杜鹃花开放式组织培养体系的建立及其遗传稳定性分析

刘雨轩; 胡宗琪; 汪雪蓉; 李冬花; 王泓钦; 赖恭梯; 甘代奎; 赖呈纯; 陈桂信

doi:10.19303/j.issn.1008-0384.2022.003.009

杜鹃花开放式组织培养体系的建立及其遗传稳定性分析

doi: 10.19303/j.issn.1008-0384.2022.003.009

刘雨轩^{1, 2,},
胡宗琪¹,
汪雪蓉¹,
李冬花¹,
王泓钦¹,
赖恭梯²,
甘代奎³,
赖呈纯^2, ,,
陈桂信^1, ,

1.
福建农林大学园艺学院，福建　福州　350002
2.
福建省农业科学研究院农业工程技术研究所，福建　福州　350003
3.
屏南县林业局，福建　宁德　352300

基金项目: 福建省林业科技项目（闽林科便函[2018]26号）

详细信息

作者简介:
刘雨轩（1997-），女，硕士研究生，研究方向：园艺植物遗传育种（Email：lyxuan23@163.com）

通讯作者:
赖呈纯（1975−），男，博士，副研究员，研究方向：园艺植物生物技术与食品生物技术（Email：lccisland@ 163.com）

陈桂信（1967−），男，博士，副教授，研究方向：园艺植物遗传育种与生物技术研究（Email：guixinchen@163.com）

中图分类号: S 685.21
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出版历程
- 收稿日期: 2021-09-01
- 修回日期: 2021-10-27
- 刊出日期: 2022-03-31

A Preliminary Study on Open Tissue Culture and Genetic Stability of Rhododendron Plantlets

1.
College of Horticulture , Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Institute of Agricultural Engineering and Technology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China
3.
Pingnan County Forestry Bureau, Ningde, Fujian　352300, China

摘要

摘要: 目的通过向培养基中添加不同种类的抑菌剂，建立锦绣杜鹃开放式组织培养，并以开放式组织培养初代和增殖阶段培育的试管芽苗为材料，探究开放式组织培养体系的遗传稳定性，以期为将来杜鹃花的工厂化育苗提供技术支持。方法以锦绣杜鹃当年生枝条的顶芽和带腋芽茎段为试验材料，对外植体的消毒方式以及开放式组织培养初代与增殖培养的阶段培养基抑菌剂的使用浓度进行探索，利用ISSR分子标记技术，检测各时期不同处理的试管芽苗与母本材料间的遗传稳定性。结果外植体材料的适宜消毒方式为10% H₂O₂消毒10～15 min，顶芽存活率61.67%，污染率33.33%，茎段存活率23.33%，污染率68.33%；开放式组织培养阶段，NaClO适宜作为抑菌剂，最适添加量为0.01%，材料存活率62.22%，此时新生叶片细长，数量多，增殖系数为3.067。ISSR分子标记分析表明，不同消毒方式处理的外植体材料、开放式组织培养的各试管芽苗材料以及母本材料间的遗传相似系数均为0.919～0.995，材料间的变异率低，遗传稳定性较好。结论初步探索了开放式组织培养用于杜鹃花组织培养工厂化育苗，锦绣杜鹃外植体的适宜消毒方式为10% H₂O₂消毒10～15 min，顶芽材料的存活率比带腋芽茎段材料高；在开放式组织培养的初代培养和增殖培养阶段，适宜添加0.01% NaClO作为培养基抑菌剂；外植体的不同消毒处理与开放式组织培养，对试管材料遗传稳定性的影响不大。
- 锦绣杜鹃 /
- 开放式组织培养 /
- 试管芽苗 /
- 遗传稳定性 /
- ISSR分子标记
Abstract: Objective An open tissue culture with added bacteriostatic agents in medium for disinfection and disease prevention was established and genetic stability of the plantlets verified for the development of commercialized propagation of rhododendron. Method Terminal buds and stem segments with attached axillary buds were cut from current year branches of Rhododendron pulchrum Sweet plants. Methods using bacteriostatic agents in varied concentrations to disinfect the cut tissues and prevent infection during proliferation stages in an open tissue culture were evaluated. Genetic of the materials in transition from parent to plantlet was scrutinized using an ISSR molecular marker technology to ensure a reliable stability in the process. Result The explants could be adequately disinfected with 10% H₂O₂ for 10 to 15m. A survival rate of terminal buds at 61.67% with a contamination rate of 33.33% and that of stem segments at 23.33% with a contamination rate of 68.33% were achieved. For the open tissue culture, 0.01% addition of NaClO in the medium was found sufficient to achieve the bacteriostatic effect with a survival rate of 62.22% on the plantlets, which had abundant, slender, new leaves and a proliferation coefficient of 3.067. The ISSR molecular maker analysis showed high genetic similarity coefficients ranging from 0.919 to 0.995 between the disinfected explants, the test-tube buds, and the exophyte plantlets. Conclusion This study preliminarily explored the open tissue culture for the feasibility of rhododedron tissue factory nursery. The cut rhododendron tissues for the culture was satisfactorily disinfected with 10% H₂O₂ in 10-15 m. The survival rate of terminal buds after the treatment was higher than that of stem segments. In the early stage of the open tissue culture, a 0.01% addition of NaClO in medium provided sufficient bacteriostatic effect without significant reduction on the survival rate and proliferation coefficient of the treated plantlets. The genetic distance between the parent and the explants remained close, indicating little variation introduced by the propagating operation. The disinfection method and culture procedure appeared feasible for the development of rhododendron seedling generation at nurseries.
- Rhododendron pulchrum Sweet /
- open tissue culture /
- test-tube seeding /
- genetic stability /
- ISSR molecular marker

