山苍子植株再生体系的优化

doi:10.11707/j.1001-7488.20220813

摘要/Abstract

摘要：

目的: 研究优化山苍子茎段愈伤组织诱导和植株再生体系的方法，提高不定芽的分化率和生根率，为山苍子良种的工厂化繁育及遗传转化体系的建立提供技术支撑。方法: 本研究以山苍子茎段节间(不带芽)为外植体，通过比较不同植物生长调节剂对其愈伤组织的诱导、增殖以及不定芽的分化、生根的影响，确定山苍子愈伤组织诱导和植株再生的最适培养基。在此基础上，对获得的再生植株进行移栽试验，比较不同基质对移栽成活率的影响。结果: 山苍子茎段愈伤组织诱导的最适培养基为1/2 MS + 0.2 mg·L^-1 6-BA + 0.3 mg·L^-1 NAA + 30 g·L^-1蔗糖+ 7 g·L^-1琼脂，其诱导率为100%；愈伤组织增殖的最适培养基为1/2 MS + 0.5 mg·L^-1 6-BA + 1.0 mg·L^-1 NAA + 30 g·L^-1蔗糖+7 g·L^-1琼脂，愈伤组织增殖系数达3.43；诱导不定芽分化的最适培养基为1/2 MS + 0.5 mg·L^-1 6-BA + 0.1 mg·L^-1 IBA + 30 g·L^-1蔗糖+7 g·L^-1琼脂，不定芽分化率为45.59%，平均不定芽数为8.14；生根阶段最适培养基为1/2 MS + 0.5 mg·L^-1 IBA + 20 g·L^-1蔗糖+ 7 g·L^-1琼脂，不定芽生根率高达93.33%，平均根数为6.74；将生长良好的再生植株进行驯化后，移栽至营养土∶珍珠岩=3 ∶1(V/V)的混合基质中，其成活率达到83.33%。结论: 本研究优化了山苍子离体再生体系，显著提高了山苍子不定芽的分化率和生根率以及再生植株的移栽成活率，可为山苍子良种的工厂化繁育提供技术支撑。

关键词: 山苍子, 茎段, 愈伤组织, 植株再生, 驯化移栽

Abstract:

Objective: The tissue culture technology of Litsea cubeba is not perfect and cannot satisfy the needs of industrialized breeding. In view of this, this research intends to optimize the stem callus induction and plant regeneration systems of L. cubeba, so as to improve the differentiation and rooting rates of adventitious buds, and provide technical supports for the establishment of industrial breeding and genetic transformation system of L. cubeba elite varieties. Method: In this study, the stem internodes (without bud) of L. cubeba were used as explants, and the optimum media for callus induction and plant regeneration of L. cubeba were determined through comparing the effects of different plant growth regulators on callus induction, proliferation, adventitious bud differentiation and rooting. On this basis, the transplanting tests of regenerated plants were performed to compare the effects of different substrates on the transplanting survival rates. Result: The optimum medium for callus induction of stem segments was 1/2 MS containing 0.2 mg·L^-1 6-BA, 0.3 mg·L^-1 NAA, 30 g·L^-1sucrose and 7 g·L^-1 agar, and the induction rate was 100%. The optimal medium for callus proliferation was 1/2 MS containing 0.5 mg·L^-1 6-BA, 1.0 mg·L^-1 NAA, 30 g·L^-1 sucrose and 7 g·L^-1 agar with a proliferation coefficient up to 3.43. The optimal medium for adventitious bud differentiation was 1/2 MS containing 0.5 mg·L^-1 6-BA, 0.1 mg·L^-1 IBA, 30 g·L^-1 sucrose and 7 g·L^-1 agar with a differentiation rate of 45.59% and an average number of 8.14 of adventitious buds. The optimum medium for rooting of adventitious buds was 1/2 MS containing 0.5 mg·L^-1 IBA, 20 g·L^-1 sucrose and 7 g·L^-1 agar, the rooting rate was 93.33% and the average number of roots was 6.74. After domestication, the regenerated plants were transplanted into the mixed substrate with nutrient soil and perlite (volume ratio 3 ∶1), and the survival rate reached to 83.33%. Conclusion: This research optimizes the in vitro regeneration system of L. cubeba, significantly improves the differentiation and rooting rates of adventitious buds and the transplanting survival rate of regenerated plants, which can provide a technical support for the industrialized breeding of L. cubeba elite varieties.

