羊草繁殖生物学特性的研究
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摘要
羊草(Leymus chinensis(Trin.)Tzvel.)又称碱草，隶属禾本科，赖草属，因其营养价值高，富含蛋白质，适口性好，抗旱，耐盐碱，耐贫瘠，抗逆性强，适应性广等优点，对我国发展草原畜牧业和退化草地、荒漠化治理方面具有举足轻重的作用。近年来，由于自然环境变劣，荒漠化加剧，以及过度放牧等不利影响，加之羊草本身固有的“三低”问题(即结实率低、出苗率低、产草量低)已对羊草生物多样性维持构成了严重的威胁，严重限制了我国人工草地建设和天然草地的改良和沙化治理的步伐。加强羊草生物学研究，开展羊草种质生物多样性保护，成为当前研究的紧迫课题。经对国内外相关文献的查新发现：国外发表的文献匮乏，国内的报导大多集中在草原生态等宏观领域，在羊草繁殖生物学方面缺乏系统的研究。本文以本课题组从国内外收集的羊草资源为材料，从以下几个方面进行了初步探索：
一、羊草繁殖性状与遗传多样性分子标记指标的相关分析。利用分子标记与形态标记对随机抽取的17份羊草种质进行了种质评估的比较研究。结果表明：两种方法均在17份供试材料中鉴别出9份羊草种质，说明分子标记方法用于羊草种质资源鉴定是可行的，并具有快速、准确、不受环境条件限制等优点。在40个10Mer的随机引物当中筛选出21个有效引物，以之对9份羊草材料进行RAPD分析，共扩增出115条带，其中95条带表现出多态性，多态比率82.61％，并筛选出S1213-900，S1213-1700，S1215-5500，S1396-1370，S1384-900，S1202-5180，S1220-2200，S1381—1580，S1211-2800，S1211-800为羊草种质所具有的10个特异性标记，据此可将羊草种质与披肩草、赖草加以区分。同时在羊草种内亦发现13条可区分供试羊草种质的特有标记。形态标记与分子标记相关性分析结果显示：羊草种质的小穗数，种子千粒重，叶色，有性繁殖量和结实率5个形态学指标与遗传多样性指标---特有带百分率及遗传距离之间，存在一定相关性。同时对羊草种质资源在收集和评价过程中存在的问题进行了探讨。
二、羊草不同基因型无性繁殖特性比较研究。以本课题组从吉林、内蒙古等省份收集的10个基因型羊草为供试材料，在相同的生态因子作用下，以吉生1号羊草为对照，对10个基因型羊草的叶数增量、芽数增量、芽高度、芽间
距、芽重量、根量六个无性系形态性状指标进行测评。结果表明：基因型的差
异也是影响羊草无性系生长发育的重要影响因子。因此，在今后的羊草无性繁
殖生物学研究中，应综合考虑环境因子和基因型因于对羊草无性繁殖生长发育
的影响。在所测评的10个基因型中，各基因型的形态性状指标差异很大，栽3
基因型较其他基因型优于对照吉生1号，此结论可为今后培育羊草新种提供重
要资料。
三、羊草幼穗离体培养方法的建立。其方法是取羊草幼穗为外植体，经
0l％升汞溶液表面消毒后，接种到含 Zing／L的 2个D的 MS培养基上，置于恒
温 25OC条件下诱导愈伤组织。在加有 ling／LZ，4D的 MS培养基上继代 2次后，
转移到含 ling／L KT和 0．smg／LNAA的 MS培养基上分化培养得到再生芽。在除
去激素后的基本培养基上获得了生根的试管苗。试管苗移栽到温室后生长正
常。羊草试管苗的分化因基因型和外源激素条件的不同而异。
四、羊草有性生殖特性的研究。在自然条件下进行了羊草自交、异交结实
性实验，采用FDA染色法检测羊草小袍子活性，并观测羊草雌蕊、雄蕊发育的
时空特点。结果表明：在大田中羊草异交结实率远大于其自交结实率；成熟花
药中有活性的花粉达到92二％以上；同时，在发育时间顺序和空间结构上，羊
草的雌蕊、雄蕊并不妨碍自体授粉。因此，初步结论认为羊草具自交不和性。
Leymus chinensis (Trin.) Tzvel.,member of gramineae.Leymus,is a very important forage species. It was not only the king of gramineae forage due to high protein content,palatability to cattle and adaptability to surroundings,but also a major candidate in artificial grassland construction and improving ecotype as well. In recent years,however,owing to the exacerbated environment problems,overload on grassland and the "three low problems"(low setting percentage,low germination percentage,low grass productivity),the population of L. chinensis has become more and more degenerated which restrict the artificial grassland construction,natural grassland improvement and control of deserted land. Thus,it is very urgent to strength research and protect the biological diversity of L. chinensis. So far,there are limited reports abroad in this area,meanwhile,the papers published in China mostly concentrate on macroscopic aspects such as grassland ecology et. Furthermore,less systematic research work and reports are focused
on reproduction biology. In this paper,initial research work have been done from four aspects as listed as below with collected L. chinensis. germaplasm resources from out and all over the country:
Firstly:Correlation analysis between reproduction character and genetic diversity molecular marker index of L. chinensis. Seventeen accessions were selected arbitrarily from collected germplasm resources. The identification on species of L. chinensis was carried out based on both RAPD and morphological data. The results suggested that only nine of them were object species occupying 53% among all experiment materials. The conclusion revealed it was reliable that RAPD marker was used in identification and evaluation on germplasm resource of L.chineses. Compared with morphological data,RAPD marker offered an efficient means to determine this species with the advantages such as rapidly,reliably and no influenced by environment events. Twenty-one primers were screened from 40 ten mer arbitrary primers,and a total of 115 DNA fragments were amplified ranging from 0.2-5.5 kb in 9 L.chineses,among which 95 (82.61%) were polymorphism. Ten specific bands-S1213-900,S1213-1700,S1215-5500,S1396-1370,S1384-900,S1202-5180,S
