地皮消愈伤组织诱导及植株高效再生体系的建立

doi:10.11983/CBB14207

植物学报 ›› 2016, Vol. 51 ›› Issue (1): 89-97.DOI: 10.11983/CBB14207

地皮消愈伤组织诱导及植株高效再生体系的建立

吕美萍¹, 王元忠², 黄衡宇^1,^*()

¹云南中医学院中药材优良种苗繁育工程研究中心, 昆明 650500
²云南省农业科学院药用植物研究所, 昆明 650200

收稿日期:2014-12-08 接受日期:2015-05-30 出版日期:2016-01-01 发布日期:2016-02-01
通讯作者: 黄衡宇
作者简介:? 共同第一作者
基金资助:
基金项目: 国家自然科学基金(No.31260077)和云南省高校重点实验室项目

Callus Induction and High Efficiency Plant Regeneration System Establishment of Pararuellia delavayana

Meiping Lü¹, Yuanzhong Wang², Hengyu Huang^1*

¹Engineering Research Center for Reproducing Fine Varieties of Chinese Medicinal Plants, Yunnan University of Chinese Traditional Medicine, Kunming 650500, China
²Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Kunming 650200, China

Received:2014-12-08 Accepted:2015-05-30 Online:2016-01-01 Published:2016-02-01
Contact: Huang Hengyu
About author:? These authors contributed equally to this paper

摘要/Abstract

摘要： 以地皮消(Pararuellia delavayana)无菌实生苗带叶茎尖和带节茎段为外植体, 探讨不同植物激素种类及组合对愈伤组织诱导、芽丛发生及植株再生的影响。结果表明, 地皮消组织培养和植株再生的适宜外植体为带节茎段, 其在MS+1.0 mg·L^-16-BA+0.5 mg·L^-1NAA+0.1 mg·L^-1 KT培养基中培养17天后, 约85.38%的外植体产生出分化能力较强的愈伤组织; 25天后约97.55%的愈伤组织开始分化出绿色芽丛; 30天后不定芽分化系数可达15.38。不定芽增殖继代6次后出现玻璃化现象, 且随着继代次数的增加, 玻璃化现象加重, 增殖率明显下降; 采用MS和B₅培养基交替使用可改善试管苗玻璃化现象并保持较高的增殖率。不定芽生根的适宜培养基为MS+0.5 mg·L^-1 NAA, 生根率可达100%。再生苗移栽成活率达95%以上。该研究建立了地皮消无性快速繁殖体系, 为保护地皮消野生资源及种苗繁育提供了有效途径, 也为其遗传转化研究奠定了基础。

关键词: 地皮消, 带节茎段, 愈伤组织, 玻璃化, 优化培养

Abstract: The effectiveness of different combinations of growth regulators and concentrations on callus induction, bud induction and plant regeneration were studied using explants excised from seedlings of Pararuellia delavayana. The optimum explants for callus induction and plant regeneration were stem explants with nodes. The percentage of callus induction was up to 85.38% when stem explants with nodes were cultured on MS medium containing 1.0 mg·L^-1 6-BA, 0.5 mg·L^-1 NAA and 0.1 mg·L^-1 KT after 17 days, The percentage of bud formation from callus was up to 97.55% after 25 days, and the frequency of adventitious bud formation was 15.38 after 30 days. Vitrification emerged after 6 subculture cycles, and the phenomenon was aggravated with increasing generation, but the proliferation rate decreased. MS and B₅ media were used interchangeably to solve the problem. The optimal medium for rooting was MS+0.5 mg·L^-1 NAA; the rooting rate was 100% and the survival rate of transplants was >95%. We established a rapid propagation system and provided an effective solution for protecting wild resources and sprout multiplication of P. delavayana, while providing an experimental base for research in genetic transformation.

Key words: Pararuellia delavayana, stem with nodes, callus, vitrification, optimal culture

吕美萍, 王元忠, 黄衡宇. 地皮消愈伤组织诱导及植株高效再生体系的建立. 植物学报, 2016, 51(1): 89-97.

Meiping Lü, Yuanzhong Wang, Hengyu Huang. Callus Induction and High Efficiency Plant Regeneration System Establishment of Pararuellia delavayana. Chinese Bulletin of Botany, 2016, 51(1): 89-97.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB14207

https://www.chinbullbotany.com/CN/Y2016/V51/I1/89

图/表 10

表1 地皮消愈伤组织诱导与芽丛发生L9 (34)正交设计

Table 1 L9 (34) orthogonal design for callus induction and bud induction of Pararuellia delavayana

Level	Factor
Level	A (6-BA, mg·L ^-1)	B (NAA, mg·L^-1)	C (KT, mg·L^-1)
1	0.5	0.1	0.01
2	1.0	0.5	0.05
3	1.5	1.0	0.10

图1 地皮消愈伤组织诱导与芽丛发生 (A) 培养7天后腋芽迅速生长; (B) 培养17天, 接触培养基的外植体基部出现绿色愈伤组织; (C) 培养25天, 愈伤组织上出现绿色芽点; (D) 培养35天, 产生致密的芽丛

