大花蕙兰杂交育种研究
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摘要
本试验在北京林业大学科技股份有限公司花卉分公司从日本引进的35个大花蕙兰(Cymbidium hybridum)品种基础上进行，研究工作主要有以下几个方面的内容。一是品种的分类，为科研和生产实践提供较为系统与合理的分类指导依据；二是通过核型分析和系谱检索，明确大花蕙兰的遗传背景，包括其倍性、品种来源、亲缘关系等，为育种时亲本的选配提供理论依据；三是大花蕙兰的花粉活力和人工授粉、果实生长等的研究，开展大花蕙兰杂交育种的基础研究工作，了解大花蕙兰杂交过程中可能出现的问题；四是对得到杂交组合的种子进行非共生萌发研究，从基本培养基、培养条件、添加物等方面入手，建立完整的非共生萌发技术体系；五是利用分子标记技术探索杂种后代的早期鉴定的可行性，筛选出多态性较高的引物，建立高效的扩增体系，通过谱带的判读研究大花蕙兰的某些遗传规律和现象。主要的研究结果如下：
1．形态学分类建立起以亲本来源为一级分类依据，花葶生长姿态、花色、花径(两侧萼瓣间距)、唇瓣形状分别为二级、三级、四级、五级分类指标的分类系统。将引进的大花蕙兰分为二群(附生地生杂交品种群、附生杂交品种群)，两系(直立花葶系、下垂花葶系)，五组(红色组橙色组黄色组绿色组白色组)，三类(大花类、中花类、小花类)，两型(圆唇型尖唇型)，之后的系谱分析基本验证了这个分类系统的科学性。数学分类的方法选用41个性状指标进行观测，对所得到的数据聚类分析，得到以花径为一级分类选择。花色为二级分类选择的分类结果，该结果能部分解释品种的倍性。
2．染色体观察的结果证明大花蕙兰的染色体基数为20(X=20)，引进的品种具有多样的倍性，其中二倍体、三倍体、四倍体的比例大致相当，还有少量的非整倍体。核型分析说明大花蕙兰为对称型核型，核型大多为B型，有少量的A型和C型，这和兰属的其它种是一致的。结合杂交结果，发现当倍性不同的亲本杂交时，倍性高的亲本作为父本具有较高的座果率。核型似近系数表明大花蕙兰品种间的核型比较近似，似近系数0.8839～0.9908。分析不同似近系数的品种作为亲本杂交的结果，发现随两亲本的似近系数增高，杂交成功率和座果率都有增高的趋势。成功的杂交组合亲本似近系数多在0.950～0.980左右。综合考虑核型似近系数和倍性，可以为亲本选配提供依据。
检索英国皇家园艺学会(RHS)60年(1941～2001)的品种(GREX)登录情况，确定引进的大花蕙兰品种涉及13个原生种。美花兰(C．insigne)、碧玉兰(C．lowianum)、独占春(C．eburneum)和虎头兰(C．hookerianum)在这些品种中的遗传贡献较大，其余如红柱兰(C．erythrostylum)、西藏虎头兰(C．tracyanum)等的遗传贡献因品种而异，充分说明了大花蕙兰不同品种的来源和相互之间的关系。根据各原生种对品种的遗传贡献对品种进行分析，引进品种的遗传距离在0.025～0.536之间，杂交时随两亲本遗传距离的增加，杂交的成功率和座果率都有降低的趋势，成功杂交组合亲本的遗传距离多在0.2～0.3左右，这和核型似近系数的结果一致。
检索品种在以往的杂交育种历史中作为亲本参与杂交的情况，可以帮助很好地了解这些品种的育种价值，一定程度上避免亲本选配时的盲目性。
3．花粉离体萌发试验得到大花蕙兰花粉离体萌发的有效培养基为蔗糖30％+硼酸0.03％+甘氨酸0.03％+GA_350mg.L~(-1)+CaCl_20.15％+琼脂0.3％。花粉的活力在干燥和低温的条件下，可以保藏6—12个月。
大花蕙兰适宜的授粉时间为开花后的1～3天，品种间杂交成功率较低，座果组合仅占全部组合的20％左右。其果实生长发育为单“S”形的生长曲线，人工授粉后50～60天和150～170天分别有两次落果高峰，这两次落果高峰分别对应于受精过程和胚的发育。果实脱落的原因主要在于品种亲缘关系的远近和倍性的差异。
4．非共生萌发试验证明大花蕙兰种子萌发适宜的培养基为1／2MS培养基，固体培养或液体培养均可用于大花蕙兰的种子萌发。适宜的培养基pH为5.4，培养基中不需要添加植物生长调节物质。0.2％的活性炭(AC)有益于大花蕙兰的种子萌发，抑制萌发过程中的褐变。培养需在有光条件下进行，培养基中糖的浓度为25g／L。高糖培养基抑制种子的萌发，说明大花蕙兰的球形胚已经脱离了原胚的生长阶段。种子萌发具有明显的群体效应，培养基表面有胚种子的播种密度10—15粒／cm~2萌发率最高。原球茎的茎叶和根的生长发育需在培养基中添加0.6mg.L~(-1)的NAA。通过炼苗移栽，形成共计1560株的杂交后代群体。
5．筛选出多态性较高的8对引物：E03／M08、E04／M02、E04／M03、E05／M01、E07／M01、E07／M02、E07／M06、E08／M01。证明AFLP可以有效地对大花蕙兰的杂种进行早期鉴定，父母本的特征条带大多可以结合到杂种的谱带中。有的杂种谱带会出现特异性条带的增加或者双亲特征谱带的丢失。
通过3年多的大花蕙兰品种间杂交育种实践，总结出从亲本选配，杂交、非共生
萌发、杂种鉴定等环节具有指导意义的指标或技术体系。我国目前大花蕙兰的育种工作才刚刚开始，此研究得到的结果对这项工作的深入进行具有重要的价值。
The research was based on 35 cultivars of cymbidium (Cymbidium hybridum) imported from Japan by Beijing Forestry University Forest Scinence Company Limited.The most studies focused on five aspects: The first was taxonomy of cymbidium cultivars.A reasonable system of taxonomy is important to the research and production of cymbidium. The second was to probe into cymbidium genetic background including the ploidy, the origon, the relationship of these imported