药用石蒜不同居群遗传多样性及驯化繁育技术
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摘要
本研究针对能提取加兰他敏(该药用于治愈阿尔茨海默病有明显疗效)的石蒜(Lycoris radiata)进行野生种质资源收集,运用现代分子标记技术(ISSR),对来源于9个省22个不同居群进行遗传多样性分析,在不同树种套种林下进行驯化培育,引用灰色关联度评价体系分析生长、生理、生物量、营养成分和加兰他敏含量之间的关联度及其与微环境因子的关联度,建立无性繁殖技术体系,开展优质资源初步选育等工作。取得以下主要研究成果:
(1)石蒜野生居群种质资源开展基础研究,发现来自亚热带南部的居群生长节律期明显早于来自中北部居群,南部居群的繁殖系数、子鳞茎质量也均优于中北部居群,所有居群繁殖系数均值为5.81,子鳞茎质量均值为1.64 g。子鳞茎5个生物学性状相比,根系数量差异最大(3.42~7.98根),其次是高度差异(1.27~2.01 cm ),再次是质量差异(1.33~1.94 g ),最后是直径差异(1.03~1.23 cm )。初步选育YL16、YL13、YL22等3个加兰他敏接近或超过300μg·g~(-1)居群,建议作为一类优质资源开发利用。
利用石蒜叶片基因组DNA为模板,建立其ISSR分析的最优化反应体系及应用程序:25μL反应体系中,有20 ng模板DNA、0.5μmol·L~(-1)随机引物、150μmol·L~(-1) dNTPs、2.0mmol·L~(-1) Mg2+、1.0 U Taq DNA聚合酶。反应程序为:94℃预变性300 s ;然后45个循环:每个循环94℃变性45s,55℃退火60s,72℃延伸120s;循环结束后72℃延伸420s。利用ISSR分子标记技术对14个野生石蒜居群进行遗传多样性分析,结果表明:物种遗传多样性很高,多态位点百分率为92.31%,Shannon指数h为0.4597,Nei指数I为0.3025;居群水平遗传多样性较低,多态位点百分率平均为49.65%,Shannon指数h平均为0.2620,Nei指数I平均为0.1763;居群间的遗传分化系数Gst为0.5035,基因流Nm为0.6983。而AMOVA分子变异分析显示:居群间遗传分化程度高,46.12%的变异发生在居群内,53.88%的变异发生在居群间。生境的片段化使居群间的基因流受阻,可能是居群间高遗传分化和居群水平低遗传多样性的主要原因。
(2)建立石蒜与3种落叶树种、1种杜英套种驯化培育试验。套种引起微环境中光照效应、气温效应、湿度效应、土壤水分和土壤养分等存在差异。套种林下光照强度日变化曲线为抛物线,林下东西部测点分别在11点、13点最大值,同一树种林下不同测点光照强度曲线呈U状图;气温曲线为单峰抛物线,最高值在午后1:00,同一树种林下不同测点气温曲线呈小U型;不同树种套种林下不同测点湿度日变化曲线类似于字母U型。土壤含水量差异如下:杜英(25.004%)>栾树(20.997%)>重阳木(19.469%)>桤木(18.796%);土壤养分有不同程度的提高,提升的幅度依次为:速效P(-23.53~100%)>水解N(2.73~56.21%)>有机质(6.59~41.07%)>速效K(2.37~31.62%)。
微环境对石蒜生长影响相当复杂。套种林下石蒜叶片叶绿素在不同生长发育季节为动态变化过程,但不同测点值基本平稳(1.84~2.16 mg·g~(-1)),叶绿素a/b的比值在1.38~2.05之间变化;套种构成的荫蔽程度差异表现在叶绿素荧光参数FV/FM(均值0.793)明显高于全光照下(0.779),其中重阳木林下、栾树林下、桤木林下、杜英林下的FV/FM分别是对照的101.54%、101.71%、101.71%和102.48%。套种在栾树下干、鲜生物量最大;套种后加兰他敏比对照提高,由高到低排序依次是:杜英(21.39μg·g~(-1))>桤木(16.61μg·g~(-1))>重阳(14.80μg·g~(-1) >栾树(13.53μg·g~(-1))。年度生长发育期内加兰他敏出现二个高峰(休眠中期7月97.536μg·g~(-1)和叶旺盛生长11月51.6343μg·g~(-1))、二个低峰(休眠初期4月6.3277μg·g~(-1)和花后的8月22.9554μg·g~(-1))。加兰他敏含量从第1年的5.6475μg·g~(-1)上升第5年的116.2253μg·g~(-1),第3~5年含量是快速增长期,建议加强后期管理,减少鳞茎自然分蘖,在休眠中期7月进行资源采集和制药提取。
通过对石蒜的生长性状、生理指标、生物量、营养成分与环境因子关联度分析,发现影响以上指标的主导环境因子存在差异性,但发现相对湿度和土壤水分是制约其生长的关键环境因子。对石蒜生长指标与环境因子回归分析,发现叶面积、鳞茎高度、根鲜重、鳞茎鲜重、鳞茎干重、蛋白质、可溶性糖、淀粉及还原糖与环境因子回归显著水平均低于0.05,相关性显著;叶鲜重、叶干重、根干重、第三年度鳞茎加兰他敏含量与环境因子回归显著水平分别均低于0.01,相关性极显著,建立的回归方程均可用;鳞茎宽度、第二年度鳞茎加兰他敏含量与环境因子回归显著水平大于0.05,相关性不显著,建立的回归方程不可用。经对石蒜性状之间的关联度分析,发现不同性状均有与其密切相关的性状,根据一个性状与其他性状关联度可推测其他性状特点,为石蒜优质资源培育提供一定技术指导。
(3)田间实验设计3种施肥措施能增加石蒜的干、鲜物质。肥料种类对增加鳞茎体积由好到差排序:钾肥>复合肥>氮肥。影响石蒜生物产量构成因素效果的肥料种类排序也是钾肥>氮肥>复合肥,实践中发现钾肥连续施用导致鳞茎自然分蘖增多,可提供制药原料的鳞茎数量减少。
