草业学报 ›› 2024, Vol. 33 ›› Issue (7): 172-181.DOI: 10.11686/cyxb2023294
• 研究论文 • 上一篇
符勇耀1(), 蔡莉1, 李丰耀1, 杨文俊1, 徐文姬1, 姜思佳2(), 杨利平1()
收稿日期:
2023-08-29
修回日期:
2023-10-26
出版日期:
2024-07-20
发布日期:
2024-04-08
通讯作者:
姜思佳,杨利平
作者简介:
yangliping1962@126.com基金资助:
Yong-yao FU1(), Li CAI1, Feng-yao LI1, Wen-jun YANG1, Wen-ji XU1, Si-jia JIANG2(), Li-ping YANG1()
Received:
2023-08-29
Revised:
2023-10-26
Online:
2024-07-20
Published:
2024-04-08
Contact:
Si-jia JIANG,Li-ping YANG
摘要:
兰州百合是我国最重要的可食用甜百合。为培育优质的百合新种质,用不同浓度的秋水仙素离体诱变兰州百合小鳞茎,通过不定芽诱导获得变异幼苗。利用染色体倍性分析、幼苗早期形态观察及ISSR分子标记对其进行鉴定。结果表明,用0.15%秋水仙素处理小鳞茎48 h诱导效果最好,变异率为58.06%。选择2个形态变异且生长较快的幼苗进行流式细胞仪分析和根尖压片计数,发现其中1个变异株系含36条染色体的细胞数比例达到了80%,接近纯合三倍体。形态学观察显示,2个变异幼苗的叶片、鳞茎及根系与对照相比存在显著差异,且气孔保卫细胞大于对照,气孔密度降低。ISSR标记检测表明,2个诱变株系与对照相比均发生遗传变异,变异率分别为19.64%和20.91%。研究结果可为获得兰州百合三倍体新种质及秋水仙素诱变育种提供重要参考依据。
符勇耀, 蔡莉, 李丰耀, 杨文俊, 徐文姬, 姜思佳, 杨利平. 兰州百合多倍体离体诱导及其分子细胞鉴定[J]. 草业学报, 2024, 33(7): 172-181.
Yong-yao FU, Li CAI, Feng-yao LI, Wen-jun YANG, Wen-ji XU, Si-jia JIANG, Li-ping YANG. Induction and molecular and cellular identification of polyploid Lilium davidii var. unicolor[J]. Acta Prataculturae Sinica, 2024, 33(7): 172-181.
秋水仙素浓度 Colchicine concentration (%) | 处理时间 Treated time (h) | 处理数 Treated number (No.) | 成活数 Survived number (No.) | 成活率 Survival rate (%) | 变异数 Varied number (No.) | 变异率 Mutation rate (%) |
---|---|---|---|---|---|---|
0.10 | 24 | 119 | 111 | 93.18 | 65 | 54.62 |
48 | 27 | 27 | 100.00 | 9 | 33.33 | |
72 | 38 | 15 | 39.47 | 0 | 0 | |
96 | 42 | 7 | 16.67 | 3 | 7.14 | |
0.15 | 24 | 28 | 25 | 89.29 | 12 | 42.86 |
48 | 31 | 29 | 93.55 | 18 | 58.06 | |
72 | 54 | 44 | 81.48 | 3 | 5.56 | |
96 | 30 | 22 | 73.33 | 1 | 3.33 | |
0.20 | 24 | 30 | 24 | 80.00 | 11 | 36.67 |
48 | 28 | 27 | 96.43 | 4 | 14.29 | |
72 | 47 | 31 | 65.96 | 1 | 2.13 | |
96 | 90 | 80 | 88.89 | 4 | 4.44 | |
0.30 | 24 | 36 | 24 | 66.67 | 3 | 8.33 |
48 | 41 | 25 | 60.98 | 5 | 12.20 | |
72 | 27 | 2 | 7.41 | 0 | 0 | |
96 | 35 | 4 | 11.43 | 3 | 8.57 | |
0.35 | 24 | 106 | 91 | 85.85 | 27 | 25.47 |
