春兰‘黄梅’×‘黄荷’杂交F<sub>1</sub>根状茎的组培快繁研究

漆子钰; 周育真; 艾叶; 彭东辉

doi:10.19303/j.issn.1008-0384.2019.09.006

春兰‘黄梅’×‘黄荷’杂交F₁根状茎的组培快繁研究

漆子钰^{1, 2,},
周育真¹,
艾叶¹,
彭东辉^1, ,

1.
福建农林大学园林学院，福建　福州　350002
2.
江西省宜春市园林局，江西　宜春　336000

基金项目: 福建省种业创新与产业化工程林业项目（ZYCX-LY-2017005）

详细信息

作者简介:
漆子钰（1992−），女，硕士，助理工程师，研究方向：园林植物与应用研究（E-mail：qiziyu2013@126.com）

通讯作者:
彭东辉（1971−），男，博士，教授，研究方向：园林植物与应用研究（E-mail：fjpdh@126.com）

中图分类号: S 682.31
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出版历程
- 收稿日期: 2019-05-14
- 修回日期: 2019-07-17
- 刊出日期: 2019-08-31

Tissue Culture for Rapid Propagation of A Cymbidium goeringii Hybrid

QI Zi-yu^{1, 2,},
ZHOU Yu-zheng¹,
AI Ye¹,
PENG Dong-hui^1, ,

1.
College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Yichun Garden Bureau, Yichun, Jiangxi　336000, China

摘要

摘要:
目的筛选春兰组培各阶段的最佳培养条件，建立优良的春兰快繁体系，为春兰大规模生产开发提供科学依据。
方法以春兰‘黄梅’×‘黄荷’F₁无菌播种形成的根状茎为试验材料，通过基本培养基（1/2MS、改良1/2MS、HyponexⅡ）和植物生长调节剂（6-BA、NAA、TDZ、IBA）等因子不同组合，围绕增殖、分化、生根等阶段建立其再生体系，并对培育出的组培苗进行移栽练苗。
结果增殖最适培养基为3.0 g·L⁻¹ HyponexⅡ+0.5 mg·L⁻¹ 6-BA+3.0 mg·L⁻¹ NAA+1.0 g·L⁻¹ 活性炭（AC）+30.0 g·L⁻¹ 白砂糖（Su）+7.0 g·L⁻¹ 琼脂（Ag），生长速度和增殖系数分别为7.31和6.02；分化最适培养基为2.0 mg·L⁻¹ 6-BA+0.3 mg·L⁻¹ NAA+30.0 g·L⁻¹ Su+7.0 g·L⁻¹ Ag，分化率、芽诱导个数和株高分别为90.00%、5.44个和2.53 cm；生根最适培养基为1.5 mg·L⁻¹ IBA+2.0 g·L⁻¹蛋白胨+1.0 g·L⁻¹ AC+25.0 g·L⁻¹ Su+7.0 g·L⁻¹ Ag，生根率、生根数和平均根长分别为100.00%、8.03条和3.00 cm；春兰组培苗练苗移栽60 d后存活率达96.53%。
结论新型基本培养基HyponexⅡ（3.0 g·L⁻¹）对春兰增殖培养有高效促进作用，高水平的IBA（1.5 mg·L⁻¹）有利于春兰组培苗生根壮苗。
- 春兰 /
- 增殖培养 /
- 分化培养 /
- 生根培养
Abstract:
Objective Optimum conditions for an efficient, rapid propagation from rhizomes of a Cymbidium goeringii hybrid were investigated.
Method Rhizomes of the aseptically seeded F₁ hybrid between two C. goeringii cultivars, Huang-mei and Huang-he, were cultivated on various media (i.e., 1/2MS、modified 1/2MS, and HyponexⅡ) with added plant growth regulators (i.e., 6-BA, NAA, TDZ, and/or IBA) to monitor the plant growth throughout propagation, differentiation, and rooting of the regeneration process. The seedlings were subsequently transplanted outdoor to determine their survival rate.
Result In the propagation stage, the optimal medium was 3.0 g HyponexⅡ·L⁻¹ + 0.5 mg 6-BA·L⁻¹ + 3.0 mg NAA·L⁻¹ + 1.0 g activated carbon (AC)·L⁻¹ + 30.0 g sugar (Su)·L⁻¹ + 7.0 g agar (Ag)·L⁻¹ to achieve a growing rate of 7.31 and a multiplication coefficient of 6.02. The optimal medium for differentiation was 2.0 mg 6-BA·L⁻¹ + 0.3 mg NAA·L⁻¹ + 30.0 g Su·L⁻¹ + 7.0 g Ag·L⁻¹ to deliver an averaged 90.00% differentiation, 5.44 buds per rhizome segment, and 2.53 cm height of seedling. For optimum rooting, the medium was 1.5 mg IBA·L⁻¹ + 2.0 g peptone·L⁻¹ + 1.0 g AC·L⁻¹ + 25.0 g Su·L⁻¹ + 7.0 g Ag·L⁻¹ to result in 100.00% root-generation with an averaged 8.03 roots at 3.00 cm long per plant. The survival rate of plantlets 60 d after transplantation was up to 96.53%.
Conclusion HyponexⅡ applied at 3.0 g·L⁻¹ was found highly efficient for the C. goeringii rhizome regeneration; and, the addition of 1.5 mg·L⁻¹ of IBA in the medium significantly enhanced the rooting for the seedlings.
- Cymbidium goeringii /
- propagation culture /
- differentiation culture /
- rooting culture

