Study on the identification method of pollen viability of passionflower germplasm resources
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摘要:
目的 明确西番莲属植物花粉最佳离体培养基、离体培养最适观测时间、花粉的最适染色法和花粉贮藏条件,快速精准鉴定西番莲属种质资源的花粉活力,为其种质资源利用与新品种选育提供科学依据。 方法 以6份西番莲属种质资源的花粉为试验材料,设计正交试验,探究不同浓度的蔗糖、H3BO3、Ca(NO3)2和聚乙二醇-4000(PEG-4000)对花粉离体萌发率的影响;利用最佳花粉离体培养基,筛选花粉离体培养最适观测时间;采用I2-KI染色法、TTC染色法与亚历山大染色法,探究最适的西番莲花粉染色法;通过不同贮藏温度与时间处理,筛选花粉的最佳贮藏条件。 结果 正交试验筛选出最佳培养基配方为100 g·L−1 蔗糖+0.04 g·L−1 H3BO3+0.01 g·L−1 Ca(NO3)2+150 g·L−1 PEG-4000+200 mg·L−1 MgSO4·7H2O+100 mg·L−1 KNO3,pH值5.5;花粉离体培养1 h为最适观测时间;TTC染色法染色效果好且结果与离体萌发率无显著差异,可有效检测西番莲花粉活力;花粉贮藏24 h以内,25 ℃与4 ℃的贮藏温度下花粉活力分别为30.48 %、26.69 %,花粉贮藏时间在1 d~7 d,4 ℃下花粉活力仍保持在26.54 %~27.05 %。 结论 筛选出西番莲属植物花粉离体萌发的最佳培养基组合、离体培养最适观测时间、花粉最适染色法与花粉贮藏最佳条件,为今后西番莲属植物花粉活力的快速筛选和品种改良提供理论依据。 Abstract: :objective Clarifying the best in vitro medium for Passiflora plant pollens, the optimal observation time for in vitro culture, the optimal pollens staining method and pollens storage conditions will help to quickly and accurately identify the pollens viability of Passiflora germplasm resources. It provides scientific basis for the utilization of germplasm resources and the breeding of new varieties. methods The effects of sucrose, H3BO3, Ca(NO3)2 and polyethylene glycol-4000 (PEG-4000) on pollens germination rate in vitro were studied by orthogonal experiment with 6 passionflower pollens samples as experimental materials. The best pollens culture medium was used to screen the optimal observation time for pollens culture in vitro. I2-KI staining, TTC staining and Alexander staining were used to explore the most suitable passionflower powder staining method. The best storage conditions of pollen were selected by different storage temperature and time. results The optimum medium formula was selected by orthogonal test as follows: 100 g·L−1 sucrose+0.02 g·L−1 H3BO3+0.04 g·L−1 Ca(NO3)2+150 g·L−1 polyethylene glycol (PEG-4000)+200 g·L−1 MgSO4·7H2O+100 g·L−1 KNO3, pH 5.5. The optimum observation time for pollens in vitro culture was 1 h. TTC staining method had good staining effect and had no significant difference with in vitro germination rate, which could effectively detect the vitality of passionflower powder. Within 24 hours of pollens storage, the pollens viability at 25 ℃ and 4 ℃ was 30.48 % and 26.69 % respectively and the pollens viability remained 26 % ~ 29 % at 4 ℃ for 7 days. conclusion The optimum medium combination, in vitro culture time, pollens staining method and pollens storage conditions for passionflower pollens germination in vitro were selected. to provide a theoretical basis for rapid screening and variety improvement of passionflower pollens vigor in the future. -
Key words:
- Passionflower /
- pollens viability /
- pollens medium /
- pollens germination in vitro /
- staining /
- pollens storage
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表 1 正交因素及水平
Table 1. Orthogonal factors and levers
水平
LeveA.蔗糖
Sucrose /(g·L−1)B.H3BO3/
(g·L−1)C.Ca(NO3)2/
(g·L−1)D.PEG-4000/