48 | 37 | 11 | 29.73 | 1 | 2.70 | |
72 | 27 | 14 | 51.85 | 3 | 11.11 | |
96 | 31 | 2 | 6.45 | 2 | 6.45 |
表1 不同浓度秋水仙素处理不同时间对兰州百合的影响
Table 1 Effects of colchicine concentration and treatment time on L. davidii var. unicolor
秋水仙素浓度 Colchicine concentration (%) | 处理时间 Treated time (h) | 处理数 Treated number (No.) | 成活数 Survived number (No.) | 成活率 Survival rate (%) | 变异数 Varied number (No.) | 变异率 Mutation rate (%) |
---|---|---|---|---|---|---|
0.10 | 24 | 119 | 111 | 93.18 | 65 | 54.62 |
48 | 27 | 27 | 100.00 | 9 | 33.33 | |
72 | 38 | 15 | 39.47 | 0 | 0 | |
96 | 42 | 7 | 16.67 | 3 | 7.14 | |
0.15 | 24 | 28 | 25 | 89.29 | 12 | 42.86 |
48 | 31 | 29 | 93.55 | 18 | 58.06 | |
72 | 54 | 44 | 81.48 | 3 | 5.56 | |
96 | 30 | 22 | 73.33 | 1 | 3.33 | |
0.20 | 24 | 30 | 24 | 80.00 | 11 | 36.67 |
48 | 28 | 27 | 96.43 | 4 | 14.29 | |
72 | 47 | 31 | 65.96 | 1 | 2.13 | |
96 | 90 | 80 | 88.89 | 4 | 4.44 | |
0.30 | 24 | 36 | 24 | 66.67 | 3 | 8.33 |
48 | 41 | 25 | 60.98 | 5 | 12.20 | |
72 | 27 | 2 | 7.41 | 0 | 0 | |
96 | 35 | 4 | 11.43 | 3 | 8.57 | |
0.35 | 24 | 106 | 91 | 85.85 | 27 | 25.47 |
48 | 37 | 11 | 29.73 | 1 | 2.70 | |
72 | 27 | 14 | 51.85 | 3 | 11.11 | |
96 | 31 | 2 | 6.45 | 2 | 6.45 |
图1 兰州百合多倍体诱导过程植株形态A:未改变的小鳞茎;B,C:发生形态变异的小鳞茎;D,E:培育发生形态变异的器官;F:变异器官继代再生长出芽;G:对照植株幼苗;H:诱变株系LZ-1;I:诱变株系LZ-2。A: Induced but unchanged bulblets; B, C: Bulblets undergoing morphological variation; D, E: Cutting organs undergoing morphological variation; F: The subculture buds from mutated organs; G: The control seedlings; H: Induced line LZ-1; I: Induced line LZ-2.
Fig.1 Plant morphology during polyploid induction of L. davidii var. unicolor
图2 兰州百合染色体倍性测试A~C:流式细胞仪分析染色体倍性,A为对照植株,B为诱变株系LZ-1,C为诱变株系LZ-2;D~F:根尖压片分析染色体数目,D为对照植株,E为诱变株系LZ-1,F为诱变株系LZ-2。A-C: Analysis of the chromosome ploidy by flow cytometry, A is control plants, B is mutant lines LZ-1, C is mutant lines LZ-2; D-F: Chromosome tableting using by root tips, D is control plants, E is mutant lines LZ-1, F is mutant lines LZ-2.
