Effects of EMS Treatment on Leaf Regeneration and Heat Tolerance of Tissue-cultured Rhododendron chrysanthum Pall. Seedlings

With tissue cultured-seedlings of Rhododendron chrysanthum Pall. in Changbai Mountain as test material, the effect of EMS treatment on the heat toler- ance of R. chrysanthum, as well as the possible heat tolerance mechanism, was in- vestigated in this study so as to lay a foundation for the popularization, application and germplasm resource innovation of R. chrysanthum. The results showed that when the leaves of the tissue cultured seedlings were treated with medium with 0.1% of EMS for 2 d, the survival rate of the leaves was 48.92%, thus the con- centration of EMS was close to the semi-lethal dose, and the EMS concentration and treatment time could be used as screening conditions for heat-tolerant mutants. The plantlets regenerated from EMS-treated leaves were treated with different tem- peratures, and related physiological indicators were determined. The results showed that the chlorophyll content, MDA content, Pro content, SOD activity, POD activity and soluble sugar content of regeneration plantlets in the treatment group were sig- nificantly different from those in the control group. At the same temperature, the chlorophyll content, soluble sugar content, Pro content, SOD activity and POD ac- tivity were significantly higher and the MDA content was significantly lower in the treatment group compared with the control. In conclusion, EMS treatment improved the heat tolerance of tissue-cultured R. chrysanthum seedlings.

Uniform Design for Optimizating Regeneration Shoots Directly from Tender Leaves and Plant Regeneration System of Rhododendron chrysanthum Pall.

[Objective] The experimental was aimed to screen the optimum regeneration shoot induction media and rooting media for tender leaves of Rhododendron chrysanthum Pall.[Method] The tender leaves of Rhododendron chrysanthum Pall were taken as explants to select the optimum bud induction media and rooting media through uniform design and the screening results were verified.[Result] The optimum media for regeneration shoot of Rhododendron chrysanthum Pall contained 1/4 MS,3.70 mg/L ZT, 0.02 mg/L IAA and 1.00 mg/L KT and its induction rate was 95.5% and the rooting media contained modified MS, 0.10 mg/L IAA and 0.07 mg/L NAA and its rooting rate was 98%. [Conclusion] Through this experiment, regeneration systems for regeneration shoot and regenerated plant from tender leaves of Rhododendron chrysanthum Pall were created successfully.

Study on Multiplication, Rooting and Transplanting of Tissue Culture Plantlets of Rhododendron chrysanthum Pall

[Objective] This study aimed to shorten the multiplication culture and root- ing culture periods of Rh. chrysanthum Pall. [Method] The Rh. chrysanthum Pall tis- sue culture plantlets collected from Changbai Mountain were used as material, and the effects of different hormone combinations and coconut milk on the proliferation and differentiation of Rh. chrysanthum Pall tissue culture plantlets were investigated. In addition, the rooting medium and transplanting matrix for Rh. chrysanthum Pall tissue culture plantlets were explored. [Result] The medium composed of modified MS, iBA （3 mg/L） and ZT （1.5 mg/L） was the optimum medium for subculture mul- tiplication of Rh. chrysanthum Pall tissue culture plantlets. The multiplication multiple and average plant height were significantly improved by adding coconut milk into the medium （150 mg/L）. [Conclusion] For Rh. chrysanthum Pall tissue culture plantlets, the optimum rooting culture medium was composed of modified MS （1/4） and IBA （5.0 mg/L）, and the tissue culture plantlets began to root 8 d after the inoculation. The root induction treatment was carried out after a 15-d sand culture, and the suitable matrix was composed of tufty soil, humus soil and perlite （2：1：1） with a survival rate of 95.66%.