Key words: Litsea cubeba, stem segments, callus, plant regeneration, domestication and transplantation

中图分类号:

S722.3⁺7

王阳,张付豪,窦敏,许婷,陈昊. 山苍子植株再生体系的优化[J]. 林业科学, 2022, 58(8): 126-135.

Yang Wang,Fuhao Zhang,Min Dou,Ting Xu,Hao Chen. Optimization of Plant Regeneration System of Litsea cubeba[J]. Scientia Silvae Sinicae, 2022, 58(8): 126-135.

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处理编号 Treatment No.	植物生长调节剂质量浓度 Mass concentration of plant growth regulator/(mg·L^-1)			诱导率 Induction rate (%)	平均褐化率 Average browning rate (%)	愈伤组织生长状况 The growth of the callus
处理编号 Treatment No.	6-BA	NAA	2, 4-D	诱导率 Induction rate (%)	平均褐化率 Average browning rate (%)	质地 Texture	诱导量 Induced amount	状态 State
CK	0.0	0.0	0.0	0	0.00±0.00f	—	—	—
1	0.2	0.0	0.5	100	0.00±0.00f	较致密 More compact	少 Few	一般 General
2	0.2	0.3	0.0	100	0.00±0.00f	致密 Compact	多 Many	好 Good
3	0.2	0.5	1.0	100	7.78±6.94ef	致密 Compact	少 Few	好 Good
4	1.0	0.0	1.0	100	27.78±11.70c	较致密 More compact	极少 Minute	一般 General
5	1.0	0.3	0.5	100	23.33±6.67c	致密 Compact	少 Few	一般 General
6	1.0	0.5	0.0	100	21.11±5.09c	致密 Compact	少 Few	一般 General
7	2.0	0.0	0.0	100	66.67±11.55a	疏松 Friable	极少 Minute	差 Poor
8	2.0	0.3	1.0	100	61.11±1.92ab	疏松 Friable	极少 Minute	差 Poor
9	2.0	0.5	0.5	100	52.22±8.39b	疏松 Friable	极少 Minute	差 Poor
K₁	2.59	31.48	29.26
K₂	24.08	28.15	25.18
K₃	60.00	27.04	32.22
R	57.41	4.44	7.04

处理编号 Treatment No.	植物生长调节剂质量浓度 Mass concentration of plant growth regulator/(mg·L^-1)		增殖系数 Proliferation coefficient	愈伤组织质地及生长状态 Callus texture and growth status
处理编号 Treatment No.	6-BA	NAA	增殖系数 Proliferation coefficient	愈伤组织质地及生长状态 Callus texture and growth status
1	0.5	0.2	2.33±0.48bc	疏松颗粒状，生长状态一般 Loose granular，general growth
2	1.0	0.2	1.81±0.32cd	疏松颗粒状，生长状态一般 Loose granular，general growth
3	1.5	0.2	1.10±0.05d	疏松水渍状，生长状态差 Loose water stains，poor growth
4	0.5	0.5	2.55±0.26b	致密型，生长状态良好 Compact，good growth
5	1.0	0.5	2.14±0.39bc	疏松颗粒状，生长状态良好 Loose granular，good growth
6	1.5	0.5	1.25±0.10d	疏松水渍状，生长状态差 Loose water stains，poor growth
7	0.5	1.0	3.43±0.89a	致密型，生长状态良好 Compact，good growth
8	1.0	1.0	3.80±0.22a	疏松颗粒状，生长状态良好 Loose granular，good growth
9	1.5	1.0	1.30±0.09d	疏松水渍状，生长状态差 Loose water stains，poor growth
K₁	2.77	1.75
K₂	2.58	1.98
K₃	1.21	2.84
R	1.56	1.09