盆栽试验发现钾肥能提高根系活力、根系CEC差异和加兰他敏含量,三者年度变化曲线十分相似。增施钾肥0.3 g·kg~(-1)、0.6 g·kg~(-1),鳞茎的根系活力、根系CEC差异和加兰他敏含量比对照均有小幅增长;增施钾肥0.9 g·kg~(-1)时,鳞茎的根系活力、根系CEC差异和加兰他敏含量均达到最大值(97.35 ug·g~(-1)h ~(-1)、9.67 cmol·kg~(-1)、276.4054μg·g~(-1));施钾肥继续增长到1.2 g·kg~(-1)时,上述指标反而降低。
(4)石蒜自然繁殖系数为2.495~2.656。在不同生长发育期进行无性繁殖,繁殖系数呈现双高峰曲线图,其值则在0.80~6.8之间变动;欲获得较高无性繁殖系数,建议在休眠期的6月、7月及初叶期的9月繁殖;欲获得较大质量的子鳞茎宜在初叶期的9月和落叶期的4月繁殖;欲获得横径较大的子鳞茎宜在6、7、9和4月份繁殖;在3~7月和9月进行繁殖,获得子鳞茎的根系发育良好。中国石蒜(L.chinensis)母鳞茎个体之间繁殖系数差异从1~19个不等,繁殖系数均值为7.09,母鳞茎质量不能作为挑选作为获得繁殖系数较多的标志。
通过对石蒜离体培养诱导胚性愈伤组织及其分化。优化培养条件后建立起高效的离体再生繁殖体系。结果表明:改良MS+BA 5 mg·L~(-1)+NAA 3 mg·L~(-1)组合胚性愈伤组织诱导率最高,3个月诱导率可达100%;改良MS+BA 1.8 mg·L~(-1)+ KT 0.7 mg·L~(-1)+NAA 2.5 mg·L~(-1) +peptone 100 mg·L~(-1)处理对胚性愈伤增殖和分化效果较好,且分化苗可再次被诱导出胚性愈伤组织;MS+BA 1.5 mg·L~(-1)+KT 0.5 mg·L~(-1)+NAA 2 mg·L~(-1) +YE 100 mg·L~(-1)+Sugar 8%是较好的优化处理组合,能有效缓解胚性愈伤组织玻璃化严重问题以及维持相对稳定的增殖与分化速度。
Lycoris radiata is special for its value to contain galanthamine, which is the main component of the cure for the alzheimer’s disease. Genetic diversity was evaluated among/within 22 wild L. radiata populations naturally distributed in nine provinces by ISSR (inter-simple sequence repeat) markers. The collected L. radiata plants were domesticated by inter-planting with different tree species. With the help of the Grey Relation Analysis, the degree of association between the galanthamine content and the growth characteristics, physiological indices, biomass, and nutrient components was evaluated, along with the relation of the galanthamine content to the micro-environmental factors. The system of vegetative propagation was established to select and breed high-quality L. radiata germplasm. The main results are as follows:
(1) A basic research on wild populations indicated that the annual growth rhythm of populations from the southern part of the subtropical zone was earlier than that from the north-central part. The vegetative propagation coefficient and young bulbs mass of populations from the southern part were similarly better than those from the north-central part. The average vegetative propagation coefficient of populations was 5.08, with the young bulb mass of 1.64 g. Comparison of five biological characters of young bulbs revealed that the number of roots varied from 3.42 to 7.98, the height of bulb from 1.27 to 2.01 cm, the mass of bulb from 1.33 to 1.94 g, and the diameter of bulb from 1.03 to 1.23 cm. Three preliminarily bred populations YL16, YL13, and YL22, whose galanthamine content was as high as, if not more than, populations with the content of galanthamine of 300μg.g~(-1), are suggested to be exploited as high quality resources.
