两种野生杜鹃属植物染色体制片方法比较及其核型分析
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摘要
大白杜鹃(Rhododendron decorum Franch.)隶属常绿杜鹃组(Sect ponticum G. Dou)云锦杜鹃亚组(Subsect Fortumes, S. Jcumer),黄钟杜鹃(Rhododendron lanatum)隶属于常绿杜鹃组下的黄钟杜鹃亚组(Subsect. Lanata Chamb.)。目前国内外对两种杜鹃的核型分析方面的研究尚未见报道。本文对两种杜鹃的染色体数目,相对长度以及适合两种杜鹃的染色体制片技术进行了初步研究,为进一步在细胞学上为杜鹃属植物分类及种质保护利用方面提供必要的依据和技术支持。现将结果报告如下：
1.在对两种杜鹃的制片过程中,低浓度的秋水仙素,8-羟基喹啉,冰水混合物均会破坏微管的形成,获得中期分裂相较多的细胞。但是秋水仙素处理时间过长或浓度过高,会造成染色体缩短变形,甚至形成多倍体,因此采用秋水仙素进行处理需要进行大量的摸索,且秋水仙素价格昂贵,使用不当对人体会造成神经毒性；用8-羟基喹啉如果处理时间不当,使用8-羟基喹啉溶液同样会导致染色体高度浓缩、变短,不利于进行核型分析。相对于前两种预处理液而言,冰水混合物对染色体形态基本无影响,并且简单易行。实验发现,两种杜鹃的根尖在0-4℃低温条件下用冰水混合物处理24h可以得到大量清晰的中期染色体图像用于核型分析。因此应用冰水混合物在0-4℃处理24h是安全,方便,有效的材料预处理方法,用1mol·L-1HCl解离10min,5mol·L-1HCl解离4min,大白杜鹃用改良苯酚品红染色72h,96h,黄钟杜鹃用改良苯酚品红染色96h,制片效果较好。
2.在两种杜鹃属植物中,大白杜鹃和黄钟杜鹃体细胞的染色体数目均为2n=26条。
3.两种杜鹃属植物中,大白杜鹃体细胞染色体的相对长度为：1.67-5.62,黄钟杜鹃的相对长度为：2.47-5.10。
4.在不同温度下,两种杜鹃属植物的发芽天数与发芽率不同。大白杜鹃在变温条件下发芽率较恒温高,发芽天数较恒温短。在变温15-20℃,20-25℃条件下,大白杜鹃的发芽率分别为81%和83%,发芽天数分别为9和8天；在20℃,25℃恒温条件下,大白杜鹃的发芽率分别为76%和73%,发芽天数分别为10和12天,因此大白杜鹃在20℃恒温下的发芽率较在25℃恒温的发芽率高,发芽天数较25℃恒温的短。本实验中大白杜鹃在25℃时的发芽天数为12天,与耿玉英的报道在25℃时大白杜鹃的发芽天数为8天不一致。黄钟杜鹃在变温15-20℃,20-25℃条件下,发芽率分别为82%和77%,发芽天数分别为11和14天；在20℃,25℃恒温条件下,黄钟杜鹃的发芽率分别为81%和73%,发芽天数分别为13和15天,因此黄钟杜鹃在变温15-20℃发芽率最高,发芽天数最短。
5.在四种取材长度中,两种杜鹃属植物根尖的最佳取材长度为0.5-1.0mm,在0.5mm,2.0mmm时均不利于观察,与其他植物取材长度在1cm左右不同。
6.由于杜鹃染色体太小,本实验只对两种杜鹃的染色体数目和核型进行了初步的研究,对于两种杜鹃之间的关系还有待进一步研究。
Rhododendron franch is under the Sect ponticum and Subsect Fortumes, the different of Rhododendron franch and Rhododendron lanatum is that Rhododendron lanatum is not only under the Sect ponticum,but it also under the Subsect. Lanata Chamb. At present, the field of study karyotype analysis that about two kinds of Rhododendron simsii Planch is a blank, at home and abroad. This article mainly carried out preliminary studies on the number and the relative length of chromosomes, and chromosome production technology that suitable for two rhododendron for further reseach that on classification of Rhododendron and germplasm resources of protection and utilization in caryologia to provide necessary and technical support. Now the result is reported on the following:
1. In the production process Of the two Rhododendron, low concentration of Autumn narcissus alkali,8-hydroxyquinoline, a mixture of ice-water will destroy the microtubules, get more middle split cells. But it can cause chromosomal shorten distortion or even formed polyploid that used high levels of autumn narcissus element or treated in long time, the processing method of Autumn narcissus alkali is not only need a lot of exploration, but the price is also too high, the autumn, and the inappropriate use of Autumn narcissus alkali will cause neurotoxicity; If the processing time is undeserved, the method of using 8-hydroxyquinoline will also lead to chromosome highly concentrated,shorten, and it adverse to promote karyotype analysis. Compared with the former two pretreatment, ice-water mixture had no effect on the basic form of chromosome, and it is a simple and feasible method. Experiments have found that two of the Rhododendron root in 0-4℃low temperature conditions with ice-water treating 24hours can be got a lot of clear middle chromosome image used in karyotype analysis. So using ice-water mixture in 0-4℃and processing 24 hours is a safe, convenient and effective material pretreatment method, with 1 mol·L-1 HCl are obtained from 10 min,5 mol·L-1 HCl are obtained from the 4 min, the producer effect is better that Rhododendron decorum is used of improved phenol magenta dyeing 72 h,96 h, Rhododendron lanatum is used of improved phenol magenta dyeing 96 h.
