文心兰花粉离体萌发培养基的筛选及萌发率的测定

罗远华; 陈燕; 方能炎; 林榕燕; 钟淮钦; 黄敏玲

doi:10.19303/j.issn.1008-0384.2023.11.005

文心兰花粉离体萌发培养基的筛选及萌发率的测定

doi: 10.19303/j.issn.1008-0384.2023.11.005

福建省农业科学院作物研究所/福建省农业科学院花卉研究中心/　福建省特色花卉工程技术研究中心，福建　福州　350013

基金项目: 福建省农业科学院对外合作项目（DWHZ-2022-09）；福建省农业科学院科技创新团队建设项目（CXTD2021010-2）；福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021016）

详细信息

作者简介:
罗远华（1981 —），男，硕士，助理研究员，主要从事花卉育种与栽培研究，E-mail：luoyh426@163.com

通讯作者:
黄敏玲（1960 —），女，研究员，主要从事花卉育种与生物技术研究，E-mail：huangml168@163.com

中图分类号: S682.31
计量
- 文章访问数: 210
- HTML全文浏览量: 112
- PDF下载量: 43
- 被引次数: 0
出版历程
- 收稿日期: 2023-08-21
- 修回日期: 2023-10-13
- 网络出版日期: 2023-12-21
- 刊出日期: 2023-11-28

Optimized Medium and Culture Conditions for Germination of Oncidium Pollens

Crop Research Institute/Flower Research Center/Fujian Engineering Research Center for Characteristic Floriculture, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的研究不同培养基组分、培养温度及培养时间对文心兰花粉离体萌发的影响，以期筛选出文心兰花粉离体萌发的适宜培养基及培养条件，为文心兰种质资源创新利用提供科学依据。方法以盛花期当天开放的花朵为试验材料，采用液体培养基，比较蔗糖、H₃BO₃、Ca(NO₃) ·4H₂O、MgSO₄·7H₂O及KNO₃对文心兰花粉离体萌发的影响，正交试验筛选文心兰花粉离体萌发培养基的最佳组合及培养条件；在此基础上，测定收集保存的68份文心兰种质资源的花粉离体萌发率。结果一定质量浓度的蔗糖、H₃BO₃、Ca(NO₃) ·4H₂O、MgSO₄·7H₂O及KNO₃对文心兰花粉的离体萌发具有一定的促进作用，各因素的最佳作用效果从高到低依次为H₃BO₃、Ca(NO₃)·4H₂O、蔗糖、MgSO₄·7H₂O及KNO₃。正交试验表明适宜的液体培养基组合为100 g·L⁻¹蔗糖+10 mg·L⁻¹H₃BO₃+50 mg·L⁻¹Ca(NO₃)·4H₂O+20 mg·L⁻¹MgSO₄·7H₂O+30 mg·L⁻¹KNO₃，各成分的质量浓度与单因素试验的结果一致；培养条件为25 ℃、黑暗培养48 h。文心兰花粉离体萌发测定结果表明，60.29%的种质资源的花粉能离体萌发，萌发率普遍较低，为0.74%～49.65%。结论筛选出文心兰花粉离体萌发适宜的液体培养基组合及培养条件，不同文心兰种质资源的花粉离体萌发率存在明显差异，本研究结果可为后续文心兰种质资源的创新利用及他人研究等提供参考。
- 文心兰 /
- 花粉 /
- 离体萌发 /
- 液体培养基 /
- 萌发率
Abstract: Objective Medium formulation and culture conditions were optimized for in vitro pollen germination of Oncidium spp. Methods On a liquid culture medium, in vitro germination rates of the pollens collected from the flowers of various Oncidium germplasms on the day of full bloom were determined. Based on the effects of sucrose, H₃BO₃, Ca(NO3)·4H2O, MgSO4·7H2O, and KNO3 on the germination rate, an orthogonal experiment was applied to optimize the medium formulation and culture conditions. Subsequently, on the optimized medium under selected conditions, in vitro pollen germination rates of 68 collected Oncidium germplasms were determined. Results The degrees of importance of the medium ingredients to the pollen germination were found to be H₃BO₃＞Ca(NO3)·4H2O＞sucrose＞MgSO4·7H2O＞KNO3. The optimized formulation consisted of 100 g·L⁻¹ of sucrose, 10 mg·L⁻¹ of H₃BO₃, 50 mg·L⁻¹ of Ca(NO3)·4H2O, 20 mg·L⁻¹ of MgSO4·7H2O, and 30 mg·L⁻¹ of KNO3. The pollen culture was conducted at 25 ℃ for 48h in darkness to deliver a generally low germination rate of 0.74% to 49.65% on 60.29% of the Oncidium germplasms. Conclusion The liquid medium and culture conditions for an in vitro pollen germination of Oncidium were optimized. The germination rates differed significantly among the species.
- Oncidium spp. /
- pollen /
- in vitro germination /
- liquid medium /
- germination rate

