刘剑锋,刘建华,程云清,钟雪,陈智文
LIU Jianfeng1,LIU Jianhua2,CHENG Yunqing1,ZHONG Xue1,CHEN Zhiwen1(1.Jilin Normal University

• 1:吉林师范大学生态环境研究所
• 2:武汉科技大学附属天佑医院

摘要(Abstract)：

目的:获得高山红景天同源四倍体新材料。方法:用高山红景天愈伤组织原生质体为材料,进行了40%PEG6000介导的原生质体融合,对融合后原生质体进行了低密度、低融点琼脂包埋培养。按原生质体大小对融合原生质体进行了活体标记,建立了起源于单个原生质体的单细胞姊妹系,获得同源四倍体材料。结果:新生子细胞、中期细胞及其原生质体的直径RD与RM均与公式RD=0.793 7RM大致相符。未融合的二倍体、两原生质体融合产物直径的变化范围分别为16.7μm≤R<21.3μm,21.0μm≤R′<26.8μm,两者直径范围存在部分重叠。融合原生质体培养时,接种密度以1×104个/mL为宜,在此密度条件下植板率为20.3%,单细胞起源的姊妹系微克隆生长迅速,易于进行标记、彼此分离并继续培养;染色体计数的结果表明,二倍体、四倍体单细胞姊妹系再生植株染色体数目分别为26,52,不同节位叶片经流式细胞仪检测了DNA含量,证实没有嵌合体的存在。结论:本研究为高山红景天多倍体育种提供了科学依据。
Objective: To acquire homozygous tetraploid germplasm of Rhodiola sachalinensis.Method: PEG-mediated protoplast fusions were conducted using callus of Rh.sachalinensis as materials.Protoplast fusion products were embedded and cultured in low-density,low-melting-point agar and marked according to the protoplast size,and single-celled sister lines were established to acquire genetically homozygous tetraploid germplasm.Result: RD and RM of newborn daughter cells or protoplasm,metaphase cells or protoplasm were approximately in line with the formula RD=0.793 7RM.The change range in diameter of the diploid cells without fusion,two protoplasts fusion product were: 16.7 μm≤R<21.3 μm,21.0 μm≤R′<26.8 μm respectively.There is an overlap between the two diameter ranges.The protoplast inoculation density of 1× 104 cells·mL-1 was appropriate when protoplasts were anchored by low-intensity,low-melting-point agar.Under the conditions of this density,plating efficiency was high and single cell origin of the sister lines microclones grew rapidly,and it was easy to mark the single cell microclones,and separate from each other to subculture.The chromosome counts results showed that chromosome numbers of diploid and tetraploid of single cell lines were 26 and 52,respectively.The result from flow cytometry assay showed that there is no presence of chimerism in single-cell regeneration plantlets.Conclusion: The results of this study provide a scientific basis for polyploid breeding of Rh.sachalinensis.

关键词(KeyWords)： 高山红景天;原生质体;聚乙二醇;四倍体
Rhodiola sachalinensis;protoplast;PEG;tetraploid

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(30801516);; 吉林省科技发展计划项目(20090165);; 吉林师范大学科研创新团队项目(JSDCXTD200807);吉林师范大学研究生科研创新计划项目(S09010102)

作者(Author): 刘剑锋,刘建华,程云清,钟雪,陈智文
LIU Jianfeng1,LIU Jianhua2,CHENG Yunqing1,ZHONG Xue1,CHEN Zhiwen1(1.Jilin Normal University

参考文献(References)：

• [1]刘剑锋,阎秀峰,程云清,等.高山红景天(Rhodiola sachalin-ensisA.Bor)茎尖包埋-玻璃化法超低温保存与植株再生[J].浙江大学学报:农业与生命科学版,2007,33(3):265.
• [2]阎秀峰,王洋,尚辛亥.温室栽培光强和光质对高山红景天生物量和红景天苷含量的影响[J].生态学报,2003,23(5):841.
• [3]阎秀峰,王洋,于涛,等.红景天的栽培采收方法:中国,200410013755.1[P].2005-02-16.
• [4]石铁源,郑明焕,李熙英,等.高山红景天育苗技术[J].林业科技,2006,31(2):60.
• [5]段英姿,客绍英,曹静.秋水仙碱诱导南丹参多倍体的研究[J].中国中药杂志,2006,31(6):445.
• [6]王朝梁,陈中坚,孙玉琴,等.秋水仙碱诱导三七多倍体的初步研究[J].中国中药杂志,2007,32(12):1222.
• [7]王跃华.川黄柏多倍体诱导研究[J].中国中药杂志,2006,31(6):448.
• [8]谭芳,沈火林,王帅,等.芹菜与CMS胡萝卜原生质体非对称性融合的初步研究[J].园艺学报,2009,36(8):1169.
• [9]Hao Y J,Deng X X.Single-cell-derived sibling lines are estab-lished as an experimental system to assess chromosome numbervariations in embryogenic callus cultures of sweet orange[J].Plant Cell Tissue Organ Cult,2003,73:275.
• [10]Guo W W,Deng X X.Intertribal hexaploid somatic hybrid plantsregeneration from electrofusion between diploids ofCitrus sinensisand its sexually incompatible relative,Clausena lansium[J].Theor Appl Genet,1999,98:581.
• [11]Hao Y J,You C X,Deng X X.Cell size as a morphologicalmarker to calculate the mitotic index and ploidy level of citruscallus[J].Plant Cell Rep,2002,20:1123.
• [12]刘剑锋,阎秀峰,程云清,等.高山红景天叶片愈伤组织诱导与植株再生[J].东北林业大学学报,2007,35(2):33.
• [13]刘剑锋,程云清,陈智文.高山红景天叶肉原生质体分离培养与植株再生[J].中草药,2009,40(7):1127.
• [14]Akym A,Djamila C,Robert H,et al.Protoplast fusion in ba-nana(Musaspp.):comparison of chemical(PEG:polyethy-lene glycol)and electrical procedure[J].Plant Cell Tissue Or-gan Cult,2005,83:145.
• [15]刘剑锋,阎秀峰,程云清,等.高山红景天愈伤组织的玻璃化法保存及植株再生[J].北京林业大学学报,2007,29(2):147.
• [16]Seguí-Simarro J M,Nuez F.Pathways to doubled haploidy:chro-mosome doubling during androgenesis[J].Cytogenet GenomeRes,2008,120:358.
• [17]Jones J R,Ranney TG,Eaker TA.Anovel method for inducingpolyploidy inRhododendronseedlings[J].J Amer RhododendronSoc,2008,62(3):130.
• [18]Jain S M.Tissue culture-derived variation in crop improvement[J].Euphytica,2001,118:153.
• [19]王正询,刘鸿先.香蕉苗试管繁殖染色体数量畸变的研究[J].遗传学报,1997,24(6):550.