凤仙花属部分植物种质资源收集与保存研究
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摘要
凤仙花属(Impatiens L.)植物全世界有900余种,我国已知有220余种。凤仙花属植物种类繁多,花形奇特,变化丰富。该属植物的多样性为选育花卉优良品种提供了丰富的基因资源。部分野生种类本身就具有很高的观赏价值,亟待引种栽培和推广。目前,该属的开发与利用很少,对于种质资源保存方面的的研究则几乎是空白。凤仙花属植物野外分布的狭域性、地域的特有性及对环境要求的严苛性使其极易受到环境变化和人为活动的影响。对凤仙花属植物进行研究并对该属种质资源进行保存意义重大。
鉴于此,本研究在收集我国原产的凤仙花属植物的基础上,对其观赏性状、开发利用价值和种间亲缘关系进行评价与分析,通过种子萌发特性和耐荫性的研究总结其一般栽培技术,以缓慢生长保存和超低温保存的方式建立凤仙花属植物种质资源离体保存体系。为该属资源利用和保护提供基础。获得下述研究结果:
1.通过野外采集,首次较大规模收集中国原产凤仙花属植物38种,2变种,7未定名种。绝大部分种类在地域分布上具有极强的狭域性。应用层次分析法建立了适用于凤仙花属植物观赏性状与开发利用价值评价的模型,对40种凤仙进行了综合评价,筛选出表现较优良的8种凤仙,分别是:麻栗坡凤仙花(I. malipoensis)、耳叶棒凤仙花(I. claviger var. auriculata)、棒凤仙花(I. claviger)、大叶凤仙花(I. apalophylla)、龙州凤仙花(I. morsei)、小萼凤仙花(Impatiens sp.)、丰满凤仙花(I. obesa)和大旗瓣凤仙花(I. macrovexilla)。在种质资源保存的基础上,可优先考虑以上几种凤仙花的开发利用。
2.应用AFLP方法对凤仙花属的种间亲缘关系进行了探讨。采用11对引物对40种凤仙进行分析,共获得1003个位点,多态性达97.11%。结果表明部分生物学性状(蒴果形状、苞片位置、萼片数和花序类型)及生境相似的种亲缘关系较近,生境及部分生物学性状作为分类依据的可靠性较高;地理分布作为分类依据的可靠性较低。
3.对龙州凤仙花、丰满凤仙花、大旗瓣凤仙花和华凤仙(I. chinensis)种子的萌发特性进行了研究。华凤仙适宜的萌发条件是:30℃浸种8h在30℃条件下萌发,发芽率达到70%;龙州凤仙花适宜的萌发条件是:20℃浸种4h在20℃条件下萌发,发芽率为34%。丰满凤仙花在20℃萌发条件下发芽率较高,达到80%;大旗瓣凤仙花在20℃和30℃的萌发温度下,发芽率均接近100%。华凤仙与龙州凤仙花萌发至24d,丰满凤仙花与大旗瓣凤仙花分别萌发至15d左右和6d左右即可达到或接近最大发芽率。
4.在三种光强条件下测定丰满凤仙花、大旗瓣凤仙花及华凤仙的生长指标(株高、叶片数、叶面积、叶片厚度和比叶重)、叶绿素指标和光合参数的变化,得到三种凤仙耐荫性的排序:大旗瓣凤仙花>华凤仙>丰满凤仙花。光合参数的测定是较简便与准确判断凤仙花属植物耐荫性的方法。通过对测得的光合参数的研究,将引种正常生长的21种凤仙花属植物按光照需求由大到小排序如下:滇水金凤(I. uliginosa)>微绒毛凤仙花(I. tomentella)>黄金凤(I. siculifer)>棒凤仙花>未定名种(兴义)(Impatiens sp.)>白花凤仙花(I. wilsonii)>菱叶凤仙花(I. rhombifolia)>耳叶棒凤仙花>红稚凤仙花(I. oxyanthera)>未定名种(融安)(Impatiens sp.)>瑞丽凤仙花(I. ruiliensis)>小萼凤仙花(Impatiens sp.)>绿萼凤仙花(I. chlorosepala)>瑶山凤仙花(I. macrovexilla var. yaoshanensis)>大叶凤仙花>未定名种(临桂)(Impatiens sp.)>龙州凤仙花>滇南凤仙花(I. duclouxii)>管茎凤仙花(I. tubulosa)>麻栗坡凤仙花>锐齿凤仙花(I. arguta)。对生境光照适应性较强的种类有:黄金凤、白花凤仙花、绿萼凤仙花;对生境光照要求较为严格的种类有:棒凤仙花、瑶山凤仙花及未定名种(兴义);其它种类对于环境光照的要求居中。根据方程Pn=Pmax(1-C0 e-aPFD/ Pmax)拟合的光合-光响应曲线比二项式拟合曲线能够更合理的分析光响应曲线的全过程,解决了二项式拟合曲线存在的问题(超过光饱和点后对光合速率迅速下降的预测,光饱和点偏大,暗呼吸速率为正值),是凤仙花属植物较为理想的光合过程分析模型。
