摘要
灰毡毛忍冬(Lonicera macranthoides Hand.-Mazz)又名拟大花忍冬、大银花等,是忍冬科忍冬属多年生半常绿小灌木植物,其花蕾绿原酸含量在全国各种药用忍冬植物中最高,是湖南、重庆、贵州等地药用忍冬植物生产的主栽品种。虽然我国药用忍冬植物产业取得了长足的发展,但是,仍存在着良种缺乏,品种混杂,干花产量和药用活性成份绿原酸含量低,繁殖技术落后,育苗周期长,繁殖系数小等系列问题,从而制约了我国药用忍冬植物产业健康可持续发展。本文以灰毡毛忍冬新品种‘金翠蕾’、‘银翠蕾’和‘白云’为研究对象,研究了灰毡毛忍冬新品种ISSR分子标记和组织培养,系统地分析了新品种组织培养增殖分化及生根过程中的内源激素、内源多胺的变化规律和调控作用,旨有效地从分子水平鉴别灰毡毛忍冬新品种,建立新品种高效组培快繁技术体系,促进我国药用忍冬植物产业又好又快发展。主要研究结果如下：
(1)灰毡毛忍冬新品种ISSR分子标记的研究。以灰毡毛忍冬叶片基因组DNA为模板,研究了退火温度、Taq DNA聚合酶的用量及模板DNA、引物、dNTPs、Mg2+浓度等6种因素对ISSR—PCR扩增的影响,建立了灰毡毛忍冬ISSR-PCR的优化反应体系和扩增程序。利用优化的反应体系,从100条ISSR引物中筛选出10条能够扩增出清晰、稳定条带的引物。以该10条引物对3个灰毡毛忍冬新品种和其它19个药用忍冬植物品种基因组DNA扩增,共扩增出108条带,其中多态性条带96条,多态性条带比率为88.9%,说明药用忍冬植物的遗传多样性丰富。并利用该体系构建3个灰毡毛忍冬新品种和其它19个药用忍冬植物品种DNA指纹图谱,可从分子水平将3个新品种与现在药用忍冬植物品种进行鉴别；利用UPGMA和主成分分析,将22个供试药用忍冬植物品种划分为忍冬种群和灰毡毛忍冬种群2个大类,每一大类又分为北方种群与南方种群。研究发现,不同药用忍冬植物品种之间的遗传差异与地理环境的差异存在相关性。
(2)灰毡毛忍冬新品种组织培养的研究。以灰毡毛忍冬新品种金翠蕾、银翠蕾和白云为研究对象,采用单因素、双因素试验和正交试验设计,开展了外植体选择、灭菌处理、基本培养基、植物激素及其配比、D-生物素浓度、活性炭浓度、培养温度、移栽基质、移栽容器、组培生根苗消毒处理、降低生产成本措施等系列试验,建立了组织培养的无菌体系,获得了适宜的初代培养基：金翠蕾和银翠蕾为MS+6-BA1.0mg·L-1+NAA0.1mg·L-1,白云则为MS+6-BA1.0mg·L-1+IBA0.1mg·L-’；筛选出适宜的继代培养基：金翠蕾为改良MS+6-BA0.5mg·L1+NAA0.1mg·L-1+D-生物素1.0mg·L-1,银翠蕾为改良MS+6-BA0.5mg·L-1+NAA0.05+D-生物素1.0mg·L-1,白云则为改良MS+6-BA1.0mg·L1+IBA0.1mg-L-1+D-生物素0.5mg·L-1,平均增殖系数4.65；研究发现适当低温有利于诱导灰毡毛忍冬组培苗生根,找到了其组培苗生根培养的适宜温度为20℃；筛选出3个品种适宜的生根培养基是1/2MS+IBA3.0mg-L-1+AC210mg·L-1,平均生根率97.4%。同时以金翠蕾叶片为外植体,诱导出了愈伤组织和不定芽,筛选出了叶片诱导愈伤组织的最佳培养基为：B5+6-BA1.0mg·L-1+2,4-D0.5mg·L-1,愈伤组织诱导率为86.7%；诱导不定芽的最佳培养基为：B5+KT0.2mg·L1+NAA1.0mg·L-1,不定芽诱导率为73,4%。研究出组培苗“塑料杯单株一步移栽”技术,适宜移栽基质为黄心土+糠壳灰+细砂(1：2：2),平均移栽达成活率达95%以上。用凉开水替代蒸馏水,白砂糖代替蔗糖配制培养基,可显著降低了组培苗生产成本,提高了灰毡毛忍冬新品种工厂化育苗的生产效益。
(3)灰毡毛忍冬新品种组培苗扦插繁殖技术的研究。以灰毡毛忍冬新品种‘金翠蕾’组培苗为试验材料,开展了不同的扦插基质、扦插时期、植物生长调节剂、ABT6浓度及浸泡时间对灰毡毛忍冬新品种组培苗扦插繁殖的影响试验,研究结果表明,扦插基质以泥炭土+珍珠岩(体积比1：3)为宜,适宜的扦插时期是5月和9月,适宜的植物生长调节剂是300mg·L-1的ABT6浸泡插穗120min,扦插成活率达95%以上,大幅度降低了灰毡毛忍冬新品种组培苗生产成本。
(4)灰毡毛忍冬新品种组织培养过程内源激素变化的研究。以金翠蕾组培苗为试验材料,分析了组培苗增殖分化、生根及愈伤组织和不定芽诱导过程的内源激素含量变化,研究了6-BA浓度对继代培养组培苗、IBA浓度对生根培养组培苗内源激素含量的影响。结果表明：①灰毡毛忍冬组培苗继代增殖分化主要受内源ZR、ABA和GA3调控,较高浓度的ZR、GA3和较低ABA有利于灰毡毛忍冬组培苗继代增殖分化,适当高浓度的IAA对丛芽增殖分化和生长也有利,但作用不明显,而较高IPA含量有利于灰毡毛忍冬外植体启动培养,但与增殖分化关系不密切。内源激素平衡关系中是GA3/ZR值主导组培苗增殖分化,较高的GA3/ZR值有利于启动组培苗增殖分化和生长,而增殖分化盛期则需要较低的GA3/ZR值, GA3/ABA值也表现了类似效应。②在灰毡毛忍冬组培苗不定根的形成过程中,不同的内源激素对不定根形成的作用不同。较高浓度内源IAA、ABA和较低浓度的内源GA3、IPA、ZR有利于根原基的分化形成,促进了组培苗不定根的形成。而较高浓度的内源IAA、GA3、ZR、IPA和较低浓度的内源ABA有利于根原基的生长发育。内源激素的平衡关系也协同参与灰毡毛忍冬组培苗根原基分化形成及生长发育。较低的(ZR+IPA)/IAA值和较高的IAA/ABA、ABA/GA3值有利于根原基的分化形成,较高(ZR+IPA)/IAA值和较低ABA/GA3值有助于根原基生长发育。③不同的内源激素在灰毡毛忍冬愈伤组织和不定芽诱导过程中的作用不同。低浓度的内源GA3、IAA、ZR、ABA有利于愈伤组织的诱导,而高浓度的内源GA3、IAA、ZR促进了愈伤组织的生长。不定芽诱导主要受内源IAA和ABA调控,低水平的内源IAA、ABA、ZR、GA3含量有助于愈伤组织分化成不定芽。
