柑橘愈伤组织DNA含量变异、体细胞胚胎发生及同源四倍体的诱导研究
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摘要
柑橘愈伤组织的保存是柑橘种质资源保存的一条重要途径。具有节省人力、物力、财力以及避免自然灾害和病虫害等优点。但是，柑橘愈伤组织存在广泛的遗传变异。为确保所保存愈伤组织的遗传稳定性，需要对其进行鉴定。在细胞学鉴定方面，前人多采用压片技术，该技术费时、检测的细胞少。流式细胞仪的使用为愈伤组织遗传稳定性的鉴定提供了一条非常方便、快速、精确的途径。
培育无核三倍体是柑橘育种学家一直追求的目标。利用二倍体与同源四倍体杂交是培育无核三倍体柑橘的一种有效方法。但自然界还没有一个同源四倍体种。创造同源四倍体为培育无核三倍体柑橘提供重要的中间材料。主要的研究结果如下：
1、本试验在4年中连续3次使用流式细胞仪，对继代培养多年的柑橘愈伤组织的细胞DNA含量进行测定。结果发现，待测的35种愈伤组织均存在细胞DNA含量加倍的现象。并且，随着时间的推移，除佩奇橘柚(Citrus reticulata×Citrus paradisi)，沙漠蒂甜橙(Citrus sinensis)，鲁斯脐橙(Citrus sinensis)和印度酸橘(Citrus reticulata)等4种愈伤组织的DNA含量加倍的细胞有减少的趋势外，其它愈伤组织的DNA含量加倍的细胞均有增加的趋势。不同基因型之间的DNA含量存在差异显著性。数据分析表明，在待测的35种愈伤组织中，有71.4％的基因型的愈伤组织细胞出现了多倍体增加的趋势。
2、对35种柑橘愈伤组织进行体细胞胚诱导，发现只有9种愈伤组织具有不同程度的胚胎发生能力，其它26种愈伤组织均不能再生。相关性分析表明，柑橘愈伤组织细胞DNA含量变异百分率与体细胞胚胎发生能力之间的相关性较小，相关系数为r=-0.1008(P<0.01)，未达到显著水平。另外，柑橘愈伤组织继代时间与体细胞胚胎发生能力之间的相关性也不显著。柑橘愈伤组织生长量与体细胞胚胎发生能力之间有一定的相关性，相关系数为r=-0.3683；高温与低温处理对体细胞胚胎发生能力的影响不明显；不同碳源对具有体细胞胚胎发生能力的柑橘愈伤组织的影响各异，但对于失去再生能力的愈伤组织而言，不同碳源似乎对其体细胞发生不起作用。
3、细胞周期分析可为研究柑橘愈伤组织理化诱变及其再生提供依据。本试验以暗柳橙(Citrus sinensis)，长沙橘(Citrus reticulata)，澳洲指橘(Microcitrus)和纳维来特脐橙(Citrus sinensis)等4种培养多年的柑橘愈伤组织为试材，使用流式细胞仪分析其特定时间段的细胞周期各时相分布情况，进而找到柑橘愈伤组织理化诱变的最佳时期即分裂最旺盛时期。结果表明，待测4种基因型柑橘愈伤组织在
Callus conservation is an important way to preserve germ plasm resources of citrus. Its merits are saving resources and avoiding natural disasters, plant diseases and insect pests. But comprehensive genetic variation occurred in citrus callus. So callus needs to be identified to ensure the genetic stability. The squash technique was used in the past, but the technique wastes time and identifies few cells. The use of flow cytometry provides a convenient, fast and precise way to identify genetic stability of callus.
Breeding of seedless triploid is the objective of citrus breeders. Hybridization of diploid and tetraploid is an effective way to breed seedless triploid citrus. But no autotetraploid variety occurs in nature. Creating autotetraploid provides bridge materials to breed seedless triploid. The main results were as follows:
1. The cell DNA content of thirty-five citrus calli of different genotypes were measured for 3 times by flow cytometry during 4 years. The results showed that the percentages of DNA content variation of citrus calli progressively increased except Page tangelo, Shamouti sweet orange, Russ navel and Cleopatra. The data suggested that 71.4% out of thirty-five citrus calli had increasing polyploids.There existed significant difference in the variation among genotypes.
2. A study carried out on the induction of somatic embryogenesis revealed that 9 citrus calli had competence for somatic embryogenesis and 26 did not. Correlation analyses suggested that the effect of DNA content variation on competence for somatic embryogenesis of citrus calli was not significant and the coefficient of correlation was minus 0.1008 (P<0.01) ; The effect of subculture duration on competence for somatic embryogenesis was also not significant. There existed correlation between growth speed and competence for somatic embryogenesis of citrus calli and their coefficient of correlation was minus 0.3683. Heat shock and low temperature had minor influence to competence for somatic embryogenesis. Different carbon source had different influence to citrus calli which had competence for somatic embryogenesis, but different carbon source had little influence on citrus calli which lost competence for somatic embryogenesis.
