小花型夏菊品种花器官愈伤辐射诱变研究与再生苗遗传变异的RAPD分析
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摘要
首先以‘金莲’、‘白矮生’、‘矮红’3个小花型夏菊品种的舌状花和管状花作为外植体进行组织培养研究；然后利用10、15、20Gy的~(60)Co γ射线对这3个小花型夏菊品种的舌状花和管状花诱导的愈伤组织进行辐射处理；最后利用RAPD技术从分子水平上对‘金莲’辐射再生苗的遗传变异程度进行分析。研究结果表明：
1．基因型及外植体类型影响花器官的愈伤诱导率、分化率以及愈伤平均分化芽的数目。‘金莲’品种的愈伤诱导率、分化率以及愈伤平均分化芽的数目均最好，管状花和舌状花分别为74.25％、30.61％、4.91和61.71％、27.28％、3.82。‘矮红’品种最差，分别为72.50％、19.03％、0.60和58.39％、11.02％、0.27，3个品种的愈伤分化率在5％水平上达到显著性差异，平均分化芽数差异在1％水平上达到显著差异。外植体类型对三个指标也有很大影响，管状花外植体均明显好于舌状花外植体，如‘白矮生’品种管状花的三个指标分别为71.66％、21.10％、2.53；舌状花三个指标分别为57.33％、13.21％、1.68，管状花的指标均显著高于舌状花的指标。
2．~(60)Co γ射线对3个品种不同外植体诱导产生的愈伤组织的褐化、分化能力均有影响。随着辐射剂量的增加，不同来源的愈伤组织褐化程度均加重，分化能力减弱，当辐射剂量达到15Gy时，‘矮红’品种所有愈伤都失去分化能力，‘白矮生’舌状花产生的愈伤失去分化能力。当辐射剂量为20Gy时，只有‘金莲’品种的愈伤仍然具有分化能力，但分化能力与对照相比已很弱，管状花和舌状花产生的愈伤分化率和平均分化芽数目分别为对照的57.11％、28.51％和47.65％、31.68％。此外，基因型和不同来源的愈伤组织，对γ射线敏感性也有差异，‘矮红’品种的愈伤最敏感，舌状花产生的愈伤比管状花产生的愈伤辐射敏感性强。
3．RAPD技术作为一种有效的分子鉴定手段可以有效、快速地检测出辐射再生苗基因组的变异程度。RAPD研究结果表明，再生辐射苗的基因组遗传变异程度与所接受γ射线剂量成正相关性，即愈伤受到辐射剂量越大，则由该愈伤分化的苗变异程度就越大。10、15和20Gy 3个处理的再生苗的遗传变异程度与对照相比，其变异系数分别为0.223、0.335和0.393。通过RAPD技术筛选出了一批变异植株，其田间表现情况正在进一步观察。
The studies include three related parts. The first one was concerned with the study on regeneration of calli derived from ray flowers and tabular flowers of three summer-flowering Chrysanthemum morifolium cultivars with small inflorescences, named 'Golden lotus', 'White dwarf' and 'Red dwarf respectively. The second one studied the effects of gamma rays on the browning and differentiation capacity of calli derived from different explants of three cultivars. The last one was variation analysis of regeneranis from tabular-flower-derived calli of 'Golden lotus' treated with 10, 15 and 20 Gy gamma rays, respectively using RAPD.
The results are as follows.
Firstly, both genotype and types of explants greatly influence induction and differentiation rate of calli and mean buds per callus. As far as Chrysanthemum morifolium 'Golden lotus' was concerned, induction and differentiation rate of calli and mean buds per callus were the highest among the three cultivars. Its tabular-flower-derived calli induction rate, differentiation rate and mean buds per callus were 74.25%, 30.61% and 4.91, and those of ray-flower-derived calli were 61.71%, 27.28% and 3.82, respectively. While the data of 'Red dwarf were the lowest , the tabular-flower-derived calli induction rate, differentiation rate and mean buds per callus were 72.50%, 19.03% and 0.61, and those of the ray-flower-derived callus were 58.39%, 11.02%, and 0.27, respectively. Callus differentiation rate of three cultivars had a significant difference at level of 5%, and so did mean buds per callus at level of 1%. Tabular flowers were superior to ray flowers in the capacities of callus induction and differentiation. T
ake 'White dwarf as an example, its induction and differentiation rate of tabular-flower-derived calli and mean buds per callus were 71.66%, 21.10% and 2.53, while those of ray-flower-derived callus were 57.33%, 13.21% and 1.68, respectively.