cultivars.The third was about the vitality of cymbidium pollen, artificial pollination and the growth of cymbidium capsule.The major objective was to find the potential obstacles in hybridization of cymbidium. The fourth was to establish the system of asymbiotic propagation. The fifth was molecular mark analysis of cymbidium parents and filial generations.The results were to identify some polymorphism primers and design a efficient system of DNA amplification. The genetic maps could be used to test earky filial generations and explain some genetic phenomena of cymbidium.The results are indicated as follows:
1 .The parents' origin was introduced as the top grade taxonomic standard.The ordinal grades were the verticality of flower stalk, color of flower,the diameter of flower, the shape of labellum etc.The imported cultivars were divided into two branches(the crossbreed from epiphytic and the crossbreed from epiphytic and terrestrial species),two groups(vertical flower stalk and pendent flower stalk),five teams ( red flower,orange flower,yellow flower,green flower,white flower) , three types(large flower,medium flower,small flower), two forms(tapering labellum and smooth abellum).The genealogical analysis proved that this taxonamic system is scientific.In numerical taxonomy,41 characters were observed.The numerical values were used in clustering by UPGMA(Unweighted Pair Group Method with Arithmetic average).In this taxonomical system,the diameter were seemed as the first grade guide.and the flower coler was second.The result of numerical taxonomy could partly distinguish the ploidy of variety.
2. The basic chromosome number is 20(X=20).The ploidy of cymbidium variety was multiplex.The proportion of diploid, triploid, tetraploid was almost equal in these cultivars.There is one aneuploid in the experimental cultivars.All of these are allopolyploids. Analysis and comparison on karyotype of these cultivars showed that karyotype of cymbidium was symmetrical., The karyotypes were belong to B type.The karyotypes of A or C were seldom observed in cymbidium cultivars.This is accordant with other species of cymbidium genus.The result of crossing was associated with the ploidies of parents to some extent.If the ploidies of parents were not same, the higher ploidy variety had better be used as father. The karyotype comparability coefficient of these cultivars were in 0.8839～
0.9908 .The rate of harvest mating increased belong with karyotype comparability coefficient .The karyotype comparability coefficient of parents of successicve mating was about 0.950～0.980.In the mating of cymbidium, ploidy and karyotype comparability coefficient were two important parameters be referenced.
