42份菊科花卉的离体保存及其遗传多样性的RAPD分析
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摘要
本研究收集了42份菊科花卉种质资源。以菊科花卉带腋芽的茎段为外植体,建立菊科花卉的再生体系;以‘追鱼’菊花试管苗为材料,探讨试管苗限制生长保存方法并采用RAPD技术进行遗传稳定性的检测,以期为菊科花卉的离体繁殖和离体种质保存提供科学依据;对收集的42份菊科花卉进行试管保存,并利用RAPD技术进行了的遗传多样性分析。主要研究结果如下:
1.菊科花卉再生体系的建立。
以‘追鱼’菊花的茎段和‘追鱼’菊花试管苗为材料,分别研究其消毒、增殖、生根、移栽的最佳条件。试验结果表明:最佳消毒方法为75%酒精(30s)+0.1%的HgCl2(3 min)+无菌水+0.1%的HgCl2(3 min);最佳增殖培养基为MS+0.3㎎/L 6-BA+0.15㎎/LNAA,增殖系数达到9.05;最佳生根培养基为1/2MS +0.2㎎/L NAA;最佳移栽基质为蛭石:泥炭土(1:2)的混合基质。
2.菊科花卉试管苗离体保存方法的探讨。
以‘追鱼’菊花试管苗生长3个月后的茎切段为材料,比较了不同浓度的蔗糖、琼脂、山梨醇和蔗糖、生长抑制物质、光照强度和光质等因素对试管苗限制生长保存的影响。结果表明:在MS培养基中添加40g/L蔗糖与15g/L山梨醇的处理保存效果最好,保存10个月时,存活率为77.8%,适宜的继代周期是8个月;光照强度对‘追鱼’菊花的离体保存影响不显著;添加30mg/LABA也有利于试管苗的离体保存。
3.菊科花卉试管苗限制生长保存前后遗传稳定性的分析
以限制生长法保存了15个月与保存前的菊花试管苗为材料,采用RAPD-PCR标记技术对菊花试管苗保存前后的遗传变异进行了检测。结果表明:保存前后的菊花RAPD图谱基本一致,说明保存后没有发生DNA水平的变异,遗传稳定性好,同时也说明这种限制生长保存方法可以适用于菊花的短期离体保存。
4.42份菊科花卉种质资源的试管苗限制生长保存
采用优化的保存方法对所收集的菊科花卉种质资源进行试管苗限制生长保存。每隔7个月继代1次,菊属,蟛蜞菊属的试管苗保存效果良好,保存后进行移栽,生长正常。其中万寿菊属的试管苗在保存过程中易出现玻璃化现象。
5.42份菊科花卉种质资源遗传多样性的RAPD分析
采用RAPD技术对42份菊科花卉种质资源进行了遗传多样性分析。结果表明:11个随机引物共产生127条扩增带,其中,扩增出的多态性谱带126条,多态性比率达99.2%,这表明42份菊科花卉的遗传多态性高,遗传资源的丰富。根据扩增条带多态性,构建了菊科花卉的亲缘关系树状图,当相似系数为0.362时,42份菊科花卉资源可分为4组,百日草、天人菊、金鸡菊的品种各为一组,菊花、万寿菊、蟛蜞菊的品种为一组;当相似系数为0.628时,分为10组,所有菊花品种为1组,其他品种分为9组;当相似系数为0.794时,共划分22组,33份菊花品种可以划分成13组,代表保存菊花的13个菊花种质群。
In this study,42 accessions of compositae germplasm resources were collected for in vitro conservation. Stem-segments with axillary buds of compositae plants were used as the explants for establishing the in vitro regeneration system. The plantlets from“Zhuiyu”chrysanthemum were applied as the conservation materials to investigate the restricting growth conservation method, and the RAPD technique was used to detect the genetic stability of the conserved plantlets., which provided more scientific basis for the micropropagation and in-vitro preservation of compositae plants. 42 accessions of compositae plants were conserved in vitro and analysed by RAPD technique for discussing genetic diversity. The main results were as follows:
1. Establishment of the regeneration system of Compositae plants
The stem segments from the“Zhuiyu”chrysanthemum and its plantlets were used as materials to study the best conditions of the surface sterilization, proliferation, rooting and transplanting. The results showed that: the best method of surface sterilization was treated with 75% alcohol (30s) +0.1% of HgCl2 (3 min) + sterile water (24 h) +0.1% of HgCl2 (3 min); the best proliferation medium was the MS medium supplemented with 0.3 mg ? L-1 6-BA and0.15 mg ? L-1 NAA, and the proliferation coefficient was 9.05; the best rooting medium was 1/2MS +0.2㎎ / L NAA, NAA effect on root induction was better than IBA; and the best transplanting medium was the mixture of vermiculite and peat (1:2).