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图 1 离体培养各个阶段

注：A为增殖培养第1 d；B为增殖培养第15 d；C为增殖培养120 d；D为分化培养第1 d；E为分化培养第15 d；F为分化培养120 d；G为生根培养120 d；H为组培苗练苗移栽60 d。

Figure 1. Tissue cultures at various development stages

Note: A is multiplication culture on 1^st day; B, 15^th day; and, C, 120^th day. D is differentiation culture on 1^st day; E, 15^th day; and, F, 120^th day. G is rooting culture on 120^th day. H indicates plantlets on 60^th day after transplantation.

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表 1 根状茎增殖培养配方正交设计L₉（3⁴）

Table 1 Orthogonal design L₉(3⁴) on culture media for rhizome propagation

水平 Level	因素 Factor
水平 Level	基本培养基 Basic medium	6-BA/(mg·L⁻¹)	NAA/(mg·L⁻¹)
1	1/2MS	0.1	2.0
2	Revised 1/2MS	0.3	3.0
3	Hyponex Ⅱ3.0 g·L⁻¹	0.5	5.0
注：改良1/2MS为不含有微量元素的1/2MS培养基。 Note: Modified ½ MS was regular ½ MS without added microelements.

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表 2 根状茎分化培养试验设计

Table 2 Experimental design on culture media for rhizome differentiation

处理号 Number	NAA/(mg·L⁻¹)	6-BA/(mg·L⁻¹)	TDZ/(mg·L⁻¹)
1	0.3	1.0	0.0
2	0.3	2.0	0.0
3	0.3	3.0	0.0
4	0.3	0.0	0.5
5	0.3	0.0	1.0
6	0.3	0.0	1.5
7	0.5	1.0	0.0
8	0.5	2.0	0.0
9	0.5	3.0	0.0
10	0.5	0.0	0.5
11	0.5	0.0	1.0
12	0.5	0.0	1.5

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表 3 组培苗生根培养全因素设计

Table 3 All factor design on culture media for plantlet rooting

处理号 Number	IBA/(mg·L⁻¹)	蛋白胨 Peptone/(g·L⁻¹)
1	0.5	1.0
2	0.5	2.0
3	0.5	3.0
4	1.0	1.0
5	1.0	2.0
6	1.0	3.0
7	1.5	1.0
8	1.5	2.0
9	1.5	3.0

