兰属中几种兰花杂交后代组培与核型研究
摘要
本试验是在泰安市泰山林业科学研究院从韩国引进的大花蕙兰(Cymbidium hybridum)品种与泰山义祥兰园栽培的国兰优良品种基础上进行的,研究工作主要有以下三个方面的内容。一是对得到杂交组合的幼胚(未成熟的种子)进行非共生萌发研究,从果实的不同发育时期、萌发的基本培养基方面入手,建立完整的非共生萌发技术体系;二是对杂种胚萌发形成的原球茎进行组培快繁技术研究,从基本培养基、大量元素、蔗糖浓度、添加物等方面入手,筛选出后代原球茎增殖快繁的最适培养基,为科研和生产实践提供较为系统与合理的参考依据;三是通过核型分析对杂交后代进行早期鉴定,明确杂交后代的遗传背景,包括其倍性、染色体形态、核型参数等,为杂交育种时后代的选育提供参考与依据。主要的研究结果如下:
1.以大花蕙兰与国兰杂种胚为外植体进行初代培养,筛选出杂种胚非共生萌发的最佳采收时间及萌发最适基本培养基。胚龄以150天左右为最佳采收时间,萌发率最高;最适萌发培养基为B11+蔗糖20 g/L +琼脂8.0 g/L+AC3.0g/L。
2.以大花蕙兰与国兰杂交原球茎为外植体进行继代培养,筛选出原球茎增殖分化的最适培养基。不同杂交组合后代在不同培养基中的增殖倍数各不相同,但继代培养的最适基本培养基均为KC+蔗糖20 g/L+琼脂8.0 g/L+香蕉泥150g/L。外源激素对增殖分化有促进作用,其中生长素类以NAA为效果最佳,细胞分裂素类以6-BA为最佳;NAA浓度为0.2mg/L时杂交原球茎增殖效果为最佳,6-BA和NAA的浓度配比为4~5∶1时,分化效果最好; 1.0mg/L的GA对继代培养有促进作用;综上所述,继代培养的最佳培养基为KC+NAA0.2mg/L +6-BA0.8mg/ L +GA1.0mg/L+蔗糖20 g/L +琼脂8.0 g/L +香蕉泥150g/L。
3.在杂交兰生根培养中,大量元素与蔗糖浓度对生根影响极其显著。最适无机盐浓度为1/4MS,60天后组培苗生根率达到100%,平均生根数最多为2.4条,平均根长最长为1.79cm;蔗糖浓度为30 g/L,根生长状况最佳,平均生根数最多为3.43条,平均根长最长为1.34 cm。杂交兰最适生根培养基为1/4MS+ NAA0.2mg/L+琼脂8.0 g/L +蔗糖30 g/L +香蕉泥150g/L。
4.杂交兰组培苗移栽时对根部进行断根处理,可刺激新根的生成,提高幼苗成活率,移栽至水苔基质中100天后成活率达81%,平均苗高为11.89cm,与未进行断根处理的相比,苗的长势也明显见好。
5.通过对4种大花蕙兰父本与杂交后代进行核型分析,对杂交后代进行早期鉴定。结果表明:大花型父本‘绿宝石’与‘天使’体细胞染色体数均为2n=4x=80,为四倍体,其分别与二倍体春剑和蕙兰杂交后代体细胞染色体数均为2n=3x=60,可鉴定其杂交后代为真杂种;小花型父本‘夏绿’与‘圣蕾芬’体细胞染色体数2n=2x=40,为二倍体,其与春剑杂交后代经核型分析,在染色体相对长度、平均臂比、及不对称系数等指标上,后代都趋于父母本的中间值,杂交后代染色体形态具有双亲的特点,也可鉴定为真杂种。
6.杂交组合后代实际核型与理论核型均存在一定的差异,表明人工杂交打破了亲本染色体组间长期进化形成的共适体系。所有亲本及后代染色体的结构主要由中部和近中部着丝点染色体组成,最长染色体与最短染色体的比值在2.01～4.2之间,臂比大于2的染色体所占的比率也都不高,均不超过35%。按Stebbins(1971)的核型分类标准,所有亲本及杂交后代除‘夏绿’为2C型,其余均为2B型。由此可见,兰属植物的染色体数目是比较保守的,变异不大,而且核型也具有较高的对称性。
The research was based on Cymbidium hybridum imported from Korea by Taishan Academy of Forestry in Taian and Cymbidium orchid varieties cultivated in Taishan Yi Xiang garden.The study mainly included the following three aspects. The first was to research the non-symbiotic germination on crossing embryos (immature seeds). From the fruits of different developmental stages, the basic medium germination aspects, the system of asymbiotic propagation was established. The second was to research tissue culture technique of the PLBs germinated from hybrid embryos. From the basic medium, a large number of elements, sucrose concentration, additives and other aspects, the optimum medium was selected for the propagation of the offspring PLBs to provide a systematic and rational frame of reference for research and production practice. The third was to identify the filial generations early and clear the genetic background, including the ploidy,chromosome morphology, karyotype parameters, to provide reference and basis for breeding of hybrid offspring when cross breeding.
