红松胚性愈伤组织瞬时转化体系的建立

贺欣; 徐秀月; 亢溢敏; 杨玲

doi:10.12302/j.issn.1000-2006.202501010

红松胚性愈伤组织瞬时转化体系的建立

贺欣, 徐秀月, 亢溢敏, 杨玲

南京林业大学学报（自然科学版） ›› 2026, Vol. 50 ›› Issue (1) : 12-22.

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南京林业大学学报（自然科学版） ›› 2026, Vol. 50 ›› Issue (1) : 12-22. DOI: 10.12302/j.issn.1000-2006.202501010

专题报道(Ⅰ):发展林业新质生产力系列专题——林木体细胞胚胎发生专题(二)

红松胚性愈伤组织瞬时转化体系的建立

贺欣 ¹ ,
徐秀月 ¹ ,
亢溢敏 ¹ ,
杨玲 ¹^,²^,^*

作者信息 +

Establishment of a transient transformation system for embryogenic callus tissue of Pinus koraiensis

HE Xin ¹ ,
XU Xiuyue ¹ ,
KANG Yimin ¹ ,
YANG Ling ¹^,²^,^*

Author information +

文章历史 +

摘要

【目的】优化根癌农杆菌介导的红松(Pinus koraiensis)胚性愈伤组织瞬时转化体系。【方法】以增殖培育10 d的红松胚性愈伤组织为受体材料,利用携带RUBY的pCambia1300载体进行瞬时转化,根据RUBY的表达量筛选最佳侵染液浓度、侵染时间、共培养时间和恢复培养时间。将promoter_PkDof5_.₆-EGFP、promoter_PkSHR-RUBY、35S-PkDof5.6-EGFP、35S-PkSHR-EGFP、35S-PkSHR-RUBY载体分别转染红松胚性愈伤组织,验证瞬时转化体系的条件。【结果】瞬时转化后,RUBY表达显著。当侵染菌液浓度(以A₆₀₀表征)达到0.4、侵染10 min、共培养1 d,无恢复培养期时,RUBY片段的拷贝数最高。基因和启动子验证瞬时转化的定量及半定量结果表示,该条件可以实现红松胚性愈伤组织的高效转化。【结论】本研究成功建立了一种简单、快速、高效、低成本的根癌农杆菌介导的红松胚性愈伤组织瞬时转化体系,并优化了该体系的主要因素,得到最佳的转化条件为:侵染菌液浓度(A₆₀₀)为0.4,侵染10 min,共培养1 d,无恢复培养期。该体系对红松体胚发生机理的大规模基因功能分析具有重要意义,并可为红松良种选育及规模化扩繁提供参考。

Abstract

【Objective】 This study aims to optimize a transient transformation system for Pinus koraiensis embryogenic callus mediated by Agrobacterium tumefaciens. 【Method】Using the ten-day cultured embryonic callus of P. koraiensis as the receptor material, transient transformation was carried out with the pCambia1300 vector carrying RUBY. The optimal concentration of infection solution, infection time, co-cultivation time, and recovery culture time were screened based on the expression level of RUBY. The vectors promoter_PkDof5_.₆-EGFP, promoter_PkSHR-RUBY, 35S-PkDof5.6-EGFP, 35S-PkSHR-EGFP, and 35S-PkSHR-RUBY were respectively transfected into the embryonic callus of P. koraiensis to verify the conditions of the transient transformation system.【Result】After transient transformation, RUBY expression was significant. When the concentration of the inoculum (A₆₀₀) reached 0.4, with an infection time of ten minutes and co-cultivation for one day without recovery culture period, the copy number of the RUBY fragment was at its highest. Genes and promoters specific validation of the quantitative and semi-quantitative results of transient transformation indicates that this condition can achieve efficient transformation of mature callus from P. koraiensis. 【Conclusion】This study successfully established a simple, rapid, efficient and low-cost Agrobacterium-mediated transient transformation system for embryogenic callus of P. koraiensis, and optimized the main factors of this system. The best transformation conditions were found to be: an infection bacterial suspension concentration (A₆₀₀) of 0.4, infection for ten minutes, co-cultivation for one day, with no recovery culture period. This system is of great significance for large-scale gene functional analysis of the somatic embryogenesis mechanism in P. koraiensis and accelerates the breeding and large-scale propagation of high-quality P. koraiensis varieties.