Interspecific Cross Compatibility of Rhododendron in Changbai Mountain

In order to explore the interspecific cross compatibility of Rhododendron and the feasibility of interspecific cross in germplasrn innovation, reciprocal crosses among five species of Rhododendron in Changbai Mountain were made, In addition, the hybrid embryos were cultured in vitro. The results showed that the species of Rhododendron in Changbai Mountain had significant differences in reciprocal cross compatibility. When Rh. chrysanthum Pall. was used as female parent, it showed high cross compatibilities with Rh. mucronulatum Turcz. （92.55%）, Rh. mucronulamm Turcz. f, album Nakai （94.14%） and Yabuli Rhododendron （92.42%）, and no signifi- cant differences were found in fruit setting rate by serf-poUination. The germination rates of hybrid embryos were 13.60%, 11.59% and 4.12%, respectively. The hybrid seedlings had weak growth vigor and albino status and were trended to be aborted. After 2-3 generations of subculture, the growth vigor of hybrid seedlings were re- covered gradually, and their differentiation rate was also significantly higher than that of Rh. chrysanthum Pall. seedlings. But the hybrid seedlings were still yellowish green. In reciprocal crosses, the fruit setting rates were significantly reduced. The combination with the highest fruit setting rate （36.51%） was Yabuli Rhododendron x Rh chrysanthum Pall The fruit setting rates of reciprocal crosses between Rh chrysan- thum Pall and Rh. mucronulatum Turcz. and Rh.： chrysanthum Pall. and Rh. schlip- penbachii Maxim were 6.52% and 0, respectively. However, the hybrid embryo＇s germination rate （39,81%） of Yabuli Rhododendron x Rh. chrysanthum Pall. was significantly higher than that of their original cross. Moreover, the hybrid seedlings grew strongly and could be directly transplanted without subculture.

牛皮杜鹃的组培快繁及种质试管保存技术

以牛皮杜鹃休眠芽为外植体,筛选最适合萌发、再分化、生根和种质试管保存的培养基。结果表明最适合休眠芽萌发的培养基为:1/4MS+GA31.5mg·L-1+维生素C20mg·L-1;基部直接再分化培养基为:1/4MS+6-BA4.0mg·L-1+NAA0.3mg·L-1;生根培养基为:MS+IBA0.46mg·L-1+活性炭450mg·L-1;种质试管保存培养基为:1/6MS(大量元素)+1/4MS(微量元素)+1/2MS(铁盐)+1/4MS(有机物质)+B93.5mg·L-1。牛皮杜鹃组织培养的不同阶段需要不同的培养基,常温条件下,利用低营养矮化常温的方法在试管内保存种质资源。

TDZ对牛皮杜鹃叶片分化及继代增殖的影响

以牛皮杜鹃组培苗为试材,研究以改良MS为基本培养基,不同质量浓度TDZ、ZT与IBA组合,对叶片愈伤组织诱导及分化、继代增殖的影响。结果表明:TDZ对组培苗叶片愈伤组织诱导及分化的效果显著优于ZT,但在继代培养时增殖效果不如ZT;TDZ 0.3mg/L+IBA 0.5mg/L处理的叶片分化效果最好,分化率高达97.44%;不定芽在改良MS+ZT 0.2mg/L+IBA 0.5mg/L中继代培养后,每瓶分化的不定芽数最多(30.2个)。

长白山牛皮杜鹃组培苗与野生苗药用成分含量分析

采用HPLC法与紫外分光光度法分别测定了长白山牛皮杜鹃组培苗与野生苗中主要药用成分芦丁与单宁酸的含量。结果表明,芦丁在野生苗两年生老叶中含量最高,为2.54mg/g,野生苗嫩叶含量为1.68mg/g,愈伤组织含量为0.16mg/g,组培苗含量为0.08mg/g,组培叶含量为0.03mg/g,根中未检测到芦丁成分。单宁酸在野生苗老叶中含量最高,为91.54mg/g,野生苗嫩叶含量为45.87mg/g,野生苗根中为24.58mg/g,愈伤组织中含量为13.51mg/g,组培苗中含量为10.32mg/g,组培叶中含量为17.46mg/g。野生苗中芦丁与单宁酸含量均高于组培苗。

不同海拔长白山牛皮杜鹃叶片适应性结构的对比分析

以长白山牛皮杜鹃为试材,研究比较了1 500、1 800、2 100、2 400m4个不同海拔高度牛皮杜鹃种群的叶片表面形态结构特征和叶片功能性状。结果表明:不同海拔高度的牛皮杜鹃叶片上表皮细胞随海拔高度的升高而排列比较疏松,叶片下表皮细胞排列比较有规则。叶干物质含量随海拔增加而增加,低海拔与高海拔的叶干物质含量差异显著;比叶面积随着海拔增加而减少,海拔1 800m叶片比叶面积低于其它3个海拔高度;叶厚随海拔增加而增加,海拔1 800m叶片厚度高于其它3个海拔。