处理编号 Treatment No.	植物生长调节剂质量浓度 Mass concentration of plant growth regulator/(mg·L^-1)		不定芽分化率 Adventitious bud differentiation rate (%)	平均不定芽数 Average adventitious bud number	不定芽分化及生长状态 Adventitious bud differentiation and growth status
处理编号 Treatment No.	6-BA	IBA	不定芽分化率 Adventitious bud differentiation rate (%)	平均不定芽数 Average adventitious bud number
CK	0	0	0.00±0.00f	0.00±0.00f	未分化 Undifferentiated
1	0.5	0.1	45.59±3.82a	8.14±0.82a	分化旺盛，长势好 Exuberant differentiation，good growth
2	1.0	0.1	25.56±5.09bc	5.75±0.25bc	分化旺盛，长势一般 Exuberant differentiation，general growth
3	2.0	0.1	18.89±1.92d	5.38±0.21bc	分化一般，长势一般 General differentiation，general growth
4	0.5	0.5	31.11±5.09b	6.06±0.35b	分化旺盛，长势好 Exuberant differentiation，good growth
5	1.0	0.5	23.33±3.34cd	4.82±0.79cd	分化一般，长势一般 Normal differentiation，general growth
6	2.0	0.5	17.78±3.85d	4.08±0.22d	分化一般，长势一般 Normal differentiation，general growth
7	0.5	1.0	0.00±0.00f	0.00±0.00f	未分化 Undifferentiated
8	1.0	1.0	7.78±1.92e	2.67±1.04e	分化差，长势一般 Poor differentiation，general growth
9	2.0	1.0	5.55±3.85ef	1.78±0.69e	分化差，长势差 Poor differentiation，poor growth
K₁	25.57	30.01
K₂	18.89	24.07
K₃	14.07	4.44
R	11.50	25.57
k₁	4.73	6.42
k₂	4.41	4.99
k₃	3.75	1.48
r	0.98	4.94

处理编号 Treatment No.	IBA/(mg·L^-1)	活性炭/(g·L^-1)	平均生根率 Average rooting rate (%)	平均根数 The average number of roots	根系生长状况 Root growth status
CK	0	0	23.33±11.55f	2.11±0.77c	根数少、细长 Few and slender roots
1	0.2	0	66.67±5.77b	4.94±0.31b	根数多、粗壮 Many and stout roots
2	0.5	0	93.33±5.77a	6.74±0.79a	根数多、粗壮 Many and stout roots
3	1.0	0	73.33±11.55b	6.13±0.76a	根数多、粗壮 Many and stout roots
4	0.2	0.5	56.67±15.28bcd	2.33±0.50c	根数少、细长 Few and slender roots
5	0.5	0.5	63.33±15.28bc	2.39±0.22c	根数少、细长 Few and slender roots
6	1.0	0.5	60.00±20.00bc	2.89±0.72c	根数少、细长 Few and slender roots
7	0.2	1.0	40.00±10.00de	2.20±0.37c	根数少、细长 Few and slender roots
8	0.5	1.0	53.33±5.77bcd	2.19±0.02c	根数少、细长 Few and slender roots
9	1.0	1.0	43.33±5.77cde	2.17±0.14c	根数少、细长 Few and slender roots
K₁	54.45	77.78
K₂	70.00	60.00
K₃	58.89	45.55
R	15.55	32.23
k₁	3.16	5.94
k₂	3.77	2.54
k₃	3.73	2.19
r	0.61	3.75

处理编号 Treatment No.	移栽基质 Transplanting substrate	移栽数量 Transplanting number	成活数量 Survival number	成活率 Survival rate (%)
1	营养土 Nutrient soil	18	13	72.22
2	营养土∶珍珠岩= 3∶1(V/V) Nutrient soil∶ perlite = 3∶1(V/V)	18	15	83.33
3	红壤土∶营养土∶珍珠岩= 2∶1/1(V/V) Red soil∶ nutrient soil∶ perlite = 2∶1∶1(V/V)	18	5	27.78
4	红壤土∶营养土∶珍珠岩= 1∶1∶1(V/V) Red soil∶ nutrient soil∶ perlite = 1∶1∶1(V/V)	18	7	38.89