HTML全文

图 1 不同培养基成分对香水文心花粉离体萌发的影响

不同小写字母表示不同质量浓度间差异显著（P＜0.05）。图2同。

Figure 1. Effect of medium ingredients on in vitro pollen germination of Onc. Sharry Baby Sweet Fragrance

Data with different lowercase letters indicate significant differences between different mass concentrations at P＜0.05. Same for Fig.2.

下载: 全尺寸图片幻灯片

图 2 不同培养条件对香水文心花粉离体萌发的影响

Figure 2. Effect of culture conditions on in vitro pollen germination of Onc. Sharry Baby Sweet Fragrance

下载: 全尺寸图片幻灯片

表 1 单因素间香水文心花粉离体萌发率的比较

Table 1. In virto germination rates of Onc. Sharry Baby Sweet Fragrance by various single factors

因素 Factors	质量浓度 Mass concentration/ (mg·L⁻¹)	花粉萌发率 Pollen germination rate/%
蔗糖 Sucrose	50×10³	4.94±0.38 ef
	100×10³	9.73±0.57 c
	150×10³	9.46±0.32 c
H₃BO₃	5×10³	7.53±0.95 d
	10×10³	17.12±1.45 a
	20×10³	12.58±1.50 b
Ca(NO₃) ·4H₂O	40×10³	6.16±0.56 e
	50×10³	13.34±0.68 b
	60×10³	9.30±0.71 c
MgSO₄·7H₂O	20×10³	4.76±0.72 f
	30×10³	4.69±0.52 f
	40×10³	2.71±0.46 g
KNO₃	30×10³	3.01±0.20 g
	40×10³	1.75±0.55 gh
	50×10³	1.19±0.13 h
同列数据后不同小写字母表示在 0.05 水平差异显著。表2和表3同。 Data with different lowercase letters on same column indicate significant difference at 0.05 level. Same for Tables 2 and 3.

下载: 导出CSV

表 2 文心兰花粉离体萌发的正交试验结果

Table 2. Pollen germination rates of Oncidium in orthogonal test

编号 No.	蔗糖 Sucrose/(g·L⁻¹)	H₃BO₃/ (mg·L⁻¹)	Ca(NO₃)·4H₂O/ (mg·L⁻¹)	MgSO₄·7H₂O/ (mg·L⁻¹)	KNO₃/ (mg·L⁻¹)	花粉萌发率 Pollen germination rate/%
编号 No.	蔗糖 Sucrose/(g·L⁻¹)	H₃BO₃/ (mg·L⁻¹)	Ca(NO₃)·4H₂O/ (mg·L⁻¹)	MgSO₄·7H₂O/ (mg·L⁻¹)	KNO₃/ (mg·L⁻¹)	香水文心 Onc.Sharry Baby ‘Sweet Fragrance’	蜜糖 Onc.Sweet Sugar
1	50	5	40	20	30	17.64±1.35 f	10.78±0.66 e
2	50	10	60	20	30	25.67±1.58 c	23.16±1.26 c
3	50	20	50	20	30	23.95±1.67 cd	22.43±0.64 c
4	100	5	60	20	30	21.89±1.65 de	16.48±1.57 d
5	100	10	50	20	30	43.25±1.43 a	32.91±1.37 a
6	100	20	40	20	30	34.08±2.06 b	28.69±1.23 b
7	150	5	50	20	30	23.87±1.75 cd	17.91±0.72 d
8	150	10	40	20	30	40.37±1.59 a	30.71±1.77 ab
9	150	20	60	20	30	36.02±1.20 b	24.69±1.78 c