2. In the plant of two Rhododendron, the Chromosome numbers of Rhododendron decorum and Rhododendron lanatum is 26.
3. In the plant of two Rhododendron,The relative length of Rhododendron decorum is 1.67-5.62, and Rhododendron lanatum is 2.47-5.10.
4. In different temperature, the sprouting days and germination rate of two Rhododendron is different. Rhododendron decorum in the variable temperature conditions is higher than constant temperature, the sprout germination days is shorter than constant temperature. In the variable temperature 15-20℃,20-25℃conditions, the germination rate of Rhododendron decorum were respectively for 81% and 83%,the sprouting days were 9 days and 8 days; In 20℃,25℃constant temperature conditions, the germinative rate of Rhododendron decorum were respectively 76% and 73%, the sprouting days were 10 days and 12 days, So the germination percentage of Rhododendron decorum which under 20℃constant temperature is higher than in 25℃constant temperature,and the sprouting days is shorter than in 25℃constant temperature. In this experiment, the sprouting days of Rhododendron decorum in 25℃were 12 days, this conclusion is different from the conclusion of Geng Yuying who reported that the sprouting days of Rhododendron decorum is 8 dyas in temperature 25℃. In the variable temperature 15-20℃,20-25℃conditions, the germination rate of Rhododendron lanatum were respectively for 82% and 77%,the sprouting days were 11 days and 14 days; In 20℃,25℃constant temperature conditions, the germinative rate of Rhododendron lanatum were respectively 81% and 73%, the sprouting days were 13 days and 15 days, So the germination percentage of Rhododendron lanatum which under 15-20℃variable temperature is highest and the sprouting days is shortest.
5.In four different length, the best is that to draw materials from Rhododendron decorum and Rhododendron lanatum in 0.5-1.0mm, in 0.5mm or 2.0mm are disbennifit to observe the chromosome.This ultimateness is obviously different from other plants that need draw materials from roottip about 1cm.
6. Because of the smaller chromosome of Rhododendron, this paper is only study the number of chromosome and karyotype analysis in two Rhododendron elementary. Nevertheless, the relationship between the two types of Rhododendron still need further research.
1.在对两种杜鹃的制片过程中,低浓度的秋水仙素,8-羟基喹啉,冰水混合物均会破坏微管的形成,获得中期分裂相较多的细胞。但是秋水仙素处理时间过长或浓度过高,会造成染色体缩短变形,甚至形成多倍体,因此采用秋水仙素进行处理需要进行大量的摸索,且秋水仙素价格昂贵,使用不当对人体会造成神经毒性；用8-羟基喹啉如果处理时间不当,使用8-羟基喹啉溶液同样会导致染色体高度浓缩、变短,不利于进行核型分析。相对于前两种预处理液而言,冰水混合物对染色体形态基本无影响,并且简单易行。实验发现,两种杜鹃的根尖在0-4℃低温条件下用冰水混合物处理24h可以得到大量清晰的中期染色体图像用于核型分析。因此应用冰水混合物在0-4℃处理24h是安全,方便,有效的材料预处理方法,用1mol·L-1HCl解离10min,5mol·L-1HCl解离4min,大白杜鹃用改良苯酚品红染色72h,96h,黄钟杜鹃用改良苯酚品红染色96h,制片效果较好。
2.在两种杜鹃属植物中,大白杜鹃和黄钟杜鹃体细胞的染色体数目均为2n=26条。
3.两种杜鹃属植物中,大白杜鹃体细胞染色体的相对长度为：1.67-5.62,黄钟杜鹃的相对长度为：2.47-5.10。
4.在不同温度下,两种杜鹃属植物的发芽天数与发芽率不同。大白杜鹃在变温条件下发芽率较恒温高,发芽天数较恒温短。在变温15-20℃,20-25℃条件下,大白杜鹃的发芽率分别为81%和83%,发芽天数分别为9和8天；在20℃,25℃恒温条件下,大白杜鹃的发芽率分别为76%和73%,发芽天数分别为10和12天,因此大白杜鹃在20℃恒温下的发芽率较在25℃恒温的发芽率高,发芽天数较25℃恒温的短。本实验中大白杜鹃在25℃时的发芽天数为12天,与耿玉英的报道在25℃时大白杜鹃的发芽天数为8天不一致。黄钟杜鹃在变温15-20℃,20-25℃条件下,发芽率分别为82%和77%,发芽天数分别为11和14天；在20℃,25℃恒温条件下,黄钟杜鹃的发芽率分别为81%和73%,发芽天数分别为13和15天,因此黄钟杜鹃在变温15-20℃发芽率最高,发芽天数最短。
5.在四种取材长度中,两种杜鹃属植物根尖的最佳取材长度为0.5-1.0mm,在0.5mm,2.0mmm时均不利于观察,与其他植物取材长度在1cm左右不同。
6.由于杜鹃染色体太小,本实验只对两种杜鹃的染色体数目和核型进行了初步的研究,对于两种杜鹃之间的关系还有待进一步研究。
Rhododendron franch is under the Sect ponticum and Subsect Fortumes, the different of Rhododendron franch and Rhododendron lanatum is that Rhododendron lanatum is not only under the Sect ponticum,but it also under the Subsect. Lanata Chamb. At present, the field of study karyotype analysis that about two kinds of Rhododendron simsii Planch is a blank, at home and abroad. This article mainly carried out preliminary studies on the number and the relative length of chromosomes, and chromosome production technology that suitable for two rhododendron for further reseach that on classification of Rhododendron and germplasm resources of protection and utilization in caryologia to provide necessary and technical support. Now the result is reported on the following:
