复幼措施对水曲柳生长与组培再生的影响

doi:10.7525/j.issn.1673-5102.2026.01.008

摘要/Abstract

摘要：

组织培养是珍贵用材树种水曲柳（Fraxinus mandshurica）无性繁殖的重要方式之一，但年龄效应造成的外植体生理状态老化，显著降低组培效率，严重制约水曲柳优良品种的快速无性扩繁与推广应用，目前在林木中通过复幼克服年龄效应提高组培效率的研究未见报道。该文研究不同复幼措施对4年生水曲柳植株生长与组培再生的影响，结果表明：截干+遮阴处理后，新生枝的高度和径粗分别降低37.50%和42.90%，新生枝和茎段外植体数量分别增加2.6倍和2.8倍，组培再生能力显著提升。茎段外植体启动率、新芽分化率和增殖系数均升高，启动率最高达到82.42%，是未处理CK（25.30%）的3.26倍；分化率由9.90%提升到61.11%；增殖系数由0提升到6.00。进一步通过生理生化指标和相关基因表达变化探究复幼措施克服年龄效应的潜在机制，发现复幼处理后，带腋芽茎中蔗糖和可溶性糖含量分别显著减少23.17%和46.24%，而生长素（IAA）和细胞分裂素（CTK）含量分别显著增多70.01%和57.22%。基于miR156A/C基因表达随植物的成熟而下调的趋势，进一步通过miR156-SPL-AP2/ERFs-ASA1基因通路的转录分析，揭示采穗母树经过截干+遮阴处理后，复幼及芽再生相关基因FmmiR156A、FmmiR156C和FmASA1表达量较CK分别提高10.01倍、13.53倍和12.58倍，FmSPL2和FmSPL9表达量较CK分别降低93.05%和88.30%，揭示上述指标可能是促进水曲柳复幼从而提高外植体组培再生效率的关键因素。该研究为解决水曲柳等林木年龄效应问题和提高组培繁殖能力提供切实可行的方法，为水曲柳良种/新品种的规模繁育和推广提供技术支撑。

关键词: 水曲柳, 组织培养, 年龄效应, 复幼处理, 生理生化指标, 基因表达

Abstract:

As an essential method for clonal propagation of the valuable timber species Fraxinus mandshurica， tissue culture is hindered by physiological aging of explants due to age-related effects， which significantly reduces tissue culture efficiency and severely restricts the rapid clonal propagation and widespread application of superior F. mandshurica varieties. Currently， there are no reports on overcoming the age effect through rejuvenation to improve tissue culture efficiency in forest trees. This study investigated the effects of different rejuvenation treatments on growth performance and tissue culture regeneration of 4-year-old F. mandshurica plants. The results showed that after trunk cutting + shading treatment， the height and diameter of new shoots decreased by 37.50% and 42.90%， respectively， while the number of new shoots and stem segment explants increased by 2.6-fold and 2.8-fold， which significantly enhanced tissue culture regeneration capacity. The initiation rate of stem segment explants， new shoot differentiation rate， and propagation coefficient of the shoots under treatment all increased compared to the untreated control（CK）. The highest initiation rate reached 82.42%， which was 3.26 times as high as that of the CK（25.30%）. The differentiation rate increased from 9.90% to 61.11%. The propagation coefficient increased from 0 to 6.00. Further investigation into the mechanism by which rejuvenation measures overcome the age effect was conducted through the analysis of physiological and biochemical indicators and related gene expression changes. It was found that after rejuvenation treatment， sucrose and soluble sugar content significantly decreased by 23.17% and 46.24%， while the content of auxin（IAA） and cytokinin（CTK） significantly increased by 70.01% and 57.22%， respectively. Based on the trend of miR156A/C gene expression downregulating with plant maturation， transcriptional analysis of the miR156-SPL-AP2/ERFs-ASA1 gene pathway further revealed that after trunk cutting + shading treatment， the expression levels of rejuvenation and bud regeneration-related genes FmmiR156A， FmmiR156C， and FmASA1 increased by 10.01， 13.53， and 12.58 times compared to CK， while the expression levels of FmSPL2 and FmSPL9 decreased by 93.05% and 88.30% compared to CK. These findings indicated that the above indicators may be key factors promoting rejuvenation in F. mandshurica， thereby improving the tissue culture regeneration efficiency of explants. This study provided practical methods for addressing the age effect and improving tissue culture propagation capacity in F. mandshurica and other forest trees species， offering technical support for the large-scale propagation and promotion of superior varieties and new cultivars of F. mandshurica.