3. Cell cycle analysis can provide reference for studying the physical and chemical mutagenesis and regeneration to Citrus calli. Cell cycle phase distribution percentages of four citrus calli (Anliucheng sweet orange, Changsha mandarin, Microcitrus,
培育无核三倍体是柑橘育种学家一直追求的目标。利用二倍体与同源四倍体杂交是培育无核三倍体柑橘的一种有效方法。但自然界还没有一个同源四倍体种。创造同源四倍体为培育无核三倍体柑橘提供重要的中间材料。主要的研究结果如下：
1、本试验在4年中连续3次使用流式细胞仪，对继代培养多年的柑橘愈伤组织的细胞DNA含量进行测定。结果发现，待测的35种愈伤组织均存在细胞DNA含量加倍的现象。并且，随着时间的推移，除佩奇橘柚(Citrus reticulata×Citrus paradisi)，沙漠蒂甜橙(Citrus sinensis)，鲁斯脐橙(Citrus sinensis)和印度酸橘(Citrus reticulata)等4种愈伤组织的DNA含量加倍的细胞有减少的趋势外，其它愈伤组织的DNA含量加倍的细胞均有增加的趋势。不同基因型之间的DNA含量存在差异显著性。数据分析表明，在待测的35种愈伤组织中，有71.4％的基因型的愈伤组织细胞出现了多倍体增加的趋势。
2、对35种柑橘愈伤组织进行体细胞胚诱导，发现只有9种愈伤组织具有不同程度的胚胎发生能力，其它26种愈伤组织均不能再生。相关性分析表明，柑橘愈伤组织细胞DNA含量变异百分率与体细胞胚胎发生能力之间的相关性较小，相关系数为r=-0.1008(P<0.01)，未达到显著水平。另外，柑橘愈伤组织继代时间与体细胞胚胎发生能力之间的相关性也不显著。柑橘愈伤组织生长量与体细胞胚胎发生能力之间有一定的相关性，相关系数为r=-0.3683；高温与低温处理对体细胞胚胎发生能力的影响不明显；不同碳源对具有体细胞胚胎发生能力的柑橘愈伤组织的影响各异，但对于失去再生能力的愈伤组织而言，不同碳源似乎对其体细胞发生不起作用。
3、细胞周期分析可为研究柑橘愈伤组织理化诱变及其再生提供依据。本试验以暗柳橙(Citrus sinensis)，长沙橘(Citrus reticulata)，澳洲指橘(Microcitrus)和纳维来特脐橙(Citrus sinensis)等4种培养多年的柑橘愈伤组织为试材，使用流式细胞仪分析其特定时间段的细胞周期各时相分布情况，进而找到柑橘愈伤组织理化诱变的最佳时期即分裂最旺盛时期。结果表明，待测4种基因型柑橘愈伤组织在
Callus conservation is an important way to preserve germ plasm resources of citrus. Its merits are saving resources and avoiding natural disasters, plant diseases and insect pests. But comprehensive genetic variation occurred in citrus callus. So callus needs to be identified to ensure the genetic stability. The squash technique was used in the past, but the technique wastes time and identifies few cells. The use of flow cytometry provides a convenient, fast and precise way to identify genetic stability of callus.
Breeding of seedless triploid is the objective of citrus breeders. Hybridization of diploid and tetraploid is an effective way to breed seedless triploid citrus. But no autotetraploid variety occurs in nature. Creating autotetraploid provides bridge materials to breed seedless triploid. The main results were as follows:
1. The cell DNA content of thirty-five citrus calli of different genotypes were measured for 3 times by flow cytometry during 4 years. The results showed that the percentages of DNA content variation of citrus calli progressively increased except Page tangelo, Shamouti sweet orange, Russ navel and Cleopatra. The data suggested that 71.4% out of thirty-five citrus calli had increasing polyploids.There existed significant difference in the variation among genotypes.
2. A study carried out on the induction of somatic embryogenesis revealed that 9 citrus calli had competence for somatic embryogenesis and 26 did not. Correlation analyses suggested that the effect of DNA content variation on competence for somatic embryogenesis of citrus calli was not significant and the coefficient of correlation was minus 0.1008 (P<0.01) ; The effect of subculture duration on competence for somatic embryogenesis was also not significant. There existed correlation between growth speed and competence for somatic embryogenesis of citrus calli and their coefficient of correlation was minus 0.3683. Heat shock and low temperature had minor influence to competence for somatic embryogenesis. Different carbon source had different influence to citrus calli which had competence for somatic embryogenesis, but different carbon source had little influence on citrus calli which lost competence for somatic embryogenesis.
3. Cell cycle analysis can provide reference for studying the physical and chemical mutagenesis and regeneration to Citrus calli. Cell cycle phase distribution percentages of four citrus calli (Anliucheng sweet orange, Changsha mandarin, Microcitrus,
引文
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