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

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.
宁德市蕉城区柑桔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，硕士，主要从事果树育种研究，E-mail：xjhui577@163.com

中图分类号: S667.9
计量
- 文章访问数: 28
- HTML全文浏览量: 17
- PDF下载量: 0
- 被引次数: 0
出版历程
- 收稿日期: 2024-02-06
- 修回日期: 2024-03-01
- 网络出版日期: 2024-03-28

Study on the identification method of pollen viability of passionflower germplasm resources

XIANG Yanyan^{1, 2
,},
HUANG Weijie^{1, 2},
LAI Jiang Long^{1, 2},
TANG Li Mei^{1, 2},
LIN Tingxing^{3
, ,},
LI Liang²,
WEI Xiuqing²,
XU Jiahui^2
,

1.
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
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 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。
- 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 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.
- Passionflower /
- pollens viability /
- pollens medium /
- pollens germination in vitro /
- 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。

注：a、b、c、d分别代表1 h、2 h、3 h、4 h等不同时间点花粉萌发观测结果。

Figure 1. Effect of pollen ex vivo culture time on pollen germination rate.

Note:a, b, c and d represent the observation results of pollen germination at different time points such as 1 h, 2 h, 3 h and 4 h respectively.

下载: 全尺寸图片幻灯片

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. Different methods were used to detect the microscopic examination effect of passionflower pollen viability.

A: Blue crown; B: White flowers; C: Xiang mi; D: Ya mi; E: Qin mi 9; F: Tai nong.

下载: 全尺寸图片幻灯片

图 3 钦蜜9号不同贮藏温度下花粉萌发率的变化

注：同行数据用不同小写字母标识表示在0.05水平差异显著。

Figure 3. Changes in pollen germination rate of 'Qinmi 9' at different storage temperatures

Note:The same column of data is marked with different lowercase letters, indicating significant differences at the 0.05 level.

下载: 全尺寸图片幻灯片

表 1 正交因素及水平

Table 1. Orthogonal factors and levers

水平 Leve	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

下载: 导出CSV

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/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±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.

下载: 导出CSV

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 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	37059.104	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 Error	15405.773	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.

下载: 导出CSV

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/ %
种质 Germplasm	I₂-IK	TTC	亚历山大 Alexandria	离体萌发率 In vitro germination rate/ %
台农 Tai nong	83.83±3.76 a	67.69±5.29 a	81.19±2.78 a	74.13±2.89 a
钦蜜9号 Qin mi	81.79±7.75 b	70.29±4.47 b	97.95±1.14 a	79.76±7.80 b
雅蜜 Ya mi	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±6.33 bc	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 Xiang mi	84.32±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.

下载: 导出CSV

参考文献(31)

