本文選題：蘋果屬　切入點(diǎn)：超低溫技術(shù)　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：蘋果是全球第二大果樹。中國是世界最大的蘋果生產(chǎn)國。蘋果屬種質(zhì)資源是蘋果傳統(tǒng)育種和基因工程育種的基礎(chǔ)。超低溫保存被認(rèn)為是長期保存植物種質(zhì)資源最理想的方法。病毒病是制約蘋果可持續(xù)發(fā)展的重要因素,栽培無毒苗木是目前生產(chǎn)上防止病毒病害的有效方法,培育無毒苗是栽培無毒苗的前提條件,建立高效的脫毒技術(shù)是培育無毒苗的關(guān)鍵技術(shù)。本研究旨在:1.建立廣譜、高效的蘋果葉片不定芽再生技術(shù)體系;2.利用葉片再生不定芽的莖尖建立蘋果超低溫保存技術(shù)體系;3.利用葉片再生不定芽的莖尖培養(yǎng)和莖尖超低溫療法脫除蘋果病毒,并揭示不同脫毒技術(shù)的脫毒機(jī)理;4.研究不同帶毒狀況對(duì)蘋果試管苗營養(yǎng)生長、生理代謝及莖尖超低溫保存的影響。獲得的主要結(jié)果如下:1.以‘Gala’和‘Fuji’兩個(gè)品種及‘M9’和‘M26’兩個(gè)砧木為試材,取4周苗齡的試管苗頂端1-3片完全展開葉,培養(yǎng)在含有不同濃度的Thidiazuron(TDZ)和0.5mg L-1 Indole-3-Butytric acid(IBA)組合的再生培養(yǎng)基上,22±2 oC暗培養(yǎng)3周后,轉(zhuǎn)到光照下培養(yǎng),8周后的再生結(jié)果表明,‘Gala’和‘Fuji’的最佳再生培養(yǎng)基為2 mg L-1TDZ+0.5 mg L-1 IBA和2-3 mg L-1 TDZ+0.5 mg L-1 IBA,再生率為100%,‘Gala’的不定芽數(shù)/外植體為6.4,‘Fuji’為2.9-3.1。‘M9’和‘M26’的最佳再生培養(yǎng)基為3 mg L-1 TDZ+0.5-1.5 mg L-1 IBA,再生率為100%,‘M9’的不定芽數(shù)/外植體為3.8-4.7,‘M26’為4.2-4.6。將葉片再生技術(shù)用于測(cè)試蘋果屬另外3個(gè)種的5個(gè)基因型,所有測(cè)試基因型均可獲得100%的再生率和0.6-8.7的不定芽數(shù)/外植體(平均數(shù)為4.5)。Inter-simple sequence repeat(ISSR)檢測(cè)‘Gala’不定芽再生植株未發(fā)現(xiàn)異常條帶。2.以‘Gala’為試材,從11周齡的葉片不定芽取3 mm(帶6片葉原基)的莖尖,用3%的硅藻酸鈉在0.1M的氯化鈣溶液中包埋莖尖成小球(直徑約5 mm),將小球在含有0.5M蔗糖的培養(yǎng)基上預(yù)培養(yǎng)5天后,超凈臺(tái)中空氣干燥6h,置入冷凍管(10個(gè)小球/冷凍管),直接投入液氮保存,水浴(38oC)解凍后2 min后,培養(yǎng)在含有0.25 mg L-1BA+0.01 mg L-1 IBA的再生培養(yǎng)基上再生植株。超低溫保存后莖尖的再生率為79.3%。將該技術(shù)用于測(cè)試蘋果屬4個(gè)種的8個(gè)基因型。結(jié)果表明,除‘Greensleeves'外,其他7個(gè)基因型的再生率分別為:‘Fuji’62.5%,‘Himekami’27.5%,‘Wangshanhong’43.8%,‘M9’74.0%,‘M26’52.5%,‘Pingdinghaitang’50.0%,‘Balenghaitang’66.3%。利用ISSR檢測(cè)‘Gala’超低溫保存再生植株未發(fā)現(xiàn)異常條帶。3.以‘Gala’為試材,在葉片不定芽再生的不同時(shí)期取不同大小的莖尖進(jìn)行培養(yǎng)。結(jié)果表明,不定芽發(fā)育時(shí)期及莖尖大小對(duì)成活率沒有明顯影響,但會(huì)影響再生率和ASPV的脫毒率。不定芽再生2-3周時(shí),0.3 mm的莖尖(含2片葉原基)的再生率為10-15%,ASPV脫除率為100%;不定芽再生3-4周時(shí),0.3 mm的莖尖(含3片葉原基)的再生率為53-55%,ASPV脫除率為95-100%;不定芽再生4周時(shí),0.4 mm的莖尖(含4片葉原基)的再生率為82%,但ASPV脫除率僅為20%。不定芽再生的各時(shí)期,任何莖尖大小都不能脫除ASGV。組織學(xué)觀察和免疫組織化學(xué)病毒定位結(jié)果解釋了葉片不定芽再生不同時(shí)期及不同大小的莖尖培養(yǎng)能脫除ASPV,不能脫毒ASGV的原因。4.以‘M9’和‘M26’試管苗頂芽為試材,研究了莖尖培養(yǎng)和超低溫療法脫除ASPV和ASGV的效果。莖尖培養(yǎng)時(shí),莖尖大小與再生率成正比,與ASPV脫毒率成反比,任何莖尖大小都不能脫除ASGV。莖尖超低溫療法時(shí),0.5 mm的莖尖(含2片葉原基)不能再生,1.5 mm的莖尖(帶5-6個(gè)葉原基)的再生率明顯高于1.0 mm(帶3-4個(gè)葉原基)的莖尖,兩種大小莖尖對(duì)ASPV的脫毒率相似(80-85%)。無論莖尖大小,都不能脫除ASGV。組織學(xué)觀察和免疫組織化學(xué)病毒定位結(jié)果揭示了超低溫療法能有效脫除ASPV,而不能脫毒ASGV的機(jī)理。