5.对引种凤仙花属植物的物候期进行观察记录;总结主要的繁殖方式有:播种繁殖、扦插繁殖与分株繁殖;按照播种苗与采集成株分别总结了栽培方法和病虫害防治规律。引种成功的凤仙花属植物共计11种。主要引种限制因子为温度和湿度。
6.通过筛选适宜的侧芽(0.1%升汞两次,每次3~4min)与种子灭菌方式、继代培养基和移栽基质配方,建立了新几内亚凤仙花(I. platypatala)、大旗瓣凤仙花、丰满凤仙花、锐齿凤仙花、龙州凤仙花、瑶山凤仙花、菱叶凤仙花、鸭跖草状凤仙花(I. commellinoides)、瑞丽凤仙花、红稚凤仙花及华凤仙11种凤仙花的组培体系。新几内亚凤仙、丰满凤仙花、龙州凤仙花及华凤仙适宜的继代培养基配方:MS+6-BA 1.0mg·L-1+NAA 0.1mg·L-1;锐齿凤仙花、瑶山凤仙花、大旗瓣凤仙花、鸭跖草状凤仙花及红稚凤仙花适宜的继代培养基配方:MS+6-BA 1.0mg·L-1+NAA 0.05mg·L-1;菱叶凤仙花和瑞丽凤仙花适宜的继代培养基配方:MS+6-BA 0.5mg·L-1+NAA 0.05mg·L-1,组培苗生长健壮,增殖正常。
新几内亚凤仙、红稚凤仙花和华凤仙适宜的移栽基质配比是:珍珠岩:草炭=2:1;丰满凤仙花和龙州凤仙花适宜的移栽基质配比是:珍珠岩:蛭石=2:1;锐齿凤仙花、瑶山凤仙花、鸭跖草状凤仙花和瑞丽凤仙花适宜的移栽基质配比是:珍珠岩:草炭=4:1;菱叶凤仙花适宜的移栽基质配比是:蛭石:草炭=1:1。
7.通过改变培养基的大量元素含量、添加调节培养基渗透压的物质、生长抑制剂及改变培养温度等方式,大大延长了组培苗离体保存的时间。不同种对不同保存方式的反应不一。均以4℃培养延长保存时间的效果最为明显,新几内亚凤仙花保存至270d,丰满凤仙花保存至210d,大旗瓣凤仙花保存至240d,华凤仙保存至180d。保存后的组培苗出瓶移栽,除新几内亚凤仙花和大旗瓣凤仙花添加甘露醇的处理在分枝数上与对照有明显差异外,其它与对照在株高、叶片数及分枝数均无明显差异。
8.建立了新几内亚凤仙和丰满凤仙花的玻璃化法超低温保存体系。技术程序如下:切取生长健壮的组培苗2mm左右的茎尖,于20℃下在2mol·L-1甘油+0.4mol·L-1乙二醇混和液中处理20min(丰满凤仙花)或30min(新几内亚凤仙),然后在0℃下用PVS2玻璃化溶液处理30min(丰满凤仙花)或40min(新几内亚凤仙),换新鲜PVS2溶液,快速浸入液氮,40℃水浴中解冻60s,用MS+1.2mol·L-1蔗糖液体培养基洗涤2次,每次10min。最后,转至继代培养基上进行恢复培养,液氮冻存后的茎尖存活率为19%(丰满凤仙花)或23%(新几内亚凤仙)。冻存后的茎尖再生成苗增殖系数与正常组培苗对照没有明显差异。
There are over 900 species in genus Impatiens in the world, of which approximately 220 species distribute in China. Diversity of Impatiens provides abundant gene resources for breeding herb flower species. But up to now our precious germplasm resources are still left in wild without sufficient researches on the breeding and horticultural culture, especially on the preservation of germplasm. Narrow distribution, rich endemism and which need a strict circumstance of Impatiens appear to correlate with human activity and environment changing closely.
