抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究

柯欣; 费琪; 夏馨蕊; 叶建仁; 朱丽华

doi:10.12302/j.issn.1000-2006.202312024

柯欣, 费琪, 夏馨蕊, 叶建仁, 朱丽华

南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (1) : 87-94.

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南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (1) : 87-94. DOI: 10.12302/j.issn.1000-2006.202312024

研究论文

抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究

作者信息 +

The factors influencing the embryogenic callus initiation and somatic embryo yield in Pinus elliottii resistant to pine needle brown spot disease

Author information +

文章历史 +

摘要

【目的】建立和优化抗性湿地松(Pinus elliottii)体胚发生体系,为湿地松优良抗性材料的大规模快速繁育奠定基础。【方法】以抗性湿地松4个家系的未成熟合子胚为材料,探究不同家系对胚性愈伤组织诱导的影响;选取增殖效果好、具有胚性胚柄细胞团(ESM)的胚性愈伤组织开展体胚成熟试验,从基因型、植物磺肽素(phytosulfokine,PSK)浓度及继代次数方面对体胚成熟条件进行研究。【结果】抗性湿地松4个家系之间胚性愈伤组织诱导率差异显著(P<0.05),其中,30号家系诱导率最高,为(29.8±5.6)%;32号家系诱导率最低,仅为(7.3±3.4)%,最终共获得37个可稳定增殖的胚性细胞系。35个供试细胞系中25.7%的细胞系可产生体胚,其中2007-3和2007-5成熟体胚产量显著高于其他细胞系,成熟子叶型胚数量平均分别为(91.3±7.0)和(89.8±8.4)个/mL,其余74.3%的细胞系未产生体胚。添加PSK对抗性湿地松体胚产量具有显著影响,其质量浓度为1 mg/L时,可显著提高体胚产量。随着继代次数的增加,成熟体胚数量呈下降趋势。其中细胞系2007-3成熟体胚数量在继代8次后逐渐下降,但第20代仍保持胚性;2007-5的成熟体胚数量随继代次数增多而下降,直至20代完全丧失胚性。【结论】家系对胚性愈伤组织的诱导影响显著。基因型和PSK含量可显著影响抗性湿地松体胚产量,同时胚性愈伤组织的胚性随继代次数的增加而降低。本研究优化了抗性湿地松体胚发生技术体系,可为湿地松优良基因型体胚繁育提供技术支撑。

Abstract

【Objective】The somatic embryogenesis induction system of resistant Pinus elliottii is established and optimized to provide technical support for the large-scale rapid propagation of high-resistant materials of P. elliottii. 【Method】 The immature zygotic embryos from four families of P. elliottii are used to explore the effects of different families on the embryogenic callus initiation rate. The embryogenic callus with good proliferation rate and generating large quantity of embryo suspensor mass is selected to test somatic embryo maturation. The maturation conditions of somatic embryos are optimized based on genotype, phytosulfokine (PSK) concentration, and subculture times. 【Result】A significant difference is observed in the initiation frequencies of embryogenic callus among the four families (P < 0.05). Family No. 30 has the highest initiation rate (29.8 ± 5.6)%, while family No. 32 has the lowest (7.3 ± 3.4)%. A total of 37 embryogenic cell lines with stable proliferation are obtained. Of these, 25.7% of 35 cell lines produce matured somatic embryos, and the yields (number of somatic embryos) in 2007-3 and 2007-5 are significantly higher than that of other cell lines, with an average yield of (91.3 ± 7.0) and (89.8 ± 8.4) cotyledonary somatic embryos per mL, respectively, while the remaining 74.3% does not produce any somatic embryos. The addition of PSK significantly affects the yield of somatic embryos of resistant P. elliottii, and embryo production is considerably increased when the PSK concentration is 1 mg/L. The number of mature somatic embryos decreases with the increase in subcultures. The number of mature somatic embryos of cell lines 2007-3 gradually decreases after eight subcultures but maintains embryogenic competence in the 20^th subculture. For cell lines 2007-5, the number of mature somatic embryos reduces as subcultures rise, ultimately losing maturation capacity after 20 subcultures. 【Conclusion】The initiation of embryogenic callus is greatly influenced by the genotype. Genotype and PSK concentration significantly affect the somatic embryo yield of resistant P. elliottii. The yield of somatic embryos decreases with the increase in subcultures. The somatic embryogenesis technology system of resistant P. elliottii is optimized to provide technical support for the somatic embryogenesis of excellent genotypes of P. elliottii.