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表 4 不同处理对根状茎增殖培养的影响

Table 4 Effects of medium composition on rhizome propagation

处理号 Number	A：基本培养基 Basic medium	B：6-BA/(mg·L⁻¹)	C：NAA/(mg·L⁻¹)	生长速度 Speed of growth	增殖系数 Proliferation coefficient
1	1	1	1	2.53±0.35 Bd	7.40±0.42 Aa
2	1	2	2	4.19±0.14 Bbcdd	6.69±0.0 5 Aab
3	1	3	3	4.76±0.31 ABbc	7.46±0.66 Aa
4	2	1	2	2.91±0.18 Bcd	6.27±0.31 Aab
5	2	2	3	3.46±0.15 Bbcd	7.20±0.30 Aa
6	2	3	1	4.19±0.47 Bbcd	5.85±0.07 Aab
7	3	1	3	5.21±0.41 ABb	5.41±0.54 Ab
8	3	2	1	5.10±0.53 ABbc	5.93±0.10 Aab
9	3	3	2	7.31±0.27 Aa	6.02±0.82 Aab
ka1	3.83	3.55	3.94	Ra值排序：A＞B＞C 最优水平：A₃B₃C₂ Rb值排序：A＞C＞B 最优水平：A₁B₂C₃
ka2	3.52	4.25	4.80
ka3	5.87	5.42	4.48
Ra	2.35	1.87	0.87
kb1	7.18	6.36	6.39
kb2	6.44	6.61	6.33
kb3	5.79	6.45	6.69
Rb	1.39	0.25	0.37
注：ka表示生长速度；Ra表示生长速度的极差；kb表示增殖系数；Rb表示增殖系数的极差。大写字母表示在0.01水平下的差异，小写字母表示0.05水平下的差异，下同。 Note: ka stands for speed of growth; Ra, variance of ka; kb, multiplication coefficient; and, Rb, variance of kb. Capital letters indicate significantly different at P＜0.01, lowercase significantly different at P＜0.05. Same for Table 5&6.

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表 5 NAA、6-BA和TDZ对根状茎芽分化的影响

Table 5 Effects of varied concentrations of NAA, 6-BA, and TDZ on rhizome differentiation

处理号 Number	分化率 Differentiation rate /%	芽诱导个数 Number of Induced bud /个	株高 Plant height/cm
1	98.00±6.32 Aa	4.07±0.53 BCDEbcde	2.70±0.81 Aa
2	90.00±10.44 ABabcd	5.44±0.78 Aa	2.53±0.75 Aa
3	94.55±12.93 ABab	4.90±0.52 ABab	1.51±0.42 DEFcd
4	78.08±19.10 Bcd	4.48±0.71 ABCDbc	1.70±0.36 CDEc
5	91.67±13.37 ABabc	4.78±0.63 ABCab	1.26±0.27 EFde
6	77.14±15.41 Bd	4.07±0.50 BCDEbcde	1.06±0.16 Fe
7	94.00±13.50 ABab	3.55±0.36 CDEcde	2.77±0.90 Aa
8	87.27±13.48 ABabcd	3.31±0.33 DEde	2.49±0.79 ABa
9	80.00±16.33 ABcd	3.09±0.33 Ee	2.07±0.55 BCb
10	46.00±9.66 Ce	3.15±0.48 Ee	1.77±0.47 CDbc
11	88.33±10.30 ABabcd	4.18±0.47 BCDEbcd	1.51±0.30 DEFcd
12	81.43±11.43 ABbcd	3.80±0.45 BCDEcde	1.47±0.33 DEFcd

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表 6 不同IBA和蛋白胨对组培苗生根的影响

Table 6 Effects of varied concentrations of IBA and peptone on rooting of seedlings

处理号 Number	生根率 Rooting rate/%	生根数 Number of roots/条	平均根长 Average root length /cm
1	72.73±16.61 Aa	5.25±0.76 ABb	2.65±1.19 Bc
2	82.86±12.87 Aa	5.89±1.16 ABab	2.80±1.20 ABbc
3	83.33±18.25 Aa	4.53±0.61 Bb	2.77±1.13 Bbc
4	89.47±15.29 Aa	5.25±1.00 ABb	2.75±1.30 Bbc
5	88.24±15.94 Aa	5.29±1.79 ABb	2.91±1.27 ABabc
6	77.42±12.62 Aa	6.87±1.09 ABab	3.18±1.12 Aa
7	97.14±9.24 Aa	6.46±1.17 ABab	3.19±1.20 Aa
8	100.00±0 Aa	8.03±1.38 Aa	3.00±1.16 ABab
9	96.43±7.91 Aa	6.31±1.37 ABab	2.75±1.08 Bbc