In view of above fact, using collected Impatiens species in China, this paper evaluated ornamental character and development value of them by Analytic Hierarchy Process, studied on interspecific relationship, seed germination characteristics and shading tolerance, which was based on summarizing cultivation techniques, and established germplasm preservation system in vitro by methods of slow growth preservation and cryopreservation. The main results were summarized as follows:
1. 38 species, 2 varieties and 7 indeterminate species were collected for the first time, some of them are shown as sharp decreasing because of habitats destruction and invasive plant. Using analytic hierarchy process to evaluate ornamental characteristic and development value of 40 collceted species, the result showed that I. malipoensis, I. claviger var. auriculata, I. claviger, I. apalophylla, I.morsei, I. obesa and I. macrovexilla have better ornamental value and can be used for landscaping directly and preferentially on the premise of germplasm preservation. Development and utilization of aforeside 8 species should be considerated preferentially.
2. The optimal reaction system of AFLP for Impatiens was established, the genetic diversity and relationship were analyzed between 40 Impatiens species. The PCR-based multi-locus DNA fingerprinting technique Amplified Fragment Length Polymorphism(AFLP)analysis of 40 species with 11 AFLP primer combinations produced multi-locus DNA fingerprint with a total of 1003 DNA fragments. Polymorphism rate was 97.11%. Result showed that species which had high similarity at parts of biological characteristics and habitat had nearer relationship. As classification foundation, habit and parts of biological characteristics had more reliability. More methods should be used in taxonomic studies of Impatiens. Result provided valuable evidence for estabilishing taxonomic system and crossbreeding in molecular level.
3. Study on seed germination charactesistics of I. chinensis, I. morsei, I. obesa, I. macrovexilla showed that optimal germination condition of I. chinensis is seed soaking for 8h at 30℃, and germinating at 30℃, the germination rate was 70%. Optimal germination condition of I.morsei is seed soaking for 4h at 20℃, germinating at 20℃, germinating rate was 34%. Germination rate of I. obesa at 20℃was higher than at 30℃, the germination rate was 80%. Germination rate of I. macrovexilla was close to 100% both at 20℃and 30℃. Germination rate of I. obesa and I. morsei have already approximated to maximum on 24th day, I. obesa was on 15th day and I. morsei was on 6th day.
4. At provided three kinds of light intensity, photosynthetic capacity of three Impatiens species(I. chinensis, I.macrovexilla, I. obesa) were discussed. Plant height, stem ameter, leaf number, leaf area, leaf thickness, chlorophyll content and photosynthesis capability were measured. The results showed that the shading adapability of I. macrovexilla was best, then was I. chinensis and I. obesa. Determination of photosynthetic indexes was more convenient and accurate way to judge shade tolerance. Photosynthetic characteristics of 21 Impatiens species had been tested. Three groups were identified based on light adaptability. Among them, I. siculifer, I. wilsonii and I. chlorosepala have better adaptability, I. claviger, I. yaoshanensis and indeterminate species(XingYi) had worse adaptability. Sorting according to requirement for light was, I. uliginosa>I. tomentella>I. siculifer>I. claviger>indeterminate species(XingYi)>I. wilsonii>I. rhombifolia>I. claviger var. auriculata>I. oxyanthera> indeterminate species(Rong An)>I. ruiliensis>I. chlorosepala>I. macrovexilla var. yaoshanensis>I. apaliphylla>indeterminate species ( Lin Gui ) >I. morsei>I. duclouxii>I. tubulosa>I.malipoensis>I.arguta. The light response curve fitting by equation [Pn=Pmax(1-C0 e-aPFD/ Pmax)] was a fairy ideal model to analyze the whole process of light reponse.
5. Based on observation of collecting Impatiens species phenophase, cultivation techniques and control of diseases and insect pests were summarized according to seedling stage, seedling separation stage, seedling formation period and blooming stage. Mode of reproduction included seed propagation, cutting propagation and division propagation. Introduction of 11 species were succeeded. Restriction factors of introduction were tempreture and humidity.
