2种溲疏叶片不定芽诱导的组织培养体系构建

doi:10.11707/j.1001-7488.LYKX20230500

摘要/Abstract

摘要：

目的: 通过溲疏属植物叶片诱导不定芽，建立完整的组织培养体系，为构建溲疏属植物遗传转化体系和分子育种提供参考，也为从头驯化野生溲疏属植物种质资源的创新提供理论依据和实践指导。方法: 以2种陕西秦岭常见野生溲疏属植物（小花溲疏和钩齿溲疏）的带叶茎段作为外植体，通过初代培养、继代培养和生根培养建立其快速繁殖体系，再利用培养植株的叶片诱导愈伤组织和不定芽诱导的方式建立其叶片诱导不定芽体系。结果: 外植体最佳消毒方式为75%乙醇表面消毒10 s，0.1%升汞表面消毒3 min，其成活率为100%，污染率为0。小花溲疏丛生芽增殖最佳的培养基配方为MS + 2 mg?L^?1 6-BA，其增殖系数为2.8。钩齿溲疏丛生芽最佳培养基配方为MS + 5 mg?L^?1 6-BA，其增殖系数为4.53。生根培养最佳的培养基配比为1/2 MS + 0.8 mg?L^?1 IBA，其小花溲疏的生根系数为1.74，钩齿溲疏的生根系数为7.86。选择30天生根苗从上至下第2~4节叶片作为诱导愈伤组织的外植体材料。小花溲疏叶片诱导愈伤组织最佳的培养基配比为WPM + 1 mg?L^?1 2,4-D，其诱导率为63%，钩齿溲疏叶片诱导愈伤组织最佳的培养基配比为WPM + 0.1 mg?L^?1 2,4-D，其诱导率为70%。结论: 以小花溲疏和钩齿溲疏的叶片作为外植体，成功建立了诱导不定芽形成和植株再生体系。小花溲疏叶片不定芽分化最佳的培养基配比为MS + 2 mg?L^?1 6-BA + 0.3 mg?L^?1 IBA，钩齿溲疏叶片不定芽分化最佳的培养基配比为MS + 5 mg?L^?1 6-BA + 0.05 mg?L^?1 NAA。

关键词: 溲疏属, 不定芽, 耐荫, 灌木, 从头驯化

Abstract:

Objective: Seeds of Deutzia are small, and their survival rate is low. Because the branches of Deutzia are hollow, branch propagation is difficult. This study aims to establish a complete tissue culture system through the induction of adventitious buds from the leaves of plants in the genus Deutzia, and lay the foundation for the genetic transformation system and molecular breeding. It also provides theoretical basis and practical guidance for the innovation of de novo domestication of wild Deutzia. Method: In this study, branches with leaves of D. parviflora and D. baroniana, two common species of Deutzia in the Qinling Mountains of Shaanxi Province, were used as explants. The explants were used for primary culture, subculture, rooting culture, and then, the leaves of the cultured plantlets were used to induce callus and adventitious buds. Result: The optimal disinfection method of explants was to disinfect the surface with 75% alcohol for 10s and 0.1% mercuric chloride for 3 min. The survival rate was 100% and the contamination rate was 0. The optimal medium for D. parviflora subculture was MS + 2 mg?L^?1 6-BA, and the proliferation coefficient was 2.8. The optimal medium for D. baroniana subculture was MS + 5 mg?L^?1 6-BA^?1, and the proliferation coefficient was 4.53. The optimal medium for rooting culture was 1/2 MS + 0.8 mg?L^?1 IBA, with the proliferation coefficient of 1.74 for D. parviflora and 7.86 for D. baroniana. The explants for callus induction were the leaves on the 2–4 nodes from top to bottom of 30 day old root seedlings. The optimal medium for D. parviflora leaves to induce callus was WPM + 1 mg?L^?1 2,4-D, with the induction rate of 63%. The optimal medium for D. baroniana leaves to induce callus was WPM + 0.1 mg?L^?1 2,4-D, and the induction rate was 70%. Conclusion: This study has successfully established a regeneration system for adventitious shoot induction and plant formation using the leaves of D. parviflora and D. baroniana as explants. The optimal medium for D. parviflora adventitious bud differentiation from leaves was MS + 2 mg?L^?1 6-BA + 0.3 mg?L^?1 IBA. The optimal medium for the adventitious bud differentiation from D. baroniana leaves was MS + 5 mg?L^?1 6-BA+ 0.05 mg?L^?1 NAA.