The retrieval of cymbidium registeration by entry the website of British Royal Horticulture society(RHS).The imported cultivars were registered in 1941～2001. Thirteen primeval species were involved in breeding of these cultivars. Cymbidium insigne, C.lowianum, C.eburneum, C.hookerianum contributed greatly in the breeding them.The contribution of C.erythrostylum, C.tracyanum etc.was less or more in different cultivars.Clustering according to the contribution showed that the genetic distances of these cultivars were in 0.025～0.536. The rate of harvest mating decreased belong with the genetic distance of parents. The genetic distance of parents of successicve mating was about 0.2～0.3.This result was accordant with that of karyotype comparability coefficient.
The action of cultivars in cross-breeding history could help estimate the breeding value of them.
3. Vitality of cymbidium pollen was estimated by germinating on artificial medium.The result show that the effective medium wassugar 30% + boracic acid0.03%+ glycin0.03% +GA_350mg.L~(-1)+CaCl_20.15%+ agar0.3%. In the dry and low temperature condition,the pollen could be preserved for 6~ 12 months.
The optimal artificial pollination time was the 1~3 days after the flower blooming .The rate of harvest mating was about 20%. The capsule growing curve was a single 'S'pattern.The apexes of capsules falling were after 50～60 days and 150—170 days from artificial pollination.The two times respectively corresponded to the fertilization and embryo development.The reasons of capsule falling were the various ploidies and the far or near relationships of cultivars.
4. Asymbiotic propagation experiment proved that the 1/2MS medium was adequate for the germination of cymbidium seeds.Not only solid medium but also liquid medium had good effects.The medium pH was 5.4.The plant hormones such as 6-BA,GA3 could inhibit the germination of seeds. 0.2% active carbon (AC) was beneficial to germination and abatement the browning of PLB (protocorm-like-body) tissue or medium.The light was an essential factor for the seed to germinate. The sugar concentration was 25g/L in the medium.The 80g/L and 120g/L sugar could do harmful to germination.This indicated that the cymbidium globular embryo developed over the stage of proembryos. 10～15 seeds with well-developed embryo per square centimeter(cm~2) was optimal sowing density .The mass effect was very distinct in cymbidium seeds germination. 0.6mg.L~(-1) NAA could accelerate the growth of leaves and roots. 1560 plants of filial generation were got in this experiment.
5. In order to identify parentage and provide the evidence for crossbreeding in molecule level.8-paired primers were selected to be used in AFLP analysis (E03/M08、 E04/M02、 E04/M03、 E05/M01、 E07/M01、 E07/M02、 E07/M06、 E08/M01). The results show that the AFLP could efficiently identify the filial generation in early time.The characteristic bands of parents could exist in the bands of
filial generations.In the bands of filial generation, a few characteristic bands would disappear but new bands could appear.
Three years work about hybridization of cymbidium cultivars established a technique system including the choosing and mating of parents, asymbiotic propagation, identification of filial generation etc .The technique and theoretics will be helpful to the breeding of cymbidium, which in china stands at the beginning line. Some conclusions of the research are important to the in-depth study in this field.
1．形态学分类建立起以亲本来源为一级分类依据，花葶生长姿态、花色、花径(两侧萼瓣间距)、唇瓣形状分别为二级、三级、四级、五级分类指标的分类系统。将引进的大花蕙兰分为二群(附生地生杂交品种群、附生杂交品种群)，两系(直立花葶系、下垂花葶系)，五组(红色组橙色组黄色组绿色组白色组)，三类(大花类、中花类、小花类)，两型(圆唇型尖唇型)，之后的系谱分析基本验证了这个分类系统的科学性。数学分类的方法选用41个性状指标进行观测，对所得到的数据聚类分析，得到以花径为一级分类选择。花色为二级分类选择的分类结果，该结果能部分解释品种的倍性。
