五寸石竹毛状根诱导及其植株再生

doi:10.11983/CBB15102

植物学报 ›› 2016, Vol. 51 ›› Issue (3): 363-368.DOI: 10.11983/CBB15102

五寸石竹毛状根诱导及其植株再生

施和平^*(), 王蓓, 杨树楠, 郭亚鹏

华南师范大学生命科学学院, 广东省植物发育生物工程重点实验室, 广州 510631

收稿日期:2015-06-06 接受日期:2015-09-28 出版日期:2016-05-01 发布日期:2016-05-24
通讯作者: 施和平
作者简介:
? 共同第一作者
基金资助:
广东省科技计划(No.2011B020304006)

Induction of Hairy Roots of Dianthus chinensis and Its Plant Regeneration

Heping Shi^*, Bei Wang, Shunan Yang, Yapeng Guo

Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China

Received:2015-06-06 Accepted:2015-09-28 Online:2016-05-01 Published:2016-05-24
Contact: Shi Heping
About author:
? These authors contributed equally to this paper

摘要/Abstract

摘要： 建立了五寸石竹(Dianthus chinensis)毛状根诱导及其植株再生体系。用含野生农杆碱型Ri质粒的发根农杆菌(Agrobacterium rhizogenes) 15834感染五寸石竹叶片外植体10天后, 从其形态学下端产生白色不定根; 30天后, 叶片外植体的生根率为95%; 所产生的毛状根能在无外源生长调节剂的固体和液体MS培养基上快速自主生长。遗传转化鉴定结果表明, 发根农杆菌Ri质粒的生根基因(rol)已在毛状根基因组中整合并表达。五寸石竹毛状根的根段在成分为MS+2.0 mg·L^-1 6-BA+0.2 mg·L^-1 NAA的培养基中培养15天后产生浅绿色疏松愈伤组织; 将愈伤组织转入成分为MS+1.0 mg·L^-1 6- BA+0.02 mg·L^-1 NAA的培养基中培养30天后逐渐形成不定芽。盆栽的五寸石竹毛状根再生植株与非转化植株(对照)相比节间缩短, 开花期提前18天。

Abstract: This study established an efficient system for inducing hairy roots and plant regeneration of Dianthus chinensis. Hairy roots could be induced from the basal surface of leaf explants of D. chinensis at 10 days after inoculation with the strain of Agrobacterium rhizogenes ATCC15834 harbouring the wild agropine-type plasmid. The percentage of rooting leaf explants was 95% at 30 days after inoculation. Hairy roots could grow rapidly and autonomously in liquid or solid plant-growth regulator-free MS medium. The transformation was confirmed by PCR amplification of the rol gene of the Ri plasmid from D. chinensis hairy roots. Hairy roots could form light green callus after culture on MS + 2.0 mg·L^-1 6-BA + 0.2 mg·L^-1 NAA for 15 days. Adventitious shoots were gradually produced from calli after culture on MS + 1.0 mg·L^-1 6-BA + 0.02 mg·L^-1 NAA for 30 days. Compared to the control, pot-grown plants regenerated from hairy roots had shorter internodes and flowering was earlier by 18 days.

施和平, 王蓓, 杨树楠, 郭亚鹏. 五寸石竹毛状根诱导及其植株再生. 植物学报, 2016, 51(3): 363-368.

Heping Shi, Bei Wang, Shunan Yang, Yapeng Guo. Induction of Hairy Roots of Dianthus chinensis and Its Plant Regeneration. Chinese Bulletin of Botany, 2016, 51(3): 363-368.

