Cryopreservation And Viruse Eradication In Shoot Tips Of Apple

Apple(Malus)is globally an ecnomicially important fruits crop. Conservation of plant germplasm is a basis for further plant breeding by both classic and biotechnological stratedgies. The present study described an efficient droplet-vitrification cryopreservation, by which 5 apple genotypes were successfully cryopreserved, with the lowest(36%) and highest(63%) shoot regrowth rates obtained in‘Wangshanhong’ and ‘Gala’. The mean shoot regrowth rate of the 5 apple genotypes was aboyt 46%. The major steps included: apple shoot tips are treated by preculture medium for 1 day, expored to plant vitrification solution 2(PVS2) for 40 min, and then transferred in droplets made of PVS2(6μL) and carried on aluminium foils, each drop containing 1 shoot tip, followed by a direct immersion in LN for storage. Histological observartions showed that only when mont of the cells in apical dome, and some cells in the yongest leaf primordia 1 and 2 survival liquid nitrogen, shoot regrowth can occur in the cryopreserved shoot tips. Assessments by ISSR, RAPD, and Flow cytometry did not detect any alternations at DNA and ploidy levels in the plants regenerated from cryopreservation, compared with those from in vitro shoots(control), indicating cryopreservation dose not cause changes in genetic integrity.Virus diseases have long threatened sustainable development of apple production. Unlike other diseases caused by fungi and bacteria, once the plants are infected by virus, there are no chemical sprays to control the diseases. In practice, virus diseases can be controlled by using virus-free materials. Availability of efficient approaches is a key for cultivation of virus-free plants. The present study attempted to eradicate Apple stem grooving virus(ASGV) and Apple stem pitting virus(ASPV). The results found cryotherapy of shoot tips resulted in production of 57.6% and 62.8% of ASPV-free plants in rootstocks ‘M26’ and ‘M9’, regardless of the size of shoot tips used. Although meristem culture can also eradicate ASPV, the size of meristems was critical. Neither cryotherapy nor meristem culture cannot eliminate ASGV. Virus localization using immunological and biochemical analysis showed that ASPV was not present in the apical dome and leaf primordia 1-2, while ASGV was easily detected in apical dom. Data of virus localization and distribution of surviving cells provided experimental explanations as to why cryotherapy and meristem culture can eradicate ASPV, but not ASGV.The results obtained in the present study would provide technical supports for establishment of cryo-banking of apple germplasm and open a new avenue for production of apple virus-free plants.