| Apple(Malus) is globally the second largest economically important fruit crop. China is the biggest apple-producing country of the whole world. Availability of and easy access to genetic resources are a prerequisite for crop improvement in both classic and biotechnological strategies. Cryopreservation has been widely recognized as an ideal means for long-term conservation of plant genetic resources. Virus-induced diseases have long threatened sustainable production of apple. Apple stem grooving virus(ASGV) and Apple stem pitting virus(ASPV) are the two major viruses that cause serious damage to apple production in the world. Practically, use of virus-free plant material is an effective method to combat virus and allows exchange of plant germplasm between countries and regions. Development of efficient and simple approaches for production of virus-free is necessary. The objectives of the present study were, therefore, to(1) establish an efficient and wide spectrum protocol of plant regeneration via adventitious shoots using leaf segments;(2) develop efficient and simple cryopreservation of Malus germplasm using adventitious shoots;(3) attempt to eradicate ASPV and ASGV using meristem culture and cryotherapy; and(4) investigate effects of virus infection on vegetative growth, physiological metabolism and recovery of shoot tips following cryopreservation in Malus plants. Results obtained in the present study were summarized as followings:1. Two apple cultivars ‘Gala’ and ‘Fuji’, and two rootstocks ‘M9’ and ‘M26’ were used for optimizing the combination of TDZ and IBA for adventitious shoot regeneration from leaf segments. The first three fully opened leaves were excised from 4-week-old in vitro stock cultures. Leaf segments were kept at 22±2 oC in the dark for 3 weeks before transfer to the light conditions, for adventitious shoot regeneration. 2 mg L-1 TDZ + 0.5 mg L-1 IBA and 2-3 mg L-1 TDZ + 0.5 mg L-1 IBA was found the most suitable for ‘Gala’ and ‘Fuji’, on which all leaf segments showed organogenic response, and 6.4 and 2.9 adventitious shoots/explant were produced in the former and latter. The best shoot regeneration of ‘M9’ and ‘M26’ was obtained when cultured on 3 mg L-1 TDZ + 0.5-1.5 mg L-1 IBA, on which organogenic response frequency was 100%, and approximately 4 adventitious shoots/explant were produced in ‘M9’ and ‘M26’. When the selected combination of TDZ and IBA were further used for testing shoot regeneration of other 5 Malus genotypes, all genotypes shown 100% organogenic response frequency and approximately 1.9, 4.9, 2.0, 3.5 and 0.6 adventitious shoots/explant were produced in ‘Himekami’, ‘Greensleeves’, ‘Wangshanhong’, ‘Pingdinghaitang’ and ‘Balenghaitang’, respectively. ISSR analysis did not find any polymorphic bands in regenerants derived from leaf segments of ‘Gala’.2. ‘Gala’ was used for establishment of encapsulation-dehydration cryopreservation protocol. Shoot tips(3 mm in length) containing 6 leaf primordia(LP) were excised from 11-week-old adventitious shoots and were encapsulated into alginate beads(5 mm in diameter). The beads were precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h, prior to direct immersion in liquid nitrogen. Following thawing in a water bather set up at 38 oC for 3 min, cryopreserved shoot tips were post-cultured for recovery. About 79.3 % of cryopreserved shoot tips regenerated into shoots. Application of this cryopreservation protocol to other 8 Malus genetypes showed that all of them could be sucessfully cryopreserved, except ‘Greensleeves’. Shoot regrowth rates were 74.0% for ‘M9’, 66.3% for ‘Balenghaitang’, 62.5% for ‘Fuji’, 52.5% for ‘M26’, 50.0% for ‘Pingdinghaitang’, 43.8% for‘Wangshanhong’and 27.5% for ‘Himekami’. ISSR analysis did not find any polymorphic bands in regenerants recovered from cryopreserved shoot tips of ‘Gala’.3. Shoot tips derived from adventitious shoot were used for eradication of ASPV and ASGV from virus-infected in vitro shoots of ‘Gala’. Leaf segments were excised from in vitro 4-week-old stock shoots and cultured on shoot regeneration medium containing 9.1 μM TDZ and 0.25 μM IBA to induce adventitious shoot formation. Shoot tips of different size and different developmental stage were excised from adventitious shoots and cultured on shoot tip culture medium containing 1.1 μM BA and 0.05 μM IBA. Results showed size and developmental stage of shoot tips excised from adventitious shoots did not influence survival rate, but significantly affected shoot regrowth rate and ASPV-free frequency. Shoot regrowth rates increased from 10 to 15 % in 0.3 mm shoot tips containing two LP excised after 2-3 weeks of shoot regeneration, to 53-55 % in those containing three LP excised after 3-4 weeks. The highest shoot regrowth rate(82 %) was obtained in shoot tips of 0.4 mm shoot tips containing four LP excised after 4 weeks. ASPV-free frequencies(95-100%) were high in 0.2-0.4 mm shoot tips containing two to three LP excised after 2-4 weeks, but low(20 %) in 0.4 mm shoot tips containing four LP excised after 4 weeks. None of the shoots regenerated from the shoot tips were ASGV-free, regardless of the size and developmental stage at which shoot tips were excised. Histological studies and virus localization provided explanations to the varying frequencies of the virus eradication using different size of shoot tips that were excised from adventitious shoots at different developmental stages.4. Shoot tips of apical buds were used to eradicate ASPV and ASGV from virus-infected in vitro shoots of apple rootstocks ‘M9’and ‘M26’ using shoot tip culture and cryotherapy. In shoot tip culture; shoot tips were proportional to shoot recovery but inverselyproportional to ASPV-free rate. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryotherapy; shoot tips(0.5 mm in length) containing two LP did not resume shoot growth. Although 1.0-mm and 1.5-mm shoot tips gave similarly high ASPV-free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryotherapy were observed in both‘M9’and‘M26’. Histological studies and virus localization provided explanations to the varying frequencies of ASPV eradication using cryotherapy. Ploidy levels analyzed by flow cytometry(FCM) were maintained in virus-free plantlets regenerated from cryotherapy.5. Vegetative growth, physiological metabolism and cryopreservation efficiency were compared among healthy, ASGV single-infected, and ASPV and ASGV co-infected in vitro cultures of ‘Gala’. For vegetative growth, virus infected shoots required longer time for bud break, proliferated greater number of shoots, and reduced fresh weight and dry weight of proliferating shoots. Virus infection decreased relative electrolyte leakage, but increased levels of total soluble protein, total soluble sugar and proline. There were no detected differences in survival rates in shoot tips that were excised from shoots ≥1cm and ≥0.5cm, <1cm, regardless of their virus status. Significant lower survival rates were found in shoot tips that were excised from <0.5cm shoots produced by ASGV single-infected and ASGV+ASPV co-infected cultures. Shoot regrowth rates were similar in shoot tips that were excised from healthy shoots ≥1cm and ≥0.5cm, <1cm, and significant lowerin shoot tips that were excised from ASGV single-infected shoots. The lowest shoot regrowth rates were found in shoot tips that were excised from ASGV+ASPV co-infected shoots.The results obtained in the present study provided technical supports for establishment of Maluscryo-bankings. Virus eradication using shoot tips from adventitious shoots and cryotherapy opened new avenues for production of virus-free plants. Cryotherapy for successful ASPV eradication suggested cryopreservation could simultaneously achieve two goals: virus eradication and long-term preservation of plant germplasm. Differences in recovery of cryopreserved shoot tips produced by the healthy and virus-infected stock shoots emphasized the importance of use of healthy plant material for cryopreservation. |
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