Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana

A non-tissue culture approach

Kenneth A Feldmann, M. David Marks

Research output: Contribution to journalArticle

323 Citations (Scopus)

Abstract

Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%-2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%-63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalMGG Molecular & General Genetics
Volume208
Issue number1-2
DOIs
StatePublished - Jun 1987
Externally publishedYes

Fingerprint

Agrobacterium
Kanamycin
Kanamycin Kinase
Seedlings
Arabidopsis
Seeds
Plant Tumor-Inducing Plasmids
Genes
Bony Callus
Kanamycin Resistance
Agrobacterium tumefaciens
R Factors

Keywords

  • Agrobacterium tumefaciens
  • Arabidopsis thaliana
  • Mendelian segregation
  • Transformation

ASJC Scopus subject areas

  • Genetics

Cite this

Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana : A non-tissue culture approach. / Feldmann, Kenneth A; David Marks, M.

In: MGG Molecular & General Genetics, Vol. 208, No. 1-2, 06.1987, p. 1-9.

Research output: Contribution to journalArticle

@article{a75d6f6b2de948d8aacffe021e1808b5,
title = "Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach",
abstract = "Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62{\%} segregated for one functional insert, 29{\%} for two unlinked or linked functional inserts, 5{\%} for three unlinked inserts, 1{\%} for four unlinked inserts, whereas 3{\%} appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0{\%}-2{\%} of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8{\%}-63{\%} of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.",
keywords = "Agrobacterium tumefaciens, Arabidopsis thaliana, Mendelian segregation, Transformation",
author = "Feldmann, {Kenneth A} and {David Marks}, M.",
year = "1987",
month = "6",
doi = "10.1007/BF00330414",
language = "English (US)",
volume = "208",
pages = "1--9",
journal = "Molecular Genetics and Genomics",
issn = "1617-4615",
publisher = "Springer Verlag",
number = "1-2",

}

TY - JOUR

T1 - Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana

T2 - A non-tissue culture approach

AU - Feldmann, Kenneth A

AU - David Marks, M.

PY - 1987/6

Y1 - 1987/6

N2 - Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%-2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%-63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.

AB - Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%-2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%-63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.

KW - Agrobacterium tumefaciens

KW - Arabidopsis thaliana

KW - Mendelian segregation

KW - Transformation

UR - http://www.scopus.com/inward/record.url?scp=34250101341&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34250101341&partnerID=8YFLogxK

U2 - 10.1007/BF00330414

DO - 10.1007/BF00330414

M3 - Article

VL - 208

SP - 1

EP - 9

JO - Molecular Genetics and Genomics

JF - Molecular Genetics and Genomics

SN - 1617-4615

IS - 1-2

ER -