Key words: Deutzia, adventitious bud, shade-tolerant, shrubs, de novo domestication

中图分类号:

S722.3⁺7

王亚鑫,尚鑫,郝英男,宁小艺,马跃,甘思楠,李进宇,张鑫. 2种溲疏叶片不定芽诱导的组织培养体系构建[J]. 林业科学, 2024, 60(10): 164-174.

Yaxin Wang,Xin Shang,Yingnan Hao,Xiaoyi Ning,Yue Ma,Sinan Gan,Jinyu Li,Xin Zhang. Establishment of Tissue Culture Systems for Inducing Adventitious Buds from Leaves of Two Deutzia Species[J]. Scientia Silvae Sinicae, 2024, 60(10): 164-174.

图/表 18

表1

表2

图1

表3

表4

图3

图2

图4

表5

图5

表6

表7

图6

图7

表8

图8

表9

图9

参考文献 0

	柴慈江, 赵桐琳, 史燕山, 等. 矮溲疏组培快繁中的茎芽增殖与生根培养研究. 安徽农业科学, 2010, 38 (17): 8871- 8873. doi: 10.3969/j.issn.0517-6611.2010.17.016
	Chai C J, Zhao T L, Shi Y S, et al. Studies on micro-shoot multiplication and rooting during in vitro propagation of Deutzia hybrida 'Boule'. Journal of Anhui Agricultural Sciences, 2010, 38 (17): 8871- 8873. doi: 10.3969/j.issn.0517-6611.2010.17.016
	冯润东, 孙宏刚. 八仙花组织培养繁殖技术. 北华大学学报(自然科学版), 2011, 12 (3): 350- 352. doi: 10.3969/j.issn.1009-4822.2011.03.025
	Feng R D, Sun H G. Propagation technology of tissue culture of Hydrangea macrophylla. Journal of Beihua University (Natural Science), 2011, 12 (3): 350- 352. doi: 10.3969/j.issn.1009-4822.2011.03.025
	韩　娟, 李亚鹏, 田彦挺, 等. 刺槐离体叶片高效再生体系的优化. 林业科学, 2023, 59 (4): 68- 78. doi: 10.11707/j.1001-7488.LYKX20210980
	Han J, Li Y P, Tian Y T, et al. Optimization of efficient regeneration system of Robinia pseudoacacia leaves in vitro. Scientia Silvae Sinicae, 2023, 59 (4): 68- 78. doi: 10.11707/j.1001-7488.LYKX20210980
	韩文跃, 盖帅孜, 刘增文, 等. 陕西省关中地区城市绿化灌木的景观与生态服务功能调查分析. 水土保持通报, 2023, 43 (3): 61- 68.
	Han W Y, Gai S Z, Liu Z W, et al. Ecological service function evaluation of urban greening shrubs in Guanzhong region, Shaanxi Province. Bulletin of Soil and Water Conservation, 2023, 43 (3): 61- 68.
	简六梅, 肖英杰, 严建兵. 从头驯化: 作物品种设计与培育的新方向. 遗传, 2023, 45 (9): 741- 753.
	Jian L M, Xiao Y J, Yan J B. De novo domestication: a new way for crop design and breeding. Hereditas, 2023, 45 (9): 741- 753.
	李东霞, 徐中亮, 符海泉, 等. 2, 4-D对椰枣合子胚愈伤组织诱导的影响. 分子植物育种, 2022, 20 (15): 5072- 5079.
	Li D X, Xu Z L, Fu H Q, et al. Effects of 2, 4-dichlorophenoxyacetic acid on callus induction of zygotic embryo in date palm. Molecular Plant Breeding, 2022, 20 (15): 5072- 5079.
	李奕萱, 舒　钰. 2017. 大花木绣球组织培养体系. 东北林业大学学报, 45(7): 66–68.
	Li Y X, Shu Y, 2017. Tissue culture system of Hydrangea paniculata var. grandiflora. Journal of Northeast Forestry University, 45(7): 66–68. ［in Chinese］
	刘叶飞, 赵海霞, 姜希萍, 等. 野大麦高效组培快繁及农杆菌介导的愈伤侵染体系建立. 植物学报, 2023, 58 (3): 440- 448. doi: 10.11983/CBB22221
	Liu Y F, Zhao H X, Jiang X P, et al. Establishment of Highly Efficient tissue culture and agrobacterium-mediated callus infection systems for Hordeum brevisubulatum. Chinese Bulletin of Botany, 2023, 58 (3): 440- 448. doi: 10.11983/CBB22221
