Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies

D. Natorajan, H. Y. Yong, Ismanizan Ismail, Zamri Zainal

Research output: Contribution to journalArticle

Abstract

Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (β-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.

Original languageEnglish
Pages (from-to)448-455
Number of pages8
JournalEmirates Journal of Food and Agriculture
Volume22
Issue number6
Publication statusPublished - Dec 2010

Fingerprint

agroinfiltration
trapping
Glucuronidase
promoter regions
leaves
gene dosage
sampling
DNA Transposable Elements
Gene Dosage
DNA Fingerprinting
Nucleic Acid Regulatory Sequences
Lycopersicon esculentum
Vacuum
Southern Blotting
DNA fingerprinting
methodology
Reporter Genes
reporter genes
transposons
Southern blotting

Keywords

  • Agrobacterium tumefaciens
  • Agroinfiltration
  • Non-Destructive GUS detection
  • T-DNA insertion

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Animal Science and Zoology
  • Food Science
  • Applied Microbiology and Biotechnology

Cite this

Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies. / Natorajan, D.; Yong, H. Y.; Ismail, Ismanizan; Zainal, Zamri.

In: Emirates Journal of Food and Agriculture, Vol. 22, No. 6, 12.2010, p. 448-455.

Research output: Contribution to journalArticle

@article{81106a291dc64e72ae6c28e55220064d,
title = "Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies",
abstract = "Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (β-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.",
keywords = "Agrobacterium tumefaciens, Agroinfiltration, Non-Destructive GUS detection, T-DNA insertion",
author = "D. Natorajan and Yong, {H. Y.} and Ismanizan Ismail and Zamri Zainal",
year = "2010",
month = "12",
language = "English",
volume = "22",
pages = "448--455",
journal = "Emirates Journal of Food and Agriculture",
issn = "2079-052X",
publisher = "United Arab Emirates University",
number = "6",

}

TY - JOUR

T1 - Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies

AU - Natorajan, D.

AU - Yong, H. Y.

AU - Ismail, Ismanizan

AU - Zainal, Zamri

PY - 2010/12

Y1 - 2010/12

N2 - Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (β-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.

AB - Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (β-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.

KW - Agrobacterium tumefaciens

KW - Agroinfiltration

KW - Non-Destructive GUS detection

KW - T-DNA insertion

UR - http://www.scopus.com/inward/record.url?scp=84880681207&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84880681207&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:84880681207

VL - 22

SP - 448

EP - 455

JO - Emirates Journal of Food and Agriculture

JF - Emirates Journal of Food and Agriculture

SN - 2079-052X

IS - 6

ER -