Chin Bull Bot ›› 2016, Vol. 51 ›› Issue (3): 363-368.doi: 10.11983/CBB15102

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Induction of Hairy Roots of Dianthus chinensis and Its Plant Regeneration

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

  1. 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:2016-05-24 Published:2016-05-01
  • Contact: Shi Heping E-mail:shihp@scnu.edu.cn
  • About author:

    ? These authors contributed equally to this paper

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.

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'

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)"

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)"

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 1st leaf from apex (cm)
Internode Length of 2nd leaf from apex (cm)
Internode Length of 3rd 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 1st leaf from apex (cm)
Length of 2nd leaf from apex (cm)
Length of 3rd 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 1st leaf from apex (cm)
Width of 2nd leaf from apex (cm)
Width of 3rd 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
[1] 范惠琴, 姚泉洪, 彭日荷, 黄晓敏, 李贤 (2001). 发根农杆菌Ri质粒转化康乃馨初步研究. 上海农业学报 17, 35-38.
[2] 侯丽丽, 施和平, 余武, 曾宝强, 周卓辉 (2014). 烟草毛状根多倍体诱导及其植株再生. 生物工程学报 30, 581-594.
[3] 施和平, 朱远锋, 王蓓, 孙将兵, 黄胜琴 (2014). 香石竹毛状根诱导、离体培养及其植株再生. 生物工程学报 30, 1742-1750.
[4] 吴晓凤, 施和平, Tsang Po Keung Eric (2008). 褐脉少花龙葵毛状根的诱导、培养及其澳洲茄胺的产生. 分子细胞生物学报 41, 183-191.
[5] 余义勋, 刘娟旭, 包满珠 (2006). 香石竹植株再生及基因工程研究进展. 植物学通报 23, 23-28.
[6] Boase MR, Winefield CS, Lill TA (2004). Transgenic regal pelargoniums that express the rol C gene from Agro- bacterium rhizogenes exhibit a dwarf floral and vegetative phenotype.In Vitro Cell Develop Biol-Plant 40, 46-50.
[7] Bogdanovic MD, Todorovic SI, Banjanac T, Dragicevic MB, Verstappen FWA, Bouwmeester HJ, Simonovic AD (2014). Production of guaianolides in Agrobacterium rhizogenes-transformed chicory regenerants flowering.In Vitro Ind Crops Prod 60, 52-59.
[8] Casanova E, Valdes AE, Zuker A, Fernandez B, Vainstein A, Trillas MI, Moysset L (2004). Rol C-transgenic carna- tion plants: adventitious organogenesis and levels of en- dogenous auxin and cytokinins.Plant Sci 167, 551-560.
[9] Choi PS, Kim YD, Choi KM, Chung HJ, Choi DW, Liu JR (2004). Plant regeneration from hairy root cultures trans- formed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell Rep 22, 828-831.
[10] Christensen B, Sriskandarajah S, Serek M, Muller R (2008). Transformation of Kalanchoe blossfeldiana with rol-genes is useful in molecular breeding towards com- pact growth.Plant Cell Rep 27, 1485-1495.
[11] Christey MC (2001). Use of Ri-mediated transformation for production of transgenic plants.In Vitro Cell Develop Biol-Plant 37, 687-700.
[12] Edwards K, Johnstone C, Thompson C (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.Nucleic Acids Res 19, 1349.
[13] Furner I, Huffman G, Amasino R, Garfinkel DJ, Gordon MP, Nester EW (1986). An Agrobacterium transforma- tion in the evolution of the genus Nicotiana. Nature 319, 422-427.
[14] Giri A, Narasu ML (2000). Transgenic hairy roots: recent trends and applications.Biotechnol Adv 18, 1-22.
[15] Godo T, Tsujii O, Ishikawa K, Mii M (1997). Fertile transgenic plants of Nierembergia scoparia Sendtner obtained by a mikimopine type strain of Agrobacterium rhizogenes. Sci Hortic 68, 101-111.
[16] Kamada H, Saitou T, Harada H (1992). No requirement of vernalization for flower formation in Ri-transformed Cicho- rium plants.Plant Tissue Cult Lett 9, 206-213.
[17] Koike Y, Hoshino Y, Mii M, Nakano M (2003). Horticultural characterization of Angelonia salicariifolia plants trans- formed with wild-type strains of Agrobacterium rhizo- genes.Plant Cell Rep 21, 981-987.
[18] Limari MA, Sun LY, Douat C, Helgeson J, Tepfer D (1998). Natural Genetic transformation by Agrobacterium rhizogenes: annual flowering in biennials, Belgian endive and carrot.Plant Physiol 118, 543-550.
[19] Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15, 473-497.
[20] Shi HP, Long YY, Sun TS, Tsang PKE (2011). Induction of hairy roots and plant regeneration from the medicinal plant Pogostemon cablin.Plant Cell Tissue Organ Cult 107, 251-260.
[21] Sun LY, Touraud G, Charbonnier C, Tepfer D (1991). Modification of phenotype in Belgian endive (Cichorium intybus) through genetic transformation by Agrobacterium rhizogenes: conversion from biennial to annual flowering. Transgenic Res 1, 14-22.
[22] Tepfer M, Casse-Delbart F (1987). Agrobacterium rhizo- genes as a vector for transforming higher plants.Microbiol Sci 4, 24-28.
[23] Winefield C, Lewis D, Arathoon S, Deroles S (1999). Alteration of Petunia plant form through the introduction of the rol C gene from Agrobacterium rhizogenes. Mol Breed 5, 543-551.
[24] Zia M, Mirza B, Malik SA, Chaudhary MF (2010). Expression of rol genes in transgenic soybean (Glycine max L.) leads to changes in plant phenotype, leaf morphology and ?owering time.Plant Cell Tissue Organ Cult 103, 227-236.
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