Effects of Exogenous Flavonoids on Embryogenic Callus Proliferation and Somatic Embryogenesis of Korean Pine

doi:10.7525/j.issn.1673-5102.2025.01.014

Abstract

Abstract:

Flavonoids play important roles in plant somatic embryogenesis. In order to explore the effect of flavonoids on somatic embryogenesis of Korean pine（Pinus koraiensis）， the embryogenic calluses were used as materials. The proliferation rate of embryogenic callus and the number of somatic embryos， and oxidative stress indexes in embryogenic callus were assessed after adding flavonoids（quercetin， naringenin， catechin） in different concentrations and phenylalanine aminolytic lyase inhibitor（2-aminoindan-2-phosphonate hydrochloride）. The results showed that the proliferation rate of embryogenic callus was significantly decreased with increasing levels of flavonoids， while the number of somatic embryos induced increased first then decreased， and the number of somatic embryos reached the peak under the treatment of exogenous flavonoids at 20 μmol?L^-1. After treatment with flavonoids， the total reactive oxygen species（ROS） level， hydrogen peroxide（H₂O₂） content and malondialdehyde（MDA） content were reduced in embryogenic callus； the superoxide dismutase（SOD） activity was increased； the catalase（CAT）， peroxidase（POD）， ascorbate peroxidase（APX） and glutathione peroxidase（GPX） activities were decreased. In summary， exogenous flavonoids might regulate cell proliferation and callus differentiation by affecting the level of oxidative stress indexes in embryogenic callus， hence， promote the formation of somatic embryos of Korean pine.

Key words: Pinus koraiensis, embryogenic callus, flavonoids, somatic embryogenesis, oxidative stress index

CLC Number:

S722.8

Wenhui GUO, Yue WANG, Lin WU, Jianfei YANG, Ling YANG, Peng ZHANG, Haibo WU, Hailong SHEN. Effects of Exogenous Flavonoids on Embryogenic Callus Proliferation and Somatic Embryogenesis of Korean Pine[J]. Bulletin of Botanical Research, 2025, 45(1): 130-138.