Key words: Fraxinus mandshurica, tissue culture, age effect, rejuvenation treatment, physiological and biochemical indicators, gene expression

中图分类号:

Q945.4

熊成明, 王巧欣, 何利明, 刘建飞, 彭广州, 胡安红, 李新宇, 詹亚光. 复幼措施对水曲柳生长与组培再生的影响[J]. 植物研究, 2026, 46(1): 91-100.

Chengming XIONG, Qiaoxin WANG, Liming HE, Jianfei LIU, Guangzhou PENG, Anhong HU, Xinyu LI, Yaguang ZHAN. Impact of Rejuvenation Measures on Growth and Tissue Culture Regeneration of Fraxinus mandshurica[J]. Bulletin of Botanical Research, 2026, 46(1): 91-100.

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参考文献 32

[1]	张鹏.不同发育阶段水曲柳种子的休眠与萌发生理［D］.哈尔滨：东北林业大学，2008.
	ZHANG P.The dormancy and germination physiology of Manchurian ash seeds in different development stage［D］.Harbin：Northeast Forestry University，2008.
[2]	刘婷，李明月，朱美如，等.不同水曲柳无性系种子休眠差异［J］.植物研究，2024，44（5）：711-720.
	LIU T， LI M Y， ZHU M R，et al.Differences in seed dormancy among different clones of Fraxinus mandshurica ［J］.Bulletin of Botanical Research，2024，44（5）：711-720.
[3]	丁一巍.水曲柳组培苗生根技术研究及根发育基因PLT表达分析［D］.哈尔滨：东北林业大学，2019.
	DING Y W.Study on shoot rooting technology in vitro and PLT gene expression analysis during root development of Fraxinus mandshurica ［D］.Harbin：Northeast Forestry University，2019.
[4]	郭长花.白杨年龄与位置效应的生理生化机制研究［D］.北京：北京林业大学，2008.
	GUO C H.Study on physiological and biochemical mechanism of age effect and position effect in white poplar［D］.Beijing：Beijing Forestry University，2008.
[5]	苏畅.基于白桦全基因组重亚硫酸盐测序（bisulfite-sequencing）的甲基化图谱分析［D］.哈尔滨：东北林业大学，2015.
	SU C.Shotgun bisulfite sequencing of the Betula platyphylla genome reveals the tree’s DNA methylation patterning［D］.Harbin：Northeast Forestry University，2015.
[6]	LIU J F， FU K Z， LI B Y，et al.A novel approach for enhancing sprouting to increase yield and its mechanism in Fraxinus mandshurica Rupr［J］.Scientia Horticulturae，2025，339：113897.
[7]	栾启福，姜景民，沈凤强，等.影响杂交松扦插成活率因素的研究［J］.中国农学通报，2011，27（16）：32-35.
	LUAN Q F， JIANG J M， SHEN F Q.Study on several factors of influencing survival rate of stem cutting of pine hybrids［J］.Chinese Agricultural Science Bulletin，2011，27（16）：32-35.
[8]	黄焱，季孔庶，汤庚国.珍珠黄杨不同扦插继代的荧光特性［J］.东北林业大学学报，2008，36（10）：9-11.
	HUANG Y， JI K S， TANG G G.Chlorophyll fluorescence parameters in different successive continuous cutting generations of Buxus sinica var. parvifolia ［J］.Journal of Northeast Forestry University，2008，36（10）：9-11.
[9]	HE L M， YAN J L， LU H，et al.Phenotypic differences of leaves and transcriptome analysis of Fraxinus mandshurica×Fraxinus sogdiana F1 variety［J］.Forests，2023，14（8）：1554.
[10]	李传胄.水曲柳物候性状高通量评价及萌动差异耐寒杂种优势形成机制研究［D］.哈尔滨：东北林业大学，2024.
