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Evaluation of Antibiotic-Based Selection Methods for Camelina sativa Stable Transformants

Ontiveros-Cisneros, Abraham LU orcid ; Moss, Oliver LU ; Van Moerkercke, Alex LU and Van Aken, Olivier LU (2022) In Cells 11(7).
Abstract

Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for... (More)

Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve Discosoma sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of Catharanthus roseus using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for Arabidopsis thaliana. We also demonstrate a successful method to cross Camelina sativa and provide qRT-PCR results to prove its effectiveness.

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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Antibiotic selection, BASTA, Camelina, Camelina crossing, Hygromycin, QRT-PCR
in
Cells
volume
11
issue
7
article number
1068
publisher
MDPI AG
external identifiers
  • scopus:85126944289
  • pmid:35406632
ISSN
2073-4409
DOI
10.3390/cells11071068
project
Plants as biofactories for insect sex pheromones production
language
English
LU publication?
yes
id
fb3f375c-07ec-4bd1-a5e8-0e65007d7b49
date added to LUP
2022-04-19 13:01:59
date last changed
2025-05-05 00:16:32
@article{fb3f375c-07ec-4bd1-a5e8-0e65007d7b49,
  abstract     = {{<p>Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve Discosoma sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of Catharanthus roseus using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for Arabidopsis thaliana. We also demonstrate a successful method to cross Camelina sativa and provide qRT-PCR results to prove its effectiveness.</p>}},
  author       = {{Ontiveros-Cisneros, Abraham and Moss, Oliver and Van Moerkercke, Alex and Van Aken, Olivier}},
  issn         = {{2073-4409}},
  keywords     = {{Antibiotic selection; BASTA; Camelina; Camelina crossing; Hygromycin; QRT-PCR}},
  language     = {{eng}},
  number       = {{7}},
  publisher    = {{MDPI AG}},
  series       = {{Cells}},
  title        = {{Evaluation of Antibiotic-Based Selection Methods for Camelina sativa Stable Transformants}},
  url          = {{http://dx.doi.org/10.3390/cells11071068}},
  doi          = {{10.3390/cells11071068}},
  volume       = {{11}},
  year         = {{2022}},
}