Figures/Tables 5

Fig.1

Fig.2

Fig.3

Fig.4

Fig. 5

References 31

1	高芳，沈海龙，刘春苹，等.红松成熟胚愈伤组织诱导外植体选择及培养条件优化［J］.南京林业大学学报（自然科学版），2017，41（3）：43-50.
	GAO F， SHEN H L， LIU C P，et al.Optimization of culture conditions and selection of suitable explants for callus induction from mature embryo of Pinus koraiensis ［J］.Journal of Nanjing Forestry University（Natural Science Edition），2017，41（3）：43-50.
2	LELU-WALTER M A， THOMPSON D， HARVENGT L，et al.Somatic embryogenesis in forestry with a focus on Europe：state-of-the-art，benefits，challenges and future direction［J］.Tree Genetics & Genomes，2013，9（4）：883-899.
3	周雪燕，高海燕，李召珉，等.基于生长与结实评价红松种子园亲本［J］.植物研究，2020，40（3）：376-385.
	ZHOU X Y， GAO H Y， LI Z M，et al.Evaluating parents of Pinus koraiensis seeds orchard with growth and fruiting［J］.Bulletin of Botanical Research，2020，40（3）：376-385.
4	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］.Forest Engineering，2021，37（1）：13-17.
5	闫平玉，王鹏，杨维满，等.红松种子园结实性状分析及优良无性系选择［J］.森林工程，2020，36（6）：19-29.
	YAN P Y， WANG P， YANG W M，et al.Analysis of seeding characters of Korean pine seed orchard and selection of excellent clones［J］.Forest Engineering，2020，36（6）：19-29.
6	PENG C X， GAO F， WANG H，et al.Physiological and biochemical traits in Korean pine somatic embryogenesis［J］.Forests，2020，11（5）：577.
7	GAO F， PENG C X， WANG H，et al.Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis ［J］.Journal of Forestry Research，2021，32：483-491.
8	任毓辉，聂帅，彭春雪，等.红松胚性愈伤组织增殖的激素配比、糖源类型和增殖周期效应研究［J］.植物研究，2022，42（4）：704-712.
	REN Y H， NIE S， PENG C X，et al.Effects of hormone combinations，carbon source type and proliferation cycle on embryogenic callus proliferation of Pinus koraiensis ［J］.Bulletin of Botanical Research，2022，42（4）：704-712.
9	PENG C X， GAO F， WANG H，et al.Optimization of maturation process for somatic embryo production and cryopreservation of embryogenic tissue in Pinus koraiensis ［J］.Plant Cell，Tissue and Organ Culture，2021，144（1）：185-194.
10	PENG C X， GAO F， WANG H，et al.Suspension culture and somatic embryogenesis of Korean pine［J］.Phyton-International Journal of Experimental Botany，2022，91（1）：223-238.
11	GAO F， PENG C X， WANG H，et al.Key techniques for somatic embryogenesis and plant regeneration of Pinus koraiensis ［J］.Forests，2020，11（9）：912.
12	NIE S， YAN Y， WANG Y，et al.Proper doses of brassinolide enhance somatic embryogenesis in different competent Korean pine cell lines during embryogenic callus differentiation［J］.Frontiers in Plant Science，2024，15：1330103.
13	JOYNER P M.Protein adducts and protein oxidation as molecular mechanisms of flavonoid bioactivity［J］.Molecules，2021，26（16）：5102.
14	SAJID M， CHANNAKESAVULA C N， STONE S R，et al.Synthetic biology towards improved flavonoid pharmacokinetics［J］.Biomolecules，2021，11（5）：754.
15	吕朝耕，王升，杨婉珍，等.2-氨基磷酸茚处理对新疆紫草悬浮细胞次生代谢物积累的影响［J］.中国中药杂志，2017，42（8）：1566-1571.
	LÜ C G， WANG S， YANG W Z，et al.Secondary metabolic responses to treatment of 2-aminoindan-2-phosphonic acid in cell lines from Arnebia euchroma ［J］.China Journal of Chinese Materia Medica，2017，42（8）：1566-1571.
16	MAXIMOVA S N， FLOREZ S， SHEN X L，et al.Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L.，the chocolate tree［J］.BMC Plant Biol，2014，14（1）：185.