	诺　敏. 2022. 大花绣球‘无尽夏’快繁体系的建立. 呼和浩特: 内蒙古农业大学.
	Nuo M, 2022. Establishment of rapid propagation system of Hydrangea macrophylla ‘Endless Summer’. Hohhot: Mongolia Agricultural University. ［in Chinese］
	祁振声, 纪惠芳. 2008. 发掘传统中药延续中华文脉—为《本草经》中的木兰、溲疏、蜀羊泉正名. 北京林业大学学报(社会科学版), 7(2): 23–34.
	Qi Z S, Ji H F, 2008. Researching Chinese traditional medicines and extending Chinese cultural context: corrected names of ‘MuLan’, ‘SouShu’, ‘ShuYangquan’ in Emperor Shen Nong’s materia medica. Journal of Beijing Forestry University (Social Sciences), 7(2): 23–34. ［in Chinese］
	史宝胜, 刘冬云, 杨新兵, 等. 2006a. 野生小花溲疏繁殖方法研究. 中国园艺学会第七届青年学术讨论会论文集: 602–605.
	Shi B S, Liu D Y, Yang X B, et al. 2006a. Study on breeding methods of wild Deutzia parviflora. Proceedings of the 7th Youth Symposium of Chinese Horticultural Society. 602–605. ［in Chinese］
	史宝胜, 刘冬云, 杨新兵, 等. 野生大花溲疏的组织培养和快速繁殖. 植物生理学通讯, 2006b, 42 (1): 68.
	Shi B S, Liu D Y, Yang X B, et al. Tissue culture and rapid propagation of the wild Deutzia grandiflora Bge. Plant Physiology Communications, 2006b, 42 (1): 68.
	孙　蕾, 邓春莉, 周胜芳, 等. 2023. 蝴蝶兰组培快繁研究进展. 分子植物育种(网络首发论文), 1–9. https://kns.cnki.net/kcms/detail/46.1068.S. 20230318.1244.002.html
	Sun L, Deng C L, Zhou S, et al. 2023. Research progress on Phalaenopsis tissue culture and rapid propagation. Molecular Plant Breeding (Online debut paper), 1-9. ［in Chinese］https://kns.cnki.net/kcms/detail/46. 1068.S.20230318.1244.002.html
	王成韬, 钟秋平, 李永成. 海南粗榧外植体灭菌方法研究. 热带作物学报, 2014, 35 (1): 110- 114. doi: 10.3969/j.issn.1000-2561.2014.01.018
	Wang C T, Zhong Q P, Li Y C. Explants sterilization conditions of Cephalotaxus mannii Hk. f. Chinese Journal of Tropical Crops, 2014, 35 (1): 110- 114. doi: 10.3969/j.issn.1000-2561.2014.01.018
	王　帆. 2022. 连翘遗传转化体系的建立. 郑州: 河南农业大学.
	Wang F. 2022. Establishment of genetic transformation system for Forsythia suspensa. Zhengzhou: Henan Agricultural University. ［in Chinese］
	王　镨, 宋家祥, 付群英. 药用植物决明的愈伤组织诱导与生长增值的培养条件研究. 中草药, 1997, (12): 739- 742. doi: 10.3321/j.issn:0253-2670.1997.12.015
	Wang P, Song J X, Fu Q Y. Studies on induction and growth of calluses of Jueming (Cassia abtusifolia). Chinese Herbal Medicine, 1997, (12): 739- 742. doi: 10.3321/j.issn:0253-2670.1997.12.015
	王亚如. 2021. 杜鹃组织培养和扦插繁殖技术研究. 长沙: 中南林业科技大学.
	Wang Y R. 2021. The research on optimization of rapid propagation of Rhododendron. Changsha: Central South University of Forestry & Technology. ［in Chinese］
	王耀瑭, 武荣花, 王利民. 2022. 培养基及光照时间对楸树组织培养的影响. 分子植物育种(网络首发论文) 1–9. https://kns.cnki.net/kcms/ detail/46.1068.S.20220916.1350.056.html
	Wang Y T, Wu R H, Wang L M. 2022. Effects of culture medium and illumination time on tissue culture of Catalpa. Molecular Plant Breeding (Online debut paper), 1-9 ［in Chinese］https://kns.cnki.net/kcms/ detail/46.1068.S.20220916.1350.056.html