The results are indicated as follows:
1. Hybrid embryos between Cymbidium hybridum and Cymbidium as the explants are first cultured to select non-symbiotic germination optimum harvest time and the optimal germination basic medium. Embryo age of 150 days or so is the best harvest time, and the germination rate was the highest. The optimal germination medium was B11 + sucrose 20 g / L + agar 8.0 g / L + AC3.0g / L.
2. Hybrid PLBs between Cymbidium hybridum and Cymbidium as the explants for subculture , the best medium was selected for PLBs proliferation and differentiation. Different combinations had different propagation multiple in different medium, however, the optimal subculture basic medium were KC + banana 150g / L. Exogenous hormones on the proliferation and differentiation are promoted, in which NAA as the auxin class to the best cytokinin to 6-BA is the best. NAA concentration of 0.2mg / L was the best to the hybrid PLBs proliferation. 6-BA and NAA concentration ratio was 4 to 5:1, which was most profitable to differentiate; GA1.0mg / L on the subculture was also promoted. In summary, the best subculture medium was KC + NAA0.2mg / L +6- BA0.8mg / L + GA1.0mg / L + banana 150g / L + sucrose 20 g / L + agar 8.0 g / L.
3. In the hybrid PLBs root culture, macroelement and sucrose concentration on rooting is extremely significant. The optimal concentration of mineral salts was 1/4MS.The rooting rate was up to100% after 60 days, while average number of root up to 2.4, the average root length of up to 1.79cm. Root growth status was the best when the sucrose concentration was 30 g / L, average number of root up to 3.43, the average root length up to 1.34 cm.. Therefore, the hybrid PLBs rooting medium was 1/4MS + NAA0.2mg/L +sucrose30 g / L + banana 150g / L+ sucrose 20 g / L + agar 8.0 g / L.
4. Hybrid plantlets were to quit dealing with the roots when transplanted to sphagnum substrate , which survival rate was 81% after 100 days and the average height was 11.89cm. From this we can deduce that root pruning treatment can stimulate the formation of new roots and improve survival rate of seedlings. Seedling vigor was significantly brought the matter to be dealt with than without.
5. Through karyotype analysis of Cymbidium hybridum male parent and filial generations, early identification of hybrids was made. The experiment results showed that Cym. Longibracteatum and Cym. KLR565 are tetraploid. They are respectively crossed with C.Longibracteatum and C. faberi ,and somatic chromosome number of their filial generation are 2n = 3x = 60.Therefore, filial generations can be identified as true hybrids. Somatic chromosome number of C. Summer Queen and C. Saint Raphin are2n = 2x = 40, diploid. They are crossed with C.Longibracteatum and their filial generations had common with their parents in chromosome morphology. Their relative chromosome length, average arm ratio, asymmetry index and other indicators tend to the intermediate value of parents, which can identify them to be true.
