秋水仙素诱导杂交兰四倍体及倍性鉴定
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摘要
兰属(Cymbidium)花卉花型优美,花色艳丽,是一类具有重要观赏价值和经济价值的花卉,在世界各地具有广阔的市场前景。国内外关于兰属花卉种间杂交培育新品种的报道较多,而多倍体育种方面研究相对较少。本研究以兰属杂交兰(大花蕙兰×墨兰)与蕙兰进一步杂交所获得的F1代杂交兰根状茎为材料,以秋水仙素为诱变剂,对杂交兰多倍体诱导和鉴定技术进行了系统的研究,旨在建立杂交兰多倍体诱导和鉴定技术体系。
主要研究结果如下：
1.以杂交兰根状茎为材料,分别采用浓度为0.02%、0.05%、0.10%、0.20%的秋水仙素对其无菌苗的根状茎浸泡处理24h、48h和72h诱导杂交兰四倍体,试验结果表明,以0.10%的秋水仙素处理48h诱导效果最佳,变异率为36%。变异的根状茎较未处理的根状茎膨大明显,茎段粗壮,变圆分节,着生有绒毛,生长势变缓。
2.对秋水仙素诱导获得杂交兰变异材料进行倍性鉴定,成功获得了染色体加倍植株。染色体观察表明,四倍体植株染色体数为2n=4x=80,二倍体对照为2n=2x=40。加倍后的四倍体植株较二倍体植株粗壮,叶片变厚,颜色变深,茎基部粗壮,颜色深,生长势变缓,根系变粗,根状茎增粗,保卫细胞增大,叶绿体数目增多,气孔密度减小等特点,可作为新材料加以利用。
3.由于杂交兰生长周期较长,生根需要较长时间,而根状茎较易获得,因此本文以杂交兰根状茎茎尖为材料,比较茎尖的不同采集时间、预处理方法、解离时间和染色方法对杂交兰根状茎茎尖染色体制片的影响。结果表明,取材时间为上午10：00-11：00,预处理方式为8-羟基喹啉处理6-7h,解离采用5mol/L HCl在25℃下30min,染色液以卡宝品红染色液染色15-20min染色体制片效果最佳。
Cymbidium flower has beautiful floral patterns and bright colors. It is a kind of flower with important ornamental and economic value and possesses a broad market prospects all over the world. There are a lot of reports about interspecific hybridization of Cymbidium genus plant to breed new varieties both at home and abroad, while relatively few studies on polyploid breeding of Cymbidium. In this research, the rhizome of F1 hybrid of Cymbidium interspecific hybrids between Cymbidium hybrida×C. sinense and C. faberi was used as material, and the colchicine as mutagen to produce polyploid. The technique for polyploid induction and identification of Cymbidium interspecific hybrids had been systematically established.The results were as follows:
1. The rhizomes of aseptic plantlets of Cymbidium interspecific hybrids were treated with colchicine at a concentration of 0.02%,0.05%,010% and 0.20%, respectively for 24, 48 and 72 hours. The results indicated that the best induction effect was obtained by the treatment of 0.10% colchicine for 48 hours, and its variation rate was 36%. Compared with the rhizomes without treatment, the mutated rhizomes were significantly enlarged, became stout, round and segmented with villi, and slowed the growth rate.
2. The mutated materials of Cymbidium interspecific hybrids induced by the colchicine were identified for ploidy. And the chromosome number of mutated plants was successfully doubled. Through observation, the chromosome number of tetraploid plants was 2n=4x=80, while that of the diploid control was 2n=2x=40. Compared with the diploid plants, the tetraploid plants were characterized by thicker and darker leaves, stout stem base with deep green color, slow growth rate, enlarged roots, rhizomes and guard cells with increased chloroplasts and decreased stomata density. They can be used as new materials in breeding Cymbidium.
3. Because Cymbidium interspecific hybrids have a long growth cycle and need a long time for rooting, it is difficult to get more root tips and shoot tips for chromosome analysis, but it is easy to get the Cymbidium rhizome. The tip of rhizome of Cymbidium interspecific hybrids was used as test material for chromosome analysis in this study. It was aimed at comparing the influences of different sampling time, methods of pretreatment, dissocication time and methods of dyeing on the squash technique for rhizome tip of Cymbidium interspecific hybrids. The results showed that the best effect for chromosome squash was obtained under the treatment of 10:00-11:00 a.m. for sampling,8-hydroxyquinoline pretreatment for 6～7h,5 mol/L HCl at 25℃for 30 minutes for dissociation and carbol fuchsin as dye liquor for 15～20 minutes.