(5)灰毡毛忍冬新品种组织培养过程多胺变化的研究。以金翠蕾组培苗为试验材料,分析了组培苗增殖分化、生根过程的内源多胺含量变化,研究了6-BA浓度对继代培养组培苗、IBA浓度对生根培养组培苗内源多胺含量的影响。结果表明：①不同的内源多胺在灰毡毛忍冬组培苗增殖分化过程中有不同效应。组培苗增殖分化主要与Spd、Put含量和Put/Spm值关系密切,与Spm含量和Put/Spd值、Put/(Spd+Spm)值关系不大；高水平的Spd、Put含量和Put/Spm值有利于组培苗增殖分化的启动,而增殖分化盛期则需要维持较低水平的Put、Spd含量和Put/Spm值。②Put、Spd、Spm及其平衡关系在灰毡毛忍冬组培苗根原基的分化形成与生长发育过程中的作用各异。高水平的Put、Spm含量和Put/Spd值、Put/(Spd+Spm)值有利于组培苗根原基的分化形成,而低水平的Put、Spm含量和Put/Spd值、Put/(Spd+Spm)值则促进根原基的生长发育。低水平的Spd含量和Put/Spm值,既可有利于组培苗根原基的分化形成,又可促进根原基生长发育。
Lonicera macranthoides Hand. Mazz, also named large flower honeysuckle or large silver flower, is a perennial and semi-evergreen shrub plant and belongs to the family Caprifoliaceae. It produces the highest chlorogenic acid content among all the cultivars of honeysuckles and is the main cultivar of medicinal honeysuckle in Hunan, Chongqing and Guizhou. The honeysuckle industry in China has made considerabel development, however, problems such as lack of thoroughbred, cultivars complexity, low dried flowers yield, low chlorogenic acid content, backward in propagation technology, long cycles of seedling and low propagation coefficient still exist, which restricts the healthy and sustainable development of honeysuckle industry. In this paper, Lonicera macranthoides cv'Jincuilei','Yincuilei'and'Baiyun'has been studied, also on their ISSR molecular markers and rapid tissue culture. The change and regulatory mechanisms of endogenous hormones and polyamines of these new varieties have been systematically analyzed in the process of tissue proliferation and trooting. The purpose of the research is that new varieties of Lonicera macranthoides will be identified effectively from the molecular level, Establish a efficient tissue culture and rapid propagation system of new varieties, and thus facilitate sustainable development of honeysuckle industry in our country. The main results of the research are as follows:
(1) Research on ISSR molecular marker in new varieties of Lonicera macranthoides. Based on the genomic DNA extracted from the leaves of Lonicera macranthoides, the factors influencing ISSR were optimized and the effect of6factors such as annealing temperature, Taq DNA polymerase dosage, DNA templates concentration, primer concentration, dNTPs concentration and Mg2+concentration on ISSR amplification were tested. A reaction system and amplified procedure suitable for Lonicera macranthoides were established. Using these optimal amplification conditions,10stable and repeatable ISSR primers were selected from total100primers. The genome DNA of3new varieties of Lonicera macranthoides and19medicinal honeysuckle varieties were amplified with the10ISSR primers and108bands were amplified totally, including96polymorphism bands with polymorphism rate of88.9%, indicating medicinal honeysuckle plant has the abundant genetic diversity. DNA fingerprinting of3new varieties of Lonicera macranthoides were constructed, Three new