导出引用

柯欣, 费琪, 夏馨蕊, 等. 抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究[J]. 南京林业大学学报（自然科学版）. 2025, 49(1): 87-94 https://doi.org/10.12302/j.issn.1000-2006.202312024

KE Xin, FEI Qi, XIA Xinrui, et al. The factors influencing the embryogenic callus initiation and somatic embryo yield in Pinus elliottii resistant to pine needle brown spot disease[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2025, 49(1): 87-94 https://doi.org/10.12302/j.issn.1000-2006.202312024

中图分类号： Q943;S722

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	李传道, 韩政敏, 叶建仁, 等. 松针褐斑病在湿地松幼林中的发展[J]. 南京林业大学学报(自然科学版), 1987, 11(1):1-7. LI C D, HAN Z M, YE J R, et al. Development of brown-spot needle blight in slash pine plantations[J]. J Nanjing For Univ(Nat Sci Ed), 1987, 11(1):1-7.DOI: 10.3969/j.jssn.1000-2006.1987.01.001. 本文引用 [1]

[2]	徐康, 程强强, 杨春霞, 等. 速生湿地松良种胚性愈伤组织诱导与增殖[J]. 广西植物, 2021, 41(2):283-291. XU K, CHENG Q Q, YANG C X, et al. Induction and proliferation of embryogenic callus on improved varieties of fast-growing Pinus elliottii[J]. Guihaia, 2021, 41(2):283-291.DOI: 10.11931/guihaia.gxzw201908048. 本文引用 [2]

[3]	叶建仁, 李传道. 我国湿地松抗松针褐斑病研究进展[J]. 林业科学研究, 1996, 9(2):189-195. YE J R, LI C D. Research progress of Pinus elliottii resistance to pine needle brown spot in China[J]. For Res, 1996, 9(2):189-195. 本文引用 [2]

[4]	季孔庶, 王潘潘, 王金铃, 等. 松科树种的离体培养研究进展[J]. 南京林业大学学报(自然科学版), 2015, 39(1):142-148. JI K S, WANG P P, WANG J L, et al. Review on in vitro culture of tree species in Pinaceae[J]. J Nanjing For Univ (Nat Sci Ed), 2015, 39(1):142-148.DOI: 10.3969/j.issn.1000-2006.2015.01.001. 本文引用 [1]

[5]	季孔庶, 王章荣, 王明庥, 等. 针叶树种扦插繁殖的研究进展及其对策[J]. 世界林业研究, 1996, 9(4):17-22. JI K S, WANG Z R, WANG M X, et al. Research progress and countermeasures of cutting propagation of coniferous species[J]. World For Res, 1996, 9(4):17-22. 本文引用 [1]

[6]	黄健秋, 卫志明. 针叶树体细胞胚胎发生的研究进展[J]. 植物生理学通讯, 1995(2):85-90. HUANG J Q, WEI Z M. Advances in somatic embryogenesis of conifers[J]. Plant Physiol Commun, 1995(2):85-90. 本文引用 [1]

[7]	胡继文, 郭文冰, 邓乐平, 等. 松树体细胞胚胎发生技术的发展与应用[J]. 林业与环境科学, 2018, 34(4):152-161. HU J W, GUO W B, DENG L P, et al. The development and application on pine somatic embryogenesis[J]. For Environ Sci, 2018, 34(4):152-161.DOI: 10.3969/j.issn.1006-4427.2018.04.026. 本文引用 [1]

[8]	孙志强, 孙占育, 席梦利. 针叶树体细胞胚胎发生研究进展[J]. 林业科技开发, 2010, 24(4):1-5. SUN Z Q, SUN Z Y, XI M L. Review on somatic embryogenesis of coniferous tree[J]. J For Eng, 2010, 24(4):1-5.DOI: 10.3969/j.issn.1000-8101.2010.04.001. 本文引用 [1]

[9]	CHEN Y M, FEI Q, XIA X R, et al. Pinus massoniana somatic embryo maturation,mycorrhization of regenerated plantlets and its resistance to Bursaphelenchus xylophilus[J]. Front Plant Sci, 2023, 14:1130471.DOI: 10.3389/fpls.2023.1130471. 本文引用 [1]

[10]	JAIN M S, DONG N, NEWTON R J. Somatic embryogenesis in slash pine (Pinus elliottii) from immature embryos cultured in vitro[J]. Plant Sci, 1989,65(2):233-241.DOI: 10.1016/0168-9452(89)90070-8. 本文引用 [1]