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图 1 不同消毒处理对顶芽材料的影响

注：柱形图上方的不同小写字母表示不同处理组内差异显著测验（P＜0.05），小写字母的不同下标表示不同处理组。图2、3同。

Figure 1. Effects of disinfection treatments on terminal buds

Note: Different lower case letters above the column indicate the significant differences in Duncan’s new Multiple-Range test in different processing groups, p＜0.05, different subscripts of lowercase letters indicate different processing groups. The same as Fig2-3.

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图 2 不同消毒处理对带腋芽茎段材料的影响

Figure 2. Effects of disinfection treatments on stem segments with axillary buds

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图 3 不同抑菌剂对材料生长的影响

注：1：CK；2：0.3% S106；3：0.01% NaClO；4：0.015% NaClO；5：0.02% NaClO；6：25 mg·L⁻¹ 代森锰锌；7：50 mg·L⁻¹ 代森锰锌；8：75 mg·L⁻¹ 代森锰锌；9：100 mg·L⁻¹ 代森锰锌。

Figure 3. Effect of antibacterial agents on growth of cut plant tissues

1: CK; 2: 0.3% S106; 3: 0.01% NaClO; 4: 0.015% NaClO; 5: 0.02% NaClO; 6: 25 mg·L⁻¹ mancozeb; 7: 50 mg·L⁻¹ mancozeb; 8: 75 mg·L⁻¹ mancozeb; 9: 100 mg·L⁻¹ mancozeb。

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图 4 抑菌剂对材料生长的影响

注：A：CK中生长的材料；B：0.3% S106 中生长的材料；C：0.01% NaClO中生长的材料。

Figure 4. Effect of bacteriostatic agent on growth of cut plant tissues

Note: A: material grown in CK; B: material grown in 0.3% S106; C: material grown in 0.01% NaClO.

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图 5 引物UBC 845电泳结果

注：1~30：锦绣杜鹃样品（顺序同表1）；M：2 000 bp marker

Figure 5. Electrophoretic map of UBC primer 845

Note：1~30: sample of R. pulchrum (in same order as shown in Table 1)；M：2 000 bp marker.

