苹果属（Malus）试管苗植株再生、茎尖超低温保存和脱毒技术研究

发布时间：2018-03-24 07:38

  本文选题：苹果属　切入点：超低温技术　出处：《西北农林科技大学》2016年博士论文

【摘要】：苹果是全球第二大果树。中国是世界最大的苹果生产国。苹果属种质资源是苹果传统育种和基因工程育种的基础。超低温保存被认为是长期保存植物种质资源最理想的方法。病毒病是制约苹果可持续发展的重要因素,栽培无毒苗木是目前生产上防止病毒病害的有效方法,培育无毒苗是栽培无毒苗的前提条件,建立高效的脱毒技术是培育无毒苗的关键技术。本研究旨在:1.建立广谱、高效的苹果叶片不定芽再生技术体系;2.利用叶片再生不定芽的茎尖建立苹果超低温保存技术体系;3.利用叶片再生不定芽的茎尖培养和茎尖超低温疗法脱除苹果病毒,并揭示不同脱毒技术的脱毒机理;4.研究不同带毒状况对苹果试管苗营养生长、生理代谢及茎尖超低温保存的影响。获得的主要结果如下:1.以‘Gala’和‘Fuji’两个品种及‘M9’和‘M26’两个砧木为试材,取4周苗龄的试管苗顶端1-3片完全展开叶,培养在含有不同浓度的Thidiazuron(TDZ)和0.5mg L-1 Indole-3-Butytric acid(IBA)组合的再生培养基上,22±2 oC暗培养3周后,转到光照下培养,8周后的再生结果表明,‘Gala’和‘Fuji’的最佳再生培养基为2 mg L-1TDZ+0.5 mg L-1 IBA和2-3 mg L-1 TDZ+0.5 mg L-1 IBA,再生率为100%,‘Gala’的不定芽数/外植体为6.4,‘Fuji’为2.9-3.1。‘M9’和‘M26’的最佳再生培养基为3 mg L-1 TDZ+0.5-1.5 mg L-1 IBA,再生率为100%,‘M9’的不定芽数/外植体为3.8-4.7,‘M26’为4.2-4.6。将叶片再生技术用于测试苹果属另外3个种的5个基因型,所有测试基因型均可获得100%的再生率和0.6-8.7的不定芽数/外植体(平均数为4.5)。Inter-simple sequence repeat(ISSR)检测‘Gala’不定芽再生植株未发现异常条带。2.以‘Gala’为试材,从11周龄的叶片不定芽取3 mm(带6片叶原基)的茎尖,用3%的硅藻酸钠在0.1M的氯化钙溶液中包埋茎尖成小球(直径约5 mm),将小球在含有0.5M蔗糖的培养基上预培养5天后,超净台中空气干燥6h,置入冷冻管(10个小球/冷冻管),直接投入液氮保存,水浴(38oC)解冻后2 min后,培养在含有0.25 mg L-1BA+0.01 mg L-1 IBA的再生培养基上再生植株。超低温保存后茎尖的再生率为79.3%。将该技术用于测试苹果属4个种的8个基因型。结果表明,除‘Greensleeves'外,其他7个基因型的再生率分别为:‘Fuji’62.5%,‘Himekami’27.5%,‘Wangshanhong’43.8%,‘M9’74.0%,‘M26’52.5%,‘Pingdinghaitang’50.0%,‘Balenghaitang’66.3%。利用ISSR检测‘Gala’超低温保存再生植株未发现异常条带。3.以‘Gala’为试材,在叶片不定芽再生的不同时期取不同大小的茎尖进行培养。结果表明,不定芽发育时期及茎尖大小对成活率没有明显影响,但会影响再生率和ASPV的脱毒率。不定芽再生2-3周时,0.3 mm的茎尖(含2片叶原基)的再生率为10-15%,ASPV脱除率为100%;不定芽再生3-4周时,0.3 mm的茎尖(含3片叶原基)的再生率为53-55%,ASPV脱除率为95-100%;不定芽再生4周时,0.4 mm的茎尖(含4片叶原基)的再生率为82%,但ASPV脱除率仅为20%。不定芽再生的各时期,任何茎尖大小都不能脱除ASGV。组织学观察和免疫组织化学病毒定位结果解释了叶片不定芽再生不同时期及不同大小的茎尖培养能脱除ASPV,不能脱毒ASGV的原因。4.以‘M9’和‘M26’试管苗顶芽为试材,研究了茎尖培养和超低温疗法脱除ASPV和ASGV的效果。茎尖培养时,茎尖大小与再生率成正比,与ASPV脱毒率成反比,任何茎尖大小都不能脱除ASGV。茎尖超低温疗法时,0.5 mm的茎尖(含2片叶原基)不能再生,1.5 mm的茎尖(带5-6个叶原基)的再生率明显高于1.0 mm(带3-4个叶原基)的茎尖,两种大小茎尖对ASPV的脱毒率相似(80-85%)。无论茎尖大小,都不能脱除ASGV。组织学观察和免疫组织化学病毒定位结果揭示了超低温疗法能有效脱除ASPV,而不能脱毒ASGV的机理。Flow cytometry(FCM)对超低温疗法的脱毒苗的鉴定没有发现DNA倍性的差异。5.以‘Gala’为试材,比较了无毒、单感(ASGV)和双感(ASGV+ASPV)试管苗在营养生长、生理代谢及茎尖超低温保存的差异。结果表明,带毒试管苗萌芽时间长、茎增殖数增多、株高降低、生长量减少(鲜重增长量和干重)、叶绿素含量降低、电解质渗出率、可溶性糖含量、可溶性蛋白含量及脯氨酸含量增加。茎尖超低温保存后的成活率和再生率与取茎尖的茎长度相关。其中,取自≥1 cm及1 cm,≥0.5 cm茎的无毒与带毒茎尖的成活率没有明显差异,但取自单感ASGV和双感(ASGV+ASPV)0.5 cm茎的茎尖的成活率和再生率明显降低。无毒茎(≥1 cm和1 cm,≥0.5 cm)的茎尖再生率没有明显差异;单感(ASGV)材料茎尖的再生率显著低于相同长度无茎尖的再生率;相同长度双感(ASGV+ASPV)茎尖再生率最低。本研究获得的结果为建立苹果超低温种质资源超低温库搭建了技术平台;叶片不定芽茎尖培养和茎尖超低温疗法脱毒为苹果脱毒开辟了新的途径,超低温疗法的脱毒效应表明超低温技术可以同时实现种质资源保存和脱除病原菌;首次强调材料的带毒状况是影响茎尖超低温保存的重要因素,为使用无毒材料进行超低温保存提供了实验证据。
[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鐨勮寧灏，

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