[1]	DE SOUZA SILVA G, DA SILVA CAMPELO BORGES G, PINHO DA COSTA CASTRO C D, et al. Physicochemical quality, bioactive compounds and in vitro antioxidant activity of a new variety of passion fruit cv. BRS Sertão Forte ( Passiflora cincinnata Mast. ) from Brazilian Semiarid region [J]. Scientia Horticulturae, 2020, 272: 109595. doi: 10.1016/j.scienta.2020.109595
[2]	GADIOLI I L, DE SÁ BARRETO DA CUNHA M, DE CARVALHO M V O, et al. A systematic review on phenolic compounds in Passiflora plants: Exploring biodiversity for food, nutrition, and popular medicine [J]. Critical Reviews in Food Science and Nutrition, 2018, 58(5): 785−807. doi: 10.1080/10408398.2016.1224805
[3]	DA SILVA LIMA L K, DE JESUS O N, SOARES T L, et al. Growth, physiological, anatomical and nutritional responses of two phenotypically distinct passion fruit species ( Passiflora L. ) and their hybrid under saline conditions [J]. Scientia Horticulturae, 2020, 263: 109037. doi: 10.1016/j.scienta.2019.109037
[4]	XIA Z Q, HUANG D M, ZHANG S K, et al. Chromosome-scale genome assembly provides insights into the evolution and flavor synthesis of passion fruit ( Passiflora edulis Sims) [J]. Horticulture Research, 2021, 8: 14. doi: 10.1038/s41438-020-00455-1
[5]	罗远华, 陈燕, 方能炎, 等. 文心兰花粉离体萌发培养基的筛选及萌发率的测定 [J]. 福建农业学报, 2023, 38(11):1293−1301. LUO Y H, CHEN Y, FANG N Y, et al. Optimized medium and culture conditions for germination of Oncidium pollens [J]. Fujian Journal of Agricultural Sciences, 2023, 38(11): 1293−1301. (in Chinese)
[6]	田青兰, 吴艳艳, 张英俊, 等. 西番莲自交和异交的花粉管荧光显微观察及授粉亲和性分析 [J]. 果树学报, 2023, 40(1):98−110. TIAN Q L, WU Y Y, ZHANG Y J, et al. Pollen tube fluorescence microscopic observation and pollination compatibility analysis of self and cross-pollination in passion fruit( Passiflora edulis) [J]. Journal of Fruit Science, 2023, 40(1): 98−110. (in Chinese)
[7]	FACCIUTO P G. Breeding Advances in Passiflora spp. (Passionflower) Native to Argentina [J]. Floriculture & Ornamental Biotechnology, 2011, 5(1).
[8]	白梦, 李博, 陈简村, 等. 报春苣苔属植物杂交亲和性分析及杂交障碍 [J]. 中南林业科技大学学报, 2024, 44(1):89−96. BAI M, LI B, CHEN J C, et al. Compatibility and crossbreeding barriers of hybridization in Primulina [J]. Journal of Central South University of Forestry & Technology, 2024, 44(1): 89−96. (in Chinese)
[9]	张波, 秦垦, 黄婷, 等. 宁夏枸杞花粉活力及柱头可授性研究 [J]. 湖北农业科学, 2022, 61(19):83−86. ZHANG B, QIN K, HUANG T, et al. Study on pollen viability and stigma receptivity of Lycium barbarum [J]. Hubei Agricultural Sciences, 2022, 61(19): 83−86. (in Chinese)
[10]	任少华, 张驰强, 王陈, 等. 香榧花粉活性检测及离体培养条件筛选 [J]. 种子, 2020, 39(8):104−108. REN S H, ZHANG C Q, WANG C, et al. Pollen viability detection and screening of in vitro culture conditions of torreyagrandis [J]. Seed, 2020, 39(8): 104−108. (in Chinese)
[11]	付强, 陈伟, 李江, 等. 思茅松花粉活力测定方法研究 [J]. 西部林业科学, 2020, 49(3):7−13. FU Q, CHEN W, LI J, et al. Selection of measurements on pollen viability in Pinus kesiya var. langbianensis [J]. Journal of West China Forestry Science, 2020, 49(3): 7−13. (in Chinese)
[12]	任飞艳, 陈显, 王士泉. 卵叶海桑花粉活力测定方法的比较研究 [J]. 种子, 2021, 40(3):35−39. REN F Y, CHEN X, WANG S Q. Comparative study on the determination methods of pollen viability of Sonneratia ovata [J]. Seed, 2021, 40(3): 35−39. (in Chinese)
[13]	张峥. 金叶含笑传粉生物学与杂交育种研究[D]. 长沙: 中南林业科技大学, 2023. ZHANG Z. Study on pollination biology and cross breeding of Michelia foveolata[D]. Changsha: Central South University of Forestry & Technology, 2023. (in Chinese)
[14]	刘洁云, 吴艳艳, 牟海飞, 等. 黄百香果花粉离体培养基筛选及贮藏试验 [J]. 西南农业学报, 2021, 34(8):1699−1704. LIU J Y, WU Y Y, MOU H F, et al. Medium screening for in vitro germination and storage conditions of yellow passion fruit pollen [J]. Southwest China Journal of Agricultural Sciences, 2021, 34(8): 1699−1704. (in Chinese)
