Establishment of an Agrobacterium-mediated transformation system for Periploca sepium Bunge

Ren Chen, Mayumi Gyokusen, Yoshihisa Nakazawa, Yinquan Su, Koichiro Gyokusen

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Agrobacterium-mediated transformation of Periploca sepium Bunge using proliferated clonal shoots was investigated to identify important factors affecting the transformation efficiency. Agrobacterium tumefaciens strains EHA105 and LBA4404 were used, both of which harbored a pKAFCR21 binary vector, which contained two reporter genes (GUS and sGFP, encoding β-glucuronidase and the synthetic green-fluorescent protein with S65T mutation) and two marker genes (encoding neomycin phosphotransferase II and hygromycin phosphotransferase). The factors evaluated were Agrobacterium strain, co-cultivation treatment, and antibiotic selection regime. The results revealed that the transformation efficiency could be synergistically increased to as high as 50-60% by infecting explants with Agrobacterium strain EHA105/pKAFCR21 and co-cultivating in the presence of 150 mg l-1 dithiothreitol, followed by selection at 100 mg l-1 kanamycin. Genomic DNA PCR, Southern hybridization, and quantitative real-time reverse transcription PCR analyses confirmed that the transgenes (GUS or sGFP) had presented, integrated, and expressed in all the tested transformant plants. The optimized protocol provides a basis for further genetic alteration of P. sepium for medicinal compounds and cispolyisoprene production.

Original languageEnglish
Pages (from-to)173-181
Number of pages9
JournalPlant Biotechnology
Volume27
Issue number2
DOIs
Publication statusPublished - Jan 1 2010

Fingerprint

Periploca
Sepia
Agrobacterium
hygromycin-B kinase
Kanamycin Kinase
Polymerase Chain Reaction
kanamycin kinase
Agrobacterium tumefaciens
Kanamycin
dithiothreitol
Dithiothreitol
Glucuronidase
kanamycin
Green Fluorescent Proteins
Agrobacterium radiobacter
Transgenes
Reporter Genes
green fluorescent protein
reporter genes
Southern blotting

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Agronomy and Crop Science
  • Plant Science

Cite this

Establishment of an Agrobacterium-mediated transformation system for Periploca sepium Bunge. / Chen, Ren; Gyokusen, Mayumi; Nakazawa, Yoshihisa; Su, Yinquan; Gyokusen, Koichiro.

In: Plant Biotechnology, Vol. 27, No. 2, 01.01.2010, p. 173-181.

Research output: Contribution to journalArticle

Chen, Ren ; Gyokusen, Mayumi ; Nakazawa, Yoshihisa ; Su, Yinquan ; Gyokusen, Koichiro. / Establishment of an Agrobacterium-mediated transformation system for Periploca sepium Bunge. In: Plant Biotechnology. 2010 ; Vol. 27, No. 2. pp. 173-181.
@article{5e93e6a9a1434b07aed42192f71e9b9b,
title = "Establishment of an Agrobacterium-mediated transformation system for Periploca sepium Bunge",
abstract = "Agrobacterium-mediated transformation of Periploca sepium Bunge using proliferated clonal shoots was investigated to identify important factors affecting the transformation efficiency. Agrobacterium tumefaciens strains EHA105 and LBA4404 were used, both of which harbored a pKAFCR21 binary vector, which contained two reporter genes (GUS and sGFP, encoding β-glucuronidase and the synthetic green-fluorescent protein with S65T mutation) and two marker genes (encoding neomycin phosphotransferase II and hygromycin phosphotransferase). The factors evaluated were Agrobacterium strain, co-cultivation treatment, and antibiotic selection regime. The results revealed that the transformation efficiency could be synergistically increased to as high as 50-60{\%} by infecting explants with Agrobacterium strain EHA105/pKAFCR21 and co-cultivating in the presence of 150 mg l-1 dithiothreitol, followed by selection at 100 mg l-1 kanamycin. Genomic DNA PCR, Southern hybridization, and quantitative real-time reverse transcription PCR analyses confirmed that the transgenes (GUS or sGFP) had presented, integrated, and expressed in all the tested transformant plants. The optimized protocol provides a basis for further genetic alteration of P. sepium for medicinal compounds and cispolyisoprene production.",
author = "Ren Chen and Mayumi Gyokusen and Yoshihisa Nakazawa and Yinquan Su and Koichiro Gyokusen",
year = "2010",
month = "1",
day = "1",
doi = "10.5511/plantbiotechnology.27.173",
language = "English",
volume = "27",
pages = "173--181",
journal = "Plant Biotechnology",
issn = "1342-4580",
publisher = "Japanese Society for Plant Cell and Molecular Biology",
number = "2",