varieties and other medicinal honeysuckle plant varieties were identified from the molecular level. The systematical cluster analysis conducted with UPGMA and PCA (principal component analysis) showed that22medicinal honeysuckle varieties could be classified into2groups, the first group is Lonicera japonica Thunb., the second one is L.macranthoides Hand. Mazz, and each group could be divided into north cultivars and south cultivars. Genetic differences between varieties of honeysuckle were correlated with their geographical differences.
(2) Research on tissue culture of new varieties of Lonicera macranthoides. Taking Lonicera macranthoides cv'Jincuilei','Yincuilei'and'Baiyun'as test materials, a serial of single factor, double factors and orthogonal tests were designed, and experiments were carried out on explant choice, germicidal treatment, preparation of minimal medium, plant growth regulators and its ratio, Biotin D concentration and activated carbon concentration, culture temperature, transplanting matrix and containers, rooted seedling disinfection, production cost reducing measures. Sterile system of tissue culture was established, and suitable initial mediums were obtained:MS+6-BA1.0mg·L-1+NAA0.1mg-L'1for 'Jincuilei'and'Yincuilei', MS+6-BA1.0mg·L-1+IBA0.1mg·L-1for'Baiyun'; suitable subculture mediums were screened out:improved MS+6-BA0.5mg·L-1+NAA0.1mg·L-1+Biotin D1.0mg·L-1for'Jincuilei', improved MS+6-BA0.5mg·L-1+NAA0.05mg·L-1+Biotin D1.0mg·L-1for'Yincuilei'and improved MS+6-BA1.0mg·L-1+IBA0.1mg·L-1+Biotin D0.5mg·L-1for'Baiyun', the average multiplication coefficient was4.65. It was found that appropriate low temperature was better for inducing Lonicera macranthoides tissue culture seedlings rooting, and the preference temperature is20℃. Suitable rooting mediums for the three varieties were screened out:1/2MS+IBA3.0mg·L-1+activated carbon200mg·L-1, the average rooting rate was97.4%. Meanwhile, using leaves of sterilized'Jincuilei'tissue culture seedlings as test material, callus and adventitious buds were induced. The optimum medium for leaves callus and adventitious buds inducing was B5+6-BA1.0mg·L-1+2,4-D0.5mg·L-1and B5++KT0.2mg·L-1+NAA1.0m·L-1, respectively, and the inductivity was86.7%and73%, respectively. The technique of "single plant one-step transplanting in plastic cups" was studied out and the suitable transplanting matrix was a mixture of yellow soil, rice hull ash and silver sand with the ratio of1:2:2, the average transplanting survival rate reached95%. Production cost can be significantly reduced when using plain boiled water and white granulated sugar instead of distilled water and sucrose, which increased productivity effect of factory nursery of Lonicera macranthoides new varieties.