[15]	吴斌, 黄东梅, 邢文婷, 等. 不同黄金百香果品种果肉挥发性成分分析 [J]. 中国果树, 2023, (9):57−64,143. WU B, HUANG D M, XING W T, et al. Analysis of volatile components in sarcocarp of different varieties of yellow passion fruit [J]. China Fruits, 2023(9): 57−64,143. (in Chinese)
[16]	王金花, 张健琴, 成良强, 等. 不同方法测定花生花粉活力的比较研究 [J]. 种子, 2022, 41(8):126−130. WANG J H, ZHANG J Q, CHENG L Q, et al. Comparative study on pollen vitality determination of peanut by different methods [J]. Seed, 2022, 41(8): 126−130. (in Chinese)
[17]	胡晋. 花粉的保存和生活力测定 [J]. 种子, 1992, 11(6):33−35,39. HU J. Preservation of pollen and determination of its viability [J]. Seed, 1992, 11(6): 33−35,39. (in Chinese)
[18]	刘帅, 彭方仁. 薄壳山核桃花粉活力测定方法比较 [J]. 经济林研究, 2023, 41(2):110−119. LIU S, PENG F R. Comparison of detection methods for pollen viability of Carya illinoinensis [J]. Non-wood Forest Research, 2023, 41(2): 110−119. (in Chinese)
[19]	刘蓉, 吴德军, 王因花, 等. 白蜡花粉最佳离体萌发培养基筛选 [J]. 南京林业大学学报(自然科学版), 2023, 47(3):70−76. LIU R, WU D J, WANG Y H, et al. Screening of optimal germination medium for in vitro Fraxinus [J]. Journal of Nanjing Forestry University (Natural Sciences Edition), 2023, 47(3): 70−76. (in Chinese)
[20]	刘禹廷, 蓝登明, 余伟莅, 等. 6种云杉属植物花粉活性测定 [J]. 安徽农业科学, 2015, 43(17):241−245. LIU Y T, LAN D M, YU W L, et al. The assay about the activity of six kinds of Picea pollen [J]. Journal of Anhui Agricultural Sciences, 2015, 43(17): 241−245. (in Chinese)
[21]	张晓宁, 叶航, 吴方圆, 等. 香花油茶无性系花粉离体萌发培养基及贮藏条件分析 [J]. 分子植物育种, 2023, (12):1−17 ZHANG X N, YE H, WU F Y, et al. In Vitro Germination of Camellia osmantha Pollen and lts StorageConditions Research [J]. Molecular Plant Breeding, 2023(12): 1−17. (in Chinese)
[22]	DAVIDSON R. Davidson RE, ed. Handbook of water soluble gums and resins[M]. New York: McGraw-Hill, 1980: 1-18, 31.
[23]	FERRARI T E, WALLACE D H. Germination of Brassica pollen and expression of incompatibility in vitro [J]. Euphytica, 1975, 24(3): 757−765. doi: 10.1007/BF00132915
[24]	ZHANG H Q, CROES A F. A new medium for pollen germination in vitro [J]. Acta Botanica Neerlandica, 1982, 31(1/2): 113−119.
[25]	READ S M, CLARKE A E, BACIC A. Stimulation of growth of cultured Nicotiana tabacum W₃₈ pollen tubes by poly(ethylene glycol) and Cu(II) salts [J]. Protoplasma, 1993, 177(1): 1−14.
[26]	张超仪, 耿兴敏. 六种杜鹃花属植物花粉活力测定方法的比较研究 [J]. 植物科学学报, 2012, 30(1):92−99. doi: 10.3724/SP.J.1142.2012.10092 ZHANG C Y, GENG X M. Comparative study on methods for testing pollen viability of the six species from genus Rhododendron [J]. Plant Science Journal, 2012, 30(1): 92−99. (in Chinese) doi: 10.3724/SP.J.1142.2012.10092
[27]	张彬, 芮雯奕, 郑建初, 等. 水稻开花期花粉活力和结实率对高温的响应特征 [J]. 作物学报, 2007, 33(7):1177−1181. ZHANG B, RUI W Y, ZHENG J C, et al. Responses of pollen activity and seed setting of rice to high temperature of heading period [J]. Acta Agronomica Sinica, 2007, 33(7): 1177−1181. (in Chinese)
[28]	刘帮龙, 张晓慧, 干友民, 等. 野生马蹄金花粉生活力检测方法比较 [J]. 草业科学, 2011, 28(11):1941−1944. LIU B L, ZHANG X H, GAN Y M, et al. Detection methods of pollen viability of wild Dichondra repens [J]. Pratacultural Science, 2011, 28(11): 1941−1944. (in Chinese)
[29]	胡春, 刘左军, 李富香, 等. 钝裂银莲花花粉活力测定方法的研究 [J]. 植物研究, 2013, 33(5):582−586. doi: 10.7525/j.issn.1673-5102.2013.05.014 HU C, LIU Z J, LI F X, et al. Detection methods for pollen viability of Anemone obtusiloba [J]. Bulletin of Botanical Research, 2013, 33(5): 582−586. (in Chinese) doi: 10.7525/j.issn.1673-5102.2013.05.014
[30]	高露璐, 李林芳, 马育珠, 等. 毛叶铁线莲花粉活力测定方法 [J]. 分子植物育种, 2017, 15(11):4667−4672. GAO L L, LI L F, MA Y Z, et al. Measuring methods of Clematis lanuginose pollen viability [J]. Molecular Plant Breeding, 2017, 15(11): 4667−4672. (in Chinese)
[31]	蔡昭艳, 董龙, 王葫青, 等. 百香果花不同发育阶段花粉活力、柱头可授性及其对坐果的影响 [J]. 果树学报, 2023, 40(5):969−977. CAI Z Y, DONG L, WANG H Q, et al. Pollen viability, stigma receptivity and their effect on fruit set of passionfruit at different flower developmental stages [J]. Journal of Fruit Science, 2023, 40(5): 969−977. (in Chinese)