	LI C Z.High-throughput evaluation of climatic traits and the formation mechanism of sprouting differential cold-tolerant hybrids in Fraxinus mandshurica ［D］.Harbin：Northeast Forestry University，2024.
[11]	ZHANG Z J， SUN Y H， LI Y.Plant rejuvenation：from phenotypes to mechanisms［J］.Plant Cell Reports，2020，39（10）：1249-1262.
[12]	张瑞娥.毛白杨不同优良无性系组培特性的比较研究［D］.杨凌：西北农林科技大学，2003.
	ZHANG R E.A comparatively study on the tissure culture characteristics of Populus tomentosa Carr. clones［D］.Yangling：Northwest A&F University，2003.
[13]	王佳佳，张明如，高磊，等.遮荫和氮素添加对芒萁光合特性与抗氧化酶活性的影响［J］.西南林业大学学报（自然科学），2019，39（6）：24-32.
	WANG J J， ZHANG M R， GAO L，et al.Effects of light intensity gradient and nitrogen addition on photosynthetic characteristics and antioxidant enzyme activities of Dicranopteris dichotoma ［J］.Journal of Southwest Forestry University （Natural Sciences），2019，39（6）：24-32.
[14]	叶洲辰，杨顺，于添化，等.复幼处理对小黑杨叶片营养成分及光合特性的影响［［J］.森林工程，2025，41（6）：1145-1155.
	YE Z C， YANG S， YU T H，et al.Effects of rejuvenation treatment on nutritional composition and photosynthetic characteristics of Populus simonii×P.nigra leaves［J］.Forest Engineering，2025，41（6）：1145-1155.
[15]	彭广州，刘建飞，王巧欣，等.循环复幼对水曲柳生长繁殖及生理的影响［J］.植物研究，2024，44（5）：721-729.
	PENG G Z， LIU J F， WANG Q X，et al.Effects of cycle rejuvenation on growth，reproduction and physiology of Fraxinus mandshurica ［J］.Bulletin of Botanical Research，2024，44（5）：721-729.
[16]	WU G， PARK M Y， CONWAY S R，et al.The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis ［J］.Cell，2009，138（4）：750-759.
[17]	YANG L， XU M L，KOO Y，et al.Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C ［J］.eLife，2013，2：e00260.
[18]	薛文辉，詹亚光，何利明，等.水曲柳复幼：微繁技术研究［J］.林业科技，2016，41（2）：4-8.
	XUE W H， ZHAN Y G， HE L M，et al.Rejuvenation and micropropagation study in Fraxinus mandschurica ［J］.Forestry Science & Technology，2016，41（2）：4-8.
[19]	BHATIA S， SINGH R.Interconversion of free sugars in relation to activities of enzymes catalyzing synthesis and cleavage of sucrose in growing stem tissues of sorghum［J］.Indian Journal of Experimental Biology，2001，39（10）：1035-1040.
[20]	YE B B， SHANG G D， PAN Y，et al.AP2/ERF transcription factors integrate age and wound signals for root regeneration［J］.The Plant Cell，2020，32（1）：226-241.
[21]	ZHANG T Q， LIAN H， TANG H B，et al.An intrinsic microRNA timer regulates progressive decline in shoot regenerative capacity in plants［J］.The Plant Cell，2015， 27（2）：349-360.
[22]	刘杰，孙宇涵，袁存权，等.林木根萌复幼方式的研究进展［J］.分子植物育种，2022，20（14）：4867-4872.
	LIU J， SUN Y H， YUAN C Q，et al.A review for rejuvenation of root-sprout in trees［J］.Molecular Plant Breeding，2022，20（14）：4867-4872.
[23]	刘建飞.水曲柳扦插繁殖配套技术及糖代谢调控年龄效应促进生根研究［D］.哈尔滨：东北林业大学，2025.