Flow cytometry(FCM)對(duì)超低溫療法的脫毒苗的鑒定沒有發(fā)現(xiàn)DNA倍性的差異。5.以‘Gala’為試材,比較了無毒、單感(ASGV)和雙感(ASGV+ASPV)試管苗在營養(yǎng)生長、生理代謝及莖尖超低溫保存的差異。結(jié)果表明,帶毒試管苗萌芽時(shí)間長、莖增殖數(shù)增多、株高降低、生長量減少(鮮重增長量和干重)、葉綠素含量降低、電解質(zhì)滲出率、可溶性糖含量、可溶性蛋白含量及脯氨酸含量增加。莖尖超低溫保存后的成活率和再生率與取莖尖的莖長度相關(guān)。其中,取自≥1 cm及1 cm,≥0.5 cm莖的無毒與帶毒莖尖的成活率沒有明顯差異,但取自單感ASGV和雙感(ASGV+ASPV)0.5 cm莖的莖尖的成活率和再生率明顯降低。無毒莖(≥1 cm和1 cm,≥0.5 cm)的莖尖再生率沒有明顯差異;單感(ASGV)材料莖尖的再生率顯著低于相同長度無莖尖的再生率;相同長度雙感(ASGV+ASPV)莖尖再生率最低。本研究獲得的結(jié)果為建立蘋果超低溫種質(zhì)資源超低溫庫搭建了技術(shù)平臺(tái);葉片不定芽莖尖培養(yǎng)和莖尖超低溫療法脫毒為蘋果脫毒開辟了新的途徑,超低溫療法的脫毒效應(yīng)表明超低溫技術(shù)可以同時(shí)實(shí)現(xiàn)種質(zhì)資源保存和脫除病原菌;首次強(qiáng)調(diào)材料的帶毒狀況是影響莖尖超低溫保存的重要因素,為使用無毒材料進(jìn)行超低溫保存提供了實(shí)驗(yàn)證據(jù)。
[Abstract]:Apple is the world's second largest fruit trees. China is the world's largest apple producer. Apple germplasm resources is the basis of the traditional apple breeding and genetic engineering breeding. Cryopreservation is considered to be the most ideal method for long-term preservation of plant germplasm resources. Virus disease is an important factor restricting the sustainable development of apple, cultivation of virus-free seedlings is effective the current production to prevent virus diseases, cultivating seedlings cultivation condition is non-toxic virus-free plants, establish efficient detoxification technology cultivation is the key technology of virus-free plants. This study is to: 1. to establish a broad spectrum, apple leaves, adventitious bud regeneration system; 2. the regeneration of adventitious buds from stem tip to establish the apple cryopreservation system; 3. the regeneration of adventitious buds from shoot tip culture and cryotherapy for removal of apple virus, and to reveal the detoxification mechanism of detoxification technology; 4.. The different growth status of nutrition with poison apple plantlets, influencing physiological metabolism and cryopreservation of shoot tips. The main results are as follows: 1. with the 'Gala' and 'Fuji' two varieties and 'M9' and 'M26' two rootstock as test materials, take 4 weeks old seedlings at the top 1-3 fully expanded leaf, cultured in containing different concentrations of Thidiazuron (TDZ) and 0.5mg L-1 Indole-3-Butytric acid (IBA) combination of regeneration medium, after 3 weeks of 22 + 2 oC dark culture, to light training, 8 weeks after regeneration. The results showed that "the best regeneration medium and Gala." "Fuji" is 2 mg L-1TDZ+0.5 mg L-1 IBA mg L-1 TDZ+0.5 mg L-1 and 2-3 IBA, the regeneration rate was 100%, the 'Gala' maximum number of shoots / explants was 6.4, 'Fuji' is the best regeneration of 2.9-3.1. 