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观赏植物大岩桐、特色经济作物红花组织培养再生体系的建立及百合遗传转化影响因素的初探
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摘要
花卉业是现代农业的一部分,随着社会物质和精神生活水平的提高,人们对花卉的需求日益增加,对花卉质量的要求也越来越高。在我国,大多数的观赏植物均采用常规方法繁殖,如种子繁殖、扦插等方法,繁殖系数低、苗木质量差、繁殖周期长、品种推广慢,且受季节和自然发育的限制,严重阻碍了花卉业在我国的迅速发展。植物组织培养技术具有独特的优势,不仅能够快速繁殖一些优良品种并进行脱毒,而且为定向改良花卉的某个目标性状提供了技术平台及前提基础。
     新疆是中国荒漠化大区,也是中国最大的盐土区,盐渍面积达1100万公顷,约占全国盐渍土面积的三分之一和新疆土地面积的6.6%,现有耕地三分之一次生盐渍化,轻度、中度盐渍地减产10%~50%,重度盐渍地颗粒无收,成为农业低产的主要原因,严重的阻碍了新疆农业经济的发展。
     随着分子遗传学和植物转基因技术的发展,利用生物技术提高观赏植物的抗逆性,使观赏植物在非生物胁迫环境中能保持优良品性并正常生长,实现南花北移,为充分利用新疆土地资源及新疆绿化建设做贡献。不仅大大丰富了北方植物的种类,还为花卉的规模化生产节约了能源。
     NHX(Na+/H+ antiporter)为液泡膜中的一种Na+/H+反向运输体可促进离子在液泡中的分室效应,跨液泡膜的pH为其提供能量。钠离子和氯离子分室在液泡中,不仅可作为有效的渗透调节剂,同时还可减小细胞毒性。通过在功能上恢复Na+/H+反向运输体(ScNHX1)缺陷型酵母突变体,从拟南芥中分离出了AtNHX1基因,并且与哺乳动物NHE反向运输体具有序列相似性。在转基因拟南芥、油菜和转基因西红柿中过量表达AtNHX1,可在液泡膜中积累大量的运输体,并且极大地提高了它们的耐盐性。
     灰绿藜是新疆重要的盐生植物。将克隆的灰绿藜NHX基因利用农杆菌介导法转化组织培养成功的观赏植物中,希望培育出即能保持原有优良性状同时又具有耐盐性的新品种,不仅为改良新疆土壤及促进新疆产业经济的发展做出贡献,而且为进一步改良观赏植物的花色、花香等提供了基础。
     本研究通过植物组织培养的方式,建立了观赏植物大岩桐的组织培养再生体系,对再生过程中材料的灭菌方式、培养条件、激素组合、移栽与苗期管理等影响因素进行了详细探讨,获得了完整的优化条件,为观赏植物的产业化和遗传转化奠定了坚实的基础;同时建立了新疆特色经济作物红花的组织培养再生体系,探索了影响红花种子萌发影响因素,愈伤组织的诱导,不定芽的诱导几个因素,通过愈伤组织获得完整植株,为红花的良种快速繁殖和遗传转化等提供参考。
     本研究还建立了畅销切花百合金康卡和索帮的组织培养再生体系,通过对影响两个品种再生过程中诸因素的探讨和比较,如光照,激素配比,生根,炼苗等,确定了最优再生体系,为遗传转化改良性状奠定了坚实的物质基础。选取杂交品种金康卡百合,利用农杆菌介导法进行耐盐基因NHX的遗传转化,针对影响遗传转化效率的几个因素,如是否预培养,共培养方式,浸染菌液浓度,浸染时间,抗生素临界值浓度等,进行初步探索。
Flower industry is a proportion of agriculture.With the development of level of living,the need of flower gets increasing,the requirement of flower quality gets higher and higher.In our country , most of Ornamental are reproduced by conventional methods,such as seed graft,separate seedling etc,reproducibility frequency is low,quality of seedling is not good,popularization of variety is slow,and is easy to be limited by season and natural condition.These factors clag the rapid development of flower industry Plant tissue culture technique has unique advantage,not only it can reproduce some good breeds rapidly,but also it provides technical flat and basis for improving some character of flower directionally.
     Xinjiang has the biggest poor soil area and salty region in China.11million hectares of salinity area occupy 1/3 of whole soil in the China and 6.6% of land area of Xinjiang.Currently there has 1/3 secondary salty soil of whole cultivated land in Xinjiang,the slight and middle degree of salty land would reduce output of 10%~50% and the heavy saltinity soil almost harvest nothing.This is the major cause of low yield and severe block of the economic development in Xinjiang.
     Advances in molecular genetics and plant transformation have made it feasible to assess biotechnological strategies in improving the adversity tolerance of ornamental,keeping steady-character and keeping the growth normally in the adversity environment.It carry out that southern flower is transplanted to north,it is contributed to make use of soil and virescence building in Xinjiang.Not only enrich the kinds of northern plants,but also economize energy sources for scale production.
     NHX1,a tonoplast Na+/H+ antiporter energized by the ?pH across the tonoplast,facilitates vacuolar compartmentalization of the cation.As a fundamental mechanism in salt tolerance,an active antiporter would function to sequester Na+ into the vacuole,which results inavoidance of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel,vacuolar In parallel,vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis.Overexpressing AtNHX1 can accumulate the large numbers of antiporter in the transgenic Arabadopsis,oilseed rape and tomato,at the same time improve their salt tolerance.
     Suaeda is a kind of important halophyte.Mediated by Agrobacterium tumefacines method,salt-tolerant gene NHX1 from Suaeda was transformed into ornamental,The goal is to develop salt-resistant variety which retains original good characters.Not only it is contributed to improve soil in Xinjiang,as well as accelerate industrial economic development 0f Xinjiang,but also provide basis for improving colour and potpourri of flower.
     In this study,we established regeneration System of Ornamental SinningI aspeciosa,and discussed the effect factors detailedly,such as sterilize methods,cultivate conditions,hormone compounding transplant and nursery manage etc,providing basisfor industry and genetic transformation of Ornamental.But also,we established regeneration system of special economic crop safflower,discussing the effect factors detailedly,such as seed germination,calli inducing,shoots inducing etc,providing the reference for well-bred reproducement rapidly and genetic transformation.
     In this study ,we also established regeneration System of Ornamental Conca .d and Siberia ,discussing and compare some effect factors between the two variety such as illumination,hormone compounding,rooting,transplant etc,make sure optim -zation system,providing the basis for genetic improvement.
     We choosed cross bred Conca .d lilium as material and carry genetic tasnsform -ation of NHX gene mediated by Agrobacterium tumefacines method.aiming at seve -ral key factors in genetic tasnsformation,such as precultivate,cocultivate method,ba cterium liquid concentration,immerge time,antibiotic critical value etc.
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