	翁秋媛, 2010. 邓恩桉组培快繁技术研究. 南京: 南京林业大学.
	Weng Q Y. 2019. Bud micropropagation and plantlet-transplanting techniques for breeding cold-resistant strain of Eucalyptus dunnii. Nanjing: Nanjing Forestry University. ［in Chinese］
	谢恩俊. 2019. 中黄1号茶树组培快繁及再生体系建. 贵阳: 贵州大学.
	Xie E J. 2019. Establishment of tissue culture and regeneration system of Zhonghuang No. 1 tea tree. Guiyang: Guizhou University. ［in Chinese］
	岳建华, 董　艳, 王小画, 等. 早花百子莲叶片器官发生和胚胎发生再生体系的建立. 植物学报, 2020, 55 (5): 588- 595. doi: 10.11983/CBB20019
	Yue J H, Dong Y, Wang X H, et al. A regeneration system for organogenesis and somatic embryogenesis using leaves of Agapanthus praecox as explants. Chinese Bulletin of Botany, 2020, 55 (5): 588- 595. doi: 10.11983/CBB20019
	张华纬. 不同激素对杜鹃愈伤组织诱导及增殖的影响. 生物化工, 2022, 8 (4): 48- 51, 57. doi: 10.3969/j.issn.2096-0387.2022.04.011
	Zhang H W. Effect of different plant growth regulators on callus induction and proliferation of Rhododendron. Biological Chemical Engineering, 2022, 8 (4): 48- 51, 57. doi: 10.3969/j.issn.2096-0387.2022.04.011
	赵华燕, 卢善发, 晁瑞堂. 杨树的组织培养及其基因工程研究. 植物学通报, 2001, 18 (2): 169- 176.
	Zhao H Y, Lu S F, Chao R T. Studies on tissue culture and gene engineering of poplar. Chinese Bulletin of Botany, 2001, 18 (2): 169- 176.
	中国科学院中国植物志编辑委员会. 1995. 中国植物志(第四十一卷). 北京: 科学出版社.
	Editorial committee of Flora of China, Chinese academy of sciences. 1995. Flora of China (Vol. 41). Beijing: Science Press. ［in Chinese］
	朱晓宇, 赵　楚, 辛建攀, 等. 溲疏属种质资源研究进展及其园林应用前景分析. 中国野生植物资源, 2020, 39 (7): 69- 74. doi: 10.3969/j.issn.1006-9690.2020.07.015
	Zhu X Y, Zhao C, Xin J P, et al. Research progress of Deutzia germplasm resources and analysis of its landscape application prospect. Chinese Wild Plant Resources, 2020, 39 (7): 69- 74. doi: 10.3969/j.issn.1006-9690.2020.07.015
	Chen Z L, Debernardi J M, Dubcovsky J, et al. Recent advances in crop transformation technologies. Nature Plants, 2022, 8 (12): 1343- 1351. doi: 10.1038/s41477-022-01295-8
	Fernie A R, Yan J B. De novo domestication: an alternative route toward new crops for the future. Molecular Plant, 2019, 12 (5): 615- 631. doi: 10.1016/j.molp.2019.03.016
	Maulida D, Erfa L, Sesanti R N, Hidayat H, 2020. Induction of kopyor coconut embryogenic callus using 2.4-D and TDZ. IOP Conference Series: Earth and Environmental Science, 411(1): 012013.
	Royal Horticultural Society. 2022. RHS advice & tips on garden & indoor plants \| Plant finder & selector / RHS Gardening. [2022-05-09]. https://www.rhs.org.uk/plants/search-results?query=Deutzia.
	The Angiosperm Phylogeny Group. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society, 2016, 181 (1): 1- 20.

