| Kiwifruit has long been cultivated in China for more than 1300 years.China is currently the largest producer of kiwifruit with highest yield and biggest harvest area in the world.At present,a total of 25 kiwifruit viruses have been identified.Among them,the infection of Actinidia chlorotic ringspot-associated virus(Ac CRa V)causes chlorotic mottle,ring spot and vein yellowing in kiwifurit leaves.The detection rate of Ac CRa V reached 41.6% in Shaanxi Province.Kiwifruit is mainly propagated by asexual reproduction.It can be easily infected by viruses during reproduction,and once upon infection,will remain infected for a lifetime.Practically,the use of virus-free propagule is the most economical and effective method to control virus diseases of fruit trees such as kiwifruit.At present,the commonly used methods for virus eradication include shoot tip culture,thermotherapy,chemotherapy,cryotherapy,etc.However,there is no research reporting the virus eradication from kiwifruit.Therefore,it is urgent to establish an easy and efficient methods of virus eradication,which would be beneficial to the reduced damage of viral diseases and sustainable production of kiwifruit.In order to establish the virus eradication system in kiwifruit,the cultivar ‘Yuxiang’ was used as the material in this study.The present study made use of newly introduced in vitro cultures as donor plants for the establishment virus eradication programs by using shoot tip culture(STC),combining thermotherapy with STC,and the combination of chemotherapy with STC.The main results are as follows:(1)The treatment for establishing of in vitro cultures of ‘Yuxiang’ was optimized.The optimized concentration of mercury chloride for the disinfection was 0.1%(w/v)with an exposure duration at 5 min.Under this condition,the survival rate of explants was 55%.The medium composition for leaf regeneration of ‘Yuxiang’ was also optimized.Results showed that the leaf segments cultured with 2.0 mg / L 6-BA and 0.3 mg / L NAA showed highest rate of adventitious shoot induction(82.2%).(2)The method for eliminating Ac CRa V by a two-step shoot tip culture was established.After the first round of shoot tip culture by using explants in the size of 0.5 mm and 1.0 mm,the same size of shoot tips excised from the recovered shoots were cultured again for shoot regrowth.The shoot regrowth level and the efficiency of Ac CRa V eradication were subsequently evaluated.Results showed that all the excised shoot tips could fully recover following shoot tip culture and the Ac CRa V eradication rate of 23.3% was obtained with the two-step use of 0.5 mm shoot tips.Moreover,the use of 1.0 mm shoot tips completely failed to eradicate Ac CRa V.(3)A method for eliminating Ac CRa V by combining thermotherapy with shoot tip culture was established.The 2-week-old in vitro ‘Yuxiang’ shoots infected with Ac CRa V were thermo-treated for 10,20 and 30 days before shoot tip excision.An alternate temperature regime of 36 ℃ for 16 h under the light and 32 ℃ for 8 h in darkness was applied.The virus detection revealed that,after 10 days of thermotherapy,the Ac CRa V eradication rates were100%,90% and 83.3% for shoots regenerated from shoot tips in the size of 0.5 mm,1.0 mm and 1.5 mm,respectively.After 20 days of thermotherapy,the Ac CRa V eradication rates obtained from shoot tips in the size of 0.5 mm,1.0 mm and 1.5 mm were 100%,100% and93.3%,respectively.After 30 days of thermotherapy under the same conditions,the Ac CRa V eradication rate was 100% for all the treatments.(4)A method combining chemotherapy with shoot tip culture was established to eradicate Ac CRa V.Shoot cuttings 1-2 cm in length obtained from four-week old in vitro shoots were used as materials,and cultured on the subculture medium containing various levels of ribavirin for a 60-day chemotherapy.Shoot tips were excised at the end of the chemotherapy and cultured for shoot regrowth and virus detection.Results showed that when treated by ribavirin at 15 μg/m L,Ac CRa V eradication efficacy of 100%,100% and 86.7% was obtained respectively from excised shoot tips in the size of 0.5 mm,1.0 mm and 1.5 mm.After 60 days of chemotherapy using 25 μg/m L ribavirin,all the shoots recovered from shoot tip culture were Ac CRa V-free,regardless of the shoot tip size ranging from 0.5-1.5 mm.In this study,the efficiency of Ac CRa V eradication in kiwifruit ‘Yuxiang’ was tested by applying three common methods.The results showed that all the methods tested could effectively facilitate Ac CRa V eradication.Among these methods,two-step shoot tip culture was easy to operate and obtained high level of shoot recoveries but low virus eradication rate.Combining thermotherapy or chemotherapy with shoot tip culture showed better virus eradication efficiencies.But thermotherapy with shoot tip culture requires continuous heating equipment and the cost is high;the chemical therapy combined with shoot tip culture method is simple to operate,but the processing time required is prolonged.In production,suitable methods or centralized methods can be selected according to the toxic situation and elimination conditions of the mother plant. |