6. Tissue culture system of 11 Impatiens species were established. Optimal disinfection way, subculture medium and substrate composition were screened according to different species. Optimal subculture medium of I. platypatala, I. obesa, I. morsei and I. chinensis: MS+6-BA 1.0mg·L-1+NAA 0.1mg·L-1, optimal subculture medium of I. arguta, I. macrovexilla var. yaoshanensis, I. macrovexilla, I. commellinoides and I. oxyanthera: MS+6-BA 1.0mg·L-1+NAA 0.05mg·L-1, Optimal subculture medium of I. rhombifolia and I. ruiliensis: MS+6-BA 0.5mg·L-1+NAA 0.05mg·L-1. Optimal transplanting medium of I. platypatala, I. oxyanthera and I. chinensis: perlite and peat was 2:1, optimal transplanting medium of I.obesa and I. morsei: perlite and vermiculite was 2:1, optimal transplanting medium of I. arguta, I. macrovexilla var. yaoshanensis, I. commellinoides and I. ruiliensis: perlite and vermiculite was 4:1, Optimal transplanting medium of I. rhombifolia: vermiculite and peat was 1:1.
7. Different species need different ways of slow growth preservation in vitro. More ideal treatment of all was 4℃, at this tempreture, I. platypatala had been preserved for 270d, I. obesa had been preserved for 210d, I. macrovexilla had been preserved for 240d, and I. chinensis had been preserved for 180d. After transplanting, there were no differences between tissue culture seedling after slow growth preservation and normal seedling on plant height, leaf number and branch number, except treaments of adding mannitol of I. platypatala and I. macrovexilla .
8. Shoot tips excised from healthy in vitro plants of I. platypatala and I. obesa were successfully cryopreserved by vitrification. A suitable procedure was established as follows:dissecting shoot tips of 2mm were loaded in a mixture of 2mol·L-1 glycerol plus 0.4 mol·L-1 sucrose for 20min(I. obesa) or 30min(I. platypatala) at 20℃. Following that, a highly concentrated cryoprotective solution (PVS2) was then added at 0℃. After dehydration at 0℃for 30min(I. obesa) or 40min(I. platypatala), the shoot tips were directly plunged into liquid nitrogen. After rapid thawing in a water bath at 40℃for about 60s, the tips were washed twice with 1.8ml of 1.2 mol·L-1 sucrose solution for 10min each time and then transferred onto subculture medium for recovery growth. The highest survival of shoot tips were 19%(I. obesa) or 23%(I. platypatala).
鉴于此,本研究在收集我国原产的凤仙花属植物的基础上,对其观赏性状、开发利用价值和种间亲缘关系进行评价与分析,通过种子萌发特性和耐荫性的研究总结其一般栽培技术,以缓慢生长保存和超低温保存的方式建立凤仙花属植物种质资源离体保存体系。为该属资源利用和保护提供基础。获得下述研究结果:
1.通过野外采集,首次较大规模收集中国原产凤仙花属植物38种,2变种,7未定名种。绝大部分种类在地域分布上具有极强的狭域性。应用层次分析法建立了适用于凤仙花属植物观赏性状与开发利用价值评价的模型,对40种凤仙进行了综合评价,筛选出表现较优良的8种凤仙,分别是:麻栗坡凤仙花(I. malipoensis)、耳叶棒凤仙花(I. claviger var. auriculata)、棒凤仙花(I. claviger)、大叶凤仙花(I. apalophylla)、龙州凤仙花(I. morsei)、小萼凤仙花(Impatiens sp.)、丰满凤仙花(I. obesa)和大旗瓣凤仙花(I. macrovexilla)。在种质资源保存的基础上,可优先考虑以上几种凤仙花的开发利用。