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图 6 外植体消毒材料的聚类分析

Figure 6. Cluster analysis on bacteriostatic agents

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图 7 开放式培养体系的聚类分析

Figure 7. Cluster analysis on open tissue culture

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表 1 遗传稳定性分析材料

Table 1. Materials subjected to genetic stability analysis

编号 Number	样品标签 Sample lable	样品来源及处理方式 Source and treatment method of sample	编号 Number	样品标签 Sample lable	样品来源及处理方式 Source and treatment method of sample
1	母本 Mother plant	锦绣杜鹃取样母株嫩芽，未处理 Buds of Rhododendron pulchrum Sweet., Untreated	16	CL7	材料使用400 mg·L⁻¹ ClO₂ 消毒10 min，初代培养基 Material disinfected by 400 mg·L⁻¹ClO₂ solution for 10 min, Incipience media
2	SY1	材料使用10% H₂O₂消毒 5 min，初代培养基 Material disinfected by 10% H₂O₂ solution for 5 min, Incipience media	17	CL8	材料使用400 mg·L⁻¹ ClO₂消毒 20 min，初代培养基 Material disinfected by 400 mg·L⁻¹ClO₂ solution for 20 min, Incipience media
3	SY2	材料使用10% H₂O₂消毒 10 min，初代培养基 Material disinfected by 10% H₂O₂ solution for 10 min, Incipience media	18	CL9	材料使用400 mg·L⁻¹ ClO₂消毒 30 min，初代培养基 Material disinfected by 400 mg·L⁻¹ClO₂ solution for 30 min, Incipience media
4	SY3	材料使用10% H₂O₂消毒 15 min，初代培养基 Material disinfected by 10% H₂O₂ solution for 15 min, Incipience media	19	OPC1	开放式初代培养，初代培养基 + 100 mg·L⁻¹代森锰锌 Open primary culture, Incipience media + 100 mg·L⁻¹ mancozeb
5	SY4	材料使用12% H₂O₂消毒 5 min，初代培养基 Material disinfected by 12% H₂O₂ solution for 5 min, Incipience media	20	OPC2	开放式初代培养，初代培养基 + 75 mg·L⁻¹代森锰锌 Open primary culture, Incipience media + 75 mg·L⁻¹mancozeb
6	SY5	材料使用12% H₂O₂消毒 10 min，初代培养基 Material disinfected by 12% H₂O₂ solution for 10 min, Incipience media	21	OPC3	开放式初代培养，初代培养基 + 50 mg·L⁻¹代森锰锌 Open primary culture, Incipience media + 50 mg·L⁻¹mancozeb
7	SY6	材料使用12% H₂O₂消毒 15 min，初代培养基 Material disinfected by 12% H₂O₂ solution for 15 min, Incipience media	22	OPC4	开放式初代培养，初代培养基 + 25 mg·L⁻¹代森锰锌 Open primary culture, Incipience media + 25 mg·L⁻¹ mancozeb
8	CN1	材料使用2% NaClO消毒 20 min，初代培养基 Material disinfected by 2% NaClO solution for 20 min,	23	OPC5	开放式初代培养，初代培养基 + 0.01% NaClO Open primary culture, Incipience media + 0.01% NaClO
9	CN2	材料使用2% NaClO消毒 30 min，初代培养基 Material disinfected by 2% NaClO solution for 30 min,	24	OPC6	开放式初代培养，初代培养基 + 0.015% NaClO Open primary culture, Incipience media + 0.015% NaClO
10	CL1	材料使用200 mg·L⁻¹ ClO₂消毒 10 min，初代培养基 Material disinfected by 200 mg·L⁻¹ClO₂ solution for 10 min, Incipience media	25	OPC7	开放式初代培养，初代培养基 + 0.02% NaClO Open primary culture, Incipience media + 0.02% NaClO
11	CL2	材料使用200 mg·L⁻¹ClO₂消毒 20 min，初代培养基 Material disinfected by 200 mg·L⁻¹ClO₂ solution for 20 min, Incipience media	26	OZZ1	开放式增殖培养，增殖培养基 + 0.01% NaClO Open proliferation culture, Proliferation medium + 0.01% NaClO
12	CL3	材料使用200 mg·L⁻¹ ClO₂消毒 30 min，初代培养基 Material disinfected by 200 mg·L⁻¹ClO₂ solution for 30 min, Incipience media	27	OZZ2	开放式增殖培养，增殖培养基 + 0.015% NaClO Open proliferation culture, Proliferation medium + 0.015% NaClO
13	CL4	材料使用300 mg·L⁻¹ ClO₂消毒 10 min，初代培养基 Material disinfected by 300 mg·L⁻¹ClO₂ solution for 10 min, Incipience media	28	OZZ3	开放式增殖培养，增殖培养基 + 0.02% NaClO Open proliferation culture, Proliferation medium + 0.02% NaClO
14	CL5	材料使用300 mg·L⁻¹ ClO₂消毒 20 min，初代培养基 Material disinfected by 300 mg·L⁻¹ClO₂ solution for 20 min, Incipience media	29	OZZ4	开放式增殖培养，增殖培养基 + 100 mg·L⁻¹代森锰锌 Open proliferation culture, Proliferation medium + 100 mg·L⁻¹ mancozeb
15	CL6	材料使用300 mg·L⁻¹ ClO₂消毒 30 min，初代培养基 Material disinfected by 300 mg·L⁻¹ClO₂ solution for 30 min, Incipience media	30	OZZ5	开放式增殖培养，增殖培养基 + 75 mg·L⁻¹代森锰锌 Open proliferation culture, Proliferation medium + 75 mg·L⁻¹ mancozeb

