西番莲种质资源花粉活力鉴定方法探究

项艳艳; 黄伟杰; 赖江龙; 汤礼梅; 林挺兴; 李亮; 魏秀清; 许家辉

doi:10.19303/j.issn.1008-0384.2024.03.007

西番莲种质资源花粉活力鉴定方法探究

项艳艳^{1, 2,},
黄伟杰^{1, 2},
赖江龙^{1, 2},
汤礼梅^{1, 2},
林挺兴³,
李亮²,
魏秀清²,
许家辉^2, ,

1.
福建农林大学园艺学院，福建　福州　350002
2.
福建省农业科学院果树研究所，福建　福州　350013
3.
宁德市蕉城区柑橘无检疫性病虫母本园种苗繁育场，福建　宁德　352000

基金项目: 福建省高质量发展超越“5511”协同创新工程项目（XTCXGC2021006）；福建省种业创新与产业化工程项目（zycxny2021010-1）；福建省农业科学院科技创新团队建设项目（CXTD2021008-2）；福建省科技计划公益类专项（2021R1028002）

详细信息

作者简介:
项艳艳（1997 — ），女，硕士研究生，主要从事果树育种研究，E-mail：2711768629@qq.com

通讯作者:
许家辉（1971 — ），男，研究员，主要从事果树育种研究，E-mail：xjhui577@163.com

中图分类号: S667.9
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出版历程
- 收稿日期: 2024-02-05
- 修回日期: 2024-02-29
- 网络出版日期: 2024-03-27
- 刊出日期: 2024-03-27

Determination of Viability of Passion Fruit Pollens

XIANG Yanyan^{1, 2,},
HUANG Weijie^{1, 2},
LAI Jianglong^{1, 2},
TANG Limei^{1, 2},
LIN Tingxing³,
LI Liang²,
WEI Xiuqing²,
XU Jiahui^2, ,

1.
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Fruit Research Institute ,Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
3.
Seed and Seedling Breeding Farm of Citrus Non-quarantine Venereal Insect Mother Garden. Ningde, Fujian　352000, China

摘要

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1～7 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。
- 西番莲 /
- 花粉活力 /
- 花粉培养基 /
- 花粉离体萌发 /
- 染色法 /
- 花粉贮藏
Abstract:
Objective Method for determining the viability of passion fruit pollens was explored to facilitate the utilization of existing germplasms and breeding new varieties.
Methods Medium and conditions for in vitro culture of Passiflora pollens was optimized. Appropriate staining methods to determine and storage conditions to maximize the pollen viability were investigated. Contents of sucrose, H₃BO₃, Ca(NO₃)₂, and polyethylene glycol-4000 (PEG-4000) of the medium were optimized in an orthogonal experiment on pollens of 6 germplasms. Pollen germination on the finalized medium for varied culture times was observed. The staining of I₂-KI, TTC, and Alexander methods and storage for different durations at different temperatures were compared.
Results The optimum medium was formulated with 100 g·L⁻¹ sucrose, 0.02 g·L⁻¹ H₃BO₃, 0.04 g·L⁻¹ Ca(NO₃)₂, 150 g·L⁻¹ PEG-4000, 200 g·L⁻¹ MgSO₄·7H₂O, and 100 g·L⁻¹ KNO₃, then adjusted to pH 5.5. The pollen germination was examined in 1 h of the in vitro culture. TTC staining showed satisfactory effect with no significant deviations from the in vitro observation indicating it an applicable indicator for pollen viability. In 24 h of storage at 25 ℃ the pollen viability was 30.48%; and at 4 ℃, it was 26.69% and remained 26.54%–29.05% for 7 d.
Conclusion The medium formulation and in vitro culture time as well as the staining method and storage conditions of passion fruit pollens were determined making a rapid and reliable procedure to maximize the pollen germination available.
- Passion fruit /
- pollen viability /
- culture medium /
- in vitro pollen germination /
- staining /
- pollens storage

HTML全文

图 1 花粉离体培养时间对花粉萌发率的影响

A：不同时期花粉萌发率；B：a、b、c、d分别代表1、2、3、4 h等花粉萌发观测结果。

Figure 1. Effect of culture time on pollen germination rate

A: pollen germination rate at different times. B: a, b, c, and d represent observed results on pollen germination at 1, 2, 3, and 4 h, respectively.

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图 2 不同方法检测西番莲花粉活力镜检效果。

A：蓝冠；B：白花；C：香蜜；D：雅蜜；E：钦蜜9号；F：台农。

Figure 2. Microscopic examinations on passion fruit pollen viability detected by different methods

A: Blue Crown; B: White Flowers; C: Xiangmi; D: Yami; E: Qinmi 9; F: Tainong.

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图 3 钦蜜9号不同贮藏温度下花粉萌发率的变化

不同小写字母表示在0.05水平差异显著。

Figure 3. Germination rates of Qinmi 9 pollens stored under different temperatures

Data with different lowercase letters indicate significant differences at 0.05 level.

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表 1 正交因素及水平

Table 1 Orthogonal factors and levers

水平 Level	A.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

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表 2 钦蜜9号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 culture medium of passion fruit pollens

编号 No.	因素 Factors				萌发率 Germination rate/%	花粉管长度 Pollen tube length/μm
编号 No.	A	B	C	D	萌发率 Germination rate/%	花粉管长度 Pollen tube length/μm
1	1	1	1	1	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	64.50±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 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 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
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。 Data are represented as mean ± standard deviation; those with different lowercase letters on same column indicate significant differences at P＜0.05.

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表 3 钦蜜9号离体萌发率方差分析结果

Table 3 Variance analysis on in vitro germination rate of Qinmi 9

变异来源 Source of variation	III 类平方和 Class III sum of squares	自由度 Degree of freedom	均方 Mean square	F	显著性 Significance（P）
蔗糖 Sources	9986.344	4	2496.586	8.365	0.006^**
H₃BO₃	4465.681	4	1116.420	3.740	0.53
Ca(NO₃)₂	505.625	4	126.406	0.424	0.788
PEG-4000	9836.241	4	2459.06	8.239	0.006^**
误差 Error	2387.782	8	298.473
表示在0.01水平差异极显著。 indicates extremely significant difference at 0.01 level.

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表 4 不同方法检测的西番莲花粉活力

Table 4 Passion fruit pollen viability detected by different methods

种质 Germplasm	染色率 Staining rate/%			离体萌发率 In vitro germination rate/%
种质 Germplasm	I₂-IK	TTC	亚历山大 Alexandria	离体萌发率 In vitro germination rate/%
台农 Tainong	83.83±3.76 a	67.69±5.29 a	81.19±2.78 a	74.13±2.89 a
钦蜜9号 Qinmi 9	81.79±7.75 b	70.29±4.47 b	97.95±1.14 a	79.76±7.80 b
雅蜜 Yami	87.85±3.22 b	69.97±5.92 c	95.42±2.16 a	63.71±2.84 c
白花 White Flowers	84.32±7.73 b	77.41±6.33bc	97.22±1.89 a	63.7±1.00 c
蓝冠 Blue Crown	80.41±3.26 a	64.85±4.41 b	78.50±5.28 a	60.79±4.61 b
香蜜 Xiangmi	84.32±7.70 b	63.78±1.11 c	98.75±1.88 a	71.34±6.97 c
数据表示为平均值±标准差，同行数据后不同小写字母表示在0.05水平差异显著。 Data are mean±SD; those with different lowercase letters on same row indicate significant difference at 0.05 level.

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