(g·L−1)1 0 0 0 0 2 50 0.01 0.01 50 3 100 0.02 0.02 100 4 150 0.03 0.03 150 5 200 0.04 0.04 200 表 2 百香果花粉离体培养基筛选的L25(54)正交试验设计
Table 2. L25(54) orthogonal experimental design for passion fruit pollen in-vivo medium screening
编号
No.因素
Factors萌发率
Germination rate/%花粉管长度
Pollen tube length/μmA B C D 1 1 1 1 1 0±0 j — 2 1 2 2 2 13.81±1.88 i 158.5±4.03 ab 3 1 3 3 3 48.25±3.12 g 152.92±2.73 ab 4 1 4 4 4 64.5±1.68 f 126.24±5.96 e 5 1 5 5 5 75.53±3.07 bcde 108.29±10.29 f 6 2 1 2 3 69.11±11.13 def 147.43±4.46 bc 7 2 2 3 4 79.85±1.03 abc 132.59±8.86 de 8 2 3 4 5 63.65±4.68 f 109.89±5.34 f 9 2 4 5 1 12.99±2.23 i 150.65±13.17 b 10 2 5 1 2 72.26±1.83 cdef 167.15±4.46 a 11 3 1 3 5 45.95±0.61 g 143.76±7.77 bcd 12 3 2 4 1 0±0 j — 13 3 3 5 2 76.99±5.37 abcde 126.75±5.77 e 14 3 4 1 3 67.23±7.08 f 134.33±12.72 cde 15 3 5 2 4 85.56±1.82 a 157.36±4.01 ab 16 4 1 4 2 6.5±3.04 ij — 17 4 2 5 3 28.73±17.87 h 124.43±12.31 e 18 4 3 1 4 83.08±3.5 ab 120.2±3.76 ef 19 4 4 2 5 0.27±0.47 j — 20 4 5 3 1 0.18±0.17 j — 21 5 1 5 4 0.54±0.47 j — 22 5 2 1 5 1.03±0.4 j — 23 5 3 2 1 0±0 j — 24 5 4 3 2 1.66±2.88 j — 25 5 5 4 3 25.94±3.2 h 158.54±9.45 ab k1 40.42 24.42 44.72 2.63 k2 59.57 24.68 33.75 34.24 k3 41.64 54.40 34.53 47.85 k4 23.75 29.33 32.12 62.71 k5 5.83 51.89 38.96 37.29 R 53.74 29.98 12.60 60.08 注:数据表示为平均值±标准差。同列数据用不同小写字母标识表示在0.05水平差异显著。k1、k2、k3、k4、k5分别表示各因素的五个水平的平均数;R代表极差。
Note: The data is represented as mean ± standard deviation. The same column of data is marked with different lowercase letters, indicating significant differences at the 0.05 level. when using different lowercase letters to indicate the same column of data.k1, k2, k3, k4, k5 represent the average of the three levels of each factor, respectively;R represents the range.表 3 ‘钦蜜9号’离体萌发率方差分析结果
Table 3. Results of variance analysis of in vitro germination rate of ‘Qinmi 9’
变异来源
Source of variationIII 类平方和
Class III sum of squares自由度
Degree of freedom均方
Mean squareF 显著性
Significance(P)蔗糖
Sources37059.104 4 9264.776 34.88 1.81E-18** H3BO3 12326.565 4 3081.641 11.602 1.88E-11** Ca(NO3)2 4321.592 4 1080.398 4.068 0.044316 PEG-4000 21115.99 4 5278.997 19.874 2.54E-18** 误差
Error15405.773 58 265.617 注:**表示在0.01水平差异极显著。
Note:**indicate extremely significant difference at the 0.01 level.表 4 不同方法检测的西番莲花粉活力
Table 4. Passionflower pollen viability detected by different methods
种质
Germplasm染色率
Staining rate/%离体萌发率
In vitro
germination
rate/
%I2-IK TTC 亚历山大
Alexandria台农
Tai nong83.83±3.76 a 67.69±5.29 a 81.19±2.78 a 74.13±2.89 a 钦蜜9号
Qin mi81.79±7.75 b 70.29±4.47 b 97.95±1.14 a 79.76±7.80 b 雅蜜
Ya mi87.85±3.22 b 69.97±5.92 c 95.42±2.16 a 63.71±2.84 c 白花
White flowers84.32±7.73 b 77.41±6.33 bc 97.22±1.89 a 63.7±1.00 c 蓝冠
Blue crown80.41±3.26 a 64.85±4.41 b 78.50±5.28 a 60.79±4.61 b 香蜜
Xiang mi84.32±7.70 b 63.78±1.11 c 98.75±1.88 a 71.34±6.97 c 注:数据表示为平均值±标准差,同列数据后不同小写字母表示在0.05水平差异显著。
Note: Datas are mean±SD; those with different lowercase letters on same column indicate significant difference at 0.05 level. -
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