Secondly, 60Co gamma ray has an great influence upon browning and differentiation capacity of calli derived from different explants of the three cultivars. As the doses of gamma ray increased from 0 , 10, 15 to 20 Gy, the browning rate of the calli rose, and differentiation capacity decreased gradually. When the dose of gamma ray was 15 Gy, all the calli of 'Red dwarf' lost differentiation capacity, and so did the ray-flower-derived calli of'White dwarf. When the dose was 20Gy, 'White dwarf lost differentiation capacity, and differentiation capacity of'Golden lotus' was lower than that of the control. Compared with
the control, its differentiation rate of tabular-derived calli and mean buds per callus decreased by 42.89% and 71.49%, and those of ray-flower-derived calli by 52.35% and 68.12%,respectively. In addition, tabular-flower-derived calli was more sensitive to gamma rays than ray-flower-derived calli, thus the latter was easier to brown and lose differentiation capacity than the former. What's more, as far as sensitivity to gamma rays was concerned , the calli of 'Red dwarf ' were most susceptible to gamma ray among the three cultivars, and ray-flower-derived calli more than tabular-flower-derived calli.
Finally, RAPD is an effective molecular means to identify the variation of genomic DNA of regenerants quickly and reliably. The results showed that the variation range of genomic DNA of plants regenerated from inrradiated calli was proportional to the dose of gamma rays. In other words, the higher the dose of gamma ray used , the wider the variation range of the genomic DNA of plants was. In comparison with the control, the variation coefficient of the genomic DNA of plants, which produce from tabular-flower-derived calli of 'Golden lotus" irradiated with the dose of 10, 15 and 20 Gy gamma rays, was 0.223,0.335 and 0.393, respectively. A batch of mutant plants had been screened through RAPD technique. And the phenotypic characteristics of these plants are under observation in the field.
1．基因型及外植体类型影响花器官的愈伤诱导率、分化率以及愈伤平均分化芽的数目。‘金莲’品种的愈伤诱导率、分化率以及愈伤平均分化芽的数目均最好，管状花和舌状花分别为74.25％、30.61％、4.91和61.71％、27.28％、3.82。‘矮红’品种最差，分别为72.50％、19.03％、0.60和58.39％、11.02％、0.27，3个品种的愈伤分化率在5％水平上达到显著性差异，平均分化芽数差异在1％水平上达到显著差异。外植体类型对三个指标也有很大影响，管状花外植体均明显好于舌状花外植体，如‘白矮生’品种管状花的三个指标分别为71.66％、21.10％、2.53；舌状花三个指标分别为57.33％、13.21％、1.68，管状花的指标均显著高于舌状花的指标。
2．~(60)Co γ射线对3个品种不同外植体诱导产生的愈伤组织的褐化、分化能力均有影响。随着辐射剂量的增加，不同来源的愈伤组织褐化程度均加重，分化能力减弱，当辐射剂量达到15Gy时，‘矮红’品种所有愈伤都失去分化能力，‘白矮生’舌状花产生的愈伤失去分化能力。当辐射剂量为20Gy时，只有‘金莲’品种的愈伤仍然具有分化能力，但分化能力与对照相比已很弱，管状花和舌状花产生的愈伤分化率和平均分化芽数目分别为对照的57.11％、28.51％和47.65％、31.68％。此外，基因型和不同来源的愈伤组织，对γ射线敏感性也有差异，‘矮红’品种的愈伤最敏感，舌状花产生的愈伤比管状花产生的愈伤辐射敏感性强。
3．RAPD技术作为一种有效的分子鉴定手段可以有效、快速地检测出辐射再生苗基因组的变异程度。RAPD研究结果表明，再生辐射苗的基因组遗传变异程度与所接受γ射线剂量成正相关性，即愈伤受到辐射剂量越大，则由该愈伤分化的苗变异程度就越大。10、15和20Gy 3个处理的再生苗的遗传变异程度与对照相比，其变异系数分别为0.223、0.335和0.393。通过RAPD技术筛选出了一批变异植株，其田间表现情况正在进一步观察。
The studies include three related parts. The first one was concerned with the study on regeneration of calli derived from ray flowers and tabular flowers of three summer-flowering Chrysanthemum morifolium cultivars with small inflorescences, named 'Golden lotus', 'White dwarf' and 'Red dwarf respectively. The second one studied the effects of gamma rays on the browning and differentiation capacity of calli derived from different explants of three cultivars. The last one was variation analysis of regeneranis from tabular-flower-derived calli of 'Golden lotus' treated with 10, 15 and 20 Gy gamma rays, respectively using RAPD.