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表 2 不同ZT含量对材料增殖系数的影响

Table 2. Effects of ZT content on proliferation coefficient of materials

材料 Materials	ZT含量 Content of ZT/(mg·L⁻¹)	增殖系数 Multiplication coefficient
材料 Materials	ZT含量 Content of ZT/(mg·L⁻¹)	10 d	20 d	30 d
带腋芽茎段 Stem segment with axillary bud	0	1.250	1.750	1.750
	1.5	1.400	1.643	2.000
	2.0	1.000	2.222	2.389
	2.5	1.000	2.313	2.500
	3.0	1.385	2.333	2.944
顶芽 Terminal bud material	0	1.000	1.000	1.000
	1.5	1.000	1.000	1.000
	2.0	1.000	1.000	1.000
	2.5	1.000	1.000	1.000
	3.0	1.000	1.000	1.000

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表 3 开放式增殖培养对材料的影响

Table 3. Effect of open tissue culture on materials

材料 Materials	抑菌剂含量 Content of bacteriostatic agent	增殖系数 Multiplication coefficient
材料 Materials	抑菌剂含量 Content of bacteriostatic agent	10 d	20 d	30 d
带腋芽茎段 Stem segment with axillary bud	0.01% NaClO	1.667	1.815	3.067
	0.015% NaClO	1.364	1.636	1.333
	0.02% NaClO	1.333	1.333	1.000
	100 mg·L⁻¹代森锰锌 100 m·L⁻¹ mancozeb	1.667	1.815	2.667
	75 mg·L⁻¹代森锰锌 75 mg·L⁻¹ mancozeb	1.167	2.000	2.526
顶芽 Terminal bud material	0.01% NaClO	1.000	1.000	1.000
	0.015% NaClO	1.000	1.000	1.000
	0.02% NaClO	1.000	1.000	1.000
	100 mg·L⁻¹代森锰锌 100 mg·L⁻¹ mancozeb	1.000	1.000	1.000
	75 mg·L⁻¹代森锰锌 75 mg·L⁻¹ mancozeb	1.000	1.000	1.000

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表 4 13条ISSR引物的碱基序列扩增

Table 4. Base sequence amplification of 13 ISSR primers

编号 Serial name	引物序列 Primer sequence	扩增总带数 Amplified bands/条	多态性带数 Polymorphic bands/条	多态性位点百分率 Percentage of polymoephic/%
UBC 810	（GA）₈T	7	2	28.57
UBC 811	（GA）₈C	10	0	0.00
UBC 815	（CT）₈G	8	1	12.50
UBC 816	（CA）₈T	10	4	40.00
UBC 823	（TC）₈C	8	1	12.50
UBC 826	（AC）₈C	9	0	0.00
UBC 827	（AC）₈G	6	2	33.33
UBC 834	（AG）₈YT	9	2	22.22
UBC 835	（AG）₈YC	9	3	33.33
UBC 840	（GA）₈YT	8	3	37.50
UBC 845	（CT）₈RG	10	8	80.00
UBC 847	（CA）₈RC	7	1	14.29
UBC 857	（AC）₈YG	9	2	22.22
总计 Total	−	110	29	−
平均 Average	−	8.46	2.23	26.36

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