2.应用AFLP方法对凤仙花属的种间亲缘关系进行了探讨。采用11对引物对40种凤仙进行分析,共获得1003个位点,多态性达97.11%。结果表明部分生物学性状(蒴果形状、苞片位置、萼片数和花序类型)及生境相似的种亲缘关系较近,生境及部分生物学性状作为分类依据的可靠性较高;地理分布作为分类依据的可靠性较低。
3.对龙州凤仙花、丰满凤仙花、大旗瓣凤仙花和华凤仙(I. chinensis)种子的萌发特性进行了研究。华凤仙适宜的萌发条件是:30℃浸种8h在30℃条件下萌发,发芽率达到70%;龙州凤仙花适宜的萌发条件是:20℃浸种4h在20℃条件下萌发,发芽率为34%。丰满凤仙花在20℃萌发条件下发芽率较高,达到80%;大旗瓣凤仙花在20℃和30℃的萌发温度下,发芽率均接近100%。华凤仙与龙州凤仙花萌发至24d,丰满凤仙花与大旗瓣凤仙花分别萌发至15d左右和6d左右即可达到或接近最大发芽率。
4.在三种光强条件下测定丰满凤仙花、大旗瓣凤仙花及华凤仙的生长指标(株高、叶片数、叶面积、叶片厚度和比叶重)、叶绿素指标和光合参数的变化,得到三种凤仙耐荫性的排序:大旗瓣凤仙花>华凤仙>丰满凤仙花。光合参数的测定是较简便与准确判断凤仙花属植物耐荫性的方法。通过对测得的光合参数的研究,将引种正常生长的21种凤仙花属植物按光照需求由大到小排序如下:滇水金凤(I. uliginosa)>微绒毛凤仙花(I. tomentella)>黄金凤(I. siculifer)>棒凤仙花>未定名种(兴义)(Impatiens sp.)>白花凤仙花(I. wilsonii)>菱叶凤仙花(I. rhombifolia)>耳叶棒凤仙花>红稚凤仙花(I. oxyanthera)>未定名种(融安)(Impatiens sp.)>瑞丽凤仙花(I. ruiliensis)>小萼凤仙花(Impatiens sp.)>绿萼凤仙花(I. chlorosepala)>瑶山凤仙花(I. macrovexilla var. yaoshanensis)>大叶凤仙花>未定名种(临桂)(Impatiens sp.)>龙州凤仙花>滇南凤仙花(I. duclouxii)>管茎凤仙花(I. tubulosa)>麻栗坡凤仙花>锐齿凤仙花(I. arguta)。对生境光照适应性较强的种类有:黄金凤、白花凤仙花、绿萼凤仙花;对生境光照要求较为严格的种类有:棒凤仙花、瑶山凤仙花及未定名种(兴义);其它种类对于环境光照的要求居中。根据方程Pn=Pmax(1-C0 e-aPFD/ Pmax)拟合的光合-光响应曲线比二项式拟合曲线能够更合理的分析光响应曲线的全过程,解决了二项式拟合曲线存在的问题(超过光饱和点后对光合速率迅速下降的预测,光饱和点偏大,暗呼吸速率为正值),是凤仙花属植物较为理想的光合过程分析模型。
5.对引种凤仙花属植物的物候期进行观察记录;总结主要的繁殖方式有:播种繁殖、扦插繁殖与分株繁殖;按照播种苗与采集成株分别总结了栽培方法和病虫害防治规律。引种成功的凤仙花属植物共计11种。主要引种限制因子为温度和湿度。
6.通过筛选适宜的侧芽(0.1%升汞两次,每次3~4min)与种子灭菌方式、继代培养基和移栽基质配方,建立了新几内亚凤仙花(I. platypatala)、大旗瓣凤仙花、丰满凤仙花、锐齿凤仙花、龙州凤仙花、瑶山凤仙花、菱叶凤仙花、鸭跖草状凤仙花(I. commellinoides)、瑞丽凤仙花、红稚凤仙花及华凤仙11种凤仙花的组培体系。新几内亚凤仙、丰满凤仙花、龙州凤仙花及华凤仙适宜的继代培养基配方:MS+6-BA 1.0mg·L-1+NAA 0.1mg·L-1;锐齿凤仙花、瑶山凤仙花、大旗瓣凤仙花、鸭跖草状凤仙花及红稚凤仙花适宜的继代培养基配方:MS+6-BA 1.0mg·L-1+NAA 0.05mg·L-1;菱叶凤仙花和瑞丽凤仙花适宜的继代培养基配方:MS+6-BA 0.5mg·L-1+NAA 0.05mg·L-1,组培苗生长健壮,增殖正常。
新几内亚凤仙、红稚凤仙花和华凤仙适宜的移栽基质配比是:珍珠岩:草炭=2:1;丰满凤仙花和龙州凤仙花适宜的移栽基质配比是:珍珠岩:蛭石=2:1;锐齿凤仙花、瑶山凤仙花、鸭跖草状凤仙花和瑞丽凤仙花适宜的移栽基质配比是:珍珠岩:草炭=4:1;菱叶凤仙花适宜的移栽基质配比是:蛭石:草炭=1:1。
7.通过改变培养基的大量元素含量、添加调节培养基渗透压的物质、生长抑制剂及改变培养温度等方式,大大延长了组培苗离体保存的时间。不同种对不同保存方式的反应不一。均以4℃培养延长保存时间的效果最为明显,新几内亚凤仙花保存至270d,丰满凤仙花保存至210d,大旗瓣凤仙花保存至240d,华凤仙保存至180d。保存后的组培苗出瓶移栽,除新几内亚凤仙花和大旗瓣凤仙花添加甘露醇的处理在分枝数上与对照有明显差异外,其它与对照在株高、叶片数及分枝数均无明显差异。
8.建立了新几内亚凤仙和丰满凤仙花的玻璃化法超低温保存体系。技术程序如下:切取生长健壮的组培苗2mm左右的茎尖,于20℃下在2mol·L-1甘油+0.4mol·L-1乙二醇混和液中处理20min(丰满凤仙花)或30min(新几内亚凤仙),然后在0℃下用PVS2玻璃化溶液处理30min(丰满凤仙花)或40min(新几内亚凤仙),换新鲜PVS2溶液,快速浸入液氮,40℃水浴中解冻60s,用MS+1.2mol·L-1蔗糖液体培养基洗涤2次,每次10min。最后,转至继代培养基上进行恢复培养,液氮冻存后的茎尖存活率为19%(丰满凤仙花)或23%(新几内亚凤仙)。冻存后的茎尖再生成苗增殖系数与正常组培苗对照没有明显差异。
There are over 900 species in genus Impatiens in the world, of which approximately 220 species distribute in China. Diversity of Impatiens provides abundant gene resources for breeding herb flower species. But up to now our precious germplasm resources are still left in wild without sufficient researches on the breeding and horticultural culture, especially on the preservation of germplasm. Narrow distribution, rich endemism and which need a strict circumstance of Impatiens appear to correlate with human activity and environment changing closely.