The stable and reproducible reaction system of ISSR-PCR amplification was established for L. radiata, which was: 20 ng template DNA, 1 U Taq DNA polymerase, 2.0 mmol·L~(-1) Mg2+, 200μmol·L~(-1) dNTPs and 0.5 mmol·L~(-1) primer in the 25μL reaction volume. The amplification program consisted of an initial step of 94℃for 300 s for pre-denaturation, 45 cycles of 94℃for 45 s, 55℃for 50s,and 72℃for 120 s,followed by final 1 cycle of 72℃for 420 s for extension. The genetic diversity of 14 L. radiata populations was analyzed by ISSR markers. The results showed that at the species level, the percentage of polymorphism (P) was 92.31%, Shannon’s index (h) was 0.4597, and Nei’s gene diversity (I) was 0.3025, indicating a high level of genetic diversity. At the population level, however, they were 49.65%, 0.2620, and 0.1763, respectively, suggesting a low level of genetic diversity. The gene differentiation coefficient (Gst) and gene flow among the populations were 0.5035 and 0.6983, respectively. The analysis of molecular variance (AMOVA) demonstrated that there was a relatively high level of genetic variation (46.12%) among populations and a relatively low genetic variation (53.88%) within populations. The high genetic differentiation among populations and the low genetic diversity within populations could be attributed to the habitat fragmentation and the limited gene flow among populations.
(2) The trial of domestication and cultivation of L. radiata and other four tree species, including three deciduous species and one evergreen species, was carried out. The differences, such as illumination intensity, air temperature, humidity, soil moisture, and soil nutrient, were remarkably present in the different interplant patterns. The curve of daily variation of illumination intensity under the different inter species was parabolically shaped, and the illumination intensity of the test points of eastward and westward arrived at the culmination on the 11:00 am and 1:00 pm. The curve of space division of daily illumination intensity under the different test points was in an U shape. The curve of daily variation of air temperature under the different inter species was in singlet parabolic shape, and air temperature arrived at the culmination on 1:00 pm. The curve of space division of daily air temperature was as similar as U shaped. The curve of daily variation of humidity under the different inter species was also similar to be U shape. The differences of soil moisture ranked from high to low by species as Elaeocarpus sylvestris (25.004%), Koelreuteria paniculata (20.997%), Bischofia poiycarpa (19.469%), and Alnus cremastogyne (18.796%). In a way, soil nutrients had been improved after cultivating L. radiata, with a range ranking from high to low as rapidly available phosphorus (-23.53~100%), hydrolysable nitrogen (2.73~56.21%), organic matter (6.59~41.07%), and rapidly available potassium (2.37~31.62%).