6. Filial generations practical and theoretical karyotype had differences, which showed that artificial hybridization broke the formation of long-term evolution system suitable for parental chromosomes. All parents and filial generations chromosome structure were mainly composed with metacentric and sub-metacentric chromosome. The ratio of the longest and shortest chromosomes was between 2.01 to 4.2.Chromosomes percentage of arm ratio >2:1 was not more than 35. By the karyotypic classification Stebbins (1971), all parents and filial generations are 2B except C. Summer Queen which was 2C. Thus, the number of chromosomes of Cymbidium was relatively conservative, the variation was small and the karyotype symmetry was also high.
1.以大花蕙兰与国兰杂种胚为外植体进行初代培养,筛选出杂种胚非共生萌发的最佳采收时间及萌发最适基本培养基。胚龄以150天左右为最佳采收时间,萌发率最高;最适萌发培养基为B11+蔗糖20 g/L +琼脂8.0 g/L+AC3.0g/L。
2.以大花蕙兰与国兰杂交原球茎为外植体进行继代培养,筛选出原球茎增殖分化的最适培养基。不同杂交组合后代在不同培养基中的增殖倍数各不相同,但继代培养的最适基本培养基均为KC+蔗糖20 g/L+琼脂8.0 g/L+香蕉泥150g/L。外源激素对增殖分化有促进作用,其中生长素类以NAA为效果最佳,细胞分裂素类以6-BA为最佳;NAA浓度为0.2mg/L时杂交原球茎增殖效果为最佳,6-BA和NAA的浓度配比为4~5∶1时,分化效果最好; 1.0mg/L的GA对继代培养有促进作用;综上所述,继代培养的最佳培养基为KC+NAA0.2mg/L +6-BA0.8mg/ L +GA1.0mg/L+蔗糖20 g/L +琼脂8.0 g/L +香蕉泥150g/L。
3.在杂交兰生根培养中,大量元素与蔗糖浓度对生根影响极其显著。最适无机盐浓度为1/4MS,60天后组培苗生根率达到100%,平均生根数最多为2.4条,平均根长最长为1.79cm;蔗糖浓度为30 g/L,根生长状况最佳,平均生根数最多为3.43条,平均根长最长为1.34 cm。杂交兰最适生根培养基为1/4MS+ NAA0.2mg/L+琼脂8.0 g/L +蔗糖30 g/L +香蕉泥150g/L。
4.杂交兰组培苗移栽时对根部进行断根处理,可刺激新根的生成,提高幼苗成活率,移栽至水苔基质中100天后成活率达81%,平均苗高为11.89cm,与未进行断根处理的相比,苗的长势也明显见好。
5.通过对4种大花蕙兰父本与杂交后代进行核型分析,对杂交后代进行早期鉴定。结果表明:大花型父本‘绿宝石’与‘天使’体细胞染色体数均为2n=4x=80,为四倍体,其分别与二倍体春剑和蕙兰杂交后代体细胞染色体数均为2n=3x=60,可鉴定其杂交后代为真杂种;小花型父本‘夏绿’与‘圣蕾芬’体细胞染色体数2n=2x=40,为二倍体,其与春剑杂交后代经核型分析,在染色体相对长度、平均臂比、及不对称系数等指标上,后代都趋于父母本的中间值,杂交后代染色体形态具有双亲的特点,也可鉴定为真杂种。
6.杂交组合后代实际核型与理论核型均存在一定的差异,表明人工杂交打破了亲本染色体组间长期进化形成的共适体系。所有亲本及后代染色体的结构主要由中部和近中部着丝点染色体组成,最长染色体与最短染色体的比值在2.01～4.2之间,臂比大于2的染色体所占的比率也都不高,均不超过35%。按Stebbins(1971)的核型分类标准,所有亲本及杂交后代除‘夏绿’为2C型,其余均为2B型。由此可见,兰属植物的染色体数目是比较保守的,变异不大,而且核型也具有较高的对称性。
The research was based on Cymbidium hybridum imported from Korea by Taishan Academy of Forestry in Taian and Cymbidium orchid varieties cultivated in Taishan Yi Xiang garden.The study mainly included the following three aspects. The first was to research the non-symbiotic germination on crossing embryos (immature seeds). From the fruits of different developmental stages, the basic medium germination aspects, the system of asymbiotic propagation was established. The second was to research tissue culture technique of the PLBs germinated from hybrid embryos. From the basic medium, a large number of elements, sucrose concentration, additives and other aspects, the optimum medium was selected for the propagation of the offspring PLBs to provide a systematic and rational frame of reference for research and production practice. The third was to identify the filial generations early and clear the genetic background, including the ploidy,chromosome morphology, karyotype parameters, to provide reference and basis for breeding of hybrid offspring when cross breeding.