下载: 导出CSV

表 3 68份文心兰种质资源花粉离体萌发率

Table 3. Statistical in vitro pollen germination rates of 68 Oncidium germplasms

序号 No.	种质名称 Name of material	花粉形态特征 Pollen morphology	花粉萌发率 Pollen germination rate/%	序号 No.	种质名称 Name of material	花粉形态特征 Pollen morphology	花粉萌发率 Pollen germination rate/%
1	香水235 Pi-Payk#235	形状规则，内含物充实	49.65±1.62 a	22	法国香水 French Fragrance	较多形状不规则，内含物皱缩、空泡化	6.22±0.95 jkl
2	香水文心 Sweet Fragrance	形状规则，内含物充实	43.25±1.43 b	23	香水254 Mon-Tho#254	形状规则，内含物充实	5.30±0.94 klm
3	蜜糖 Sweet Sugar	形状规则，内含物充实	32.91±1.37 c	24	XM-1	较多形状不规则，内含物皱缩、空泡化	5.10±0.74 klmn
4	小蜜蜂 Little Bee	形状规则，内含物充实	30.89±1.74 c	25	欧洲之星 Eurostar	较多形状不规则，内含物皱缩、空泡化	4.67±1.43 klmn
5	百万金币 Million Dollar	形状规则，内含物充实	20.88±3.07 d	26	粉星 Massai Pink	少量形状不规则，内含物皱缩	4.54±0.94 klmno
6	红樱桃 Red Cherry	少量形状不规则，内含物皱缩	20.42±1.89 d	27	红星 Massai Red	形状规则，内含物充实	4.50±1.33 klmnop
7	芒果文心 Mango	形状规则，内含物充实	20.27±1.59 d	28	紫薇 Ziwei	少量形状不规则，内含物皱缩	4.26±1.16 lmnop
8	红日 Red Sun	少量形状不规则，内含物皱缩	14.78±2.41 e	29	金香 Fragrant Gold	较多形状不规则，内含物皱缩、空泡化	4.09±0.36 lmnop
9	剑叶文心 Red Panther	形状规则，内含物充实	14.14±1.48 e	30	香香公主 Princess Fragrance	形状规则，内含物充实 Regular shape，inclusion sufficient	3.69±1.06 mnop
10	门神 Everglades	形状规则，内含物充实	13.89±1.79 e	31	紫香兰 Dark Matter	少量形状不规则，内含物皱缩	3.65±0.82 mnop
11	香水305 See-Daa#305	形状规则，内含物充实	11.36±1.60 f	32	黄色精灵 Golden Spirit	较多形状不规则，内含物皱缩、空泡化	3.24±0.67 mnopq
12	香水252 Mon-Tho#252	形状规则，内含物充实	10.91±1.98 fg	33	蕙心兰 Dawn	较多形状不规则，内含物皱缩、空泡化	3.13±0.56 mnopq
13	黄金天使 Golden Angel	少量形状不规则，内含物皱缩	9.76±1.38 fgh	34	黄花斑点 Alpine	较多形状不规则，内含物皱缩、空泡化	2.71±0.68 nopqr
14	香水304 Jairak Catt #304	形状规则，内含物充实	9.71±0.88 fgh	35	美少女 Beauty Lady	形状规则，内含物充实	2.56±0.60 nopqr
15	红鸟 Red bird	形状规则，内含物充实	8.69±0.76 ghi	36	香水美人 Kathrin Zoch	较多形状不规则，内含物皱缩、空泡化	2.09±0.48 opqr
16	三色香水 Tricolor	少量形状不规则，内含物皱缩	8.34±1.55 hij	37	花精灵 Flower Fairy	少量形状不规则，内含物皱缩，细胞质空泡化	2.02±0.60 pqr
17	香水607 Jairak Catt #607	形状规则，内含物充实	7.74±1.04 hij	38	铁板文心 Beauty Gold	少量形状不规则，内含物皱缩	1.91±0.51 pqr
18	香水599 Jairak Catt#599	形状规则，内含物充实	6.93±0.70 ijk	39	黄金午后 Rich Yellow	少量形状不规则，内含物皱缩，细胞质空泡化	1.12±0.19 qr
19	花猫 Carmela	少量形状不规则，内含物皱缩、空泡化	6.77±1.38 ijk	40	香水564 Jairak Fragrance#564	少量形状不规则，内含物皱缩	1.08±0.19 qr
20	情人 Lover	少量形状不规则，内含物皱缩	6.43±0.65 ijkl	41	光华 Chian-Tzy Deilight	较多形状不规则，内含物皱缩、空泡化	0.74±0.09 r
21	香水307 Pra-Lak#307	少量形状不规则，内含物皱缩	6.29±1.45 ijkl

下载: 导出CSV

参考文献(27)