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参考文献(30)

[1]	陈心启. 国兰及其品种全书[M]. 北京: 中国林业出版社, 2011: 1−10.
[2]	陈宇勒. 兰花繁育技术图解[M]. 广州: 广东科技出版社, 2009: 26−45.
[3]	李子红. 珍品兰花快速繁殖与养护[M]. 上海: 上海科学技术出版社, 2006: 5−10.
[4]	凌晓祺. 国兰花期调控与组培快繁技术优化的研究[D]. 杭州: 浙江大学, 2016. LIN X Q. Study On Flowering Regulation and Technique Optimization of Tissue Culture of Chinese Orchid[D]. Hangzhou: Zhejiang University, 2013. (in Chinese)
[5]	于永畅, 张林, 王厚新, 等. 影响春兰杂交兰‘宋梅’×‘集圆’组培苗生根的因素 [J]. 北方园艺, 2013(7):66−68. YU Y C, ZHANG L, WANG H X, et al. Some Influencing Factors of Root Differentiation of Seedlings in Hybridization of Cymbidium goeringii ‘Songmei’×‘Jiyuan’ [J]. Northern Horticulture, 2013(7): 66−68.(in Chinese)
[6]	李玉萍, 李沁晔, 罗凤霞. 宋梅×韩国桃花杂交兰组培苗壮苗和生根培养基研究 [J]. 江苏农业科学, 2018, 46(14):35−37, 44. LI Y P, LI Q Y, LUO F X. Study on tissue Culture and rooting medium of Hybrid Orchid of ‘Song Mei’×’Korea Peach Flower’ [J]. Jiangsu agricultural science, 2018, 46(14): 35−37, 44.(in Chinese)
[7]	王玉英, 李灵丽, 刘丹, 等. ‘春绿兰×虎雪兰’F₁代组培苗快繁技术 [J]. 北方园艺, 2018(3):87−91. WANG Y Y, LI L L, LIU D, et al. Rapid Propagation of Cymbidium faberi Rolfe var. Szechuanicum×(C. tracyanum var. Huanghua×C. mastersii) F1 hybrids [J]. Northern Horticulture, 2018(3): 87−91.(in Chinese)
[8]	卜朝阳, 董伟清, 闭志强, 等. 蝴蝶兰克隆苗根系生长影响因素研究 [J]. 西南农业学报, 2009, 22(6):1737−1740. DOI: 10.3969/j.issn.1001-4829.2009.06.052 PU C Y, DONG W Q, BI Z Q, et al. Affecting Factors on Root System Growth of Phalaenopsis Clone Seedlings [J]. Southwest China Journal of Agricultural Sciences, 2009, 22(6): 1737−1740.(in Chinese) DOI: 10.3969/j.issn.1001-4829.2009.06.052
[9]	曾碧玉, 许传俊, 张文惠, 等. 蝴蝶兰花梗初代培养研究 [J]. 福建农业学报, 2013, 28(2):124−128. DOI: 10.3969/j.issn.1008-0384.2013.02.006 ZENG B Y, XU C J, ZHANG W H, et al. Culturing Flower-stalks of Phalaenopsis [J]. Fujian Journal of Agricultural Sciences, 2013, 28(2): 124−128.(in Chinese) DOI: 10.3969/j.issn.1008-0384.2013.02.006
[10]	郑艳艳. 墨兰组培体系的优化及根状茎在生物反应器培养的研究[D]. 延吉: 延边大学, 2011. ZHENG Y Y. Optimization of Tissue Culture System and Proliferation of Rhizome in Bioreactor Culture of Cymbidium sinense Willd[D]. Yanji: Yanbian University, 2011. (in Chinese)
[11]	李洪林, 杨波. 春兰根状茎离体培养与快速繁殖(简报) [J]. 亚热带植物科学, 2012, 41(4):67−68. LI H L, YANG B. In vitro Culture and Rapid Propagation of Cymbidium goeringii [J]. Subtropical Plant Science, 2012, 41(4): 67−68.(in Chinese)
[12]	洪伟, 吴承祯. 试验设计与分析——原理·操作·案例[M]. 北京: 中国林业出版社, 2004: 95−115.