[11]	LIAO Y K, AMERSON H V. Slash pine (Pinus elliottii Engelm.) somatic embryogenesis I.Initiation of embryogenic cultures from immature zygotic embryos[J]. N For, 1995, 10(2):145-163.DOI: 10.1007/BF00033404. 本文引用 [1]

[12]	吴丽君, 翁秋媛, 陈达. 湿地松体胚发育成熟的影响因子研究[J]. 福建农业学报, 2013, 28(4):372-376. WU L J, WENG Q Y, CHEN D. Factors affecting maturation of somatic embryos of slash pine[J]. Fujian J Agric Sci, 2013, 28(4):372-376.DOI: 10.19303/j.issn.1008-0384.2013.04.014. 本文引用 [1]

[13]	方珞, 吴小芹. 抗松针褐斑病湿地松体细胞的悬浮培养[J]. 生物技术通报, 2019, 35(3):13-18. FANG L, WU X Q. Suspension culture of somatic cells of Pinus elliottii against brown spot needle blight of pine[J]. Biotechnol Bull, 2019, 35(3):13-18.DOI: 10.13560/j.cnki.biotech.bull.1985.2018-0694. 本文引用 [1]

[14]	YANG F, XIA X R, KE X, et al. Somatic embryogenesis in slash pine (Pinus elliottii Engelm):improving initiation of embryogenic tissues and maturation of somatic embryos[J]. Plant Cell Tissue Organ Cult (PCTOC), 2020, 143(1):159-171.DOI: 10.1007/s11240-020-01905-3. 本文引用 [2]

[15]	LELU-WALTER M A, BERNIER-CARDOU M, KLIMASZEWSKA K. Clonal plant production from self- and cross-pollinated seed families of Pinus sylvestris (L.) through somatic embryogenesis[J]. Plant Cell Tissue Organ Cult(PCTOC), 2008, 92(1):31-45.DOI: 10.1007/s11240-007-9300-x. 本文引用 [1]

[16]	蒋菁, 熊发前, 唐秀梅, 等. 赤霉素、光照及基因型对花生体细胞胚诱导和植株再生的影响[J]. 南方农业学报, 2013, 44(6):903-908. JIANG J, XIONG F Q, TANG X M, et al. Effect of gibberellin,light and genotype on peanut somatic embryogenesis and plantlet regeneration[J]. J South Agric, 2013, 44(6):903-908. 本文引用 [1]

[17]	MATSUBAYASHI Y, SAKAGAMI Y. Phytosulfokine,sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L[J]. Proc Natl Acad Sci USA, 1996, 93(15):7623-7627.DOI: 10.1073/pnas.93.15.7623. 本文引用 [1]

[18]	MATSUBAYASHI Y, TAKAGI L, SAKAGAMI Y. Phytosulfokine-alpha,a sulfated pentapeptide,stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites[J]. Proc Natl Acad Sci USA, 1997, 94(24):13357-13362.DOI: 10.1073/pnas.94.24.13357. 本文引用 [1]

[19]	IGASAKI T, AKASHI N, UJINO-IHARA T, et al. Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica[J]. Plant Cell Physiol, 2003, 44(12):1412-1416.DOI: 10.1093/pcp/pcg161. 本文引用 [4]

[20]	陈新建, 杨艳会, 陈军营, 等. 植物新型肽类生长调节物质:植物磺肽素[J]. 植物生理学通讯, 2005, 41(5):669-673. CHEN X J, YANG Y H, CHEN J Y, et al. A new peptide plant growth regulation substance,phytosulfokine (PSK)[J]. Plant Physiol J, 2005, 41(5):669-673. 本文引用 [2]

[21]	MATSUBAYASHI Y, MORITA A, MATSUNAGA E, et al. Physiological relationships between auxin,cytokinin,and a peptide growth factor,phytosulfokine-αin stimulation of asparagus cell proliferation[J]. Planta, 1999, 207(4):559-565.DOI: 10.1007/s004250050518. 本文引用 [2]

[22]	陈金慧, 张艳娟, 吴亚云, 等. 植物磺肽素在杂交鹅掌楸体胚发生中的作用[J]. 林业科学, 2013, 49(2):33-38. CHEN J H, ZHANG Y J, WU Y Y, et al. Effects of phytosulfokine on the somatic embryogenesis of Liriodendron hybrids(L.chinense × L.tulipifera)[J]. Sci Silvae Sin, 2013, 49(2):33-38.DOI: 10.11707/j.1001-7488.20130206. 本文引用 [1]

