‘正午’牡丹微繁殖技术体系的优化

doi:10.11707/j.1001-7488.20211207

摘要/Abstract

摘要：

目的: 牡丹微繁殖技术已在20多个品种中建立，但污染率高、增殖系数低、生根难和移栽成活率低等问题仍然存在，并制约着该技术的推广应用，因此开展微繁殖技术体系的优化研究具有重要意义。方法: 以‘正午’牡丹为材料，通过对不同外植体启动培养效果比较、增殖培养阶段LED(发光二级管)光源应用以及生根方式改进等试验，对微繁殖技术体系进行优化改进。结果: 启动培养阶段，黄化嫩茎外植体优于鳞芽外植体，其污染率较低(19.59%)，存活率较高(80.41%)，且基本不褐化。增殖培养阶段，与荧光灯光源相比，LED光源(光质配比70%红光+30%蓝光，光照强度50 μmol·m^-2s^-1)可以显著促进试管苗的增殖和生长，增殖系数3.79，且试管苗生长健壮。生根培养阶段，基于维生素B₂(VB₂)在黑暗条件下可以和IBA共存、但在光照条件下分解IBA的特性，将试管苗先在附加IBA 1.0 mg·L^-1和VB₂ 1.0~5.0 mg·L^-1的培养基中暗培养30天诱导根原基，再转至光照培养20天，利用VB₂加速IBA分解、消除IBA对根原基伸长生长的抑制，可以实现试管苗一步生根，其生根率(71.07%~74.23%)和根数(3.17~3.34条)与两步生根无显著差异。与两步生根法获得的生根苗相比，一步生根法获得的生根苗基部愈伤组织较少，生根苗质量更佳，其一级苗占比为75.00%，移栽成活率为85.00%。结论: 在‘正午’牡丹微繁殖中，黄化嫩茎是一种优于鳞芽的外植体来源，其污染率较低、诱导成活率较高；光质配比70%红光+30%蓝光、光照强度50 μmol·m^-2s^-1的LED光照培养，可以显著促进试管苗增殖和生长；在生根培养基中添加维生素B₂，结合光照条件控制(暗培养30天后转至光照培养20天)，可实现试管苗的一步生根，其操作流程简单、生根成本低、试管苗质量佳、移栽成活率高。本研究所建立和优化的‘正午’牡丹微繁殖技术体系，不仅可以促进该品种快繁技术的产业化应用，还可为其他牡丹品种微繁殖技术研究提供参考。

关键词: 牡丹, 微繁殖, 外植体, 增殖, LED, VB₂, 一步生根

Abstract:

Objective: Micropropagation protocols have been developed for more than 20 varieties of tree peony(Paeonia Sect. Moutan), but not applied to industrial use due to the high contamination rate, low multiplication rate, rooting difficulties and low transplanting survival rate. Therefore, it is an important issue to improve the micropropagation protocols of tree peony. Method: This study was performed to optimize the micropropagation protocol of Paeonia × lemoinei 'High Noon' through selection of explants, light-emitting diodes(LED) illumination culture and improvement of rooting methods. Result: Etiolated shoots were explants superior to scale buds which showed lower contamination rate(19.59%), higher survival rate(80.41%), and no browning during initial culture. Compared with traditional fluorescent lamp, the optimal LED illumination(light quality 70%Red+30%Blue, light intensity 50 μmol·m^-2s^-1) significantly promoted the multiplication and growth of the in vitro shoots. The multiplication rate was 3.79, and the in vitro shoots grew well. Based on the reactivity that vitamin B₂ (VB₂) can coexist with IBA under dark conditions and decompose IBA under light conditions, an efficient one-step rooting method was developed. The shoots were firstly cultured in the dark on medium supplemented with IBA 1.0 mg·L^-1 and VB₂ 1.0-5.0 mg·L^-1 for 30 days to induce rooting, and then transferred to light culture for 20 days to accelerate IBA decomposition which will help eliminate the inhibition of IBA on root elongation. The rooting percentage(71.07%-74.23%) and root number(3.17-3.34) of one-step rooting method were similar to those of two-step rooting method. Compared with two-step rooting method, the one-step rooting protocol improved the transplanting survival rate by optimizing the quality of rooted shoots, and its proportion of first grade plantlet was up to 75.00%, which produced 85.00% survival after transplanting. Conclusion: Etiolated shoots are explants superior to scale buds in initiation and establishment of in vitro culture. LED illumination(light quality 70% Red+30% Blue, light intensity 50 μmol·m^-2s^-1) can significantly promote the multiplication and growth of in vitro shoots. An efficient one-step rooting method was established in P. × lemoinei 'High Noon' micropropagation both by addition of vitamin B₂ in the rooting medium and by the control of light conditions(30 days of dark culture, and then 20 days of light culture), which produced simpler technical process, lower rooting cost, better quality of rooted shoots and higher transplanting survival rate than two-step rooting method. This study has developed an optimized micropropagation protocol for P. × lemoinei 'High Noon', greatly promoting the micropropagation of the variety, and providing a basis for the micropropagation of other tree peony varieties.