2. Study on in vitro conservation of Compositae plants
The plantlets of“Zhuiyu”chrysanthemum which had grown for 3 months were used as materials. The effects of different concentrations of sucrose, agar, sorbitol, growth inhibitor and light intensity on restricting growth conservation of plantlets were evaluated. As the results showed, MS media containing 40 g/L sucrose and 15g/L sorbitol was the optimal condition for conservation. The survival rate was up to 77.8% after preservation for 10 months. The optimal subculture time could be 8 months. At the same time, 30mg/LABA promoted conservation of plantlets as well. However, light intensity did not affect the in vitro conservation of“Zhui yu”.
3. Analysis of genetic stability of the conserved Compositae plants
Using plantlets before conservation and plantlets conserved for 10 months as materials, the genetic variation between these two treatments was detected by RAPD-PCR marker techniques. The resulted demonstrated that the patterns of ISSP were absolutely identical, which showed that there were no variations of DNA levels and the genetic stability of the conserved plantlets were very stable. This also confirmed the feasibility of the restricting conservation.
4. The in vitro restricting conservation of 42 accessions of Compositae germplasm resources
All collected compositae plants were preserved in vitro by advanced conservation method. All of them were sunbculured by every 7 months. The plantlets of Chrysanthemum and Wedelia were well preserved and growth of these plantlets was good after transplantation . Nevertheless, vitrification was easy to occur in the plantlets of Tagetes.
5. RAPD analysis of genetic diversity of 42 accessions of Compositae germplasm resources
In this experiment, the genetic diversity of 42 accessions of compositae germplasm resources was assessed by using RAPD (Random amplified microsatellite polymorphism). As results showed, 127 amplified bands were produced by 11 random primers. The ratio of polymorphic bands reached to 99.2% with 126 polymorphic bands appeared, which illustrated that 42 accessions of compositae germplasm resources had abundant polymorphism and rich genetic resources. According to the diversity of the amplified bands, a dendrogram was constructed to indicate their genetic relationships. When similarity coefficient was 0.362, 42 accessions of compositae germplasm resource could be separated into 4 groups, Zinnia group, Indian blankets group, Coreopsis basalis group and another group consisting of Chrysanthemum, marigold, Wedelia chinensis. When similarity coefficient was 0.628, all Chrysanthemum species were put in the same group, the rest species were divided into other 9 groups. While, when similarity coefficient was 0.794, 22 groups were set up. 33 chrysanthemum species were divided into 13 groups which presented that 13 different chrysanthemum germplams were preserved.