Micro-environment had a complicated impact on the growth of L. radiata. Chlorophyll content ofL. radiata’s leaf under different inter-planting species displayed continuous changes in different growth season, but different test points from different inter-planting species kept almost stable (1.84~2.16mg.g~(-1)) with the ratio of Chlorophyll a/b ofL. radiata’s leaf varying from 1.38 to 2.05. The daily variation of PSⅡphotochemistry (FV/FM,0.793) ofL. radiata’s leaf under different inter-planting species remarkably surpassed the data(FV/FM,0.779) under full-exposure. PSⅡphotochemistry(FV/FM ) ofL. radiata’s leaf under cultivating Bischofia poiycarpa, Koelreuteria paniculata, Alnus cremastogyne and Elaeocarpus sylvestris was respectively, 101.54%, 101.71%, 101.71%, and 102.48% higher than that under full-exposure. The new and dry biomass of L. radiata under cultivating Koelreuteria paniculata were the heaviest. The galanthamine content ofL. radiata’s bulbs coming from inter-planting species was higher than that coming from full-exposure, and the range of galanthamine content of L. radiata’s bulbs from high to low was Elaeocarpus sylvestri(s21.39μg·g~(-1)), Alnus cremastogyne (16.61μg·g~(-1)), Bischofia poiycarpa (14.80μg·g~(-1)), and Koelreuteria paniculata (13.53μg·g~(-1)). Galanthamine content ofL. radiata’s bulbs usually demonstrated two peak periods (97.536μg·g~(-1) in July of the dormancy stage, and 51.6343μg·g~(-1) in November of the leaf growth stage) and two bottom periods (6.3277μg·g~(-1) in April of the initial dormancy stage and 22.9554μg·g~(-1) in August after flowering). The galanthamine content ofL. radiata’s bulbs ascended from the first year (5.6475μg·g~(-1)) to the fifth year (116.2253μg·g~(-1)), with a rapid and stable phase from the third year to the fifth year.
By analyzing the grey relation between growth characteristics, physiological index, biomass modular, nutrient component, galanthamine content, and environmental factors, it was found that difference was present in the principal environmental factors affecting these indices, and humidity and soil moisture were the key environmental factors restraining the bulbs growth. Regression analysis indicated that there was a significant linear correlation between leaf area, bulbs’length, root fresh weight, bulbs’fresh weight, bulbs’dry weight, protein, soluble sugar, starch, reducing sugar, and environmental factors at the 5% level. There was a significant linear correlation between leaf fresh weight, leaf dry weight, root dry weight, and galanthamine content in the third year at the 1% level. All regression equations are available. But significant linear correlation between bulb-width, and galanthamine content in the second year could not be constructed at the 5% level. Through analyzing regression among growth characters, it was found that one character usually related to other characters, from which other characters could be speculated.
(3) Three field-experiment-schemes on fertilization could increase bulbs fresh and dry biomass. Different kinds of fertilizers could increase the bulb size and affect the bulbs biomass increment, with an effect ranging from good to poor: potassium fertilizer, compound fertilizer, and nitrogen fertilizer; potassium fertilizer, nitrogen fertilizer, and compound fertilizer. An increase in the natural tiller number of bulbs, in addition, was observed after continuously applying potassium fertilizer.
In the potting experiment, potassium fertilizer increased root activities, root CEC, and galanthamine content of bulbs, and these showed similar daily variation curves in shape. Compared with control, root activities, root CEC, and galanthamine content of bulbs were improved via increasing potassium application by 0.3 g·kg~(-1) or 0.6 g·kg~(-1). Root activities, root CEC, and galanthamine content of bulbs rose to a peak of 97.35 ug·g~(-1)h ~(-1), 9.67 cmol·kg~(-1), and 276.4054μg·g~(-1) when potassium application was increased to 0.9 g·kg~(-1). However, these indices mentioned above started to decline when potassium was applied by the dosage of 1.2 g·kg~(-1).
(4) The natural propagation coefficient of L.radiata bulbs ranged from 2.495 to 2.656. The curve of vegetative propagation coefficient took on two peaks under different development phases, varying from 0.80 to 6.8, In order to get high vegetative propagation coefficient, it is suggested that vegetative propagation should be carried out in June, July, and September. In order to get more massive young bulbs, vegetative propagation should be carried out in September and April. In order to get larger young bulbs, vegetative propagation should be carried out in June, July, September, and April. In order to get good root activities, vegetative propagation should be carried out from March to July, and in September. The number of young bulbs produced varied from in L. chinensis, with the average of vegetative propagation coefficient being 7.09.
Embryogenic callus (EC) was induced and differentiated by tissue culture in L. aurea. Embryogenic callus inductivity reached 100% in three months with the treatment of improved MS+BA 5 mg·L~(-1)+NAA 3 mg·L~(-1).Improved MS+BA 1.8 mg·L~(-1)+KT 0.7 mg·L~(-1)+NAA 2.5 mg·L~(-1)+peptone 100 mg·L~(-1) was favorable for multiplication of embryogenic callus and for promoting differentiation of somatic embryo. Under this medium the embryogenic callus could be induced from sprouts again. MS+BA 1.5 mg.L~(-1)+KT 0.5 mg.L~(-1)+NAA 2 mg.L~(-1) +YE 100 mg·L~(-1)+Sugar 8%, a better medium, could effectively alleviate the problem of seriously vitrifiation as well as sustaining a relatively stable speed of multiplication of embryogenic callus and promotion differentiation of somatic embryo.