In view of above fact, using collected Impatiens species in China, this paper evaluated ornamental character and development value of them by Analytic Hierarchy Process, studied on interspecific relationship, seed germination characteristics and shading tolerance, which was based on summarizing cultivation techniques, and established germplasm preservation system in vitro by methods of slow growth preservation and cryopreservation. The main results were summarized as follows:
1. 38 species, 2 varieties and 7 indeterminate species were collected for the first time, some of them are shown as sharp decreasing because of habitats destruction and invasive plant. Using analytic hierarchy process to evaluate ornamental characteristic and development value of 40 collceted species, the result showed that I. malipoensis, I. claviger var. auriculata, I. claviger, I. apalophylla, I.morsei, I. obesa and I. macrovexilla have better ornamental value and can be used for landscaping directly and preferentially on the premise of germplasm preservation. Development and utilization of aforeside 8 species should be considerated preferentially.
2. The optimal reaction system of AFLP for Impatiens was established, the genetic diversity and relationship were analyzed between 40 Impatiens species. The PCR-based multi-locus DNA fingerprinting technique Amplified Fragment Length Polymorphism(AFLP)analysis of 40 species with 11 AFLP primer combinations produced multi-locus DNA fingerprint with a total of 1003 DNA fragments. Polymorphism rate was 97.11%. Result showed that species which had high similarity at parts of biological characteristics and habitat had nearer relationship. As classification foundation, habit and parts of biological characteristics had more reliability. More methods should be used in taxonomic studies of Impatiens. Result provided valuable evidence for estabilishing taxonomic system and crossbreeding in molecular level.
3. Study on seed germination charactesistics of I. chinensis, I. morsei, I. obesa, I. macrovexilla showed that optimal germination condition of I. chinensis is seed soaking for 8h at 30℃, and germinating at 30℃, the germination rate was 70%. Optimal germination condition of I.morsei is seed soaking for 4h at 20℃, germinating at 20℃, germinating rate was 34%. Germination rate of I. obesa at 20℃was higher than at 30℃, the germination rate was 80%. Germination rate of I. macrovexilla was close to 100% both at 20℃and 30℃. Germination rate of I. obesa and I. morsei have already approximated to maximum on 24th day, I. obesa was on 15th day and I. morsei was on 6th day.
