山茶叶片的离体再生及内源激素与不定芽分化的关系

doi:10.16420/j.issn.0513-353x.2023-0621

摘要/Abstract

摘要：

以山茶（Camellia japonica）叶片为外植体进行愈伤组织诱导和再分化。结果表明：叶片在MS + 5.0 mg · L^-1 6-BA + 0.1 mg · L^-1 2,4-D + 5.0 mg · L^-1 PVP + 3%蔗糖培养基上愈伤组织诱导率最高（79.19%）；将愈伤组织转至原配方培养基光照培养（12 h · d^-1），可分化出不定芽，分化率为51.32%。不定芽在不定根诱导培养基（1/8MS + 0.5 mg · L^-1 IBA + 1.0 mg · L^-1 PVP + 2%蔗糖）上生根率最高（50%）；生根试管苗移栽至田间，成活率达66.67%。利用液相色谱串联质谱法（LC-MS/MS）检测激素发现，具不定芽分化能力的愈伤组织中，激素信号转导途径、玉米素生物合成代谢途径、α-亚麻酸代谢途径激素含量发生显著变化。在所有检测的内源激素中，有30种（90.9%）含量下调，仅有3种激素含量上调。结果显示，在培养条件一致的前提下，内源激素含量是决定山茶不定芽分化的主要因素。

关键词: 山茶, 再生体系, 内源激素, 不定芽分化

Abstract:

A regeneration system has been established in this study using Camellia japonica leaves as explants. The highest frequency of callus induction（79.19%）was obtained by culturing the leaf explants on the MS media containing 5.0 mg · L^-1 6-BA，0.1 mg · L^-1 2,4-D，5.0 mg · L^-1 PVP and 3% sucrose. Differentiated adventitious buds were generated by directly transferring the explants to the condition of light（12 h · d^-1）. The frequency of adventitious buds generation was 51.32%. Consequently，adventitious roots were induced from these buds using 1/8MS media containing 0.5 mg · L^-1 IBA，1.0 mg · L^-1 PVP and 2% sucrose. The highest frequency of root induction was 50%. The C. japonica seedlings rate was 66.67%. The endogenous hormone levels in the tissue-cultured materials were detected using LC-MS/MS. The results demonstrated that the plant hormone signal transduction pathway，zeatin biosynthesis pathway and α-linolenic acid metabolic pathway were changed in the callus with ability to differentiate adventitious buds. Among all the endogenous hormones detected，a total of 30 were down-regulated（90.9%），only three hormones showed up-regulation. The endogenous hormone levels largely determined the induction of callus and differentiation of adventitious buds of C. japonica explants grown under the same culture conditions.

Key words: Camellia japonica, regeneration system, endogenous hormones, differentiation of adventitious bud

王建昭, 高园, 刀梅, 张洪烨, 杨自云, 陈龙清, 吴田. 山茶叶片的离体再生及内源激素与不定芽分化的关系[J]. 园艺学报, 2024, 51(8): 1891-1905.

WANG Jianzhao, GAO Yuan, DAO Mei, ZHANG Hongye, YANG Ziyun, CHEN Longqing, WU Tian. Establishment of Regeneration in Vitro System of Camellia japonica and Relationship Between Endogenous Hormones and Adventitious Bud Differentiation[J]. Acta Horticulturae Sinica, 2024, 51(8): 1891-1905.

图/表 11

表1 山茶叶片外植体在不同培养基中的愈伤组织诱导率

Table 1 Callus induction media from Camellia japonica leaves

培养基编号 Medium number	培养基配方/（mg · L^-1） Medium component					诱导率/% Induction percentage
培养基编号 Medium number	6-BA	2,4-D	NAA	IBA	KT	诱导率/% Induction percentage
YS1	2.0	1.5	0.05	0	0	42.68
YS2	2.5	2.0	0.50	0	0	35.20
YS3	5.0	0.1	0	0	0	79.19
YS4	2.0	0.5	0	0	0	41.54
YS5	0.4	2.0	0	0	0	43.60
YS6	0.5	0	2.00	0.1	1.0	66.57