(1)石蒜野生居群种质资源开展基础研究,发现来自亚热带南部的居群生长节律期明显早于来自中北部居群,南部居群的繁殖系数、子鳞茎质量也均优于中北部居群,所有居群繁殖系数均值为5.81,子鳞茎质量均值为1.64 g。子鳞茎5个生物学性状相比,根系数量差异最大(3.42~7.98根),其次是高度差异(1.27~2.01 cm ),再次是质量差异(1.33~1.94 g ),最后是直径差异(1.03~1.23 cm )。初步选育YL16、YL13、YL22等3个加兰他敏接近或超过300μg·g~(-1)居群,建议作为一类优质资源开发利用。
利用石蒜叶片基因组DNA为模板,建立其ISSR分析的最优化反应体系及应用程序:25μL反应体系中,有20 ng模板DNA、0.5μmol·L~(-1)随机引物、150μmol·L~(-1) dNTPs、2.0mmol·L~(-1) Mg2+、1.0 U Taq DNA聚合酶。反应程序为:94℃预变性300 s ;然后45个循环:每个循环94℃变性45s,55℃退火60s,72℃延伸120s;循环结束后72℃延伸420s。利用ISSR分子标记技术对14个野生石蒜居群进行遗传多样性分析,结果表明:物种遗传多样性很高,多态位点百分率为92.31%,Shannon指数h为0.4597,Nei指数I为0.3025;居群水平遗传多样性较低,多态位点百分率平均为49.65%,Shannon指数h平均为0.2620,Nei指数I平均为0.1763;居群间的遗传分化系数Gst为0.5035,基因流Nm为0.6983。而AMOVA分子变异分析显示:居群间遗传分化程度高,46.12%的变异发生在居群内,53.88%的变异发生在居群间。生境的片段化使居群间的基因流受阻,可能是居群间高遗传分化和居群水平低遗传多样性的主要原因。
(2)建立石蒜与3种落叶树种、1种杜英套种驯化培育试验。套种引起微环境中光照效应、气温效应、湿度效应、土壤水分和土壤养分等存在差异。套种林下光照强度日变化曲线为抛物线,林下东西部测点分别在11点、13点最大值,同一树种林下不同测点光照强度曲线呈U状图;气温曲线为单峰抛物线,最高值在午后1:00,同一树种林下不同测点气温曲线呈小U型;不同树种套种林下不同测点湿度日变化曲线类似于字母U型。土壤含水量差异如下:杜英(25.004%)>栾树(20.997%)>重阳木(19.469%)>桤木(18.796%);土壤养分有不同程度的提高,提升的幅度依次为:速效P(-23.53~100%)>水解N(2.73~56.21%)>有机质(6.59~41.07%)>速效K(2.37~31.62%)。
微环境对石蒜生长影响相当复杂。套种林下石蒜叶片叶绿素在不同生长发育季节为动态变化过程,但不同测点值基本平稳(1.84~2.16 mg·g~(-1)),叶绿素a/b的比值在1.38~2.05之间变化;套种构成的荫蔽程度差异表现在叶绿素荧光参数FV/FM(均值0.793)明显高于全光照下(0.779),其中重阳木林下、栾树林下、桤木林下、杜英林下的FV/FM分别是对照的101.54%、101.71%、101.71%和102.48%。套种在栾树下干、鲜生物量最大;套种后加兰他敏比对照提高,由高到低排序依次是:杜英(21.39μg·g~(-1))>桤木(16.61μg·g~(-1))>重阳(14.80μg·g~(-1) >栾树(13.53μg·g~(-1))。年度生长发育期内加兰他敏出现二个高峰(休眠中期7月97.536μg·g~(-1)和叶旺盛生长11月51.6343μg·g~(-1))、二个低峰(休眠初期4月6.3277μg·g~(-1)和花后的8月22.9554μg·g~(-1))。加兰他敏含量从第1年的5.6475μg·g~(-1)上升第5年的116.2253μg·g~(-1),第3~5年含量是快速增长期,建议加强后期管理,减少鳞茎自然分蘖,在休眠中期7月进行资源采集和制药提取。
通过对石蒜的生长性状、生理指标、生物量、营养成分与环境因子关联度分析,发现影响以上指标的主导环境因子存在差异性,但发现相对湿度和土壤水分是制约其生长的关键环境因子。对石蒜生长指标与环境因子回归分析,发现叶面积、鳞茎高度、根鲜重、鳞茎鲜重、鳞茎干重、蛋白质、可溶性糖、淀粉及还原糖与环境因子回归显著水平均低于0.05,相关性显著;叶鲜重、叶干重、根干重、第三年度鳞茎加兰他敏含量与环境因子回归显著水平分别均低于0.01,相关性极显著,建立的回归方程均可用;鳞茎宽度、第二年度鳞茎加兰他敏含量与环境因子回归显著水平大于0.05,相关性不显著,建立的回归方程不可用。经对石蒜性状之间的关联度分析,发现不同性状均有与其密切相关的性状,根据一个性状与其他性状关联度可推测其他性状特点,为石蒜优质资源培育提供一定技术指导。
(3)田间实验设计3种施肥措施能增加石蒜的干、鲜物质。肥料种类对增加鳞茎体积由好到差排序:钾肥>复合肥>氮肥。影响石蒜生物产量构成因素效果的肥料种类排序也是钾肥>氮肥>复合肥,实践中发现钾肥连续施用导致鳞茎自然分蘖增多,可提供制药原料的鳞茎数量减少。
盆栽试验发现钾肥能提高根系活力、根系CEC差异和加兰他敏含量,三者年度变化曲线十分相似。增施钾肥0.3 g·kg~(-1)、0.6 g·kg~(-1),鳞茎的根系活力、根系CEC差异和加兰他敏含量比对照均有小幅增长;增施钾肥0.9 g·kg~(-1)时,鳞茎的根系活力、根系CEC差异和加兰他敏含量均达到最大值(97.35 ug·g~(-1)h ~(-1)、9.67 cmol·kg~(-1)、276.4054μg·g~(-1));施钾肥继续增长到1.2 g·kg~(-1)时,上述指标反而降低。