[23]	刘玲梅, 汤浩茹, 刘娟. 试管苗长期继代培养中的形态发生能力与遗传稳定性[J]. 生物技术通报, 2008(5):22-27. LIU L M, TANG H R, LIU J. Morphogenetic capacity and genetic stability of tissue in vitro cultures in long-term subculturing[J]. Biotechnol Bull, 2008(5):22-27. 本文引用 [1]

[24]

薛美凤, 郭余龙, 李名扬, 等. 长期继代对棉花胚性愈伤组织体胚发生能力及再生植株变异的影响[J]. 西南农业学报, 2002, 15(4):19-21.

XUE

M F

, GUO

Y L

, LI

M Y

, et al. Effects of long-term subculture on embryogenesis capability of cotton callus and somatic variation[J]. Southwest China J Agric Sci, 2002, 15(4):19-21.DOI: 10.16213/j.cnki.scjas.2002.04.005.

本文引用 [1]

[25]

TRONTIN

, REYMOND

, QUONIOU

, et al. An overview of current achievements and shortcomings in developing Maritime pine somatic embryogenesis and enabling technologies in France[C]// PARK Y S, BONGA J M, PARK S Y.Advances in somatic embryogenesis of trees and its application for the future forests plantations. Suwon:1st IUFRO2.09.02 Conference, 2010.

本文引用 [2]

[26]	苏秀城. 杉木无性系不同继代代数组培苗差异研究[J]. 福建林学院学报, 2000(4):353-356. SU X C. Study on the differences of the seedling of different generations from successive tissue culture of Chinese fir clone[J]. J Fujian Coll For, 2000(4):353-356. 本文引用 [1]

[27]

王艳丽, 孙婷玉, 沈李元, 等. 继代培养时间对抗性黑松体胚发生的影响[J]. 西南林业大学学报(自然科学), 2019, 39(2):78-85.

WANG

Y L

, SUN

T Y

, SHEN

L Y

, et al. Effects of subculture time on somatic embryogenesis of nematode-resistant Pinus thunbergii[J]. J Southwest For Univ (Nat Sci), 2019, 39(2):78-85.DOI: 10.11929/j.swfu.201808046.

本文引用 [1]

[28]

张彩云, 朱丽华, 谈家金, 等. 抗松针褐斑病湿地松体细胞胚胎发生与植株再生[J]. 东北林业大学学报, 2016, 44(6):17-22.

ZHANG

C Y

, ZHU

L H

, TAN

J J

, et al. Somatic embryogenesis and plantlet regeneration of disease-resistant slash pine(Pinus elliottii Engelm.) to brown spot needle blight[J]. J Northeast For Univ, 2016, 44(6):17-22.DOI: 10.13759/j.cnki.dlxb.20160510.011.

本文引用 [1]

[29]	FILONOVA L H, BOZHKOV P V, VON ARNOLD S. Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking[J]. J Exp Bot, 2000, 51(343):249-264.DOI: 10.1093/jexbot/51.343.249. 本文引用 [2]

[30]	PINTO G, SILVA S, PARK Y S, et al. Factors influencing somatic embryogenesis induction in Eucalyptus globulus Labill.:basal medium and anti-browning agents[J]. Plant Cell Tissue Organ Cult(PCTOC), 2008, 95(1):79-88.DOI: 10.1007/s11240-008-9418-5. 本文引用 [1]

[31]	胡继文, 郭文冰, 邓乐平, 等. 湿地松及其杂种的体细胞胚胎发生与植株再生[J]. 华南农业大学学报, 2019, 40(1):107-115. HU J W, GUO W B, DENG L P, et al. Somatic embryogenesis and plantlet regeneration in Pinus elliottii and its hybrids[J]. J South China Agric Univ, 2019, 40(1):107-115.DOI: 10.7671/j.issn.1001-411X.201804001. 本文引用 [1]

[32]	PULLMAN G S, JOHNSON S. Somatic embryogenesis in loblolly pine (Pinus taeda L.):improving cultureinitiation rates[J]. Ann For Sci, 2002, 59(5/6):663-668.DOI: 10.1051/forest:2002053. 本文引用 [1]