'M9' and 'M26' medium was 3 mg L-1 TDZ+0.5-1.5 mg L-1 IBA. The regeneration rate was 100%, the 'M9' The number of adventitious buds / explants for 3.8-4.7, "M26" 4.2-4.6. will be used to test the apple leaf regeneration technology belongs to the other 3 kinds of 5 genotypes, all tested genotypes can be obtained and the regeneration rate of 100% 0.6-8.7 / number of adventitious bud explants (average of 4.5).Inter-simple sequence repeat (ISSR) detection of 'Gala' adventitious bud regeneration found no abnormal bands.2. to 'Gala' as test materials, 3 mm from the leaves of 11 week old adventitious buds (with 6 leaf primordia) of stem apex, stem tip Cheng Xiaoqiu entrapped in calcium chloride solution 0.1M with acid sodium (diameter 3% diatom about 5 mm), the ball in a medium containing 0.5M sucrose pre cultured for 5 days, clean air drying 6h in Taichung, frozen tube (10 balls / frozen tube), directly into liquid nitrogen preservation, water bath (38oC) after thawing after 2 min culture medium containing 0.25 mg in regeneration regeneration L-1BA+0.01 mg L-1 IBA Plants. After cryopreservation of Shoot Tip Regeneration rate of 79.3%. for the test of apple is 4 kind of 8 genotypes. The results showed that in addition to "Greensleeves', the regeneration rate of the other 7 genotypes were as follows: 62.5%" Fuji "," Himekami "27.5%" Wangshanhong "43.8%." M9 "74%" M26 "52.5%, 50%" Pingdinghaitang "," Balenghaitang "66.3%." Gala "ISSR is used to detect the cryopreservation of regenerated plants found no abnormal bands of.3. in' Gala 'as test materials, different sizes in leaves of different period of adventitious bud regeneration of shoot tip culture. The results showed that no during the development of adventitious buds and shoot tip size has no obvious effect on the survival rate, but will affect the regeneration rate of ASPV and the virus-free rate of adventitious bud regeneration. At 2-3 weeks, 0.3 mm shoot tips (including 2 leaf primordia) the regeneration rate was 10-15%, ASPV removal rate was 100%; adventitious bud regeneration at 3-4 weeks ,0.3 mm鐨勮寧灝，

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