导出引用

贺欣, 徐秀月, 亢溢敏, 等. 红松胚性愈伤组织瞬时转化体系的建立[J]. 南京林业大学学报（自然科学版）. 2026, 50(1): 12-22 https://doi.org/10.12302/j.issn.1000-2006.202501010

HE Xin, XU Xiuyue, KANG Yimin, et al. Establishment of a transient transformation system for embryogenic callus tissue of Pinus koraiensis[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 2026, 50(1): 12-22 https://doi.org/10.12302/j.issn.1000-2006.202501010

中图分类号： 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]	刘德栋. 我国红松良种选育研究进展[J]. 防护林科技, 2017, 30(3):96-99. LIU D D. Research progress on improved seed selection of Korean pine in China[J]. Protection Forest Science and Technology, 2017, 30(3):96-99. DOI:10.13601/j.issn.1005-5215.2017.03.041. 本文引用 [1]

[2]	MALEKI S S, MOHAMMADI K, JI K S. Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens[J]. Plant Cell,Tissue and Organ Culture (PCTOC), 2018, 133(3):437-445.DOI: 10.1007/s11240-018-1388-7. 本文引用 [2]

[3]	TANG W, XIAO B, FEI Y J. Slash pine genetic transformation through embryo cocultivation with A. tumefaciens and transgenic plant regeneration[J]. In Vitro Cellular & Developmental Biology-Plant, 2014, 50(2):199-209.DOI: 10.1007/s11627-013-9551-7. 本文引用 [1]

[4]	RICHARD WENCK A, QUINN M, WHETTEN R W, et al. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)[J]. Plant Molecular Biology, 1999, 39(3):407-416.DOI: 10.1023/A:1006126609534. 本文引用 [1]

[5]	HOU H, WU Y N, YANG L, et al. A method for genetic transformation using embryonic callus of Pinus koraiensis[J]. Forests, 2024, 15(12):2058.DOI: 10.3390/f15122058. 本文引用 [1]

[6]	ZHANG G, GURTU V, KAIN S R. An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells[J]. Biochemical and Biophysical Research Communications, 1996, 227(3):707-711.DOI: 10.1006/bbrc.1996.1573. 本文引用 [1]

[7]	HE Y B, ZHANG T, SUN H, et al. A reporter for noninvasively monitoring gene expression and plant transformation[J]. Horticulture Research, 2020, 7:152.DOI: 10.1038/s41438-020-00390-1. 本文引用 [2]

[8]

古文鹏, 贾森泉, 周永明, 等. 艰难梭菌tcdA和tcdB基因绝对定量PCR方法的建立及应用[J]. 中国抗生素杂志, 2025, 50(2):137-143.

W P

, Jia

S Q

, Zhou

Y M

, et al. Establishment and application of absolute quantitative PCR method for tcdA and tcdB genes of Clostridium difficile[J]. Chinese Journal of Antibiotics, 2025, 50(2):137-143.DOI:10.13461/j.cnki.cja.007840.

本文引用 [1]

[9]

李玉珠, 余江弟, 丁菲菲, 等. 植物遗传转化中体细胞再生的分子机制及应用研究进展[J]. 草业学报, 2024, 33(2):198-211.

Y Z

, YU

J D

, DING

F F

, et al. Progress in studies of molecular mechanisms and applications of somatic cell regeneration during genetic transformation[J]. Acta Prataculturae Sinica, 2024, 33(2):198-211.DOI: 10.11686/cyxb2023147.

本文引用 [1]

[10]	李玲, 顾恒, 岳远征, 等. 木本植物瞬时转化体系的研究进展[J]. 分子植物育种, 2020, 18(23):7784-7794. LI L, GU H, YUE Y Z, et al. Advances in transient transformation systems of woody plants[J]. Molecular Plant Breeding, 2020, 18(23):7784-7794.DOI: 10.13271/j.mpb.018.007784. 本文引用 [1]

[11]	SHEPHERD C T. Transient expression of GFP in immature seed tissues[J]. Methods in Molecular Biology, 2009, 526:23-28.DOI: 10.1007/978-1-59745-494-0_2. 本文引用 [1]

[12]	CANA-QUIJADA P, BEJARANO E R, LOZANO-DURÁN R, et al. Transient expression assay in NahG Arabidopsis Plants using Agrobacterium tumefaciens[J]. Bio-protocol, 2018, 8(12):e2894.DOI: 10.21769/BioProtoc.2894. 本文引用 [1]

[13]	YANG W H, REN J Q, LIU W R, et al. An efficient transient gene expression system for protein subcellular localization assay and genome editing in Citrus protoplasts[J]. Horticultural Plant Journal, 2023, 9(3):425-436.DOI: 10.1016/j.hpj.2022.06.006. 本文引用 [2]

[14]

UMEMOTO

, YASUMOTO

, YAMAZAKI

, et al. Integrated gene-free potato genome editing using transient transcription activator-like effector nucleases and regeneration-promoting gene expression by Agrobacterium infection[J]. Plant Biotechnology, 2023, 40(3):211-218.DOI: 10.5511/plantbiotechnology.23.0530a.