[13]	唐凤鸾, 黄雪梅, 冼康华, 等. 春兰种苗繁育及开花调控研究进展 [J]. 南方农业学报, 2015, 46(6):1067−1073. DOI: 10.3969/j.issn.2095-1191.2015.6.1067 TANG F L, HUANG X M, XI K H, et al. Progress on seedling propagation and flowering regulation of Cymbidium goeringii [J]. Journal of Southern Agriculture, 2015, 46(6): 1067−1073.(in Chinese) DOI: 10.3969/j.issn.2095-1191.2015.6.1067
[14]	潘银萍, 李承秀, 王长宪, 等. 春荷鼎杂交根状茎的组培快繁的研究 [J]. 中国农学通报, 2010, 26(5):209−212. PAN Y P, LI C X, WANG C X, et al. Study of the Rootstocks about Cymbidium faberi ‘chun he ding’ in Rapid Multiplication Techniques [J]. Chinese Agricultural Science Bulletin, 2010, 26(5): 209−212.(in Chinese)
[15]	于永畅. 蕙兰‘大一品’组织培养技术及部分国兰品种抗寒性研究[D]. 泰安: 山东农业大学, 2014. YU Y C. Study on Tissue Culture of Cymbidium faberi ‘Da Yipin’ and Cold Resistance of Chinese Cymbidium[D]. Taian: Shandong Agricultural University, 2014. (in Chinese)
[16]	陈丹丹. 建兰离体培养与形态发生特征研究[D]. 南京: 南京农业大学, 2015. CHEN D D. In Vitro Culture of Cymbidium Ensifolium and the Study of Morphogenesis Characteristics[D]. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
[17]	苏妍. 寒兰杂交后代快速繁殖体系及生理生化研究[D]. 福州: 福建农林大学, 2016. SU Y. Studies on Rapid Propagation of Hybrid Offspring of Cymbidium Kanran and Changes of Physiology and Biochemistry[D]. Fuzhou: Fujian Agriculture and Forestry University, 2016. (in Chinese)
[18]	许申平, 袁秀云, 王默霏, 等. 墨兰(Cymbiduim sinense)组培快繁技术体系研究 [J]. 热带作物学报, 2018, 39(5):926−930. DOI: 10.3969/j.issn.1000-2561.2018.05.015 XU S P, YUAN X Y, WANG M F, et al. Micropropagation in vitro of Cymbidium sinensis [J]. Chinese Journal of Tropical Crops, 2018, 39(5): 926−930.(in Chinese) DOI: 10.3969/j.issn.1000-2561.2018.05.015
[19]	牛田. 春兰种质资源的SRAP分子评价及组织培养[D]. 泰安: 山东农业大学, 2014. NIU T. Molecular Evaluation of the Germplasm Resouces by SRAP and Tissue Culture[D]. Taian: Shandong Agricultural University, 2014. (in Chinese)
[20]	孙芳. 春兰蕙兰杂交育种技术研究[D]. 泰安: 山东农业大学, 2013. SUN F. Studies on the Crossbreeding between Cymbidium Goeringii and Cymbidium Faberi[D]. Taian: Shandong Agricultural University, 2013. (in Chinese)
[21]	刘幸佳. 几种国兰组织培养及春兰菌根真菌研究[D]. 杭州: 浙江农林大学, 2013. LIU X J. Study on Several Cymbidium Plants Tissue Culture and Cymbidium goeringii Mycorrhizal Fungi[D]. Hangzhou: Zhejiang A & F University, 2013. (in Chinese)
[22]	施满容. 金线莲组织培养条件优化对其有效成分含量的影响 [J]. 福建农业学报, 2018, 33(5):495−501. SHI M R. Optimizing Tissue Culture of Anoectochilus roxburghii for a Maximized Content on Effe [J]. Fujian Journal of Agricultural Sciences, 2018, 33(5): 495−501.(in Chinese)
