刺槐离体叶片高效再生体系的优化

doi:10.11707/j.1001-7488.LYKX20210980

林业科学 ›› 2023, Vol. 59 ›› Issue (4): 68-78.doi: 10.11707/j.1001-7488.LYKX20210980

刺槐离体叶片高效再生体系的优化

韩娟^1,²,李亚鹏²,田彦挺²,郭琪²,李云²,孙宇涵²,邓永平³,牛东升⁴,苏立琢⁵,李秀宇²,彭祚登^1,*

1. 国家林业和草原局刺槐工程技术研究中心　北京林业大学林学院　北京 100083
2. 国家林业和草原局刺槐工程技术研究中心　林木育种国家工程实验室　林木、花卉遗传育种教育部重点实验室　北京林业大学生物科学与技术学院　北京 100083
3. 甘肃省清水县自然资源局　清水 741400
4. 山西省吉县刺槐种子园　吉县 042200
5. 辽宁省凌海市生态建设发展中心　凌海 121228

收稿日期:2021-12-30 出版日期:2023-04-25 发布日期:2023-07-05
通讯作者: 彭祚登
基金资助:
北京林业大学青年教师科学研究中长期项目(2015ZCQ-SW-03)；国家自然科学基金项目(31971675)；国家重点研发课题（2017YFD0600503）

Optimization of Efficient Regeneration System of Robinia pseudoacacia Leavesin vitro

Juan Han^1,²,Yapeng Li²,Yanting Tian²,Qi Guo²,Yun Li²,Yuhan Sun²,Yongping Deng³,Dongsheng Niu⁴,Lizhuo Su⁵,Xiuyu Li²,Zuodeng Peng^1,*

1. Engineering Technology Research Center of Black Locust of National Forestry and Grassland Administration　College of Forestry, Beijing Forestry University　Beijing 100083
2. National Engineering Laboratory for Tree Breeding　Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education　College of Biological Sciences and Technology, Beijing Forestry University　Beijing 100083
3. Natural Resources Bureau of Qingshui County, Gansu Province　Qingshui 741400
4. Seed Garden of Robinia pseudoacacia in Jixian County, Shanxi Province　Jixian 042200
5. Linghai Ecological Construction and Development Center, Liaoning Province　Linghai 121228)

Received:2021-12-30 Online:2023-04-25 Published:2023-07-05
Contact: Zuodeng Peng

摘要/Abstract

摘要：

目的: 通过优化再生过程中的重要影响因素，建立刺槐离体叶片高效的再生体系，这将为刺槐苗木繁殖、诱变育种、遗传转化以及进一步开展刺槐分子育种等奠定良好的基础。方法: 以刺槐组培苗离体的叶片为外植体，观察叶片再生不定芽的整个过程，探究基因型、取材时期、黑暗处理时长以及植物生长调节剂对刺槐叶片再生不定芽的影响，同时筛选适合不定芽生根的IBA浓度。结果: 1）二倍体基因型普18-22再生能力最佳；2）组培苗继代45天为最佳外植体取材时期；3）黑暗处理14天为最佳黑暗处理时长；4）最佳的叶片再生不定芽的培养基为：MS+6-BA 3.0 mg·L^?1+KT 1.0 mg·L^?1+2,4-D 0.1 mg·L^?1+蔗糖30 g·L^?1+琼脂6.5 g·L^?1；普18-22不定芽诱导率达96.67%，平均每个叶片再生7.4个不定芽；5）不定芽生根最佳培养基为：1/2MS+蔗糖30 g·L^?1+琼脂6.5 g·L^?1+IBA 0.3 mg·L^?1；生根率达100%。结论: 以继代培养45天的二倍体刺槐组培苗普18-22叶片为外植体，接种至MS+6-BA 3.0 mg·L^?1+KT 1.0 mg·L^?1+2,4-D 0.1 mg·L^?1+蔗糖30 g·L^?1+琼脂6.5 g·L^?1的再生培养基中，黑暗处理14 天后转移至正常光照培养，45天后将再生的不定芽转移至生根培养基1/2MS+蔗糖30 g·L^?1+琼脂6.5 g·L^?1+IBA 0.3 mg·L^?1中，待不定芽生根后进行炼苗移栽，得到完整的再生植株。

关键词: 刺槐, 叶片, 黑暗处理, 植物生长调节剂, 再生体系

Abstract:

