君子兰生殖生物学基础及种质资源分子标记研究
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摘要
君子兰(Clivia miniata),石蒜科(Amaryllidaceae),君子兰属(CliviaLindl.),多年生草本植物。君子兰全株入药,并且具有观赏价值。该属共包括6个二倍体种,大花君子兰(C.miniata)是我国北方常见的栽培品种。本文以大花君子兰为试验材料,观察其不同花期雄核发育进程,研究其花粉生活力的测定方法,研究其开花特性、柱头可授性和自交不亲和性机制,研究其有性繁殖和无性繁殖育种技术的特点；并且以大花君子兰和垂笑君子兰为试验材料,利用微卫星分子标记技术对其进行亲缘关系分析。主要结果如下：
对61个试验样本进行DNA扩增,其中大花栽培品种51个,8个垂笑品种,2个大花和垂笑的杂交品种。来自大花君子兰的11对引物(CM3, CM9, CM12, CM54, CM65, CM103, CM137, CM253, CM289, CM357, CM425)和来自垂笑君子兰的3对引物(CN68, CN89, CN106)在目的片段区出现清晰的扩增子。聚丙烯酰胺变性凝胶电泳检测结果,其中4对引物(CM3, CM253, CM425, CN89)为单态,10对引物具有多态性。其创新点是首次采用微卫星分子标记筛选10对可靠的、有效的遗传学分子标记物,为君子兰物种的系统发育关系及遗传学多样性研究提供了有用的信息。
利用荧光显微技术观察君子兰柱头可授性和自交不亲和现象,结果表明君子兰开花2-3天后柱头具有强可授性,君子兰花粉管中不规则出现的胼胝质塞影响花粉管的正常发育,柱头上也可见胼胝质出现,导致花粉管不能正常进入柱头。浓度1%-5%的NaCl溶液能够有效地克服君子兰自交不亲和性；在NaCl溶液中添加0.3-0.5%的H3BO3溶液可以对其作用效果略有提高。其创新点是解决了君子兰自交育种的障碍,为克服自交不亲和配制杂种一代工作奠定基础。
本文明确地将君子兰的花期划分为5个时期,确定不同时期的形态特征,并且研究了各个时期花药发育特点和雄核发育进程。结果表明君子兰在现蕾期阶段小孢子处于单核期或者单核靠边期,这一时期的花粉适合做单倍体培养的外植体选材。同时筛选了花粉活力的最佳测定方法是I：-KI染色法；花粉的萌发的最佳培养基配方是MS液体培养基+NAA0.5mg/L+2,4-D0.1mg/L.其创新点在于为小孢子培养奠定了理论基础。
本文对君子兰的开花特性和花莛的生长规律进行了研究。结果表明君子兰花莛生长存在“慢-快-慢”的周期,生长曲线呈“S”型。浓度50-100mg/L的GA3溶液能够有效促进花莛长度生长。其创新点在于研究花莛快速生长的时间,为栽培工作确定最佳的肥水调控时机,以便调控君子兰花莛的长度生长。
通过对君子兰的无性繁殖方式进行研究发现“抹头”和“折箭”这两种处理方法可以促进君子兰脚芽萌发；利用君子兰未授粉子房和胚珠进行组织培养,出愈率最高的外植体形式是胎座+胚珠；最佳培养基配方是MS+琼脂0.8%+蔗糖3%+2,4-Dlmg/L+KT2mg/L;最佳培养方式是固-液双层培养。最适合播种的种子是种粒饱满的中等大小种子,并且随采随播。
Clivia miniata which belongs to Clivia Lindl, the family Amaryllidaceae is a perennial herb. All parts of Clivia mimiata are used as medicine and it also has ornamental value. The genus Clivia Lindl includes six diploid species (2n=2x=22). Clivia miniata is cultivated varieties common in Northern China. In this paper, Clivia miniata was used as experimental materials. The different florescence androgenesis processes were observed and its pollen viability was determined. The stigma receptivity and self-incompatibility mechanism were studied and flowering habit were also observed. Meanwhile, the characteristics of sexual reproduction and asexual reproduction breeding technology were studied. The close relative relationship between the wild species and cultivars was analysesed by using microsatellite molecular marker technique. The main results are as follows:
61test samples were processed by DNA amplification, of which,51Clivia miniata cultivated varieties,8Clivia nobilis wild types,2hybrid variety between Clivia miniata and Clivia nobilis. The clear amplicons appeared in the objective fragments from11pairs of primers of Clivia miniata (CM3, CM9, CM12, CM54, CM65, CM103, CM137, CM253, CM289, CM357, CM425) and3pairs of primers of Clivia nobilis (CN68, CN89, CN106). The detection results with denaturing polyacrylamide gel electrophoresis indicate that4pairs of primers (CM3, CM253, CM425, CN89) are singlet state and10pairs of primers are with polymorphism. The innovation is that screening10pairs of effective, reliable genetics and molecular marker by using microsatellite molecular marker technique firstly, which provides useful information for the phylogeny relationship and genetic diversity research of Clivia Lindl.