长白山杜鹃花属种间杂交亲和性的研究

为了探索长白山杜鹃花属种间杂交的亲和性及种间杂交在种质创新中的可行性,以5种长白山杜鹃花属植物为试材,进行种间正反杂交,并对杂种胚进行离体培养.结果表明,杜鹃花属种间正反杂交亲和力有显著差异,以牛皮杜鹃为母本,与迎红杜鹃、白花迎红杜鹃、亚布力杜鹃杂交有较高的亲和性,与母本的自然授粉坐果率无显著差异,分别为92.55％、94.14％、92.42％,杂种胚的萌发率分别为13.60％、11.59％、4.12％;杂种苗生长势微弱,呈白化状态,有败育迹象,经继代培养2～3次后,生长势得到一定的恢复,且分化率明显高于牛皮杜鹃种胚苗,但植株的颜色仍是黄绿色.而反交时,坐果率明显降低,坐果率最高的组合为亚布力杜鹃×牛皮杜鹃,为36.51％;与迎红杜鹃的杂交坐果率为6.52％;而与大字杜鹃的杂交坐果率为0,但反交时亚布力×牛皮杜鹃的种胚萌发率极显著地高于正交,为39.81％,而且种胚苗生长健壮,无需继代,可直接进行炼苗移栽.

延缓生长法在试管内保存牛皮杜鹃种质初探

以牛皮杜鹃嫩茎基部直接再生形成的丛生芽团为试材,应用均匀设计法探讨了影响牛皮杜鹃丛生芽团试管保存的主要因子和水平,同时筛选最适于牛皮杜鹃丛生芽团试管保存的培养基,结果表明:最适宜的丛生芽团试管保存培养基为:DR+根皮苷2.70 mg/L+KT0.70 mg/L,保存46个月生长率仅为1.12%。对保存前后的芽苗分化的结果进行对比可知,分化率达99.5%以上,较未保存前的分化率89.0%高,且保存后的生根速度快。本试验结果证明,在试管内采取"延缓生长"法保存牛皮杜鹃丛生芽团是切实可行的新方法。

基于均匀设计优化牛皮杜鹃嫩叶直接再生芽苗及植株再生体系

[目的]筛选最适合的牛皮杜鹃嫩叶直接再生芽苗诱导培养基和生根培养基。[方法]以牛皮杜鹃嫩叶为外植体,应用均匀设计对牛皮杜鹃嫩叶直接再生芽苗诱导培养基和生根培养基进行筛选,并对筛选结果进行了验证。[结果]最适合牛皮杜鹃嫩叶直接再生芽苗的诱导培养基为:1/4 MS+3.70 mg/L ZT+0.02 mg/L IAA+1.00 mg/L KT,诱导率达95.5%;最适生根培养基为:MS(改良)+0.10mg/L IAA+0.07 mg/L NAA,生根率达98%。[结论]通过验证试验证明,成功建立了牛皮杜鹃嫩叶直接再生芽苗及植株再生体系。

长白山牛皮杜鹃种苗速生调控技术

本研究以牛皮杜鹃组培苗为供试植物,通过多因子交叉重复试验对影响牛皮杜鹃组培苗快速生长的各主要因素及其水平的作用进行了探讨,旨在探索并建立一种牛皮杜鹃种苗快速生长的调控方法。结果表明：最适合牛皮杜鹃组培苗快速生长的主要因素及其水平是光照时间为13 h·d-1、营养液追施间隔时间为21~22 d、光照强度为700~800 lx和温度为26℃。经过25 d的调控处理,牛皮杜鹃组培苗纵向生长高度和横向直径分别达到17.6 cm和6.0 mm,对照组仅分别达到8.8 cm和2.0 mm;50 d后纵向生长高度和横向直径分别达到26.0 cm和7.4mm,而对照组仅分别达到10.2 cm和2.9 mm。初步得到牛皮杜鹃种苗速生的优化组合,该组合将有利于牛皮杜鹃种苗的快速生长。

牛皮杜鹃RAPD反应体系的优化

[目的]建立牛皮杜鹃RAPD反应体系。[方法]以野生牛皮杜鹃的叶片为材料,采用改进CTAB法提取基因组DNA,并对影响RAPD扩增反应的各因素进行优化。[结果]适合牛皮杜鹃的RAPD反应体系为:在20μlPCR反应体积中加模板DNA10ng,Mg2+浓度1.5mmol/L,引物OPC0515pmol/μl,dNTP0.15mmol/L,TaqDNA聚合酶1.0U。[结论]该研究为牛皮杜鹃的遗传多样性分析提供了有益的参考。

不同地区牛皮杜鹃叶片解剖结构比较分析

利用光学显微镜对生长在长白山小天池和敦化老白山的牛皮杜鹃叶片横切面进行观察.结果表明,生长在老白山的牛皮杜鹃叶片长度、宽度和叶柄长度都大于生长在小天池的牛皮杜鹃;内部结构上,生长在老白山的牛皮杜鹃叶片主脉直径、叶片厚度、栅栏组织厚度、海绵组织厚度的测量值均高于小天池的牛皮杜鹃,上、下表皮的厚度和细胞层数没有显著差异.