Fig.2 Chromosome ploidy analysis of L.davidii var. unicolor
样本 Sample | 细胞总数Total of cells | 含24条染色体的细胞数Number of cells with 24 chromosomes | 含36条染色体的细胞数Number of cells with 36 chromosomes | 其他染色体数量Cell of other chromosomes |
---|---|---|---|---|
对照 Control | 5 | 5 | 0 | 0 |
兰州-1 LZ-1 | 17 | 0 | 5 | 12 |
兰州-2 LZ-2 | 20 | 0 | 16 | 4 |
表2 兰州百合染色体数目观察
Table 2 Chromosome number analysis in L.davidii var. unicolor
样本 Sample | 细胞总数Total of cells | 含24条染色体的细胞数Number of cells with 24 chromosomes | 含36条染色体的细胞数Number of cells with 36 chromosomes | 其他染色体数量Cell of other chromosomes |
---|---|---|---|---|
对照 Control | 5 | 5 | 0 | 0 |
兰州-1 LZ-1 | 17 | 0 | 5 | 12 |
兰州-2 LZ-2 | 20 | 0 | 16 | 4 |
样本 Sample | 株高 Plant height (mm) | 叶片长度 Leaf length (mm) | 叶片宽度 Leaf width (mm) | 叶长宽比 Leaf length-width ratio | 鳞茎直径 Bulblet diameter (mm) | 鳞片长 Scale length (mm) | 鳞片宽 Scale width (mm) |
---|---|---|---|---|---|---|---|
对照 Control | 84.78±1.46b | 82.83±1.72b | 1.69±0.11c | 54.47±3.15a | 4.25±0.16c | 5.11±0.34b | 3.29±0.21c |
兰州-1 LZ-1 | 105.68±2.39a | 99.65±2.47a | 6.01±0.18a | 17.01±0.63b | 8.96±0.21b | 11.16±0.36a | 5.84±0.22b |
兰州-2 LZ-2 | 81.73±1.53b | 72.09±1.72c | 4.76±0.16b | 15.63±0.70b | 10.70±0.26a | 11.35±2.67a | 6.82±0.15a |
表3 兰州百合对照和变异植株的形态学特征比较
Table 3 Comparison of morphological characterization in control and mutagenic plants
样本 Sample | 株高 Plant height (mm) | 叶片长度 Leaf length (mm) | 叶片宽度 Leaf width (mm) | 叶长宽比 Leaf length-width ratio | 鳞茎直径 Bulblet diameter (mm) | 鳞片长 Scale length (mm) | 鳞片宽 Scale width (mm) |
---|---|---|---|---|---|---|---|
对照 Control | 84.78±1.46b | 82.83±1.72b | 1.69±0.11c | 54.47±3.15a | 4.25±0.16c | 5.11±0.34b | 3.29±0.21c |
兰州-1 LZ-1 | 105.68±2.39a | 99.65±2.47a | 6.01±0.18a | 17.01±0.63b | 8.96±0.21b | 11.16±0.36a | 5.84±0.22b |
兰州-2 LZ-2 | 81.73±1.53b | 72.09±1.72c | 4.76±0.16b | 15.63±0.70b | 10.70±0.26a | 11.35±2.67a | 6.82±0.15a |
图3 兰州百合变异幼苗植株形态的比较A:从左到右分别是兰州百合对照和2个诱变株系(LZ-1和LZ-2)幼苗植株形态,标尺为1.8 cm;B:从左到右分别是对照和2个诱变株系叶片形态,标尺为1.5 cm; C,D:从左到右分别是对照和2个诱变株系外层鳞片及其横切面,标尺为0.8和0.5 cm;E:从左到右分别是对照和2个诱变株系不定根,标尺为 1.0 cm;F,G:对照和LZ-2接种1个月的小鳞茎比较。A: The plant morphology of the control and two induced lines of the L. davidii var. unicolor seedling from left to right, the bar represents 1.8 cm; B: The leaf morphology of the control and two induced lines of the L. davidii var. unicolor from left to right, the bar represents 1.5 cm; C, D: The outer scales and its transverse section of the control and two induced lines of the L. davidii var. unicolor from left to right, the bars represent 0.8 and 0.5 cm; E: The roots of the control and two induced lines of the L. davidii var. unicolor from left to right, the bar represents 1.0 cm; F, G: The comparison of the bulblets of the control and LZ-2 grown for one month.