1.菊科花卉再生体系的建立。
以‘追鱼’菊花的茎段和‘追鱼’菊花试管苗为材料,分别研究其消毒、增殖、生根、移栽的最佳条件。试验结果表明:最佳消毒方法为75%酒精(30s)+0.1%的HgCl2(3 min)+无菌水+0.1%的HgCl2(3 min);最佳增殖培养基为MS+0.3㎎/L 6-BA+0.15㎎/LNAA,增殖系数达到9.05;最佳生根培养基为1/2MS +0.2㎎/L NAA;最佳移栽基质为蛭石:泥炭土(1:2)的混合基质。
2.菊科花卉试管苗离体保存方法的探讨。
以‘追鱼’菊花试管苗生长3个月后的茎切段为材料,比较了不同浓度的蔗糖、琼脂、山梨醇和蔗糖、生长抑制物质、光照强度和光质等因素对试管苗限制生长保存的影响。结果表明:在MS培养基中添加40g/L蔗糖与15g/L山梨醇的处理保存效果最好,保存10个月时,存活率为77.8%,适宜的继代周期是8个月;光照强度对‘追鱼’菊花的离体保存影响不显著;添加30mg/LABA也有利于试管苗的离体保存。
3.菊科花卉试管苗限制生长保存前后遗传稳定性的分析
以限制生长法保存了15个月与保存前的菊花试管苗为材料,采用RAPD-PCR标记技术对菊花试管苗保存前后的遗传变异进行了检测。结果表明:保存前后的菊花RAPD图谱基本一致,说明保存后没有发生DNA水平的变异,遗传稳定性好,同时也说明这种限制生长保存方法可以适用于菊花的短期离体保存。
4.42份菊科花卉种质资源的试管苗限制生长保存
采用优化的保存方法对所收集的菊科花卉种质资源进行试管苗限制生长保存。每隔7个月继代1次,菊属,蟛蜞菊属的试管苗保存效果良好,保存后进行移栽,生长正常。其中万寿菊属的试管苗在保存过程中易出现玻璃化现象。
5.42份菊科花卉种质资源遗传多样性的RAPD分析
采用RAPD技术对42份菊科花卉种质资源进行了遗传多样性分析。结果表明:11个随机引物共产生127条扩增带,其中,扩增出的多态性谱带126条,多态性比率达99.2%,这表明42份菊科花卉的遗传多态性高,遗传资源的丰富。根据扩增条带多态性,构建了菊科花卉的亲缘关系树状图,当相似系数为0.362时,42份菊科花卉资源可分为4组,百日草、天人菊、金鸡菊的品种各为一组,菊花、万寿菊、蟛蜞菊的品种为一组;当相似系数为0.628时,分为10组,所有菊花品种为1组,其他品种分为9组;当相似系数为0.794时,共划分22组,33份菊花品种可以划分成13组,代表保存菊花的13个菊花种质群。
In this study,42 accessions of compositae germplasm resources were collected for in vitro conservation. Stem-segments with axillary buds of compositae plants were used as the explants for establishing the in vitro regeneration system. The plantlets from“Zhuiyu”chrysanthemum were applied as the conservation materials to investigate the restricting growth conservation method, and the RAPD technique was used to detect the genetic stability of the conserved plantlets., which provided more scientific basis for the micropropagation and in-vitro preservation of compositae plants. 42 accessions of compositae plants were conserved in vitro and analysed by RAPD technique for discussing genetic diversity. The main results were as follows:
1. Establishment of the regeneration system of Compositae plants
The stem segments from the“Zhuiyu”chrysanthemum and its plantlets were used as materials to study the best conditions of the surface sterilization, proliferation, rooting and transplanting. The results showed that: the best method of surface sterilization was treated with 75% alcohol (30s) +0.1% of HgCl2 (3 min) + sterile water (24 h) +0.1% of HgCl2 (3 min); the best proliferation medium was the MS medium supplemented with 0.3 mg ? L-1 6-BA and0.15 mg ? L-1 NAA, and the proliferation coefficient was 9.05; the best rooting medium was 1/2MS +0.2㎎ / L NAA, NAA effect on root induction was better than IBA; and the best transplanting medium was the mixture of vermiculite and peat (1:2).
2. Study on in vitro conservation of Compositae plants
The plantlets of“Zhuiyu”chrysanthemum which had grown for 3 months were used as materials. The effects of different concentrations of sucrose, agar, sorbitol, growth inhibitor and light intensity on restricting growth conservation of plantlets were evaluated. As the results showed, MS media containing 40 g/L sucrose and 15g/L sorbitol was the optimal condition for conservation. The survival rate was up to 77.8% after preservation for 10 months. The optimal subculture time could be 8 months. At the same time, 30mg/LABA promoted conservation of plantlets as well. However, light intensity did not affect the in vitro conservation of“Zhui yu”.