[23]	王永清, 余道平. 春兰根状茎离体培养 [J]. 园艺学报, 2005, 32(4):706. DOI: 10.3321/j.issn:0513-353X.2005.04.046 WANG Y Q, YU D P. In Vitro Culture of Cymbidium goeringii Rhizomes [J]. Acta Horticulturae Sinica, 2005, 32(4): 706.(in Chinese) DOI: 10.3321/j.issn:0513-353X.2005.04.046
[24]	邢海, 孙叶芳, 郑琪, 等. 中透艺春兰根状茎的组培快繁及艺向培养研究 [J]. 现代农业科技, 2016(8):139−140, 145. DOI: 10.3969/j.issn.1007-5739.2016.08.087 XING H, SUN Y F, ZHENG Q, et al. Study om Rapid Propagation and Directional Art Culture of Rhizome of Cymbidium goeringii ‘Zhongtou yi’ [J]. Modern Agricultural Sciences and Technology, 2016(8): 139−140, 145.(in Chinese) DOI: 10.3969/j.issn.1007-5739.2016.08.087
[25]	叶秀仙, 黄敏玲, 林榕燕, 等. 素心建兰无菌播种快繁技术研究 [J]. 福建农业学报, 2015, 30(10):939−943. DOI: 10.3969/j.issn.1008-0384.2015.10.003 YE X X, HUANG M L, LIN R Y, et al. Aseptic Sowing and Rapid Propagation of Cymbidium ensifolium var.susin [J]. Fujian Journal of Agricultural Sciences, 2015, 30(10): 939−943.(in Chinese) DOI: 10.3969/j.issn.1008-0384.2015.10.003
[26]	刘昳雯, 许春梅, 王丹, 等. 莲瓣兰" 滇梅”根状茎的增殖与分化技术研究 [J]. 安徽农业科学, 2014, 42(10):2890−2892. DOI: 10.3969/j.issn.0517-6611.2014.10.021 LIU Y W, XU C M, WANG D, et al. Research of Proliferation and Differentiation of Cymbidium tortisepalum ‘Dian Mei’ Rhizome [J]. Journal of Anhui Agricultural Sciences, 2014, 42(10): 2890−2892.(in Chinese) DOI: 10.3969/j.issn.0517-6611.2014.10.021
[27]	刘帆, 朱文丽, 黄明忠, 等. 紫花苞舌兰类原球茎诱导及植株再生 [J]. 基因组学与应用生物学, 2017, 36(5):2082−2087. LIU F, ZHU W L, HUANG M Z, et al. Inducement of Philippine Ground Orchid (Spathoglottis plicata) Protocorm-Like Bodies and Plant Regeneration [J]. Genomics and Applied Biology, 2017, 36(5): 2082−2087.(in Chinese)
[28]	卜朝阳. 蝴蝶兰高效离体繁殖途径的研究 [J]. 西北农林科技大学学报(自然科学版), 2005, 33(12):69−72, 77. DOI: 10.3321/j.issn:1671-9387.2005.12.014 PU Z Y. Study on efficient Propagation Pathway of Phalaenopsis spp [J]. Journal of Northwest A&F University (Natural Science Edition), 2005, 33(12): 69−72, 77.(in Chinese) DOI: 10.3321/j.issn:1671-9387.2005.12.014
[29]	魏韩英. 春兰‘宋梅’组织培养及兰花菌根真菌的研究[D]. 杭州: 浙江农林大学, 2010. WEI H Y. Study on Tissue Culture and Mycorrhizal Fungi of Cymbidium goeringii ‘Songmae’[D]. Hangzhou: Zhejiang A & F University, 2010. (in Chinese)
[30]	Predieri S, Brizzi G, Gatti E. Effect of nitrogen supply on rooting of pear (Pyrus communis L.) cv. Conference microcuttings [J]. Advances in horticultural science, 1999, 13(2): 68−70.