2．染色体观察的结果证明大花蕙兰的染色体基数为20(X=20)，引进的品种具有多样的倍性，其中二倍体、三倍体、四倍体的比例大致相当，还有少量的非整倍体。核型分析说明大花蕙兰为对称型核型，核型大多为B型，有少量的A型和C型，这和兰属的其它种是一致的。结合杂交结果，发现当倍性不同的亲本杂交时，倍性高的亲本作为父本具有较高的座果率。核型似近系数表明大花蕙兰品种间的核型比较近似，似近系数0.8839～0.9908。分析不同似近系数的品种作为亲本杂交的结果，发现随两亲本的似近系数增高，杂交成功率和座果率都有增高的趋势。成功的杂交组合亲本似近系数多在0.950～0.980左右。综合考虑核型似近系数和倍性，可以为亲本选配提供依据。
检索英国皇家园艺学会(RHS)60年(1941～2001)的品种(GREX)登录情况，确定引进的大花蕙兰品种涉及13个原生种。美花兰(C．insigne)、碧玉兰(C．lowianum)、独占春(C．eburneum)和虎头兰(C．hookerianum)在这些品种中的遗传贡献较大，其余如红柱兰(C．erythrostylum)、西藏虎头兰(C．tracyanum)等的遗传贡献因品种而异，充分说明了大花蕙兰不同品种的来源和相互之间的关系。根据各原生种对品种的遗传贡献对品种进行分析，引进品种的遗传距离在0.025～0.536之间，杂交时随两亲本遗传距离的增加，杂交的成功率和座果率都有降低的趋势，成功杂交组合亲本的遗传距离多在0.2～0.3左右，这和核型似近系数的结果一致。
检索品种在以往的杂交育种历史中作为亲本参与杂交的情况，可以帮助很好地了解这些品种的育种价值，一定程度上避免亲本选配时的盲目性。
3．花粉离体萌发试验得到大花蕙兰花粉离体萌发的有效培养基为蔗糖30％+硼酸0.03％+甘氨酸0.03％+GA_350mg.L~(-1)+CaCl_20.15％+琼脂0.3％。花粉的活力在干燥和低温的条件下，可以保藏6—12个月。
大花蕙兰适宜的授粉时间为开花后的1～3天，品种间杂交成功率较低，座果组合仅占全部组合的20％左右。其果实生长发育为单“S”形的生长曲线，人工授粉后50～60天和150～170天分别有两次落果高峰，这两次落果高峰分别对应于受精过程和胚的发育。果实脱落的原因主要在于品种亲缘关系的远近和倍性的差异。
4．非共生萌发试验证明大花蕙兰种子萌发适宜的培养基为1／2MS培养基，固体培养或液体培养均可用于大花蕙兰的种子萌发。适宜的培养基pH为5.4，培养基中不需要添加植物生长调节物质。0.2％的活性炭(AC)有益于大花蕙兰的种子萌发，抑制萌发过程中的褐变。培养需在有光条件下进行，培养基中糖的浓度为25g／L。高糖培养基抑制种子的萌发，说明大花蕙兰的球形胚已经脱离了原胚的生长阶段。种子萌发具有明显的群体效应，培养基表面有胚种子的播种密度10—15粒／cm~2萌发率最高。原球茎的茎叶和根的生长发育需在培养基中添加0.6mg.L~(-1)的NAA。通过炼苗移栽，形成共计1560株的杂交后代群体。
5．筛选出多态性较高的8对引物：E03／M08、E04／M02、E04／M03、E05／M01、E07／M01、E07／M02、E07／M06、E08／M01。证明AFLP可以有效地对大花蕙兰的杂种进行早期鉴定，父母本的特征条带大多可以结合到杂种的谱带中。有的杂种谱带会出现特异性条带的增加或者双亲特征谱带的丢失。
通过3年多的大花蕙兰品种间杂交育种实践，总结出从亲本选配，杂交、非共生
萌发、杂种鉴定等环节具有指导意义的指标或技术体系。我国目前大花蕙兰的育种工作才刚刚开始，此研究得到的结果对这项工作的深入进行具有重要的价值。
The research was based on 35 cultivars of cymbidium (Cymbidium hybridum) imported from Japan by Beijing Forestry University Forest Scinence Company Limited.The most studies focused on five aspects: The first was taxonomy of cymbidium cultivars.A reasonable system of taxonomy is important to the research and production of cymbidium. The second was to probe into cymbidium genetic background including the ploidy, the origon, the relationship of these imported cultivars.The third was about the vitality of cymbidium pollen, artificial pollination and the growth of cymbidium capsule.The major objective was to find the potential obstacles in hybridization of cymbidium. The fourth was to establish the system of asymbiotic propagation. The fifth was molecular mark analysis of cymbidium parents and filial generations.The results were to identify some polymorphism primers and design a efficient system of DNA amplification. The genetic maps could be used to test earky filial generations and explain some genetic phenomena of cymbidium.The results are indicated as follows:
1 .The parents' origin was introduced as the top grade taxonomic standard.The ordinal grades were the verticality of flower stalk, color of flower,the diameter of flower, the shape of labellum etc.The imported cultivars were divided into two branches(the crossbreed from epiphytic and the crossbreed from epiphytic and terrestrial species),two groups(vertical flower stalk and pendent flower stalk),five teams ( red flower,orange flower,yellow flower,green flower,white flower) , three types(large flower,medium flower,small flower), two forms(tapering labellum and smooth abellum).The genealogical analysis proved that this taxonamic system is scientific.In numerical taxonomy,41 characters were observed.The numerical values were used in clustering by UPGMA(Unweighted Pair Group Method with Arithmetic average).In this taxonomical system,the diameter were seemed as the first grade guide.and the flower coler was second.The result of numerical taxonomy could partly distinguish the ploidy of variety.
