theyounger leaf primordium survived following cryopreservation.2. Cryopreservation by droplet-vitrification and vitrification. Shoot tips (2mm)containing5-6leaf primordium (LP) were excised from4-week old in vitro stock shoots ofapple ‘Gala’. The shoot tips were precultured in liquid MS containing2M glycerol and0.8M sucrose for1day. In droplet-vitrification, the precultured shoot tips were treated by plantvitrification solution2(PVS2) for40min，and then transferred in droplets made of PVS2(6μL) and carried on aluminium foils, each drop containing1shoot tip, followed by a directimmersion in LN for storage. In vitrification, the precultured shoot tips were treated withPVS2for30min, and then transferred into cryotubes (1.8mL), each containing100μL PVS2and10shoot tips, followed by a direct immersion in LN. Frozen shoot tips in bothdroplet-vitrification and vitrification were removed out and immediately placed in MSsupplemented with1.2M sucrose for20min. Shoot recovery was performed by culturingshoot tips on a post-culture medium. The droplet-vitrification and vitrification protocolsdeveloped were tested for6apple genotypes including4species and1hybrid.3. Comparison of three established cryogenic protocols in freezing effectiveness.Number of surviving cells and shoot regrowth rate were higher, and shoot recovery was fasterin shoot tips cryopreserved by encapsulation-dehydration than droplet-vitrification andvitrification. All three cryopreservation protocols produced three types of regenerants insurvival shoot tips following cryopreservation: callus formation only, leaf developmentwithout shoot formation and normal shoot development. Percentages of these three types ofregenerants were4.8%,18.7%and76.5%in encapsulation-dehydration;29.4%,11.2%and59.4%in droplet-vitrification. Effects of season change on shoot regrowth were more obviousin shoot tips cryopreserved by encapsulation-dehydration than droplet-vitrification.Bothprotocols produced the highest rates of shoot regrowth during October and December. Nopolymorphic bands were detected by inter-simple sequence repeats (ISSR) and randomamplified polymorphic DNA (RAPD) in the plantlets recovered from cryopreserved shoottips, and ploidy levels analyzed by flow cytometry (FCM) were maintained in plantletsregenerated from cryopreserved shoot tips, regardless of the cryopreservation protocols used.4．ASPV and ASGV were eradicated by cryopreservation of shoot tips (≥0.5mm) andthermotherapy combined with cryopreservation of shoot tips (≥0.5mm). With ‘M9’and‘M26’ stock culture co-infected with ASPV and ASGV, meristem culture (≥0.5mm) could noteradicate ASPV but cryopreservation of shoot tips by droplet-vitrification produced about85.1%(‘M9’) and80.3%(‘M26’) of virus-free plants when the size of shoot tips was0.5-1.0mm, and57.6%(‘M9’) and62.8%(‘M26’) with shoot tip size at1.0-2.0mm. Both thermotherapy (36oC/30oC，16-h photoperiod，60days) combined with meristem culture(≥0.5mm) and single use of cryopreservation of shoot tips (≥0.5mm) failed to eradicateASGV. However, with apple ‘Gala’, combination of thermortherapy and cryopreservationresulted in57.1%(shoot tip size at0.5-1.0mm) and50%(shoot tip size at1.0-2.0mm) ofASGV-free plants, respectively. Virus localization by immunohistochemistry showed thatASPV were only found in the fourth and the older leaf primordium and the location below themeristem dome. ASGV could be found throughout the meristem dome. Mechanisms as towhy cryopreservation can eradicate ASPV and why ASGV was difficult to be eliminatedwere proposed.The results obtained in the present study would provide technical supports forestablishment of apple cryo-bankings, and for production of virus-free plants. |
PDF Full Text Request