The results are as follows.
Firstly, both genotype and types of explants greatly influence induction and differentiation rate of calli and mean buds per callus. As far as Chrysanthemum morifolium 'Golden lotus' was concerned, induction and differentiation rate of calli and mean buds per callus were the highest among the three cultivars. Its tabular-flower-derived calli induction rate, differentiation rate and mean buds per callus were 74.25%, 30.61% and 4.91, and those of ray-flower-derived calli were 61.71%, 27.28% and 3.82, respectively. While the data of 'Red dwarf were the lowest , the tabular-flower-derived calli induction rate, differentiation rate and mean buds per callus were 72.50%, 19.03% and 0.61, and those of the ray-flower-derived callus were 58.39%, 11.02%, and 0.27, respectively. Callus differentiation rate of three cultivars had a significant difference at level of 5%, and so did mean buds per callus at level of 1%. Tabular flowers were superior to ray flowers in the capacities of callus induction and differentiation. T
ake 'White dwarf as an example, its induction and differentiation rate of tabular-flower-derived calli and mean buds per callus were 71.66%, 21.10% and 2.53, while those of ray-flower-derived callus were 57.33%, 13.21% and 1.68, respectively.
Secondly, 60Co gamma ray has an great influence upon browning and differentiation capacity of calli derived from different explants of the three cultivars. As the doses of gamma ray increased from 0 , 10, 15 to 20 Gy, the browning rate of the calli rose, and differentiation capacity decreased gradually. When the dose of gamma ray was 15 Gy, all the calli of 'Red dwarf' lost differentiation capacity, and so did the ray-flower-derived calli of'White dwarf. When the dose was 20Gy, 'White dwarf lost differentiation capacity, and differentiation capacity of'Golden lotus' was lower than that of the control. Compared with
the control, its differentiation rate of tabular-derived calli and mean buds per callus decreased by 42.89% and 71.49%, and those of ray-flower-derived calli by 52.35% and 68.12%,respectively. In addition, tabular-flower-derived calli was more sensitive to gamma rays than ray-flower-derived calli, thus the latter was easier to brown and lose differentiation capacity than the former. What's more, as far as sensitivity to gamma rays was concerned , the calli of 'Red dwarf ' were most susceptible to gamma ray among the three cultivars, and ray-flower-derived calli more than tabular-flower-derived calli.
Finally, RAPD is an effective molecular means to identify the variation of genomic DNA of regenerants quickly and reliably. The results showed that the variation range of genomic DNA of plants regenerated from inrradiated calli was proportional to the dose of gamma rays. In other words, the higher the dose of gamma ray used , the wider the variation range of the genomic DNA of plants was. In comparison with the control, the variation coefficient of the genomic DNA of plants, which produce from tabular-flower-derived calli of 'Golden lotus" irradiated with the dose of 10, 15 and 20 Gy gamma rays, was 0.223,0.335 and 0.393, respectively. A batch of mutant plants had been screened through RAPD technique. And the phenotypic characteristics of these plants are under observation in the field.
引文
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