4. At provided three kinds of light intensity, photosynthetic capacity of three Impatiens species(I. chinensis, I.macrovexilla, I. obesa) were discussed. Plant height, stem ameter, leaf number, leaf area, leaf thickness, chlorophyll content and photosynthesis capability were measured. The results showed that the shading adapability of I. macrovexilla was best, then was I. chinensis and I. obesa. Determination of photosynthetic indexes was more convenient and accurate way to judge shade tolerance. Photosynthetic characteristics of 21 Impatiens species had been tested. Three groups were identified based on light adaptability. Among them, I. siculifer, I. wilsonii and I. chlorosepala have better adaptability, I. claviger, I. yaoshanensis and indeterminate species(XingYi) had worse adaptability. Sorting according to requirement for light was, I. uliginosa>I. tomentella>I. siculifer>I. claviger>indeterminate species(XingYi)>I. wilsonii>I. rhombifolia>I. claviger var. auriculata>I. oxyanthera> indeterminate species(Rong An)>I. ruiliensis>I. chlorosepala>I. macrovexilla var. yaoshanensis>I. apaliphylla>indeterminate species ( Lin Gui ) >I. morsei>I. duclouxii>I. tubulosa>I.malipoensis>I.arguta. The light response curve fitting by equation [Pn=Pmax(1-C0 e-aPFD/ Pmax)] was a fairy ideal model to analyze the whole process of light reponse.
5. Based on observation of collecting Impatiens species phenophase, cultivation techniques and control of diseases and insect pests were summarized according to seedling stage, seedling separation stage, seedling formation period and blooming stage. Mode of reproduction included seed propagation, cutting propagation and division propagation. Introduction of 11 species were succeeded. Restriction factors of introduction were tempreture and humidity.
6. Tissue culture system of 11 Impatiens species were established. Optimal disinfection way, subculture medium and substrate composition were screened according to different species. Optimal subculture medium of I. platypatala, I. obesa, I. morsei and I. chinensis: MS+6-BA 1.0mg·L-1+NAA 0.1mg·L-1, optimal subculture medium of I. arguta, I. macrovexilla var. yaoshanensis, I. macrovexilla, I. commellinoides and I. oxyanthera: MS+6-BA 1.0mg·L-1+NAA 0.05mg·L-1, Optimal subculture medium of I. rhombifolia and I. ruiliensis: MS+6-BA 0.5mg·L-1+NAA 0.05mg·L-1. Optimal transplanting medium of I. platypatala, I. oxyanthera and I. chinensis: perlite and peat was 2:1, optimal transplanting medium of I.obesa and I. morsei: perlite and vermiculite was 2:1, optimal transplanting medium of I. arguta, I. macrovexilla var. yaoshanensis, I. commellinoides and I. ruiliensis: perlite and vermiculite was 4:1, Optimal transplanting medium of I. rhombifolia: vermiculite and peat was 1:1.
7. Different species need different ways of slow growth preservation in vitro. More ideal treatment of all was 4℃, at this tempreture, I. platypatala had been preserved for 270d, I. obesa had been preserved for 210d, I. macrovexilla had been preserved for 240d, and I. chinensis had been preserved for 180d. After transplanting, there were no differences between tissue culture seedling after slow growth preservation and normal seedling on plant height, leaf number and branch number, except treaments of adding mannitol of I. platypatala and I. macrovexilla .
8. Shoot tips excised from healthy in vitro plants of I. platypatala and I. obesa were successfully cryopreserved by vitrification. A suitable procedure was established as follows:dissecting shoot tips of 2mm were loaded in a mixture of 2mol·L-1 glycerol plus 0.4 mol·L-1 sucrose for 20min(I. obesa) or 30min(I. platypatala) at 20℃. Following that, a highly concentrated cryoprotective solution (PVS2) was then added at 0℃. After dehydration at 0℃for 30min(I. obesa) or 40min(I. platypatala), the shoot tips were directly plunged into liquid nitrogen. After rapid thawing in a water bath at 40℃for about 60s, the tips were washed twice with 1.8ml of 1.2 mol·L-1 sucrose solution for 10min each time and then transferred onto subculture medium for recovery growth. The highest survival of shoot tips were 19%(I. obesa) or 23%(I. platypatala).
引文
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