[1]	罗远华, 黄敏玲, 吴建设. 文心兰育种研究进展 [J]. 江西农业学报, 2012, 24(10):15−20. LUO Y H, HUANG M L, WU J S. Progress in Oncidium breeding study [J]. Acta Agriculturae Jiangxi, 2012, 24(10): 15−20.(in Chinese)
[2]	罗远华, 黄敏玲, 林兵, 等. 文心兰花粉活力与杂交结荚性研究 [J]. 福建农业学报, 2015, 30(3):258−263. LUO Y H, HUANG M L, LIN B, et al. Studying on pollen viability and hybrid pod setting ability of Oncidium [J]. Fujian Journal of Agricultural Sciences, 2015, 30(3): 258−263.(in Chinese)
[3]	王钦丽, 卢龙斗, 吴小琴, 等. 花粉的保存及其生活力测定 [J]. 植物学通报, 2002, 37(3):365−373. WANG Q L, LU L D, WU X Q, et al. Pollen preservation and its viability test [J]. Chinese Bulletin of Botany, 2002, 37(3): 365−373.(in Chinese)
[4]	郑宝强, 王雁, 彭镇华, 等. 杂种卡特兰花粉萌发和花粉贮藏性研究 [J]. 热带亚热带植物学报, 2012, 20(1):13−18. ZHENG B Q, WANG Y, PENG Z H, et al. Germination and preservation of rhyncholaeliocattleya pollen [J]. Journal of Tropical and Subtropical Botany, 2012, 20(1): 13−18.(in Chinese)
[5]	钱鑫, 刘芬, 牛晓玲, 等. 无距虾脊兰花粉离体萌发及储藏条件的研究 [J]. 西北植物学报, 2014, 34(2):341−348. QIAN X, LIU F, NIU X L, et al. In vitro pollen germination and storage conditions of Calanthe tsoongiana [J]. Acta Botanica Boreali-Occidentalia Sinica, 2014, 34(2): 341−348.(in Chinese)
[6]	邓茜玫, 郑宝强, 郭欣, 等. 聚石斛花粉生活力及贮藏的研究 [J]. 林业科学研究, 2014, 27(5):657−661. DENG X M, ZHENG B Q, GUO X, et al. Pollen viability and preservation of Dendrobium lindleyi [J]. Forest Research, 2014, 27(5): 657−661.(in Chinese)
[7]	杨佳慧, 吴婷, 朱俊, 等. 长瓣兜兰开花特性与繁育系统研究 [J]. 园艺学报, 2021, 48(5):1002−1012. YANG J H, WU T, ZHU J, et al. Studies on flowering characteristics and breeding system of Paphiopedilum dianthum [J]. Acta Horticulturae Sinica, 2021, 48(5): 1002−1012.(in Chinese)
[8]	张晓莹, 傅巧娟, 赵福康, 等. 大花蕙兰花粉贮藏与离体萌发研究 [J]. 浙江农业学报, 2019, 31(9):1502−1508. ZHANG X Y, FU Q J, ZHAO F K, et al. Study on preservation and germination of Cymbidium hybridum pollen [J]. Acta Agriculturae Zhejiangensis, 2019, 31(9): 1502−1508.(in Chinese)
[9]	夏春英, 谢小敏, 刘江枫, 等. 竹叶兰花粉离体萌发及其贮藏特性 [J]. 森林与环境学报, 2019, 39(5):454−459. XIA C Y, XIE X M, LIU J F, et al. In vitro germination and storage characteristics of Arundina graminifolia pollen [J]. Journal of Forest and Environment, 2019, 39(5): 454−459.(in Chinese)
[10]	夏春英, 谢小敏, 刘江枫, 等. 澳洲石斛花粉离体萌发液体培养基研究 [J]. 江苏农业科学, 2020, 48(8):149−152. XIA C Y, XIE X M LIU J F, et al. Study on optimum liquid medium concentration for pollen in vitro germination of Dendrobium kingianum [J]. Jiangsu Agricultural Sciences, 2020, 48(8): 149−152.(in Chinese)
[11]	BREWBAKER J L, KWACK B H. The essential role of calcium ion in pollen germination and pollen tube growth [J]. American Journal of Botany, 1963, 50(9): 859−865. doi: 10.1002/j.1537-2197.1963.tb06564.x
[12]	TUSHABE D, ROSBAKH S. A compendium of in vitro germination media for pollen research [J]. Frontiers in Plant Science, 2021, 12: 709945. doi: 10.3389/fpls.2021.709945
[13]	REINDERS A. Fuel for the road: Sugar transport and pollen tube growth [J]. Journal of Experimental Botany, 2016, 67(8): 2121−2123. doi: 10.1093/jxb/erw113