Fig.3 The comparison of morphology in mutant seedling plants of L. davidii var. unicolor
样本 Sample | 上表皮细胞长 Adaxial epidermal cell length (μm) | 上表皮细胞宽 Adaxial epidermal cell width (μm) | 上表皮细胞长宽比 Adaxial epidermal cell length-width ratio | 气孔密度 Stomatal frequency (No.·mm-2) | 保卫细胞长 Guard cell length (μm) | 保卫细胞宽 Guard cell width (μm) |
---|---|---|---|---|---|---|
对照Control | 256.94±5.15c | 30.30±0.61c | 8.76±0.29c | 42.07±2.27a | 19.86±0.40c | 15.62±0.20c |
兰州-1 LZ-1 | 406.85±8.49b | 41.04±0.84a | 10.10±0.26b | 24.51±1.54c | 32.33±0.21a | 27.39±0.27a |
兰州-2 LZ-2 | 448.89±13.55a | 37.62±0.76b | 12.28±0.49a | 30.49±1.66b | 27.31±0.34b | 18.38±0.22b |
表4 兰州百合对照和变异植株上表皮细胞、保卫细胞和气孔的比较
Table 4 Comparison of adaxial epidermis cell, guard cell and stomata in control and mutagenic plants
样本 Sample | 上表皮细胞长 Adaxial epidermal cell length (μm) | 上表皮细胞宽 Adaxial epidermal cell width (μm) | 上表皮细胞长宽比 Adaxial epidermal cell length-width ratio | 气孔密度 Stomatal frequency (No.·mm-2) | 保卫细胞长 Guard cell length (μm) | 保卫细胞宽 Guard cell width (μm) |
---|---|---|---|---|---|---|
对照Control | 256.94±5.15c | 30.30±0.61c | 8.76±0.29c | 42.07±2.27a | 19.86±0.40c | 15.62±0.20c |
兰州-1 LZ-1 | 406.85±8.49b | 41.04±0.84a | 10.10±0.26b | 24.51±1.54c | 32.33±0.21a | 27.39±0.27a |
兰州-2 LZ-2 | 448.89±13.55a | 37.62±0.76b | 12.28±0.49a | 30.49±1.66b | 27.31±0.34b | 18.38±0.22b |
图4 兰州百合变异幼苗的上表皮和气孔观察A~C:兰州百合对照、诱变株系LZ-1和LZ-2上表皮细胞形态,标尺为20 μm;D~F:对照、诱变株系LZ-1和LZ-2叶片气孔,标尺为20 μm;G~I:对照、诱变株系LZ-1和LZ-2叶片气孔,标尺为10 μm。A-C: The adaxial epidermal cells in the control of L. davidii var. unicolor and mutant lines LZ-1, LZ-2 respectively, scales represent 20 μm; D-F: The leaf blade stomas in the control of L. davidii var. unicolor and mutant lines LZ-1, LZ-2 respectively, scales represent 20 μm; G-I: The leaf blade stomas in the control of L. davidii var. unicolor and mutant lines LZ-1, LZ-2 respectively, scales represent 10 μm.
Fig.4 The observation of adaxial epidermal cells and leaf blade stomas in mutant seedlings of L.davidii var. unicolor
图5 兰州百合变异幼苗移栽生长比较A~C:兰州百合对照、诱变株系LZ-1和LZ-2幼苗移栽当天形态;D~F:兰州百合对照、诱变株系LZ-1和LZ-2幼苗移栽30 d形态,标尺为3 cm。A-C: The morphology of the control, induced lines LZ-1 and LZ-2 of L. davidii var. unicolor for 0 day; D-F: The morphology of the control, induced lines LZ-1 and LZ-2 of L. davidii var. unicolor for 30 days, the bars represent 3 cm.
Fig.5 The growth of transplanting seedlings in mutant L. davidii var. unicolor plants
图6 兰州百合变异植株的ISSR检测A:引物3A30;B:引物3A50;C:引物UBC811;D:引物UBC814;E:引物UBC815; F:引物UBC835;G:引物UBC842;H:引物UBC843;I:引物UBC844;J:引物UBC845;K:引物UBC857;1~3为3个独立的LZ-2样本,4~6为3个独立的LZ-1样本,7~9为3个独立的对照样本,M为Marker 2000。A: Primer 3A30; B: Primer 3A50; C: Primer UBC811; D: Primer UBC814; E: Primer UBC815; F: Primer UBC835; G: Primer UBC842; H: Primer UBC843; I: Primer UBC844; J: Primer UBC845; K: Primer UBC857; 1-3: Three independent LZ-2 samples; 4-6: Three independent LZ-1 samples; 7-9: Three independent control samples; M: Marker 2000.
Fig.6 ISSR detection for mutant plants of L. davidii var. unicolor
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