The results are indicated as follows:
1. Hybrid embryos between Cymbidium hybridum and Cymbidium as the explants are first cultured to select non-symbiotic germination optimum harvest time and the optimal germination basic medium. Embryo age of 150 days or so is the best harvest time, and the germination rate was the highest. The optimal germination medium was B11 + sucrose 20 g / L + agar 8.0 g / L + AC3.0g / L.
2. Hybrid PLBs between Cymbidium hybridum and Cymbidium as the explants for subculture , the best medium was selected for PLBs proliferation and differentiation. Different combinations had different propagation multiple in different medium, however, the optimal subculture basic medium were KC + banana 150g / L. Exogenous hormones on the proliferation and differentiation are promoted, in which NAA as the auxin class to the best cytokinin to 6-BA is the best. NAA concentration of 0.2mg / L was the best to the hybrid PLBs proliferation. 6-BA and NAA concentration ratio was 4 to 5:1, which was most profitable to differentiate; GA1.0mg / L on the subculture was also promoted. In summary, the best subculture medium was KC + NAA0.2mg / L +6- BA0.8mg / L + GA1.0mg / L + banana 150g / L + sucrose 20 g / L + agar 8.0 g / L.
3. In the hybrid PLBs root culture, macroelement and sucrose concentration on rooting is extremely significant. The optimal concentration of mineral salts was 1/4MS.The rooting rate was up to100% after 60 days, while average number of root up to 2.4, the average root length of up to 1.79cm. Root growth status was the best when the sucrose concentration was 30 g / L, average number of root up to 3.43, the average root length up to 1.34 cm.. Therefore, the hybrid PLBs rooting medium was 1/4MS + NAA0.2mg/L +sucrose30 g / L + banana 150g / L+ sucrose 20 g / L + agar 8.0 g / L.
4. Hybrid plantlets were to quit dealing with the roots when transplanted to sphagnum substrate , which survival rate was 81% after 100 days and the average height was 11.89cm. From this we can deduce that root pruning treatment can stimulate the formation of new roots and improve survival rate of seedlings. Seedling vigor was significantly brought the matter to be dealt with than without.
5. Through karyotype analysis of Cymbidium hybridum male parent and filial generations, early identification of hybrids was made. The experiment results showed that Cym. Longibracteatum and Cym. KLR565 are tetraploid. They are respectively crossed with C.Longibracteatum and C. faberi ,and somatic chromosome number of their filial generation are 2n = 3x = 60.Therefore, filial generations can be identified as true hybrids. Somatic chromosome number of C. Summer Queen and C. Saint Raphin are2n = 2x = 40, diploid. They are crossed with C.Longibracteatum and their filial generations had common with their parents in chromosome morphology. Their relative chromosome length, average arm ratio, asymmetry index and other indicators tend to the intermediate value of parents, which can identify them to be true.
6. Filial generations practical and theoretical karyotype had differences, which showed that artificial hybridization broke the formation of long-term evolution system suitable for parental chromosomes. All parents and filial generations chromosome structure were mainly composed with metacentric and sub-metacentric chromosome. The ratio of the longest and shortest chromosomes was between 2.01 to 4.2.Chromosomes percentage of arm ratio >2:1 was not more than 35. By the karyotypic classification Stebbins (1971), all parents and filial generations are 2B except C. Summer Queen which was 2C. Thus, the number of chromosomes of Cymbidium was relatively conservative, the variation was small and the karyotype symmetry was also high.
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