Figure 1 Callus induction and bud induction in Pararuellia delavayana(A) Axillary buds spring up quickly after 7 days; (B) Green callus deriving from the base of explants after 17 days; (C) Green buds from callus after 25 days; (D) Cluster buds from callus after 35 days

表2 地皮消愈伤组织诱导和芽丛发生L9 (34)正交实验设计与结果

Table 2 Results of callus induction and bud induction by L9 (34) orthogonal test in Pararuellia delavayana

No.		Hormone (mg·L^-1)				Rate of callus induced (%)	Rate of buds induced (%)
No.		A (6-BA)	B (NAA)	C (KT)	D (Error)	Rate of callus induced (%)	Rate of buds induced (%)
C1		0.5	0.1	0.01	(1)	25.12	53.03
C2		0.5	0.5	0.05	(2)	40.67	60.31
C3		0.5	1.0	0.10	(3)	49.23	64.80
C4		1.0	0.1	0.05	(3)	55.35	66.31
C5		1.0	0.5	0.10	(1)	85.38	97.55
C6		1.0	1.0	0.01	(2)	82.89	93.70
C7		1.5	0.1	0.10	(2)	58.51	89.51
C8		1.5	0.5	0.01	(3)	57.95	89.78
C9		1.5	1.0	0.05	(1)	50.97	63.62
Callus induced	K₁	38.34	46.33	55.32	53.82
	K₂	75.54	61.33	49.00	60.69
	K₃	55.81	61.03	64.37	54.18
	R₁	37.20	15.00	15.38	6.87
Buds induced	K₁	59.38	69.62	78.84	71.40
	K₂	85.85	82.55	63.41	81.17
	K₃	80.97	74.04	83.95	73.63
	R₂	26.47	12.93	20.54	9.77

表3 地皮消愈伤组织诱导方差分析结果

Table 3 Variance analysis of callus induction in Pararuellia delavayana

Source	Type III sum of square	df	Mean square	F	Significance
A (6-BA)	1966.454	2	983.227	21.923	P<0.05
B (NAA)	441.480	2	220.740	4.922	P>0.05
C (KT)	358.389	2	179.195	3.995	P>0.05
Error	89.699	2	44.850

表4 6-BA 3水平Duncan检验

Table 4 Duncan’s test in three levels of 6-BA

Levels	Mean	0.05 level
2	75.54	a
3	55.81	ab
1	38.34	b

表5 地皮消芽丛发生方差分析结果

Table 5 Variance analysis of bud induction in Pararuellia delavayana

Source	Type III sum of square	df	Mean square	F	Significance
A (6-BA)	1190.812	2	595.406	7.566	P>0.05
B (NAA)	259.114	2	129.557	1.646	P>0.05
C (KT)	685.951	2	342.976	4.358	P>0.05
Error	157.393	2	78.696

图2 地皮消芽的增殖与生长 (A) 转接10天后分化出小芽点; (B) 20天后芽丛大量繁殖; (C) 培养30天的芽丛情况; (D) 培养40天丛生苗的生长情况

Figure 2 Proliferation and growth of adventitious shoots in Pararuellia delavayana(A) Small buds differentiation after 10 days; (B) Calluses blooming after 20 days; (C) Adventitious shoots after 30 days; (D) Adventitious seedlings after 40 days

表6 地皮消在不同基本培养基中的增殖及生长情况

Table 6 Results of proliferation and growth in Pararuellia delavayana in different basic medium

Media	Regeneration coefficient (%)	Growth condition
B₅	11.05	Adventitious shoots with lower induction rate were stronger, the vitrification was reduced obviously
N₆	1.65	Plantlets with no callus induction turned reddish, the vitrification was reduced obviously but the growth slowed
MS (1/2NH₄NO₃)	7.93	Weak adventitious shoots, the vitrification was reduced obviously

图3 地皮消玻璃化现象及改善情况 (A) 增殖第4代培养40天的试管苗; (B) 第4代试管苗; (C) 第5代试管苗; (D) 第6代玻璃化苗; (E) B5培养基中生长40天的试管苗; (F) MS (NH4NO3减半)培养基中生长40天后的试管苗; (G) N6培养基中培养30天的生长情况; (H) B5和MS交替使用2次后培养40天的试管苗

Figure 3 Phenomenon and improving results of vitrification in Pararuellia delavayana(A) Plantlets of the fourth generation after 40 days; (B) Plantlets of the fourth generation; (C) Plantlets of the fifth generation; (D) Vitrification plantlets of the sixth generation; (E) Proliferous plantlets in B5 medium after 40 days; (F) Proliferous plantlets in MS (NH4NO3 halved) medium after 40 days; (G) Proliferous plantlets in N6 medium after 30 days; (H) Proliferous plantlets of B5 and MS medium used interchangeably twice after 40 days

图4 地皮消生根培养及移栽 (A) MS培养基中培养45天时的生根苗; (B) MS培养基中培养45天时的根系; (C) 移栽苗

Figure 4 Rooting culture and transplanting of Pararuellia delavayana(A) Rooting plantlets in MS medium after 45 days; (B) Root system in MS medium after 45 days; (C) Transplanting plantlets

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地皮消愈伤组织诱导及植株高效再生体系的建立

Callus Induction and High Efficiency Plant Regeneration System Establishment of Pararuellia delavayana

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