[33]	MIGUEL C, GONÇALVES S, TERESO S, et al. Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine[J]. Plant Cell Tissue Organ Cult(PCTOC), 2004, 76(2):121-130.DOI: 10.1023/B:TICU.0000007253.91771.e3. 本文引用 [1]

[34]	陈士刚, 秦彩云, 王聪慧, 等. 红松5个家系未成熟种子的胚性愈伤组织诱导研究[J]. 森林工程, 2021, 37(1):13-17. CHEN S G, QIN C Y, WANG C H, et al. Embryogenic callus induction of Korean pine immature seeds from five families[J]. For Eng, 2021, 37(1):13-17.DOI: 10.16270/j.cnki.slgc.2021.01.003. 本文引用 [1]

[35]

程子珊, 易敏, 宋才玲, 等. 湿地松体细胞胚胎发生胚性愈伤组织诱导条件优化[J]. 江西农业大学学报, 2021, 43(5):1054-1064.

CHENG

Z S

, YI

, SONG

C L

, et al. Optimization of embryogenic callus induction conditions for somatic embryogenesis of Pinus elliottii[J]. Acta Agric Univ Jiangxiensis, 2021, 43(5):1054-1064.DOI: 10.13836/j.jjau.2021114.

本文引用 [1]

[36]	DÍAZ-SALA C. Molecular dissection of the regenerative capacity of forest tree species:special focus on conifers[J]. Front Plant Sci, 2019, 9:1943.DOI: 10.3389/fpls.2018.01943. 本文引用 [1]

[37]	MALABADI R B, NATARAJA K. Influence of triacontanol on somatic embryogenesis of Pinus roxburghii Sarg[J]. Balt For, 2007, 13(1):39-44. 本文引用 [1]

[38]	CARNEROS E, CELESTINO C, KLIMASZEWSKA K, et al. Plant regeneration in Stone pine (Pinus pinea L.) by somatic embryogenesis[J]. Plant Cell Tissue Organ Cult (PCTOC), 2009, 98(2):165-178.DOI: 10.1007/s11240-009-9549-3. 本文引用 [1]

[39]	KIEŁKOWSKA A, ADAMUS A. Peptide growth factor phytosulfokine-α stimulates cell divisions and enhances regeneration from B.oleracea var. capitata L.protoplast culture[J]. J Plant Growth Regul, 2019, 38(3):931-944.DOI: 10.1007/s00344-018-9903-y. 本文引用 [1]

[40]	ASIF M, EUDES F, RANDHAWA H, et al. Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat[J]. Plant Cell Tissue Organ Cult (PCTOC), 2014, 116(1):125-130.DOI: 10.1007/s11240-013-0379-y. 本文引用 [1]

[41]	PEREIRA C, MONTALBÁN I A, PEDROSA A, et al. Regeneration of Pinus halepensis (Mill.) through organogenesis from apical shoot buds[J]. Forests, 2021, 12(3):363.DOI: 10.3390/f12030363. 本文引用 [1]

[42]	BRETON D, HARVENGT L, TRONTIN J F, et al. Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine[J]. Plant Cell Tissue Organ Cult(PCTOC), 2006, 87(1):95-108.DOI: 10.1007/s11240-006-9144-9. 本文引用 [2]

[43]	XIA X R, YANG F, KE X, et al. Somatic embryogenesis of Masson pine (Pinus massoniana):initiation,maturation and genetic stability analysis at SSR loci[J]. Plant Cell Tissue Organ Cult (PCTOC), 2021, 145(3):667-677.DOI: 10.1007/s11240-021-02036-z. 本文引用 [1]

[44]	FU C H, LI L Q, WU W J, et al. Assessment of genetic and epigenetic variation during long-term Taxus cell culture[J]. Plant Cell Rep, 2012, 31(7):1321-1331.DOI: 10.1007/s00299-012-1251-y. 本文引用 [1]

[45]	LI Q F, DENG C, ZHU T Q, et al. Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana[J]. Trees, 2019, 33(2):469-480.DOI: 10.1007/s00468-018-1793-x. 本文引用 [1]

[46]

PASSAMANI

L Z

, REIS

R S

, VALE

E M

, et al. Long-term culture with 2,4-dichlorophenoxyacetic acid affects embryogenic competence in sugarcane callus via changes in starch,polyamine and protein profiles[J]. Plant Cell Tissue Organ Cult (PCTOC), 2020, 140(2):415-429.DOI: 10.1007/s11240-019-01737-w.

本文引用 [1]