Objective: Leaves are rich in sources, easy to sample, and have little impact on the plant after collection, such they are ideal materials for plant asexual reproduction. However, the regeneration efficiency ofRobinia pseudoacacia leaves is not high enough to meet the requirements of genetic transformation. In this study, an efficient regeneration system of R. pseudoacacia leaves in vitro was established by optimizing the important factors in the regeneration process, which would lay a good foundation for R. pseudoacacia seedling reproduction, mutagenesis breeding, genetic transformation and further development of molecular breeding of R. pseudoacacia. Method: The isolated leaves of R. pseudoacacia tissue culture seedlings were used as explants, the whole process of regeneration of adventitious buds from the isolated leaves was observed to investigate the effects of genotype, sampling period, dark treatment duration as well as plant growth regulators on the regeneration of adventitious buds fromR. pseudoacacia leaves. Meanwhile, the IBA concentration suitable for rooting of adventitious buds was screened. Result: 1) Diploid genotype Pu-18-22 had the best regeneration ability; 2) Subculture of tissue culture seedlings for 45 days was the best time for selecting explants; 3) Darkness treatment for 14 days was the best darkness treatment time; 4) The optimal medium for adventitious bud regeneration was MS (culture medium) + N-(Phenylmethyl)-9H-purin-6-amine 3.0 mg·L^?1 + Kinetin 1.0 mg·L^?1 + 2,4-Dichlorophenoxyacetic acid 0.1 mg·L^?1 + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1; The adventitious bud induction rate of Pu-18-22 was 96.67%, and 7.4 adventitious buds were regenerated per leaf on average; 5) The best rooting medium for adventitious buds was 1/2MS + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1 ＋ 3-Indolebutyric acid 0.3 mg·L^?1, and the rooting rate was 100%. Conclusion: The leaves of diploid R. pseudoacacia Pu-18-22 tissue culture plantlets are used as explants that are inoculated into MS culture medium + N-(Phenylmethyl)-9H-purin-6-amine 3.0 mg·L^?1 + Kinetin 1.0 mg·L^?1 + 2,4-Dichlorophenoxyacetic acid 0.1 mg·L^?1 + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1. After 14 days of dark treatment, the leaves are transferred to normal light culture. After 45 days of the light culture, the regenerated adventitious buds are then transferred to the rooting medium of 1/2MS culture medium + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1 + 3-Indolebutyric acid 0.3 mg·L^?1, After the adventitious shoots take root, the seedlings are hardened and transplanted to obtain the complete regenerated plants.

Key words: Robinia pseudoacacia, leaf, dark treatment, plant growth regulator, regeneration system

中图分类号:

S722.3⁺7

韩娟,李亚鹏,田彦挺,郭琪,李云,孙宇涵,邓永平,牛东升,苏立琢,李秀宇,彭祚登. 刺槐离体叶片高效再生体系的优化[J]. 林业科学, 2023, 59(4): 68-78.

Juan Han,Yapeng Li,Yanting Tian,Qi Guo,Yun Li,Yuhan Sun,Yongping Deng,Dongsheng Niu,Lizhuo Su,Xiuyu Li,Zuodeng Peng. Optimization of Efficient Regeneration System of Robinia pseudoacacia Leavesin vitro[J]. Scientia Silvae Sinicae, 2023, 59(4): 68-78.

图/表 14

图1

图2

表1

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表2

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图5

图6

图7

表4

植物生长调节剂对不定芽诱导的影响①"