3. Analysis of genetic stability of the conserved Compositae plants
Using plantlets before conservation and plantlets conserved for 10 months as materials, the genetic variation between these two treatments was detected by RAPD-PCR marker techniques. The resulted demonstrated that the patterns of ISSP were absolutely identical, which showed that there were no variations of DNA levels and the genetic stability of the conserved plantlets were very stable. This also confirmed the feasibility of the restricting conservation.
4. The in vitro restricting conservation of 42 accessions of Compositae germplasm resources
All collected compositae plants were preserved in vitro by advanced conservation method. All of them were sunbculured by every 7 months. The plantlets of Chrysanthemum and Wedelia were well preserved and growth of these plantlets was good after transplantation . Nevertheless, vitrification was easy to occur in the plantlets of Tagetes.
5. RAPD analysis of genetic diversity of 42 accessions of Compositae germplasm resources
In this experiment, the genetic diversity of 42 accessions of compositae germplasm resources was assessed by using RAPD (Random amplified microsatellite polymorphism). As results showed, 127 amplified bands were produced by 11 random primers. The ratio of polymorphic bands reached to 99.2% with 126 polymorphic bands appeared, which illustrated that 42 accessions of compositae germplasm resources had abundant polymorphism and rich genetic resources. According to the diversity of the amplified bands, a dendrogram was constructed to indicate their genetic relationships. When similarity coefficient was 0.362, 42 accessions of compositae germplasm resource could be separated into 4 groups, Zinnia group, Indian blankets group, Coreopsis basalis group and another group consisting of Chrysanthemum, marigold, Wedelia chinensis. When similarity coefficient was 0.628, all Chrysanthemum species were put in the same group, the rest species were divided into other 9 groups. While, when similarity coefficient was 0.794, 22 groups were set up. 33 chrysanthemum species were divided into 13 groups which presented that 13 different chrysanthemum germplams were preserved.
引文
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De Jong J, Rademaker W,Monique F,et al.Restoring adventitiousshoot formation on Chrysanthe- mum leaf explants following cocultivation with Agrobacterium tumef aciens.Plant Cell Tis Org Cult, 1993 ,32: 263～270.
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Engelmann F.In vitro conserveation methods[A].In: Callow J A, Ford-Lloyd, BV, Newbury, H J, eds, Biotechnology and plant Genetic Resources [C]. CABI, Oxon, 1997: 119-161.
Fabbri A, et al. Random amplified polymorphic DNA analysis of olive (olea europaea L.) cultivars [J]. J Amer Soc Hort sci, 1995, 120(3): 538-542.
FAO. Report on the state of the worlds plants genetic resources [J]. 1996.
Frankel O H, Brown A H D. Current plant genetic resources-acritical appraisa1. Genetics: New Frontiers Vo1. IV. Oxford and IBH Publishing. 1984:25-36.
Galzy,R.Recberches sur la croissance de Vitis rupestris Scheekle Sain et court noue cultive in vitro a differentes temperatures [J].Ann.Phytopathoi.1969,1:149-166.
Harada T, et al. DNA-RAPDs detect genetic variation and paternity in Malus [J]. Euphytica.1993, 65(2): 87-91. http://www.sipo.gov.cn/sipo/ztxx/yczyhctzsbh/zlk/gjgy/200512/P020060403601429846913.pdf. 粮食和农业植物遗传资源国际条约. 2001
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Kirsten .W ,Jenny P, et al. Rapid detection of genetic variability in Chrysanthemum Using random primers. Heredity 71(1993):335-341.
Lundergan C, Janick J. Low temperature storage of in vitro apple shoots[J]. Hortscience, 1979, 14(4):5-14
Lundergan C,Janick J.Low temperature storage of in vitro apple shoots[A]. Hortscience,1979, 14(4): 5-14.
Lyndsey A, Withers L A. In vitro approaches to conversation of plant genetic resources [A]. In: Plant Tissue Culture and Its Agricultural Applications (Lyndsey A. Withers and P. G. Aldrrsoneds) [C]. London :Butterworths , 1986: 261-276.
Marino G,Rosati P,Sagrati F.Storage of In vitro cultures of Prunus rootstocks[J].Plant Cell Tiss Org Cult,1985,5: 73-78.
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