Abstract
Plant cell suspension cultures are used in basic research and applied biotechnology. In both cases, the transfer and stable integration of heterologous genes is a required technique. This report describes a rapid method for transformation of cell cultures of Medicago truncatula, the model species for the legume family. Accession A17 from the cultivar Jemalong is the reference genotype selected for the sequencing of the genome and therefore most studies on Medicago are carried out on this accession line. However, this line has a low embryogenic capacity and is poorly responsive to transformation protocols that rely on somatic embryogenesis. An alternative method for transformation of suspension cultures of this line, which does not depend on leaf transformation or somatic embryogenesis, was therefore needed. The method described herein uses Agrobacterium tumefaciens mediated gene transfer, allowing the transformation of Medicago callus tissue and the following establishment of liquid suspension cell cultures approximately 2 months after transformation. Kanamycin resistance was used to select for positive transformation events and the screening was facilitated by visualization of a fluorescent marker, which was fused to the gene of interest. This new protocol reduces the time between transformation and cell culture establishment, and allows the generation of transgenic suspension cultures of Medicago reference accession A17.
| Original language | English |
|---|---|
| Pages (from-to) | 445-450 |
| Number of pages | 6 |
| Journal | Plant Cell, Tissue and Organ Culture |
| Volume | 136 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 15 Mar 2019 |
Fingerprint
Keywords
- A17
- Agrobacterium transformation
- Cell suspension culture
- Fluorescent protein
- Jemalong
- Medicago truncatula
Cite this
}
Generation of transgenic cell suspension cultures of the model legume Medicago truncatula : a rapid method for Agrobacterium mediated gene transfer. / Santos, Rita B.; Pires, Ana Sofia; van der Hoorn, Renier A.L.; Schiermeyer, Andreas; Abranches, Rita.
In: Plant Cell, Tissue and Organ Culture, Vol. 136, No. 3, 15.03.2019, p. 445-450.Research output: Contribution to journal › Article
TY - JOUR
T1 - Generation of transgenic cell suspension cultures of the model legume Medicago truncatula
T2 - a rapid method for Agrobacterium mediated gene transfer
AU - Santos, Rita B.
AU - Pires, Ana Sofia
AU - van der Hoorn, Renier A.L.
AU - Schiermeyer, Andreas
AU - Abranches, Rita
PY - 2019/3/15
Y1 - 2019/3/15
N2 - Plant cell suspension cultures are used in basic research and applied biotechnology. In both cases, the transfer and stable integration of heterologous genes is a required technique. This report describes a rapid method for transformation of cell cultures of Medicago truncatula, the model species for the legume family. Accession A17 from the cultivar Jemalong is the reference genotype selected for the sequencing of the genome and therefore most studies on Medicago are carried out on this accession line. However, this line has a low embryogenic capacity and is poorly responsive to transformation protocols that rely on somatic embryogenesis. An alternative method for transformation of suspension cultures of this line, which does not depend on leaf transformation or somatic embryogenesis, was therefore needed. The method described herein uses Agrobacterium tumefaciens mediated gene transfer, allowing the transformation of Medicago callus tissue and the following establishment of liquid suspension cell cultures approximately 2 months after transformation. Kanamycin resistance was used to select for positive transformation events and the screening was facilitated by visualization of a fluorescent marker, which was fused to the gene of interest. This new protocol reduces the time between transformation and cell culture establishment, and allows the generation of transgenic suspension cultures of Medicago reference accession A17.
AB - Plant cell suspension cultures are used in basic research and applied biotechnology. In both cases, the transfer and stable integration of heterologous genes is a required technique. This report describes a rapid method for transformation of cell cultures of Medicago truncatula, the model species for the legume family. Accession A17 from the cultivar Jemalong is the reference genotype selected for the sequencing of the genome and therefore most studies on Medicago are carried out on this accession line. However, this line has a low embryogenic capacity and is poorly responsive to transformation protocols that rely on somatic embryogenesis. An alternative method for transformation of suspension cultures of this line, which does not depend on leaf transformation or somatic embryogenesis, was therefore needed. The method described herein uses Agrobacterium tumefaciens mediated gene transfer, allowing the transformation of Medicago callus tissue and the following establishment of liquid suspension cell cultures approximately 2 months after transformation. Kanamycin resistance was used to select for positive transformation events and the screening was facilitated by visualization of a fluorescent marker, which was fused to the gene of interest. This new protocol reduces the time between transformation and cell culture establishment, and allows the generation of transgenic suspension cultures of Medicago reference accession A17.
KW - A17
KW - Agrobacterium transformation
KW - Cell suspension culture
KW - Fluorescent protein
KW - Jemalong
KW - Medicago truncatula
UR - http://www.scopus.com/inward/record.url?scp=85056809742&partnerID=8YFLogxK
U2 - 10.1007/s11240-018-1525-3
DO - 10.1007/s11240-018-1525-3
M3 - Article
VL - 136
SP - 445
EP - 450
JO - Plant Cell Tissue And Organ Culture
JF - Plant Cell Tissue And Organ Culture
SN - 0167-6857
IS - 3
ER -