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图/表 4

表1 PCR扩增所用的引物及其序列

Table 1 Primer sequences used for the PCR amplification

Target gene	Primer sequence
rol B rol C	P1: 5'-GCT CTT GCA GTG CTA GAT TT-3' P2: 5'-GAA GGT GCA AGC TAC CTC TC -3' P1: 5'- CTC CTG ACA TCA AAC TCG TC-3' P2: 5'- TGC TTC GAG TTA TGG GTA CA-3'

图1 五寸石竹毛状根诱导、离体培养及其植株再生（(A) 发根农杆菌ATCC15834菌株感染12天后叶片外植体产生的毛状根; (B) 固体培养14天的毛状根; (C) 液体培养14天的毛状根; (D) 毛状根在MS+2.0 mg·L-1 6-BA+0.2 mg·L-1 NAA上培养15天后形成的愈伤组织; (E) 毛状根愈伤组织在MS+1.0 mg·L-1 6-BA+ 0.02 mg·L-1 NAA上培养20天后分化出不定芽; (F) 不定芽在MS+0.1 mg·L-1 6-BA+0.2 mg·L-1 NAA上增殖; (G) 盆栽45天的非转化植株(组培苗); (H) 盆栽45天的毛状根再生植株）

Fig. 2 Induction and in vitro culture of hairy roots and its plant regeneration of Dianthus chinensis （(A) Hairy root formation from leaf explants infected by the strain of Agrobacterium rhizogenes ATCC15834 for 12 days; (B) Solid medium culture of hairy roots for 14 days; (C) Liquid medium culture of hairy roots for 14 days; (D) Calli formation from hairy roots cultured on MS+2.0 mg·L-1 6-BA+0.2 mg·L-1 NAA for 15 days; (E) Adventitious shoots formation from calli after cultured on MS+1.0 mg·L-1 6-BA+0.02 mg·L-1 NAA for 20 days; (F) Multiplication of adventitious shoots on MS+0.1 mg·L-1 6-BA+0.2 mg·L-1 NAA; (G) Pot-grown untransformed (control) plants of Dianthus chinensis for 45 days; (H) Pot-grown regenerated plants from hairy roots for 45 days）

图2 五寸石竹毛状根rol B和rol C基因的PCR扩增产物的凝胶电泳分析（1: 1 kb DNA marker; 2-5: rol B引物扩增片段; 6-9: rol C引物扩增片段; 2, 6: 从发根农杆菌菌落扩增的片段; 3, 7: 从非转化根(对照根)基因组DNA扩增的片段; 4, 5, 8, 9: 从毛状根基因组DNA扩增的片段）

Fig. 2 Gel electrophoresis analysis of PCR fragments of rol B and rol C genes amplified from the genome DNA of ha- iry roots(Lane 1: 1 kb DNA marker; Lane 2-5: Fragments with rol B primers;Lane 6-9: Fragments with rol C primers; Lane 2 and 6: Fragments amplified from the colony of Agrobacterium rhizogenes ATCC15834; Lane 3 and 7: Fragments from genome DNA of untransformed roots; Lane 4, 5, 8, and 9: Fragments amplified from genome DNA of hairy roots)

表2 五寸石竹毛状根再生植株与非转化株生长形态的比较

Table 2 Comparison of growth morphology of untransformed and hairy root-regenerated plants of Dianthus chinensis

	Control plants	Hairy roots regenerated plants
Plant height (cm)	22.50±2.64	17.20±2.25
Internode Length of 1^st leaf from apex (cm) Internode Length of 2^nd leaf from apex (cm) Internode Length of 3^rd leaf from apex (cm)	2.26±0.74 2.67±0.92 1.70±0.73	1.60±0.35 1.96±0.60 1.53±0.14
Length of 1^st leaf from apex (cm) Length of 2^nd leaf from apex (cm) Length of 3^rd leaf from apex (cm)	2.54±0.321 4.11±0.34 5.09±0.28	3.18±0.17 4.20±0.32 4.94±0.30
Width of 1^st leaf from apex (cm) Width of 2^nd leaf from apex (cm) Width of 3^rd leaf from apex (cm)	0.50±0.08 0.94±0.07 1.16±0.07	0.58±0.07 0.9±0.15 1.00±0.06

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五寸石竹毛状根诱导及其植株再生

Induction of Hairy Roots of Dianthus chinensis and Its Plant Regeneration

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