本文引用 [1]

[15]	GENG L, LIU C, LI F. Application of the combination of Agrobacterium-mediated transformation and particle bombardment in plant genetic transformation[J]. Acta Botanica Boreali-Occidentalia Sinica, 2005, 25(1):205-210.DOI: 10.3321/j.issn:1000-4025.2005.01.037. 本文引用 [1]

[16]	XIA X H, CHEN Y F, LI C L, et al. Effect of factors on GUS transient expression of wheat immature embryo transformated by particle bombardment[J]. Journal of Triticeae Crops, 2006, 26(2):42-45.DOI: 10.3969/j.issn.1009-1041.2006.02.009. 本文引用 [1]

[17]

俞瑶, 程华, 陈素梅, 等. 基于农杆菌真空渗透法的菊花瞬时表达系统的优化[J]. 农业生物技术学报, 2023, 31(12):2654-2664.

, CHENG

, CHEN

S M

, et al. Optimization of transient expression system in Chrysanthemum morifolium based on Agrobacterium vacuum infiltration method[J]. Journal of Agricultural Biotechnology, 2023, 31(12):2654-2664.DOI: 10.3969/j.issn.1674-7968.2023.12.019.

本文引用 [1]

[18]

杨海清, 吴凡颖, 王睿琦, 等. 桃愈伤组织和果实瞬时转化体系的优化[J]. 北京农学院学报, 2024, 39(1):13-17,57.

YANG

H Q

, WU

F Y

, WANG

R Q

, et al. Optimization of transient transformation system in peach callus and fruit[J]. Journal of Beijing University of Agriculture, 2024, 39(1):13-17,57.DOI: 10.13473/j.cnki.issn.1002-3186.2024.0103.

本文引用 [1]

[19]

闵思源, 陈柏楠, 陈康, 等. 桂花与石楠叶片中瞬时表达体系的建立试验初报[J]. 南方农业, 2020, 14(17):127-128,135.

MIN

S Y

, CHEN

B N

, CHEN

, et al. Preliminary report on the establishment of transient expression system in leaves of Osmanthus fragrans and Photinia fragrans[J]. South China Agriculture, 2020, 14(17):127-128,135.DOI: 10.19415/j.cnki.1673-890x.2020.17.061.

本文引用 [1]

[20]	田淑婷, 曹晓云, 时春莹, 等. 农杆菌介导的葡萄风信子愈伤组织遗传转化体系的优化[J]. 草地学报, 2023, 31(4):1234-1241. TIAN S T, CAO X Y, SHI C Y, et al. Optimization of Agrobacterium tumefaciens-mediated genetic transformation system based on callus of grape hyacinth[J]. Acta Agrestia Sinica, 2023, 31(4):1234-1241. 本文引用 [1]

[21]	李晓军, 安轶, 黄李超, 等. 银腺杨84K茎段瞬时转化体系的建立[J]. 林业科学, 2021, 57(4):82-89. LI X J, AN Y, HUANG L C, et al. Establishment of a transient transformation system for stem segments of poplar 84K[J]. Scientia Silvae Sinicae, 2021, 57(4):82-89.DOI: 10.11707/j.1001-7488.20210409. 本文引用 [1]

[22]	BAO R, SHI W, WANG Y. Optimization and application of transient expression system in the callus of Vitis vinifera L. cv. Thompson seedless[J]. Acta Botanica Boreali-Occidentalia Sinica, 2017, 37(4): 654-664. 本文引用 [1]

[23]	GAO F, SHI Y J, WANG R R, et al. Optimization of key technologies for induction of embryogenic callus and maturation of somatic embryos in Korean pine (Pinus koraiensis)[J]. Forests, 2023, 14(4):850.DOI: 10.3390/f14040850. 本文引用 [1]

[24]	LI G, SONG P L, WANG X, et al. Establishment of Agrobacterium tumefaciens mediated transient transformation system in young leaves of Duli pear (Pyrus betulifolia)[J]. Journal of Fruit Science, 2021, 38(11).DOI: 10.13925/j.cnki.gsxb.20210187. 本文引用 [1]

[25]	安文杰, 张永侠, 原海燕. 红籽鸢尾瞬时表达体系的建立[J]. 分子植物育种, 2020, 18(19):6359-6363. AN W J, ZHANG Y X, YUAN H Y. A system establishment of transient expression on Iris foetidissima L[J]. Molecular Plant Breeding, 2020, 18(19):6359-6363.DOI: 10.13271/j.mpb.018.006359. 本文引用 [1]

[26]	ZHANG Y J, LI Y, WANG J J, et al. Effects of three kinds of Agrobacterium and different transformation conditions on the transient expression of GFP in Nicotiana benthamiana[J]. Bulletin of Botanical Research, 2022, 42(1):121-129.DOI: 10.7525/j.issn.1673-5102.2022.01.013. 本文引用 [1]