}

TY - JOUR

T1 - Establishment of an Agrobacterium-mediated transformation system for Periploca sepium Bunge

AU - Chen, Ren

AU - Gyokusen, Mayumi

AU - Nakazawa, Yoshihisa

AU - Su, Yinquan

AU - Gyokusen, Koichiro

PY - 2010/1/1

Y1 - 2010/1/1

N2 - Agrobacterium-mediated transformation of Periploca sepium Bunge using proliferated clonal shoots was investigated to identify important factors affecting the transformation efficiency. Agrobacterium tumefaciens strains EHA105 and LBA4404 were used, both of which harbored a pKAFCR21 binary vector, which contained two reporter genes (GUS and sGFP, encoding β-glucuronidase and the synthetic green-fluorescent protein with S65T mutation) and two marker genes (encoding neomycin phosphotransferase II and hygromycin phosphotransferase). The factors evaluated were Agrobacterium strain, co-cultivation treatment, and antibiotic selection regime. The results revealed that the transformation efficiency could be synergistically increased to as high as 50-60% by infecting explants with Agrobacterium strain EHA105/pKAFCR21 and co-cultivating in the presence of 150 mg l-1 dithiothreitol, followed by selection at 100 mg l-1 kanamycin. Genomic DNA PCR, Southern hybridization, and quantitative real-time reverse transcription PCR analyses confirmed that the transgenes (GUS or sGFP) had presented, integrated, and expressed in all the tested transformant plants. The optimized protocol provides a basis for further genetic alteration of P. sepium for medicinal compounds and cispolyisoprene production.

AB - Agrobacterium-mediated transformation of Periploca sepium Bunge using proliferated clonal shoots was investigated to identify important factors affecting the transformation efficiency. Agrobacterium tumefaciens strains EHA105 and LBA4404 were used, both of which harbored a pKAFCR21 binary vector, which contained two reporter genes (GUS and sGFP, encoding β-glucuronidase and the synthetic green-fluorescent protein with S65T mutation) and two marker genes (encoding neomycin phosphotransferase II and hygromycin phosphotransferase). The factors evaluated were Agrobacterium strain, co-cultivation treatment, and antibiotic selection regime. The results revealed that the transformation efficiency could be synergistically increased to as high as 50-60% by infecting explants with Agrobacterium strain EHA105/pKAFCR21 and co-cultivating in the presence of 150 mg l-1 dithiothreitol, followed by selection at 100 mg l-1 kanamycin. Genomic DNA PCR, Southern hybridization, and quantitative real-time reverse transcription PCR analyses confirmed that the transgenes (GUS or sGFP) had presented, integrated, and expressed in all the tested transformant plants. The optimized protocol provides a basis for further genetic alteration of P. sepium for medicinal compounds and cispolyisoprene production.

UR - http://www.scopus.com/inward/record.url?scp=77955311012&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77955311012&partnerID=8YFLogxK

U2 - 10.5511/plantbiotechnology.27.173

DO - 10.5511/plantbiotechnology.27.173

M3 - Article

AN - SCOPUS:77955311012

VL - 27

SP - 173

EP - 181

JO - Plant Biotechnology

JF - Plant Biotechnology

SN - 1342-4580

IS - 2

ER -