1220-2200,S1381-1580,S1211-2800,S1211-800 were found in nine genotypes of L.chineses. Furthermore,the samples of L.chineses could be distinguished out with the 10 special marker. The correlation analyzed between based on morphological data and on RAPD data showed that there were some correlation between number of spikes,weight of thousand seeds,leaf color,the level of sexual propagation,setting percentage and index of genetic diversity. Additionally,the problems in the process of collecting,arranging,evaluating for accession of L.chineses were discussed.
Secondly:Comparable research on asexual reproduction biological character of different L. chinensis. genotypes. Analysis was conducted on six cloning morphological character index-leaf
number increment bud number increment,bud height,bud distance,bud weight,root number of 10 L. chinensis. genotypes. The genotype Jisheng-1 was chosen as control and the experimental ecological factors are the same. The results showed that:genotype difference is a very important influencing factor for the cloning growth and development of L. chinensis,both environmental factor and genotype factor should be considered in the future research on asexual reproduction biology of L. chinensis. Between the 10 genotypes,morphological character index was obviously different. Furthermore,compared with other genotypes,Zai-3 genotype had better character index than Jisheng-1. All these conclusions will provide foundation for future breeding research work of L. chinensis.
Thirdly:Establishment of regeneration system of L. chinensis through culture in vitro. Immature-stachys-base explants were cultured on Murashige and Skoog's basal medium (MS) supplemented with 2 mg/L 2,4-D after surface sterilized with 0.1% HgCl2 to induce callus at 25 C constant temperature. After moved to M
一、羊草繁殖性状与遗传多样性分子标记指标的相关分析。利用分子标记与形态标记对随机抽取的17份羊草种质进行了种质评估的比较研究。结果表明：两种方法均在17份供试材料中鉴别出9份羊草种质，说明分子标记方法用于羊草种质资源鉴定是可行的，并具有快速、准确、不受环境条件限制等优点。在40个10Mer的随机引物当中筛选出21个有效引物，以之对9份羊草材料进行RAPD分析，共扩增出115条带，其中95条带表现出多态性，多态比率82.61％，并筛选出S1213-900，S1213-1700，S1215-5500，S1396-1370，S1384-900，S1202-5180，S1220-2200，S1381—1580，S1211-2800，S1211-800为羊草种质所具有的10个特异性标记，据此可将羊草种质与披肩草、赖草加以区分。同时在羊草种内亦发现13条可区分供试羊草种质的特有标记。形态标记与分子标记相关性分析结果显示：羊草种质的小穗数，种子千粒重，叶色，有性繁殖量和结实率5个形态学指标与遗传多样性指标---特有带百分率及遗传距离之间，存在一定相关性。同时对羊草种质资源在收集和评价过程中存在的问题进行了探讨。
二、羊草不同基因型无性繁殖特性比较研究。以本课题组从吉林、内蒙古等省份收集的10个基因型羊草为供试材料，在相同的生态因子作用下，以吉生1号羊草为对照，对10个基因型羊草的叶数增量、芽数增量、芽高度、芽间
距、芽重量、根量六个无性系形态性状指标进行测评。结果表明：基因型的差
异也是影响羊草无性系生长发育的重要影响因子。因此，在今后的羊草无性繁
殖生物学研究中，应综合考虑环境因子和基因型因于对羊草无性繁殖生长发育
的影响。在所测评的10个基因型中，各基因型的形态性状指标差异很大，栽3
基因型较其他基因型优于对照吉生1号，此结论可为今后培育羊草新种提供重
要资料。
三、羊草幼穗离体培养方法的建立。其方法是取羊草幼穗为外植体，经
0l％升汞溶液表面消毒后，接种到含 Zing／L的 2个D的 MS培养基上，置于恒
温 25OC条件下诱导愈伤组织。在加有 ling／LZ，4D的 MS培养基上继代 2次后，
转移到含 ling／L KT和 0．smg／LNAA的 MS培养基上分化培养得到再生芽。在除
去激素后的基本培养基上获得了生根的试管苗。试管苗移栽到温室后生长正
常。羊草试管苗的分化因基因型和外源激素条件的不同而异。
四、羊草有性生殖特性的研究。在自然条件下进行了羊草自交、异交结实
性实验，采用FDA染色法检测羊草小袍子活性，并观测羊草雌蕊、雄蕊发育的
时空特点。结果表明：在大田中羊草异交结实率远大于其自交结实率；成熟花
药中有活性的花粉达到92二％以上；同时，在发育时间顺序和空间结构上，羊
草的雌蕊、雄蕊并不妨碍自体授粉。因此，初步结论认为羊草具自交不和性。