(3) Research on cutting propagation technique of Lonicera macranthoides new varieties bred by tissue culture seedlings. Using Lonicera macranthoides'Jincuilei'as test material, experiments were carried out to study on the influence to propagation of Lonicera macranthoides new varieties bred by tissue culture seedlings with different cutting propagation matrix, cutting propagation period, plant growth regulators, concentration of ABT6and soak period. The results showed that the proper cutting propagation matrix was a mixture of peat soil and pearlite with the ratio of1:3at volume, the appropriate cutting propagation period was in May and September, the suitable plant growth regulator was300mg·L-1ABT6in what the shoots soak for120min. The average survival ratio of cutting propagation reached95%, which substantially reduced the production cost of tissue culture of Lonicera macranthoides new varieties.
(4) Research on endogenous hormones changes in process of tissue culture of Lonicera macranthoides new varieties. Using Lonicera macranthoides'Jincuilei'in vitro seedlings as test material, the endogenous hormones content changes were analyzed during in vitro seedling proliferation and differentiation, rooting and callus and adventitious buds inducing, and the influences of6-BA concentration on in vitro seedling subculture and IBA on rooting were also studied. The results showed that:①Subculture proliferation and differentiation of Lonicera macranthoides was mainly regulated and controlled by endogenous ZR, ABA and GA, and was benefited by a higher concentration of ZR and GA and lower concentration of ABA. An appropriate high concentration of IAA also indistinctively benefits proliferation and differentiation of plexus buds. A higher content of IPA is positively related to explant cultivation initiation of Lonicera macranthoides,but less closely with proliferation and differentiation. Higher GA/ZR value is dominant in endogenous hormones equilibrium relation, which benefits seedling cultivation proliferation, differentiation and growth initiation. However, it needs lower GA/ZR value in flourishing period, as well as GA/ABA value.②Endogenous hormones play different roles in the process of adventitious roots formation and growth of Lonicera macranthoides. The former is positively related by a high concentration of IAA, ABA and a lower concentration of GA, IP A, ZR, yet the latter is positively by a high concentration of IAA, GA, ZR, IP A and a lower concentration of ABA. Endogenous hormones equilibrium relation also takes part in the process of adventitious roots formation and growth of Lonicera macranthoides synergeticly. A lower (ZR+IPA)/IAA value and a higher IAA/ABA and ABA/GA value benefits their formation, and a higher (ZR+IPA)/IAA value and a lower ABA/GA value benefits their growth.③Endogenous hormones play different roles in the process of formation and growth of callus and adventitious buds induction of Lonicera macranthoides. Low concentration of GA, IAA, ZR, ABA is positively related to callus inducing, which is benefited by high concentration of GA、IAA and ZR. Adventitious buds inducing is mainly regulated by endogenous IAA and ABA, and low level content of endogenous IAA、ABA、ZR and GA promotes the formation of adventitious buds from callus.
(5) Research on polyamines change in the process of tissue culture of Lonicera macranthoides new varieties. Using Lonicera macranthoides'Jincuilei'in vitro seedlings as test material, the polyamines content changes were analyzed during in vitro seedling proliferation,differentiation and rooting, and the influences of6-BA concentration on polyamines content during in vitro seedling subculture and of IBA concentration on polyamines content during in vitro seedling rooting were studied. The results showed:①Endogenous polyamines plays different roles in the process of in vitro seedling proliferation and differentiation of Lonicera macranthoides, which is positively related by Spd and Put content and Put/Spm value, but less closely with Spm content, Put/Spd value and Put/(Spd+Spm) value. A highter Spd and Put content, Put/Spd value benefits proliferation and differentiation and growth initiation of in vitro seedling, However, it needs a lower Put and Spd content and Put/Spm value in flourishing period.②Put、Spd、 Spm content and their equilibrium relation play different roles in the process of in vitro seedling root primordium proliferation and differentiation of of Lonicera macranthoides. A high level of Spd and Put content, Put/Spm value and Put/(Spd+Spm) value benefits their differentiation and formation, yet a low level of those boost their growth. A low level of Spd content and Put/Spm value is not only positively related to differentiation of seedling cultivation root primordium, but also to their growth.
引文
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