Establishment Of Cryopreservation Protocol And Analysis Of Virus Eradication Effects In Garlic

As one of the vegetative propagation plant,garlic?Allium sativum L.?has many disadvantages in its traditional germplasm conservation.Cryopreservation is a promising technology for plant germplasm conservation,it has been widely applied in main garlic cultivars and genotypes in many countries worldwide.However,due to the significant genetic specificity of this technique,and rare research data of garlic cryopreservation in China.The application of this technique in garlic germplasm preservation was greatly restricted in China.At present,all of the shoot apices used in garlic cryopreservation are sampled from the field.Althought this kind of explant have been widely used for cryopreservation of garlic,it still have some disadvantages as source of shoot apices.For instance,they are easy to be contaminated by pathogens from field,which will lead to the failure of cryopreservation;Shoot apices are usually difficult to be excised from cloves and bulbils;the number of cloves and that of its shoot apices are limited.These factors are obstacles in the development and application of garlic cryopreservation.Cryotherapy is a novel application of plant cryopreservation techniques that allows pathogen eradication at high frequency.Even though this method has been applied in many species,research on garlic virus cryotherapy is just emerging.In addition,it is very necessary to evalute the genetic stablitily and growth adaptability of regenerated plant in cryopreservation.However,it is a remarkable fact that such studies have rarely been reported on garlic crops.This research was aimed to establish the cryopreservation protocol for Chinese garlic cultivars by shoot apices excised from adventitious buds in vitro;compare the virus eradication effects between cryotherapy and traditional methods;evaluate the genetic stablity of cryopreserved garlic in level of molecular,chromosome ploidy and field performance.The main results are summarized as followings:1.Garlic cultivar‘G064'were used in optimizing the combination of NAA and 6-BA for adventitious bud induction from stem disc,then other 9 cultivars were applied in this regeneration protocol.The average propagation coefficient of adventitious bud was 18.6,indicating that this protocol can provide sufficient and sterile materials in consistent physiological state for garlic cryopreservation research.Assessment of genetic stability in cryopreserved garlic plantlets represented that no polymorphic bands were detected by SSR.2.The garlic cryopreservation protocol was established by 10 Chinese cultivars.The main steps in this protocol were described as follows:Shoot apices 2 mm in length with 2-4leaf primordia and a basal part were excised from adventitious buds?cultured for 5-7 weeks with the pseudostem diameter in 2-3 mm?.The shoot apices were pre-cultured for 2 days with 0.3 mol/L sucrose at 24°C,incubated in a loading solution containing 2 mol/L glycerol+0.6 mol/L sucrose in Murashige and Skoog medium?MS medium?for 20 min at24±1°C,and dehydrated with plant vitrification solution 3?PVS3?for 1.5 hours at 0°C prior to direct immersion in liquid nitrogen?LN?for 1 h.The frozen shoot apices were re-warmed and unloaded with 1.2 mol/L sucrose for 20 min at room temperature and transferred to MS medium for recovery.With the optimized factors,the average survival and regeneration rate of cryopreserved garlic plantlets were 56.1%and 48.3%,respectively.Moreover,the early-harvest cultivars were proven not suitable for this protocol.The experiment about effect of shoot apices sources on the garlic cryopreservation provided the new source of garlic shoot apices for cryopreservation.Histological observation revealed that most of the cells in the shoot meristem and the 1^st-4^th leaf primordium survived,while the cells near the base of the shoot and leaves died during cryopreserved process.Evaluation of genetic stability in cryopreserved garlic plantlets showed that neither polymorphic bands nor chromosomal variation were detected by SSR and flow cytometry.3.The results of DAS-ELISA and RT-PCR showed that garlic cultivar‘G064'used in this study is complex infection by Onion Yellow Dwarf Virus?OYDV?,Leek Yellow Streak Virus?LYSV?,Garlic Latent Virus?GLV?and Garlic Virues?GarVs?.The comparision of virus eradication efficiency between cryotherapy and traditional methods represented that cryotherapy obtained the highest virus eradication rate for OYDV?76.4%?,and its regeneration rate of plantlet?65.0%?was significantly higher than that of traditional methods.4.The field performance including morphological traits,photosynthetic characteristics,growth period,bolting rate,one-clove bulb rate and bulb characteristics of cryopreserved garlic were evaluated in a comparison to normal in vitro and field-derived garlic in this study.The results showed that cryopreserved garlic represents obvious superiority in growth,photosynthetic ability and bulb characteristics.With the consideration of virus detection results,these superiority were ascribed to the virus eradication by cryotherapy.Moreover,the genetic stability of regenerated garlic in the level of morphology and photosynthetic physiology were also proven to be relatively stable by comparing in vitro-derived garlic with field-derived garlic.In general,this study demonstrated that regenerated garlic plant derived from cryopreservation can well adapt to its native environment.The results of this study not only provided technical supports for establishment of garlic cryo-bankings in China,but also promoted the development and application of cryopreservation in garlic.