Adventitious Shoot Regeneration And Virus Elimination By Shoot Tip Culture In Apple

Apple(Malus) is globally one of the most important fruit trees and simultaneously the biggest producing fruit in China. The efficiency of in-vitro propagation of apple is higher comparing with conventional propagation methods, i.e. grafting and cuttage. The leaf organogenesis is an indispensable means of tissue culture providing an effective method for in-vitro propagation of apple. The technique of leaf regeneration has also been widely applied in genetic transformation of Malus species. Viral diseases greatly threaten the apple propagation and production. The use of virus-free plant material is virtually an effective means combating problems of virus diseases and virus elimination approach is a necessity for obtaining virus-free plants. This research has used apple cultivar ‘Gala’ and rootstock‘M26’co-infected with apple virus ASPV and ASGV as test materials to(1) optimize a highly effective protocol with wide spectrum for leaf-derived adventitious buds initiation and development;(2) investigate the virus eradication efficiency using adventitious shoot tip culture of leaf segments with thermotherapy applied. Results achieved in the present study were summarized as followings:1.Two apple genotypes ‘Gala’ and ‘M26’infected with ASPV and ASGV were used as the experimental materials for compositional optimization of TDZ and IBA for leaf adventitious shoots regeneration. Three fully opened leaves in the top were excised from the in-vitro plantlet as explants and the number of normal shoots with regular meristem tip and 3leaf primordia were recorded after 3 weeks of culture in the darkness. The optimum combination of TDZ and IBA were 2mg/L with 0.5mg/L for ‘Gala’ and 3mg/L with 0.5mg/L for ‘M26’on which 4.6 and 4.0 normal shoots were recorded. Exogenous addition of CVP(Coriolus versciclor polysaccharide) of 4g/L into the optimized medium for shoot regeneration significantly enhanced leaf regeneration efficiency of ‘Gala’ and ‘M26’. The average number of adventitious shoots regenerated from ‘gala’ were improved from 4.4 to 6.8after the accretion of CVP in 4g/L compared with ‘M26’improved from 3.9 to 5.6.2. Leaf segments from ‘Gala’ and ‘M26’co-infected with ASPV and ASGV were cultured on optimized shoot regeneration medium(SRM). Adventitious shoots of normal form were excised after culture of 2, 3 and 4 weeks on SRM with 2, 3 and 4 leaf primordia respectively followed by culture on basic medium(BM) containing IBA 0.01mg/L and BA0.25mg/L for further regeneration. Detection of virus was implemented after 3 months ofregrowth on BM. Results demonstrated that size and developmental stages of adventitious shoots did affect the regrowth rate as well as the virus elimination efficiency. As for ‘Gala’,the regrowth rate of 2 weeks of culture improved from 10% to 82% after the 4 weeks of culture. Frequency of ASPV eradication reached 100% when adventitious shoot tip was separated after 2 and 3 weeks of culture and dropped dramatically to 20% after the 4 weeks of culture. When it referred to ‘M26’, the shoot tips excised after 2 weeks of culture failed to regenerate into whole plants. The regrowth rate after 3 and 4 weeks of culture were 45% and76% separately. Noticeably, adventitious shoot tip culture alone cannot obtain any M26 plantlet which is free from ASPV as well as ASGV which can be detected in every regenerated plantlet of ‘Gala’ and ‘M26’. Immunohistochemical staining of virus illustrated virus distribution in leaf segments during the regeneration process, explaining the patterns of ASPV eradication in ‘Gala’, ASPV reservation in ‘M26’and ASGV retention in both genotypes. In an attempt to eradicate viruses from ‘M26’, Leaf segments were subsequently subjected to 1 and 2 weeks’ thermotherapy under 36 ℃ after 4 weeks of culture on SRM and1 week culture on BM. Results showed the regrowth rate of 1mm shoot tips containing 4 leaf primordia dropped from 93% after 5 weeks of culture to 47% when thermotherapy of 2 weeks was added. Frequency of virus free plantlets was 100% for ASPV and 17% for ASGV after the treatment of 2 weeks’ thermotherapy. localization of ASGV in shoot tips of ‘Gala’ after melatonin treatment of 2μM illustrated the inhibiting effect ofmelatonin on the replication and movement of ASGV virus.The results obtained from this research provided new means for improving the leaf regeneration efficiency in apple through addition of polysaccharide extracts from Coriolus versciclor. The successfulattempt of virus elimination based on adventitious shoot tip culture and thermotherapy opened new avenue for producing virus free plant material.