The Clivia miniata stigma receptivity and self-incompatibility phenomenon was observed by using fluorescence microscopy technique which indicates that Clivia miniata stigma has a strong receptivity while Clivia miniata blossom2-3days and irregular callose plug appear in the Clivia miniata pollen tube affect the pollen germination.
The callose also appeared in the stigma which result in the pollen tube can not enter the stigma normally. The concentration of NaCl solution1%-5%can effectively overcome the self-incompatibility;0.3-0.5%H3BO3solution in NaCl solution can increase the effect slightly. The innovation point is to solve the Clivia inbred breeding barriers, so as to establish the foundation work for overcoming self-incompatibility with hybrids.
This paper divided the Clivia miniata bloom clearly and determines the morphological characteristics of different florescence. Also, each period of anther development characteristics and androgenesis process were studied. The results show the microspores remain at uninucleate stage or late-uninucleate when Clivia miniata in budding stage. Microspore in this period is suitable for explant material of haploid culture. The optimal determine method for pollen viability was screened and that is I2-KI staining method. The best medium of pollen germination is liquid MS medium+NAA0.5mg/L2,4-D0.1mg/L.The innovation point is laid the theoretical foundation for the microspore culture.
The characteristics of Clivia miniata blossom and growth regularity of scape were also studied. The results show that scape growth exist "slow-fast-slow" circle and the growth trendline was "S" type. The concentration of GA3solution50-100mg/L can effectively promote the scape length growth and improve the quality of the flower. Innovation is to find the scape rapid growth time and to determine the best time to fertilizer and water management for cultivation so that to control the scape length growth.
The Clivia miniata asexual propagation methods were studied in this experiment. The results show that "head disposed" and "scape broken" these two kinds of treatment methods can promote offshoot germination. The highest rate of explants form even is placenta+ovules in tissue culture of young ovaries and ovules of Clivia miniata; The best medium is MS+0.8%agarophyte+3%sucrose+2,4-Dlmg/L+KT2mg/L;The best culture method is liquid-solid double-layer culture. The most suitable sowing seed is a medium size vigor seed, and being warehoused no more than3months.
对61个试验样本进行DNA扩增,其中大花栽培品种51个,8个垂笑品种,2个大花和垂笑的杂交品种。来自大花君子兰的11对引物(CM3, CM9, CM12, CM54, CM65, CM103, CM137, CM253, CM289, CM357, CM425)和来自垂笑君子兰的3对引物(CN68, CN89, CN106)在目的片段区出现清晰的扩增子。聚丙烯酰胺变性凝胶电泳检测结果,其中4对引物(CM3, CM253, CM425, CN89)为单态,10对引物具有多态性。其创新点是首次采用微卫星分子标记筛选10对可靠的、有效的遗传学分子标记物,为君子兰物种的系统发育关系及遗传学多样性研究提供了有用的信息。
利用荧光显微技术观察君子兰柱头可授性和自交不亲和现象,结果表明君子兰开花2-3天后柱头具有强可授性,君子兰花粉管中不规则出现的胼胝质塞影响花粉管的正常发育,柱头上也可见胼胝质出现,导致花粉管不能正常进入柱头。浓度1%-5%的NaCl溶液能够有效地克服君子兰自交不亲和性；在NaCl溶液中添加0.3-0.5%的H3BO3溶液可以对其作用效果略有提高。其创新点是解决了君子兰自交育种的障碍,为克服自交不亲和配制杂种一代工作奠定基础。
本文明确地将君子兰的花期划分为5个时期,确定不同时期的形态特征,并且研究了各个时期花药发育特点和雄核发育进程。结果表明君子兰在现蕾期阶段小孢子处于单核期或者单核靠边期,这一时期的花粉适合做单倍体培养的外植体选材。同时筛选了花粉活力的最佳测定方法是I：-KI染色法；花粉的萌发的最佳培养基配方是MS液体培养基+NAA0.5mg/L+2,4-D0.1mg/L.其创新点在于为小孢子培养奠定了理论基础。
本文对君子兰的开花特性和花莛的生长规律进行了研究。结果表明君子兰花莛生长存在“慢-快-慢”的周期,生长曲线呈“S”型。浓度50-100mg/L的GA3溶液能够有效促进花莛长度生长。其创新点在于研究花莛快速生长的时间,为栽培工作确定最佳的肥水调控时机,以便调控君子兰花莛的长度生长。
通过对君子兰的无性繁殖方式进行研究发现“抹头”和“折箭”这两种处理方法可以促进君子兰脚芽萌发；利用君子兰未授粉子房和胚珠进行组织培养,出愈率最高的外植体形式是胎座+胚珠；最佳培养基配方是MS+琼脂0.8%+蔗糖3%+2,4-Dlmg/L+KT2mg/L;最佳培养方式是固-液双层培养。最适合播种的种子是种粒饱满的中等大小种子,并且随采随播。