	LIU J F.Study on a comprehensive technology of cutting propagation and promoting root formation by the age effect regulation on sugar metabolism in Fraxinus mandshurica ［D］.Harbin：Northeast Forestry University，2025.
[24]	LIVNE S， WEISS D.Cytosolic activity of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1A［J］.Plant & Cell Physiology，2014，55（10）：1727-1733.
[25]	彭广州.循环复幼促进水曲柳扦插生根繁殖研究［D］.哈尔滨：东北林业大学，2024.
	PENG G Z.Study on promoting rooting propagation of Fraxinus mandshurica cutting by cyclic rejuvenation［D］.Harbin：Northeast Forestry University，2024.
[26]	杨舜垚.湿加松平茬复幼机理研究［D］.北京：北京林业大学，2022.
	YANG S Y.The mechanism of Pinus elliottii × P . caribaea rejuvenation after stem pruning［D］.Beijing：Beijing Forestry University，2022.
[27]	刘从，田甜，李珊，等.中国木本植物幼苗生长对光照强度的响应［J］.生态学报，2018，38（2）：518-527.
	LIU C， TIAN T， LI S，et al.Growth response of Chinese woody plant seedlings to different light intensities［J］.Acta Ecologica Sinica，2018，38（2）：518-527.
[28]	YU S， CAO L， ZHOU C M，et al.Sugar is an endogenous cue for juvenile-to-adult phase transition in plants［J］.eLife，2013，2：e00269.
[29]	郑万钧.中国树木志：第4卷［M］.北京：中国林业出版社，2004：4389-4404.
	ZHENG W J.Silva Sinica：Vol.4［M］.Beijing：China Forestry Publishing House，2004：4389-4404.
[30]	薛思雷，王庆成，孙欣欣，等.遮荫对水曲柳和蒙古栎光合、生长和生物量分配的影响［J］.植物研究，2012，32（3）：354-359.
	XUE S L， WANG Q C， SUN X X，et al.Effects of shading on the photosynthetic characteristics，growth，and biomass allocation in Fraxinus mandshurica and Quercus mongolica ［J］.Bulletin of Botanical Research，2012，32（3）：354-359.
[31]	孙佳音，杨逢建，庞海河，等.遮荫对南方红豆杉光合特性及生活史型影响［J］.植物研究，2007，27（4）：439-444.
	SUN J Y， YANG F J， PANG H H，et al.Effects of shading treatment on photosynthetic response and life cycle form of Taxus chinensis var. mairei ［J］.Bulletin of Botanical Research，2007，27（4）：439-444.
[32]	陈德良，陶月良，吴友贵，等.遮荫对百山祖冷杉光合特性和叶绿素荧光参数的影响［J］.核农学报，2016，30（10）：2056-2064.
	CHEN D L， TAO Y L， WU Y G，et al.Effect of shade on the photosynthetic characteristics and chlorophyll fluorescence parameters of Abies beshanzuensis M.H.Wu.［J］.Journal of Nuclear Agricultural Sciences，2016，30（10）：2056-2064.

不同复幼处理 Different rejuvenation treatments	外植体启动率 Explant initiation rate/%	芽分化率 Differentiation rate/%	分化芽数量 Number of differentiated buds	增殖系数 Propagation coefficient	隶属函数值 D	排名 Ranking
截干+遮阴 Trunk cutting+shading	82.42	61.11	5.67	6.00	0.99	1
循环3次 Three cycles of rejuvenation	82.59	63.33	6.33	6.63	0.76	2
截干 Trunk cutting	74.03	44.44	4.33	4.23	0.76	3
埋干 Stem layering for shoot regeneration	85.74	25.56	1.33	3.77	0.63	4
循环2次 Two cycles of rejuvenation	70.37	55.56	4.67	5.87	0.61	5
循环1次 One cycle of rejuvenation	56.30	34.44	2.67	4.43	0.41	6
未处理 CK	25.30	9.90	1.33	0	0.09	7