(4)石蒜自然繁殖系数为2.495~2.656。在不同生长发育期进行无性繁殖,繁殖系数呈现双高峰曲线图,其值则在0.80~6.8之间变动;欲获得较高无性繁殖系数,建议在休眠期的6月、7月及初叶期的9月繁殖;欲获得较大质量的子鳞茎宜在初叶期的9月和落叶期的4月繁殖;欲获得横径较大的子鳞茎宜在6、7、9和4月份繁殖;在3~7月和9月进行繁殖,获得子鳞茎的根系发育良好。中国石蒜(L.chinensis)母鳞茎个体之间繁殖系数差异从1~19个不等,繁殖系数均值为7.09,母鳞茎质量不能作为挑选作为获得繁殖系数较多的标志。
通过对石蒜离体培养诱导胚性愈伤组织及其分化。优化培养条件后建立起高效的离体再生繁殖体系。结果表明:改良MS+BA 5 mg·L~(-1)+NAA 3 mg·L~(-1)组合胚性愈伤组织诱导率最高,3个月诱导率可达100%;改良MS+BA 1.8 mg·L~(-1)+ KT 0.7 mg·L~(-1)+NAA 2.5 mg·L~(-1) +peptone 100 mg·L~(-1)处理对胚性愈伤增殖和分化效果较好,且分化苗可再次被诱导出胚性愈伤组织;MS+BA 1.5 mg·L~(-1)+KT 0.5 mg·L~(-1)+NAA 2 mg·L~(-1) +YE 100 mg·L~(-1)+Sugar 8%是较好的优化处理组合,能有效缓解胚性愈伤组织玻璃化严重问题以及维持相对稳定的增殖与分化速度。
Lycoris radiata is special for its value to contain galanthamine, which is the main component of the cure for the alzheimer’s disease. Genetic diversity was evaluated among/within 22 wild L. radiata populations naturally distributed in nine provinces by ISSR (inter-simple sequence repeat) markers. The collected L. radiata plants were domesticated by inter-planting with different tree species. With the help of the Grey Relation Analysis, the degree of association between the galanthamine content and the growth characteristics, physiological indices, biomass, and nutrient components was evaluated, along with the relation of the galanthamine content to the micro-environmental factors. The system of vegetative propagation was established to select and breed high-quality L. radiata germplasm. The main results are as follows:
(1) A basic research on wild populations indicated that the annual growth rhythm of populations from the southern part of the subtropical zone was earlier than that from the north-central part. The vegetative propagation coefficient and young bulbs mass of populations from the southern part were similarly better than those from the north-central part. The average vegetative propagation coefficient of populations was 5.08, with the young bulb mass of 1.64 g. Comparison of five biological characters of young bulbs revealed that the number of roots varied from 3.42 to 7.98, the height of bulb from 1.27 to 2.01 cm, the mass of bulb from 1.33 to 1.94 g, and the diameter of bulb from 1.03 to 1.23 cm. Three preliminarily bred populations YL16, YL13, and YL22, whose galanthamine content was as high as, if not more than, populations with the content of galanthamine of 300μg.g~(-1), are suggested to be exploited as high quality resources.