Leymus chinensis (Trin.) Tzvel.,member of gramineae.Leymus,is a very important forage species. It was not only the king of gramineae forage due to high protein content,palatability to cattle and adaptability to surroundings,but also a major candidate in artificial grassland construction and improving ecotype as well. In recent years,however,owing to the exacerbated environment problems,overload on grassland and the "three low problems"(low setting percentage,low germination percentage,low grass productivity),the population of L. chinensis has become more and more degenerated which restrict the artificial grassland construction,natural grassland improvement and control of deserted land. Thus,it is very urgent to strength research and protect the biological diversity of L. chinensis. So far,there are limited reports abroad in this area,meanwhile,the papers published in China mostly concentrate on macroscopic aspects such as grassland ecology et. Furthermore,less systematic research work and reports are focused
on reproduction biology. In this paper,initial research work have been done from four aspects as listed as below with collected L. chinensis. germaplasm resources from out and all over the country:
Firstly:Correlation analysis between reproduction character and genetic diversity molecular marker index of L. chinensis. Seventeen accessions were selected arbitrarily from collected germplasm resources. The identification on species of L. chinensis was carried out based on both RAPD and morphological data. The results suggested that only nine of them were object species occupying 53% among all experiment materials. The conclusion revealed it was reliable that RAPD marker was used in identification and evaluation on germplasm resource of L.chineses. Compared with morphological data,RAPD marker offered an efficient means to determine this species with the advantages such as rapidly,reliably and no influenced by environment events. Twenty-one primers were screened from 40 ten mer arbitrary primers,and a total of 115 DNA fragments were amplified ranging from 0.2-5.5 kb in 9 L.chineses,among which 95 (82.61%) were polymorphism. Ten specific bands-S1213-900,S1213-1700,S1215-5500,S1396-1370,S1384-900,S1202-5180,S
1220-2200,S1381-1580,S1211-2800,S1211-800 were found in nine genotypes of L.chineses. Furthermore,the samples of L.chineses could be distinguished out with the 10 special marker. The correlation analyzed between based on morphological data and on RAPD data showed that there were some correlation between number of spikes,weight of thousand seeds,leaf color,the level of sexual propagation,setting percentage and index of genetic diversity. Additionally,the problems in the process of collecting,arranging,evaluating for accession of L.chineses were discussed.
Secondly:Comparable research on asexual reproduction biological character of different L. chinensis. genotypes. Analysis was conducted on six cloning morphological character index-leaf
number increment bud number increment,bud height,bud distance,bud weight,root number of 10 L. chinensis. genotypes. The genotype Jisheng-1 was chosen as control and the experimental ecological factors are the same. The results showed that:genotype difference is a very important influencing factor for the cloning growth and development of L. chinensis,both environmental factor and genotype factor should be considered in the future research on asexual reproduction biology of L. chinensis. Between the 10 genotypes,morphological character index was obviously different. Furthermore,compared with other genotypes,Zai-3 genotype had better character index than Jisheng-1. All these conclusions will provide foundation for future breeding research work of L. chinensis.
Thirdly:Establishment of regeneration system of L. chinensis through culture in vitro. Immature-stachys-base explants were cultured on Murashige and Skoog's basal medium (MS) supplemented with 2 mg/L 2,4-D after surface sterilized with 0.1% HgCl2 to induce callus at 25 C constant temperature. After moved to M
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