主要研究结果如下：
1.以杂交兰根状茎为材料,分别采用浓度为0.02%、0.05%、0.10%、0.20%的秋水仙素对其无菌苗的根状茎浸泡处理24h、48h和72h诱导杂交兰四倍体,试验结果表明,以0.10%的秋水仙素处理48h诱导效果最佳,变异率为36%。变异的根状茎较未处理的根状茎膨大明显,茎段粗壮,变圆分节,着生有绒毛,生长势变缓。
2.对秋水仙素诱导获得杂交兰变异材料进行倍性鉴定,成功获得了染色体加倍植株。染色体观察表明,四倍体植株染色体数为2n=4x=80,二倍体对照为2n=2x=40。加倍后的四倍体植株较二倍体植株粗壮,叶片变厚,颜色变深,茎基部粗壮,颜色深,生长势变缓,根系变粗,根状茎增粗,保卫细胞增大,叶绿体数目增多,气孔密度减小等特点,可作为新材料加以利用。
3.由于杂交兰生长周期较长,生根需要较长时间,而根状茎较易获得,因此本文以杂交兰根状茎茎尖为材料,比较茎尖的不同采集时间、预处理方法、解离时间和染色方法对杂交兰根状茎茎尖染色体制片的影响。结果表明,取材时间为上午10：00-11：00,预处理方式为8-羟基喹啉处理6-7h,解离采用5mol/L HCl在25℃下30min,染色液以卡宝品红染色液染色15-20min染色体制片效果最佳。
Cymbidium flower has beautiful floral patterns and bright colors. It is a kind of flower with important ornamental and economic value and possesses a broad market prospects all over the world. There are a lot of reports about interspecific hybridization of Cymbidium genus plant to breed new varieties both at home and abroad, while relatively few studies on polyploid breeding of Cymbidium. In this research, the rhizome of F1 hybrid of Cymbidium interspecific hybrids between Cymbidium hybrida×C. sinense and C. faberi was used as material, and the colchicine as mutagen to produce polyploid. The technique for polyploid induction and identification of Cymbidium interspecific hybrids had been systematically established.The results were as follows:
1. The rhizomes of aseptic plantlets of Cymbidium interspecific hybrids were treated with colchicine at a concentration of 0.02%,0.05%,010% and 0.20%, respectively for 24, 48 and 72 hours. The results indicated that the best induction effect was obtained by the treatment of 0.10% colchicine for 48 hours, and its variation rate was 36%. Compared with the rhizomes without treatment, the mutated rhizomes were significantly enlarged, became stout, round and segmented with villi, and slowed the growth rate.
2. The mutated materials of Cymbidium interspecific hybrids induced by the colchicine were identified for ploidy. And the chromosome number of mutated plants was successfully doubled. Through observation, the chromosome number of tetraploid plants was 2n=4x=80, while that of the diploid control was 2n=2x=40. Compared with the diploid plants, the tetraploid plants were characterized by thicker and darker leaves, stout stem base with deep green color, slow growth rate, enlarged roots, rhizomes and guard cells with increased chloroplasts and decreased stomata density. They can be used as new materials in breeding Cymbidium.
3. Because Cymbidium interspecific hybrids have a long growth cycle and need a long time for rooting, it is difficult to get more root tips and shoot tips for chromosome analysis, but it is easy to get the Cymbidium rhizome. The tip of rhizome of Cymbidium interspecific hybrids was used as test material for chromosome analysis in this study. It was aimed at comparing the influences of different sampling time, methods of pretreatment, dissocication time and methods of dyeing on the squash technique for rhizome tip of Cymbidium interspecific hybrids. The results showed that the best effect for chromosome squash was obtained under the treatment of 10:00-11:00 a.m. for sampling,8-hydroxyquinoline pretreatment for 6～7h,5 mol/L HCl at 25℃for 30 minutes for dissociation and carbol fuchsin as dye liquor for 15～20 minutes.
引文
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