[14]	李婷, 乔琦, 李剑峰, 等. 牡丹花粉生活力测定方法及其贮藏研究进展 [J]. 贵州农业科学, 2020, 48(9):123−126. LI T, QIAO Q, LI J F, et al. Advances in determination method and storage of Paeonia suffruticosa pollen activity [J]. Guizhou Agricultural Sciences, 2020, 48(9): 123−126.(in Chinese)
[15]	于金平, 王媛媛, 张琪, 等. 百合不同品种间花粉萌发活力检测分析 [J]. 沈阳农业大学学报, 2018, 49(1):14−19. YU J P, WANG Y Y, ZHANG Q, et al. Investigation report on pollen viability among different lily cultivars [J]. Journal of Shenyang Agricultural University, 2018, 49(1): 14−19.(in Chinese)
[16]	WANG Q L, LU L D, WU X Q, et al. Boron influences pollen germination and pollen tube growth in Picea meyeri [J]. Tree Physiology, 2003, 23(5): 345−351. doi: 10.1093/treephys/23.5.345
[17]	FANG K F, DU B S, ZHANG Q, et al. Boron deficiency alters cytosolic Ca²⁺ concentration and affects the cell wall components of pollen tubes in Malus domestica [J]. Plant Biology (Stuttgart, Germany), 2019, 21(2): 343−351. doi: 10.1111/plb.12941
[18]	聂超仁, 况红玲, 于静亚, 等. 钟花樱花粉离体萌发培养及低温保存技术研究 [J]. 西南林业大学学报(自然科学), 2018, 38(1):54−58. NIE C R, KUANG H L, YU J Y, et al. Pollen germination in vitro culture and cryopreservation technology of Cerasus campanulata [J]. Journal of Southwest Forestry University (Natural Sciences), 2018, 38(1): 54−58.(in Chinese)
[19]	CHEN J C, FANG S C. Erratum to: The long pollen tube journey and in vitro pollen germination of Phalaenopsis orchids [J]. Plant Reproduction, 2017, 30(3): 153. doi: 10.1007/s00497-017-0306-1
[20]	STEINHORST L, KUDLA J. Calcium - a central regulator of pollen germination and tube growth [J]. Biochimica et Biophysica Acta, 2013, 1833(7): 1573−1581. doi: 10.1016/j.bbamcr.2012.10.009
[21]	ZHAN N, HUANG L J. Effects of Ca²⁺ on in vitro pollen germination of three Acacia species [J]. Silvae Genetica, 2016, 65(2): 11−16. doi: 10.1515/sg-2016-0012
[22]	FAN L M, WANG Y F, WANG H, et al. In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts [J]. Journal of Experimental Botany, 2001, 52(361): 1603−1614. doi: 10.1093/jexbot/52.361.1603
[23]	LI J, HUANG Y, TAN H, et al. An endoplasmic reticulum magnesium transporter is essential for pollen development in Arabidopsis [J]. Plant Science:an International Journal of Experimental Plant Biology, 2015, 231: 212−220.
[24]	唐毓玮, 龙凌云, 黄秋伟, 等. 澳系睡莲花粉离体萌发及低温保存研究 [J]. 热带作物学报, 2020, 41(7):1380−1386. TANG Y W, LONG L Y, HUANG Q W, et al. Pollen germination in vitro and cryopreservation reserch of anecphya waterlily [J]. Chinese Journal of Tropical Crops, 2020, 41(7): 1380−1386.(in Chinese)
[25]	贺庆梅, 陈岩岩, 李启虔, 等. 宫粉羊蹄甲花粉离体萌发培养基的筛选 [J]. 南方农业学报, 2015, 46(12):2173−2179. HE Q M, CHEN Y Y, LI Q Q, et al. Screening of culture medium for in vitro pollen germination of Bauhinia variegate L [J]. Journal of Southern Agriculture, 2015, 46(12): 2173−2179.(in Chinese)
[26]	PACINI E, GUARNIERI M, NEPI M. Pollen carbohydrates and water content during development, presentation, and dispersal: A short review [J]. Protoplasma, 2006, 228(1): 73−77.
[27]	芦娟, 苏瑾, 姜成英, 等. 不同浓度的糖、硼、钙对油橄榄花粉萌发的影响 [J]. 经济林研究, 2017, 35(1):103−107. LU J, SU J, JIANG C Y, et al. Effects of different concentrations of sugar, boron and calcium on pollen germination in Olea europaea [J]. Nonwood Forest Research, 2017, 35(1): 103−107.(in Chinese)