2. The basic chromosome number is 20(X=20).The ploidy of cymbidium variety was multiplex.The proportion of diploid, triploid, tetraploid was almost equal in these cultivars.There is one aneuploid in the experimental cultivars.All of these are allopolyploids. Analysis and comparison on karyotype of these cultivars showed that karyotype of cymbidium was symmetrical., The karyotypes were belong to B type.The karyotypes of A or C were seldom observed in cymbidium cultivars.This is accordant with other species of cymbidium genus.The result of crossing was associated with the ploidies of parents to some extent.If the ploidies of parents were not same, the higher ploidy variety had better be used as father. The karyotype comparability coefficient of these cultivars were in 0.8839～
0.9908 .The rate of harvest mating increased belong with karyotype comparability coefficient .The karyotype comparability coefficient of parents of successicve mating was about 0.950～0.980.In the mating of cymbidium, ploidy and karyotype comparability coefficient were two important parameters be referenced.
The retrieval of cymbidium registeration by entry the website of British Royal Horticulture society(RHS).The imported cultivars were registered in 1941～2001. Thirteen primeval species were involved in breeding of these cultivars. Cymbidium insigne, C.lowianum, C.eburneum, C.hookerianum contributed greatly in the breeding them.The contribution of C.erythrostylum, C.tracyanum etc.was less or more in different cultivars.Clustering according to the contribution showed that the genetic distances of these cultivars were in 0.025～0.536. The rate of harvest mating decreased belong with the genetic distance of parents. The genetic distance of parents of successicve mating was about 0.2～0.3.This result was accordant with that of karyotype comparability coefficient.
The action of cultivars in cross-breeding history could help estimate the breeding value of them.
3. Vitality of cymbidium pollen was estimated by germinating on artificial medium.The result show that the effective medium wassugar 30% + boracic acid0.03%+ glycin0.03% +GA_350mg.L~(-1)+CaCl_20.15%+ agar0.3%. In the dry and low temperature condition,the pollen could be preserved for 6~ 12 months.
The optimal artificial pollination time was the 1~3 days after the flower blooming .The rate of harvest mating was about 20%. The capsule growing curve was a single 'S'pattern.The apexes of capsules falling were after 50～60 days and 150—170 days from artificial pollination.The two times respectively corresponded to the fertilization and embryo development.The reasons of capsule falling were the various ploidies and the far or near relationships of cultivars.
4. Asymbiotic propagation experiment proved that the 1/2MS medium was adequate for the germination of cymbidium seeds.Not only solid medium but also liquid medium had good effects.The medium pH was 5.4.The plant hormones such as 6-BA,GA3 could inhibit the germination of seeds. 0.2% active carbon (AC) was beneficial to germination and abatement the browning of PLB (protocorm-like-body) tissue or medium.The light was an essential factor for the seed to germinate. The sugar concentration was 25g/L in the medium.The 80g/L and 120g/L sugar could do harmful to germination.This indicated that the cymbidium globular embryo developed over the stage of proembryos. 10～15 seeds with well-developed embryo per square centimeter(cm~2) was optimal sowing density .The mass effect was very distinct in cymbidium seeds germination. 0.6mg.L~(-1) NAA could accelerate the growth of leaves and roots. 1560 plants of filial generation were got in this experiment.
5. In order to identify parentage and provide the evidence for crossbreeding in molecule level.8-paired primers were selected to be used in AFLP analysis (E03/M08、 E04/M02、 E04/M03、 E05/M01、 E07/M01、 E07/M02、 E07/M06、 E08/M01). The results show that the AFLP could efficiently identify the filial generation in early time.The characteristic bands of parents could exist in the bands of
filial generations.In the bands of filial generation, a few characteristic bands would disappear but new bands could appear.
Three years work about hybridization of cymbidium cultivars established a technique system including the choosing and mating of parents, asymbiotic propagation, identification of filial generation etc .The technique and theoretics will be helpful to the breeding of cymbidium, which in china stands at the beginning line. Some conclusions of the research are important to the in-depth study in this field.
引文
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