The stable and reproducible reaction system of ISSR-PCR amplification was established for L. radiata, which was: 20 ng template DNA, 1 U Taq DNA polymerase, 2.0 mmol·L~(-1) Mg2+, 200μmol·L~(-1) dNTPs and 0.5 mmol·L~(-1) primer in the 25μL reaction volume. The amplification program consisted of an initial step of 94℃for 300 s for pre-denaturation, 45 cycles of 94℃for 45 s, 55℃for 50s,and 72℃for 120 s,followed by final 1 cycle of 72℃for 420 s for extension. The genetic diversity of 14 L. radiata populations was analyzed by ISSR markers. The results showed that at the species level, the percentage of polymorphism (P) was 92.31%, Shannon’s index (h) was 0.4597, and Nei’s gene diversity (I) was 0.3025, indicating a high level of genetic diversity. At the population level, however, they were 49.65%, 0.2620, and 0.1763, respectively, suggesting a low level of genetic diversity. The gene differentiation coefficient (Gst) and gene flow among the populations were 0.5035 and 0.6983, respectively. The analysis of molecular variance (AMOVA) demonstrated that there was a relatively high level of genetic variation (46.12%) among populations and a relatively low genetic variation (53.88%) within populations. The high genetic differentiation among populations and the low genetic diversity within populations could be attributed to the habitat fragmentation and the limited gene flow among populations.
(2) The trial of domestication and cultivation of L. radiata and other four tree species, including three deciduous species and one evergreen species, was carried out. The differences, such as illumination intensity, air temperature, humidity, soil moisture, and soil nutrient, were remarkably present in the different interplant patterns. The curve of daily variation of illumination intensity under the different inter species was parabolically shaped, and the illumination intensity of the test points of eastward and westward arrived at the culmination on the 11:00 am and 1:00 pm. The curve of space division of daily illumination intensity under the different test points was in an U shape. The curve of daily variation of air temperature under the different inter species was in singlet parabolic shape, and air temperature arrived at the culmination on 1:00 pm. The curve of space division of daily air temperature was as similar as U shaped. The curve of daily variation of humidity under the different inter species was also similar to be U shape. The differences of soil moisture ranked from high to low by species as Elaeocarpus sylvestris (25.004%), Koelreuteria paniculata (20.997%), Bischofia poiycarpa (19.469%), and Alnus cremastogyne (18.796%). In a way, soil nutrients had been improved after cultivating L. radiata, with a range ranking from high to low as rapidly available phosphorus (-23.53~100%), hydrolysable nitrogen (2.73~56.21%), organic matter (6.59~41.07%), and rapidly available potassium (2.37~31.62%).
Micro-environment had a complicated impact on the growth of L. radiata. Chlorophyll content ofL. radiata’s leaf under different inter-planting species displayed continuous changes in different growth season, but different test points from different inter-planting species kept almost stable (1.84~2.16mg.g~(-1)) with the ratio of Chlorophyll a/b ofL. radiata’s leaf varying from 1.38 to 2.05. The daily variation of PSⅡphotochemistry (FV/FM,0.793) ofL. radiata’s leaf under different inter-planting species remarkably surpassed the data(FV/FM,0.779) under full-exposure. PSⅡphotochemistry(FV/FM ) ofL. radiata’s leaf under cultivating Bischofia poiycarpa, Koelreuteria paniculata, Alnus cremastogyne and Elaeocarpus sylvestris was respectively, 101.54%, 101.71%, 101.71%, and 102.48% higher than that under full-exposure. The new and dry biomass of L. radiata under cultivating Koelreuteria paniculata were the heaviest. The galanthamine content ofL. radiata’s bulbs coming from inter-planting species was higher than that coming from full-exposure, and the range of galanthamine content of L. radiata’s bulbs from high to low was Elaeocarpus sylvestri(s21.39μg·g~(-1)), Alnus cremastogyne (16.61μg·g~(-1)), Bischofia poiycarpa (14.80μg·g~(-1)), and Koelreuteria paniculata (13.53μg·g~(-1)). Galanthamine content ofL. radiata’s bulbs usually demonstrated two peak periods (97.536μg·g~(-1) in July of the dormancy stage, and 51.6343μg·g~(-1) in November of the leaf growth stage) and two bottom periods (6.3277μg·g~(-1) in April of the initial dormancy stage and 22.9554μg·g~(-1) in August after flowering). The galanthamine content ofL. radiata’s bulbs ascended from the first year (5.6475μg·g~(-1)) to the fifth year (116.2253μg·g~(-1)), with a rapid and stable phase from the third year to the fifth year.