Clivia miniata which belongs to Clivia Lindl, the family Amaryllidaceae is a perennial herb. All parts of Clivia mimiata are used as medicine and it also has ornamental value. The genus Clivia Lindl includes six diploid species (2n=2x=22). Clivia miniata is cultivated varieties common in Northern China. In this paper, Clivia miniata was used as experimental materials. The different florescence androgenesis processes were observed and its pollen viability was determined. The stigma receptivity and self-incompatibility mechanism were studied and flowering habit were also observed. Meanwhile, the characteristics of sexual reproduction and asexual reproduction breeding technology were studied. The close relative relationship between the wild species and cultivars was analysesed by using microsatellite molecular marker technique. The main results are as follows:
61test samples were processed by DNA amplification, of which,51Clivia miniata cultivated varieties,8Clivia nobilis wild types,2hybrid variety between Clivia miniata and Clivia nobilis. The clear amplicons appeared in the objective fragments from11pairs of primers of Clivia miniata (CM3, CM9, CM12, CM54, CM65, CM103, CM137, CM253, CM289, CM357, CM425) and3pairs of primers of Clivia nobilis (CN68, CN89, CN106). The detection results with denaturing polyacrylamide gel electrophoresis indicate that4pairs of primers (CM3, CM253, CM425, CN89) are singlet state and10pairs of primers are with polymorphism. The innovation is that screening10pairs of effective, reliable genetics and molecular marker by using microsatellite molecular marker technique firstly, which provides useful information for the phylogeny relationship and genetic diversity research of Clivia Lindl.
The Clivia miniata stigma receptivity and self-incompatibility phenomenon was observed by using fluorescence microscopy technique which indicates that Clivia miniata stigma has a strong receptivity while Clivia miniata blossom2-3days and irregular callose plug appear in the Clivia miniata pollen tube affect the pollen germination.
The callose also appeared in the stigma which result in the pollen tube can not enter the stigma normally. The concentration of NaCl solution1%-5%can effectively overcome the self-incompatibility;0.3-0.5%H3BO3solution in NaCl solution can increase the effect slightly. The innovation point is to solve the Clivia inbred breeding barriers, so as to establish the foundation work for overcoming self-incompatibility with hybrids.
This paper divided the Clivia miniata bloom clearly and determines the morphological characteristics of different florescence. Also, each period of anther development characteristics and androgenesis process were studied. The results show the microspores remain at uninucleate stage or late-uninucleate when Clivia miniata in budding stage. Microspore in this period is suitable for explant material of haploid culture. The optimal determine method for pollen viability was screened and that is I2-KI staining method. The best medium of pollen germination is liquid MS medium+NAA0.5mg/L2,4-D0.1mg/L.The innovation point is laid the theoretical foundation for the microspore culture.
The characteristics of Clivia miniata blossom and growth regularity of scape were also studied. The results show that scape growth exist "slow-fast-slow" circle and the growth trendline was "S" type. The concentration of GA3solution50-100mg/L can effectively promote the scape length growth and improve the quality of the flower. Innovation is to find the scape rapid growth time and to determine the best time to fertilizer and water management for cultivation so that to control the scape length growth.
The Clivia miniata asexual propagation methods were studied in this experiment. The results show that "head disposed" and "scape broken" these two kinds of treatment methods can promote offshoot germination. The highest rate of explants form even is placenta+ovules in tissue culture of young ovaries and ovules of Clivia miniata; The best medium is MS+0.8%agarophyte+3%sucrose+2,4-Dlmg/L+KT2mg/L;The best culture method is liquid-solid double-layer culture. The most suitable sowing seed is a medium size vigor seed, and being warehoused no more than3months.
引文
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