Key words: Paeonia Sect. Moutan, micropropagation, explant, multiplication, light-emitting diodes, vitamin B₂, one-step rooting

中图分类号:

S723.1

文书生,成仿云,钟原. ‘正午’牡丹微繁殖技术体系的优化[J]. 林业科学, 2021, 57(12): 68-78.

Shusheng Wen,Fangyun Cheng,Yuan Zhong. Optimization of the Micropropagation Protocol of Paeonia × lemoinei 'High Noon'[J]. Scientia Silvae Sinicae, 2021, 57(12): 68-78.

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LED光源组合 LED light combination	波长 Wave length/nm	光照强度 Illumination intensity/(μmol·m^-2s^-1)
W	400~750	30
R	630	R 30
R∶B 8∶2	—	R 24+B 4
R∶B 7∶3	—	R 21+B 9
R∶B 6∶4	—	R 18+B 12
B	460	B 30

外植体 Explant	污染率 Contamination(%)	褐化率 Browning(%)	存活率 Survival(%)
萌蘖芽Sucker bud	32.79±5.33 b	2.46±1.29 a	64.75±7.38 b
腋芽Axillary bud	44.71±6.41 a	2.35±1.16 a	52.94±5.72 c
黄化嫩茎Etiolated shoot	19.59±2.94 c	0.00±0.00 b	80.41±4.29 a

生根方式 Rooting method	VB₂浓度 Vitamin B₂/(mg·L^-1)	生根率 Rooting percentage(%)	平均根数 Root number	平均根长 Root length/cm	基部愈伤组织等级 Grade of callus size
两步生根 Two-step rooting	—	77.21±8.71 a	2.84±0.22 a	3.04±0.7 a	2
一步生根 One-step rooting	0	50.64±2.75 c	1.09±0.24 c	1.21±0.0 c	2
	1	74.23±3.90 a	3.34±0.45 a	3.76±0.5 a	1
	5	71.07±2.01 ab	3.17±0.28 a	3.54±0.3 a	1
	10	67.98±5.03 b	3.09±0.39 a	3.33±0.5 a	1
	15	64.44±3.17 b	2.01±0.11 b	2.20±0.6 b	1

生根方式 Rooting method	一级苗 First-grade plantlets		二级苗 Second-grade plantlets		三级苗 Third-grade plantlets
生根方式 Rooting method	占比 Percentage(%)	成活率 Survival(%)	占比 Percentage(%)	成活率 Survival(%)	占比 Percentage(%)	成活率 Survival(%)
两步生根 Two-step rooting	62.50	82.00	23.75	52.63	13.75	9.09
一步生根 One-step rooting	75.00	85.00	13.75	45.45	11.25	11.11

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