植物生长调节剂组合 Combination of plant growth regulators	不定芽诱导率 Induction rate of adventitious buds (%)	平均不定芽诱导数 Average number of adventitious buds induced	愈伤组织、不定芽生长情况 Growth status of callus and adventitious buds
SIM1	54.87±0.02f	1.5±0.1d	愈伤组织较少；不定芽分化慢，长势差，无玻璃化 Small number of calli, slow differentiation of adventitious buds, poorer growth, no vitrification
SIM2	69.63±0.05e	1.67±0.24d	愈伤组织较少；不定芽分化慢，长势较差，轻微玻璃化 Small number of calli, Slow differentiation of adventitious buds, poor growth, light vitrification
SIM3	74.86±0.03e	2.18±0.08d	愈伤组织一般；不定芽分化较慢，长势一般，轻度玻璃化 Ordinary number of calli, slow differentiation of adventitious buds, poor growth, light vitrification
SIM4	77.24±0.03cde	3.19±0.2c	愈伤组织一般；不定芽分化中等，长势良好，无玻璃化 Ordinary number of calli, medium differentiation of adventitious buds, good growth, no vitrification
SIM5	82.91±0.03bcd	4.24±0.22b	愈伤组织适中；不定芽分化较快，长势良好，轻度玻璃化 Medium number of calli, rapid differentiation of adventitious buds, good growth, light vitrification
SIM6	96.67±0.01a	7.4±0.4a	愈伤组织适中；不定芽分化快，长势旺盛，无玻璃化 Medium number of calli, faster differentiation of adventitious buds, vigorous, no vitrification
SIM7	84.24±0.01bc	4.36±0.1b	愈伤组织较多；不定芽分化较慢，长势良好，轻度玻璃化 Many number of calli, slow differentiation of adventitious buds, good growth, light vitrification
SIM8	88.64±0.02b	4.62±0.37b	愈伤组织较多；不定芽分化较慢，长势良好，轻度玻璃化 Many number of calli, slow differentiation of adventitious buds, good growth, light vitrification
SIM9	76.06±0.02de	4.23±0.17b	愈伤组织较多；不定分化慢，长势一般，中度玻璃化 Many number of calli, slow differentiation of adventitious buds, poor growth, moderate vitrification

表4

图8

图9

图10

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基因型 Genotype	不定芽诱导率 Induction rate of adventitious buds (%)	平均不定芽诱导数 Average number of Adventitious buds induced	愈伤组织、不定芽生长情况 Growth status of callus and adventitious buds
普18-22 Pu-18-22	83.19±0.88a	6.26±0.15a	愈伤组织数量中等，不定芽分化数量多，长势旺盛，无玻璃化 Moderate number of calli, many differentiated adventitious buds, vigorous growth, no vitrification
普28-15 Pu-28-15	76.37±0.18b	4.4±0.15b	愈伤组织数量中等，不定芽分化数量中等，生长较慢，无玻璃化，轻度褐化 Moderate number of calli, moderate number of differentiated adventitious buds, slow growth, no vitrification, light browning
普26-8 Pu-26-8	59.44±1.06cd	3.37±0.14cd	愈伤组织数量较少，不定芽分化数量较少，生长较慢，轻度玻璃化 Small number of calli, small number of differentiated adventitious buds, slow growth, light vitrification
243	42.67±1.63e	2.27±0.2e	水渍状愈伤组织过多，不定芽分化数量少，生长较慢，中度玻璃化，无褐化 Many waterlogged calli, small number of differentiated adventitious buds, slow growth, moderate vitrification, no browning
D18-8	61.83±1.99c	3.48±0.29c	愈伤组织数量中等，不定芽分化数量较少，长势中等，轻微玻璃化 Moderate number of calli, small number of differentiated adventitious buds, moderate growth, light vitrification

取材时期 Sampling period/d	不定芽诱导率 Induction rate of adventitious buds (%)	平均不定芽诱导数 Average number of adventitious buds induced	愈伤组织、不定芽生长情况 Growth status of callus and adventitious buds
30	63.59±1.16c	1.76±0.13c	愈伤组织轻度褐化，不定芽分化较慢、长势一般 Light browning of callus, slow differentiation of adventitious buds, ordinary growth
45	87.53±0.79a	3.52±0.03a	愈伤组织无褐化，不定芽分化较快、长势旺盛 No browning of callus, rapid differentiation of adventitious buds, vigorous growth
60	75.88±1.38b	2.58±0.14b	愈伤组织生长较慢，不定芽分化较慢，长势良好 Slow growth of callus, slow differentiation of adventitious buds, moderate growth

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刺槐离体叶片高效再生体系的优化

Optimization of Efficient Regeneration System of Robinia pseudoacacia Leavesin vitro

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处理 Treatment	植物生长调节剂 Plant growth regulators/(mg·L^?1)				接种数 Numbers of inoculations
处理 Treatment	2,4-D	6-BA	KT	TDZ	接种数 Numbers of inoculations
SIM1	0.1	1	0	0	80
SIM2	0.1	1	0.5	0.02	80
SIM3	0.1	1	1	0.04	80
SIM4	0.1	3	0	0.02	80
SIM5	0.1	3	0.5	0.04	80
SIM6	0.1	3	1	0	80
SIM7	0.1	5	0	0.04	80
SIM8	0.1	5	0.5	0	80
SIM9	0.1	5	1	0.02	80