1. In the production process Of the two Rhododendron, low concentration of Autumn narcissus alkali,8-hydroxyquinoline, a mixture of ice-water will destroy the microtubules, get more middle split cells. But it can cause chromosomal shorten distortion or even formed polyploid that used high levels of autumn narcissus element or treated in long time, the processing method of Autumn narcissus alkali is not only need a lot of exploration, but the price is also too high, the autumn, and the inappropriate use of Autumn narcissus alkali will cause neurotoxicity; If the processing time is undeserved, the method of using 8-hydroxyquinoline will also lead to chromosome highly concentrated,shorten, and it adverse to promote karyotype analysis. Compared with the former two pretreatment, ice-water mixture had no effect on the basic form of chromosome, and it is a simple and feasible method. Experiments have found that two of the Rhododendron root in 0-4℃low temperature conditions with ice-water treating 24hours can be got a lot of clear middle chromosome image used in karyotype analysis. So using ice-water mixture in 0-4℃and processing 24 hours is a safe, convenient and effective material pretreatment method, with 1 mol·L-1 HCl are obtained from 10 min,5 mol·L-1 HCl are obtained from the 4 min, the producer effect is better that Rhododendron decorum is used of improved phenol magenta dyeing 72 h,96 h, Rhododendron lanatum is used of improved phenol magenta dyeing 96 h.
2. In the plant of two Rhododendron, the Chromosome numbers of Rhododendron decorum and Rhododendron lanatum is 26.
3. In the plant of two Rhododendron,The relative length of Rhododendron decorum is 1.67-5.62, and Rhododendron lanatum is 2.47-5.10.
4. In different temperature, the sprouting days and germination rate of two Rhododendron is different. Rhododendron decorum in the variable temperature conditions is higher than constant temperature, the sprout germination days is shorter than constant temperature. In the variable temperature 15-20℃,20-25℃conditions, the germination rate of Rhododendron decorum were respectively for 81% and 83%,the sprouting days were 9 days and 8 days; In 20℃,25℃constant temperature conditions, the germinative rate of Rhododendron decorum were respectively 76% and 73%, the sprouting days were 10 days and 12 days, So the germination percentage of Rhododendron decorum which under 20℃constant temperature is higher than in 25℃constant temperature,and the sprouting days is shorter than in 25℃constant temperature. In this experiment, the sprouting days of Rhododendron decorum in 25℃were 12 days, this conclusion is different from the conclusion of Geng Yuying who reported that the sprouting days of Rhododendron decorum is 8 dyas in temperature 25℃. In the variable temperature 15-20℃,20-25℃conditions, the germination rate of Rhododendron lanatum were respectively for 82% and 77%,the sprouting days were 11 days and 14 days; In 20℃,25℃constant temperature conditions, the germinative rate of Rhododendron lanatum were respectively 81% and 73%, the sprouting days were 13 days and 15 days, So the germination percentage of Rhododendron lanatum which under 15-20℃variable temperature is highest and the sprouting days is shortest.
5.In four different length, the best is that to draw materials from Rhododendron decorum and Rhododendron lanatum in 0.5-1.0mm, in 0.5mm or 2.0mm are disbennifit to observe the chromosome.This ultimateness is obviously different from other plants that need draw materials from roottip about 1cm.
6. Because of the smaller chromosome of Rhododendron, this paper is only study the number of chromosome and karyotype analysis in two Rhododendron elementary. Nevertheless, the relationship between the two types of Rhododendron still need further research.
引文
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