[27]	常婧, 高行英, 李小东, 等. 农杆菌介导韭菜遗传转化相关因素的研究[J]. 生物技术通报, 2014, 30(1):125-131. CHANG J, GAO X Y, LI X D, et al. Research on the factors related to Agrobacterium-mediated genetic transformation of the Chinese chive[J]. Biotechnology Bulletin, 2014, 30(1):125-131. 本文引用 [1]

[28]	唐靖雯, 王宁, 伍程程, 等. 白花玉石籽石榴遗传转化体系的建立[J]. 果树学报, 2024, 41(12):2621-2633. TANG J W, WANG N, WU C C, et al. Establishment of genetic transformation system for ‘Baihuayushizi’ pomegranate[J]. Journal of Fruit Science, 2024, 41(12):2621-2633.DOI: 10.13925/j.cnki.gsxb.20240326. 本文引用 [1]

[29]

余晓敏, 王亚琴, 刘雨菡, 等. 根癌农杆菌介导万寿菊遗传转化体系的建立[J]. 植物学报, 2023, 58(5):760-769.

X M

, WANG

Y Q

, LIU

Y H

, et al. Establishment of Agrobacterium tumefaciens-mediated genetic transformation system of marigold(Tagetes erecta)[J]. Chinese Bulletin of Botany, 2023, 58(5):760-769.DOI: 10.11983/CBB22141.

本文引用 [2]

[30]

王冬月, 王如月, 孙茂桐, 等. ‘窄冠白杨1号’遗传转化体系建立与抗虫基因转化[J]. 植物研究, 2024, 44(3):361-369.

WANG

D Y

, WANG

R Y

, SUN

M T

, et al. Establishment of genetic transformation system for ‘Populus leucopyramidalis 1’ and transformation of insect resistance gene[J]. Bulletin of Botanical Research, 2024, 44(3):361-369.DOI: 10.7525/j.issn.1673-5102.2024.03.005.

本文引用 [2]

[31]

王梦真, 何锐杰, 邓雅婷, 等. 百香果高频再生及遗传转化体系的建立[J/OL]. 分子植物育种, 2024:1-9.( 2024-04-03). https://kns.cnki.net/KCMS/detail/detail.aspx?filename=FZZW20240329006&dbname=CJFD&dbcode=CJFQ.

https://kns.cnki.net/KCMS/detail/detail.aspx?filename=FZZW20240329006&dbname=CJFD&dbcode=CJFQ

WANG

M Z

, HE

R J

, DENG

Y T

, et al. Establishment of high-frequency in vitro regeneration and genetic transformation system in passion fruit[J/OL]. Molecular Plant Breeding, 2024:1-9.( 2024-04-03). https://kns.cnki.net/KCMS/detail/detail.aspx?filename=FZZW20240329006&dbname=CJFD&dbcode=CJFQ.

https://kns.cnki.net/KCMS/detail/detail.aspx?filename=FZZW20240329006&dbname=CJFD&dbcode=CJFQ

本文引用 [1]

[32]

何旭, 高源, 张群野, 等. 白城小黑杨遗传转化体系建立及其应用[J]. 植物研究, 2023, 43(5):667-678.

, GAO

, ZHANG

Q Y

, et al. Establishment and application of genetic transformation system for Populus simonii × P.nigra ‘Baicheng’[J]. Bulletin of Botanical Research, 2023, 43(5):667-678.DOI: 10.7525/j.issn.1673-5102.2023.05.004.

本文引用 [1]

[33]	刘炎, 刘克俭, 王江, 等. 农杆菌介导的针叶树种遗传转化研究进展[J]. 黑龙江农业科学, 2021(4):136-141. LIU Y, LIU K J, WANG J, et al. Research progress on Agrobacterium mediated genetic transformation of conifer species[J]. Heilongjiang Agricultural Sciences, 2021(4):136-141.DOI: 10.11942/j.issn1002-2767.2021.04.0136. 本文引用 [1]

[34]	LIU S W, MA J J, LIU H M, et al. An efficient system for Agrobacterium-mediated transient transformation in Pinus tabuliformis[J]. Plant Methods, 2020, 16:52.DOI: 10.1186/s13007-020-00594-5. 本文引用 [1]

[35]

李菲, 曹永琼, 王纲, 等. 根癌农杆菌介导彩色马蹄莲遗传转化体系的建立及优化[J]. 南方农业学报, 2024, 55(6):1692-1699.

, CAO

Y Q

, WANG

, et al. Establishment and optimization of Agrobacterium tumefaciens mediated genetic transformation system for Zantedeschia hybrida Spr[J]. Journal of Southern Agriculture, 2024, 55(6):1692-1699.DOI: 10.3969/j.issn.2095-1191.2024.06.014.

本文引用 [1]