牛皮杜鹃植株解剖结构的研究

在前人对杜鹃花研究的基础上,以长白山野生牛皮杜鹃(Rhododendron Chrysanthum Pall)为材料,采用常规石蜡切片法对根、茎、叶、花、果实及种子的解剖结构进行观察研究.

均匀设计法优化牛皮杜鹃腋芽丛生芽苗及高频植株再生体系研究

以牛皮杜鹃嫩茎段为外植体,应用均匀设计法筛选最适合的培养基。结果表明,最适合嫩茎段腋芽丛生芽苗的诱导培养基为1/2 MS+2-ip 3.50mg/L+IAA 0.02mg/L+GA3 1.00mg/L,诱导率可达96%;生根培养基为MS(改良)+IAA 0.10mg/L+NAA 0.07mg/L,生根率可达98%。以再生植株的茎节为材料进行快繁的结果表明,在30d的一个培养周期内,增殖倍数平均达60以上。通过试验,建立了牛皮杜鹃腋芽丛生芽苗及高频植株的再生体系。

3种长白山高山杜鹃叶片结构及其对环境的适应性

依托扫描电子显微技术,研究了长白山高山珍稀濒危植物牛皮杜鹃、毛毡杜鹃及苞叶杜鹃的叶片结构,分析了其对高山环境的适应性。结果表明:3种杜鹃叶片均为异面叶,具表皮附属物。上表皮由1～2层细胞组成,有角质层,无气孔器;下表皮由1层细胞组成,有气孔器分布。栅栏组织细胞排列紧密有2～8层,海绵组织细胞间隙大,中脉维管束为圆形外韧型。此外,叶片的表皮附属物类型,气孔器特点,海绵组织细胞排列方式等差异明显。3种杜鹃植物的扫描电镜结构特征,既为种间鉴别提供了依据,也反映了其对高山环境的适应性。

不同海拔牛皮杜鹃种群遗传多样性的RAPD分析

利用RAPD标记技术对采自老白山和长白山不同海拔的牛皮杜鹃进行遗传多样性分析。结果表明:产地不同(长白山和老白山)的牛皮杜鹃种群间的遗传多样性水平较高,遗传距离较大;而产地相同(长白山不同海拔)的牛皮杜鹃种群间遗传多样性水平较低,具有较小的遗传距离,即遗传背景相对相似;长白山不同海拔的4个牛皮杜鹃种群还可分为两类。

长白山不同海拔牛皮杜鹃叶片解剖结构的比较分析

采用石蜡切片法制片,利用光学显微镜观察和比较了长白山北坡9个海拔高度（1 700～2 600 m）牛皮杜鹃叶片的形态和解剖结构.结果表明,随着海拔的升高,牛皮杜鹃叶片的厚度、栅栏组织厚度、栅海比及叶片紧密度均呈增加趋势,而叶片长度、叶片宽度、叶柄长度、海绵组织厚度、主脉直径和叶疏松度呈减小趋势;叶片长宽比、表皮厚度及表皮细胞层数无明显差异.

牛皮杜鹃嫩茎段试管内高效再生方法

本研究以牛皮杜鹃嫩茎段为外植体,通过均匀设计法对影响牛皮杜鹃腋芽萌发生长同时生根的各主要因素及其水平的作用进行了实验探讨,旨在探索一种试管内牛皮杜鹃高效植株的再生方法。结果表明,最适合牛皮杜鹃嫩茎段生根同时腋芽萌发生长的培养基为：基本培养基＋KT 0.13-0.14 mg·L-1＋IBA 0.05-0.08 mg·L-1＋NAA 0.02-0.05 mg·L-1＋GA3 2.70-2.90 mg·L-1,植株再生率为99.2%。以再生植株茎节为材料进行快繁的结果表明,在28 d的培养周期内,每段平均增殖6倍以上。再生植株的移栽成活率达96.0%以上。成功建立了高效植株再生体系,所建立的高效植株再生体系适合于牛皮杜鹃种苗工厂化生产。