表2 山茶组培不定芽在不同培养基中的生根率

Table 2 Induction of adventitious root media for Camellia japonica

培养基编号 Medium number	培养基配方/（mg · L^-1）Medium component		生根率/% Induction percentage of rooting
培养基编号 Medium number	IAA	IBA	生根率/% Induction percentage of rooting
Sg1	0.5	0	0
Sg2	0.5	0	33.33
Sg3	0	0.5	50.00

图1 山茶叶片离体再生过程 a、b：YS3、YS6培养基中的愈伤组织；c：继代3次的愈伤组织；d：分化的不定芽；e：切下的不定芽继代培养；f：不定芽分节；g：Sg1生根培养基不能诱导不定根；h、i：Sg2、Sg3生根培养基诱导的不定根。

Fig. 1 Overall process of in vitro regeneration from Camellia japonica leaves a，b：Camellia japonica callus in YS3 and YS6 media；c：Callus after three times of subculture；d：Differentiated adventitious buds；e：Subculture of adventitious bud；f：Segmentation of adventitious buds；g：Adventitious roots could not be induced on Sg1 rooting media；h，i：Adventitious roots on Sg2 and Sg3 rooting media.

表3 山茶组培苗在不同基质中的移栽成活率

Table 3 Transplant survival of Camellia japonica grouped seedlings in different substrates

培养基编号 Medium number	基质配比 Matrix ratio				成活率/% Rate of survival
培养基编号 Medium number	腐殖土 Humus	珍珠岩 Perlite	蛭石 Vermiculite	生土 Raw soil	成活率/% Rate of survival
1	1	1	1	3	66.7
2	1	1	0	2	66.7
3	0	1	0	1	66.7
4	0	0	0	1	0

图2 山茶有不定芽分化（有芽）和无不定芽分化（无芽）愈伤组织含量差异内源激素聚类热图图中红色代表高含量，绿色代表低含量；1、2和3代表3个生物学重复。

Fig. 2 Endogenous hormone clustering heat map of callus content difference between Camellia japonica with adventitious bud differentiation（with bud）and without adventitious bud differentiation（without bud） Red represents high content，green represents low content. The numbers 1，2 and 3 indicate three biological repeats.

图3 山茶有不定芽分化（有芽）和无不定芽分化（无芽）愈伤组织含量差异内源激素KEGG富集图图中点的颜色越红表示富集越显著，点的大小代表富集到的差异代谢物个数的多少。

Fig. 3 Differential endogenous hormone KEGG enrichment map of callus content in Camellia japonica with adventitious bud differentiation（with bud）and without adventitious bud differentiation（without bud） The red indicates the more significant enrichment. The size of the dots represents the number of differentiated metabolites enriched.

图4 山茶不定芽分化（有芽）和无不定芽分化（无芽）愈伤组织中的生长素类激素含量 IAA-Glu：吲哚乙酸-谷氨酸；IAA-Val-Me：吲哚乙酸-缬氨酸甲酯；IPA：3-吲哚丙酸；ICAld：吲哚-3-甲醛；IAA-Ala：N-（3-吲哚乙酰基）-L-丙氨酸；TRA：色胺。柱形图表示3个生物学重复的平均值，误差棒为标准差。t检验，* P < 0.05；** P < 0.01；*** P < 0.001；**** P < 0.0001。下同。

Fig. 4 Auxin hormone content in callus with adventitious bud differentiation（with bud）and without adventitious bud differentiation（without bud）in Camellia japonica IAA-Glu：Indoleacetic acid-glutamic acid；IAA-Val-Me：Indole-3-acetyl-L-valine methyl ester；IPA：3-Indolepopionic acid；ICAld：Indole-3-formaldehyde；IAA-Ala：N-（3-Indolyl acetyl）-L-alanine；TRA：Tryptamine. The bar plot represents the mean of the 3 biological replicates，and the error bar is the standard deviation. * represents the statistical difference in the t-test. * P < 0.05；** P < 0.01；*** P < 0.001；**** P < 0.0001. The same below.