消毒处理 Disinfection treatment		成活率 Survival rate (%)	污染率 Contamination rate (%)
75%乙醇 75% Ethanol	0.1%升汞 0.1% Mercuric chloride	成活率 Survival rate (%)	污染率 Contamination rate (%)
30 s	9 min	20.0±10.7c	0.0±0.0c
30 s	6 min	53.3±13.3b	0.0±0.0c
30 s	3 min	86.7±9.1a	0.0±0.0c
30 s	1 min	93.3±6.7a	26.7±11.8b
10 s	9 min	86.7±9.1a	0.0±0.0c
10 s	6 min	100.0±0.0a	0.0±0.0c
10 s	3 min	100.0±0.0a	0.0±0.0c
10 s	1 min	100.0±0.0a	46.7±13.3a

培养基 Medium	6-BA/(mg?L^?1)	NAA/(mg?L^?1)	增殖系数 Proliferation coefficient
MS	1	0.1	0.20±0.03d
MS	2	0.1	0.33±0.03cd
MS	3	0.1	0.13±0.02d
MS	1	0	1.20±0.05b
MS	2	0	2.00±0.09a
MS	3	0	0.80±0.05bc

培养基 Medium	6-BA/(mg?L^?1)	NAA/(mg?L^?1)	增殖系数 Proliferation coefficient
MS	1	0	1.67±0.98b
MS	2	0	1.87±0.96b
MS	3	0	1.93±0.73b
MS	4	0	3.13±2.53ab
MS	5	0	4.53±3.56a

培养基 Medium	6-BA/ (mg?L^?1)	NAA/ (mg?L^?1)	IBA/ (mg?L^?1)	生根系数 Rooting coefficient	生根状态 State of rooting
MS	1	0.1	0	1.74±0.23a	粗且多 Thick and numerous
1/2MS	0	0	0.8	0.45±0.09b	细且少 Thin and few

培养基 Medium	IBA/(mg?L^?1)	生根系数 Rooting coefficient	生根状态 State of rooting
1/2MS	0.4	3.43±1.57b	细且少 Thin and few
1/2MS	0.6	4.00±1.29ab	细且少 Thin and few
1/2MS	0.8	7.86±5.98a	粗且多 Thick and numerous
1/2MS	1.0	5.00±1.00ab	细且少 Thin and few
1/2MS	1.2	4.00±1.28ab	细且少 Thin and few