17	PENG C X， GAO F， TRETYAKOVA I N，et al.Transcriptomic and metabolomic analysis of Korean pine cell lines with different somatic embryogenic potential［J］.International Journal of Molecular Sciences，2022，23（21）：13301.
18	IMIN N， GOFFARD N， NIZAMIDIN M，et al.Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures［J］.BMC Plant Biology，2008，8（1）：110.
19	GUO H H， GUO H X， ZHANG L，et al.Metabolome and transcriptome association analysis reveals dynamic regulation of purine metabolism and flavonoid synthesis in transdifferentiation during somatic embryogenesis in cotton［J］.International Journal of Molecular Sciences，2019，20（9）：2070.
20	WANG L C， LIU N， WANG T Y，et al.The GhmiR157a–GhSPL10 regulatory module controls initial cellular dedifferentiation and callus proliferation in cotton by modulating ethylene-mediated flavonoid biosynthesis［J］.Journal of Experimental Botany，2018，69（5）：1081-1093.
21	DOWNEY C D， ZOŃ J， JONES A M P.Improving callus regeneration of Miscanthus × giganteus J.M.Greef，Deuter ex Hodk.，Renvoize‘M161’callus by inhibition of the phenylpropanoid biosynthetic pathway［J］.In Vitro Cellular & Developmental Biology-Plant，2019，55（1）：109-120.
22	BAMNESHIN M， MIRJALILI M H， NAGHAVI M R，et al.Gene expression pattern and taxane biosynthesis in a cell suspension culture of Taxus baccata L.subjected to light and a phenylalanine ammonia lyase（PAL） inhibitor［J］.Journal of Photochemistry and Photobiology B：Biology，2022，234（14）：112532.
23	谢添伊.外源抗坏血酸和过氧化氢调控水曲柳褐化和体胚发生机制研究［D］.哈尔滨：东北林业大学，2021.
	XIE T Y.Mechanism of exogenous ascorbic acid and hydrogen peroxide regulating browning and somatic embryogenesis of Fraxinus mandshurica ［D］.Harbin：Northeast Forestry University，2021.
24	徐松华.逆境条件下植物体内活性氧代谢研究进展［J］.安徽农学通报，2021，27（21）：29-32.
	XU S H.Research advances of reactive oxygen species in plants under environmental stress［J］.Anhui Agricultural Science Bulletin，2021，27（21）：29-32.
25	LEWIS D R， RAMIREZ M V， MILLER N D，et al.Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks［J］.Plant Physiology，2011，156（1）：144-164.
26	夏凯丽，刘婷，杨静慧，等.黑莓体细胞胚诱导中的细胞生理特性［J］.中国南方果树，2018，47（1）：114-118.
	XIA K L， LIU T， YANG J H，et al.Cellular physiological properties in blackberry somatic embryo induction［J］.South China Fruits，2018，47（1）：114-118.
27	任宁，邹梦雯，陈冠群，等.不同成胚能力的百子莲愈伤组织中氧化还原水平的差异分析［J］.分子植物育种，2022，20（4）：1281-1288.
	REN N， ZOU M W， CHEN G Q，et al.Difference analysis of redox levels in the callus of Agapanthus praecox ssp. orientalis with different embryogenic ability［J］.Molecular Plant Breeding，2022，20（4）：1281-1288.
28	WANG Y， CAO S L， SUI X Y，et al.Genome-wide characterization，evolution，and expression analysis of the ascorbate peroxidase and glutathione peroxidase gene families in response to cold and osmotic stress in Ammopiptanthus nanus ［J］.Journal of Plant Growth Regulation，2023，42（1）：502-522.
29	LI S C.Novel insight into functions of ascorbate peroxidase in higher plants：more than a simple antioxidant enzyme［J］.Redox Biology，2023，64：102789.
30	周小华，李昆志，赵峥，等.外源抗坏血酸对水稻抗铝生理指标的影响［J］.热带作物学报，2021，42（3）：769-776.
	ZHOU X H， LI K Z， ZHAO Z，et al.Effects of exogenous ascorbic acid on physiological indexes in rice under aluminum stress［J］.Journal of Tropical Crops，2021，42（3）：769-776.
31	GAO F， WANG R R， SHI Y J，et al.Reactive oxygen metabolism in the proliferation of Korean pine embryogenic callus cells promoted by exogenous GSH［J］.Scientific Reports，2023，13（1）：2218.