图5 山茶有不定芽分化（有芽）和无不定芽分化（无芽）愈伤组织细胞分裂素类激素含量 K9G：呋喃甲基腺嘌呤-9-葡糖苷；DHZROG：双氢玉米核苷-O-糖苷；mT：3-[（9H-嘌呤-6-基氨基）甲基]苯酚；2MeScZR：2-甲硫基顺式玉米素核苷；pT9G：4-{[（9-β-D-吡喃葡糖基-9H-嘌呤-6-基）氨基]甲基}苯酚；BAPR：6-苄基腺苷；mTR：N-（3-羟基苄基）腺苷；IPR：异戊烯腺嘌呤核苷；oT：6-（2-羟基苄基氨基）；iP9G：异戊烯腺嘌呤-9-葡糖苷；tZOG：反式-玉米素-9-Β-葡糖苷；DHZ7G：双氢玉米素-7-糖苷；iP7G：异戊烯腺嘌呤-7-葡糖苷；oTR：6-（2-羟基苄基）氨基嘌呤核糖甙；pTR：N-（4-甲氧基苄基）-腺苷；cZROG：顺式玉米核苷-O-糖苷；IP：N6-异戊烯腺嘌呤；mT9G：3-{[（9-β-D-吡喃葡糖基-9H-嘌呤-6-基）氨基]甲基}苯酚；BAP9G：6-苄氨基-9-（A-D-吡喃葡糖基）嘌呤。

Fig. 5 Cytokinins in callus with adventitious bud differentiation（with bud）and without adventitious bud differentiation（without bud）in Camellia japonica K9G：Kinetin-9-glucoside；DHZROG：Dihydrozeatin-O-glucoside riboside；mT：meta-Topolin；2MeScZR：2-Methylthio-cis-zeatin riboside；pT9G：4-{[（9-β-D-Glucopyranosyl-9H-purin-6-yl）amino]methyl}phenol；BAPR：6-Benzyladenosine；mTR：meta-Topolin riboside；IPR：Isopentenyl adenine nucleoside；oT：6-（2-Hydroxybenzyl amino）；iP9G：Isopentenadenine-9-glucoside；tZOG：trans-Zeatin-O-glucoside；DHZ7G：Dihydrozeaxin-7-glycoside；iP7G：Isopentenadenine-7-glucoside；oTR：ortho-Topolin；pTR：para-Topolin riboside；cZROG：cis-Zeatin-O-glucoside riboside；IP：N6-isopentennyladenine；mT9G：meta-Topolin-9-glucoside；BAP9G：N6-Benzyladenine-9-glucoside.

图6 山茶有不定芽分化（有芽）和无不定芽分化（无芽）愈伤组织中茉莉酸类激素含量 JA-Val：茉莉酸-缬氨酸；JA-Phe：茉莉酸-苯丙氨酸；JA-ILE：茉莉酸-异亮氨酸；JA：茉莉酸；OPC-4：氧化戊烯基环戊烷丁酸。

Fig. 6 Jasmonates in callus with adventitious bud differentiation（with bud）and without adventitious bud differentiation（without bud）in Camellia japonica JA-Val：Jasmonate-valine；JA-Phe：Jasmonate-phenylalanine；JA-ILE：Jasmonate-isoleucine；JA：Jasmonate；OPC-4：Oxypentenyl cyclopentane butyric acid.

图7 山茶有不定芽分化（有芽）和无不定芽分化（无芽）愈伤组织中水杨酸类激素和独脚金醇含量 SAG：水杨酸-2-O-β-葡糖苷；SA：水杨酸；ST：独脚金醇。

Fig. 7 Salicylic acids in callus with adventitious bud differentiation（with bud）and without adventitious bud differentiation（without bud）in Camellia japonica SAG：Salicylate 2-O-β-glucoside；SA：Salicylic acid；ST：Strigol.

图8 山茶离体再生的模型图 ↑表示内源激素水平上升；↓表示内源激素水平下降。

Fig. 8 Model diagram of regeneration in vitro of Camellia japonica ↑ means increased endogenous hormone levels；↑ means reduced endogenous hormone levels.

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