By analyzing the grey relation between growth characteristics, physiological index, biomass modular, nutrient component, galanthamine content, and environmental factors, it was found that difference was present in the principal environmental factors affecting these indices, and humidity and soil moisture were the key environmental factors restraining the bulbs growth. Regression analysis indicated that there was a significant linear correlation between leaf area, bulbs’length, root fresh weight, bulbs’fresh weight, bulbs’dry weight, protein, soluble sugar, starch, reducing sugar, and environmental factors at the 5% level. There was a significant linear correlation between leaf fresh weight, leaf dry weight, root dry weight, and galanthamine content in the third year at the 1% level. All regression equations are available. But significant linear correlation between bulb-width, and galanthamine content in the second year could not be constructed at the 5% level. Through analyzing regression among growth characters, it was found that one character usually related to other characters, from which other characters could be speculated.
(3) Three field-experiment-schemes on fertilization could increase bulbs fresh and dry biomass. Different kinds of fertilizers could increase the bulb size and affect the bulbs biomass increment, with an effect ranging from good to poor: potassium fertilizer, compound fertilizer, and nitrogen fertilizer; potassium fertilizer, nitrogen fertilizer, and compound fertilizer. An increase in the natural tiller number of bulbs, in addition, was observed after continuously applying potassium fertilizer.
In the potting experiment, potassium fertilizer increased root activities, root CEC, and galanthamine content of bulbs, and these showed similar daily variation curves in shape. Compared with control, root activities, root CEC, and galanthamine content of bulbs were improved via increasing potassium application by 0.3 g·kg~(-1) or 0.6 g·kg~(-1). Root activities, root CEC, and galanthamine content of bulbs rose to a peak of 97.35 ug·g~(-1)h ~(-1), 9.67 cmol·kg~(-1), and 276.4054μg·g~(-1) when potassium application was increased to 0.9 g·kg~(-1). However, these indices mentioned above started to decline when potassium was applied by the dosage of 1.2 g·kg~(-1).
(4) The natural propagation coefficient of L.radiata bulbs ranged from 2.495 to 2.656. The curve of vegetative propagation coefficient took on two peaks under different development phases, varying from 0.80 to 6.8, In order to get high vegetative propagation coefficient, it is suggested that vegetative propagation should be carried out in June, July, and September. In order to get more massive young bulbs, vegetative propagation should be carried out in September and April. In order to get larger young bulbs, vegetative propagation should be carried out in June, July, September, and April. In order to get good root activities, vegetative propagation should be carried out from March to July, and in September. The number of young bulbs produced varied from in L. chinensis, with the average of vegetative propagation coefficient being 7.09.
Embryogenic callus (EC) was induced and differentiated by tissue culture in L. aurea. Embryogenic callus inductivity reached 100% in three months with the treatment of improved MS+BA 5 mg·L~(-1)+NAA 3 mg·L~(-1).Improved MS+BA 1.8 mg·L~(-1)+KT 0.7 mg·L~(-1)+NAA 2.5 mg·L~(-1)+peptone 100 mg·L~(-1) was favorable for multiplication of embryogenic callus and for promoting differentiation of somatic embryo. Under this medium the embryogenic callus could be induced from sprouts again. MS+BA 1.5 mg.L~(-1)+KT 0.5 mg.L~(-1)+NAA 2 mg.L~(-1) +YE 100 mg·L~(-1)+Sugar 8%, a better medium, could effectively alleviate the problem of seriously vitrifiation as well as sustaining a relatively stable speed of multiplication of embryogenic callus and promotion differentiation of somatic embryo.
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