[1]	Hairui WEN, Yuchun YANG, Yue LIU, Chunjing JIAO, Jun WANG, Fang WANG, Yelei YANG, Yitong LI, Jie WANG. Responses of Non-structural Carbohydrates and Stoichiometric Characteristics of Pinus koraiensis to Thinning [J]. Bulletin of Botanical Research, 2026, 46(2): 348-360.
[2]	Jingjing ZHANG, Haixia LUO, Yanping HONG, Shicheng ZHAO. Optimization of Agrobacterium rhizogenes-Mediated Genetic Transformation System of Solanum nigrum [J]. Bulletin of Botanical Research, 2026, 46(1): 111-119.
[3]	Qingbin JIA, Xudong YAO, Guixiang JIN, Hongying YU, Yanxia LI. Genetic Variation Analysis of Radial Growth and Superior Family Selection in Korean Pine Half-Sib Families [J]. Bulletin of Botanical Research, 2026, 46(1): 158-166.
[4]	Fang WANG, Yelei YANG, Changhua YUAN, Jun WANG, Wei WEI, Lin WU, Kuiyou LI, Kuiquan LI, Zhimin LU, Yuanxing WANG, Yuchun YANG. Variation Analysis of Growth Traits of Offspring and Superior Materials Selection of Pinus koraiensis [J]. Bulletin of Botanical Research, 2025, 45(5): 816-826.
[5]	Yanyan PAN, Xiaoguang LI, Lijun LIU, Hong LI, Xin ZHONG, Yifei ZHANG. Response of Pinus Koraiensis Growth and Rhizosphere Soil Enzyme Activity to Lepista nuda Inoculation [J]. Bulletin of Botanical Research, 2025, 45(3): 460-470.
[6]	Yitong LUAN, Niansen LI, Lujing QIAO, Cunyong JU, Tijiu CAI, Peili SUN. Niches, Interspecific Associations and Community Stability of Dominant Tree Species of Piceakoraiensis-Abies nephrolepis-Pinus koraiensis Forest [J]. Bulletin of Botanical Research, 2024, 44(5): 753-762.
[7]	Xuelai WANG, Xiaoting LIU, Liran WANG, Shitong LI, Taijin ZHANG, Jiafeng ZHANG, Jinghua XU, Guanzheng QU, Xiyang ZHAO. Coupling Evaluation of Growth and Wood Properties of Pinus koraiensis Half-sib Families [J]. Bulletin of Botanical Research, 2024, 44(4): 554-564.
[8]	Nan LI, Xiaoxia TIAN, Peichun MAO, Mingli ZHENG, LIN MENG, Lan YUN. Phenotype and Pigment Analysis of Flower Organs of Iris lactea var. chinensis [J]. Bulletin of Botanical Research, 2024, 44(1): 51-61.
[9]	Zhezhe LI, Yidan ZHANG, Bo WANG, Zhenghao WANG, Lu WANG, Beibei CHENG. Changes and Influencing Factors of Anthocyanins in Hibiscus syriacus During Flowering [J]. Bulletin of Botanical Research, 2023, 43(4): 550-561.
[10]	Na LI, Xiaonan WANG. Screening and Phenotypic Identification of Seed Coat Pigmentation Mutants in Arabidopsis thaliana [J]. Bulletin of Botanical Research, 2023, 43(1): 59-68.
[11]	Mengqiao GUO, Xuanyu CHEN, Senrong HONG, Jiao LI, Jie FAN, Xinyu CHENG. The Correlation Between Leaf Phenotype Diversity and Total Flavonoids Content of Overground Part of Tetrastigma hemsleyanum Diels & Gilg [J]. Bulletin of Botanical Research, 2022, 42(5): 876-885.
[12]	Yuhui REN, Shuai NIE, Chunxue PENG, Ling YANG, Hailong SHEN. Effects of Hormone Combinations, Carbon Source Type and Proliferation Cycle on Embryogenic Callus Proliferation of Pinus koraiensis [J]. Bulletin of Botanical Research, 2022, 42(4): 704-712.
[13]	Qiyang Gao, Yulong Huang, Wenbing Guo, Fencheng Zhao, Yang Liu. Effects of Carbon Source and Other Factors on the Proliferation of Embryogenic Callus of Elite Clone of Pinus elliottii [J]. Bulletin of Botanical Research, 2022, 42(1): 21-28.
[14]	Zhi-Gang WEI, De-An XIA, Rui-Qi Wang, Yang ZHANG, Ying-Ying Liu, Ruo-Lin LI, Chuan-Ping YANG. Provenance Test of Pinus koraiensis in Different Types of Natural Secondary Forests in Xiaoxing’an Mountains [J]. Bulletin of Botanical Research, 2021, 41(5): 807-815.
[15]	Chun-Xue PENG, Xue-Mei CUI, Hai-Long SHEN. Callus Induction and Somatic Embryogenesis and Physiological State Analysis from Mature Zygotic Embryo Explant of Syringa reticulata var. mandshurica [J]. Bulletin of Botanical Research, 2021, 41(4): 557-563.