Regeneration and transformation of Crambe abyssinica

W. Qi, I.E.M. Tinnenbroek-Capel, J. Schaart, Huang Bangquan, J. Cheng, R.G.F. Visser, E.N. van Loo, F.A. Krens

Research output: Contribution to journalArticleAcademicpeer-review

11 Citations (Scopus)

Abstract

Background: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. Results: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L-1) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. Conclusions: Present research revealed the potential of using crambe meristematic tissue for genetic transformation andin vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.
Original languageEnglish
Article number235
Number of pages12
JournalBMC Plant Biology
Volume14
DOIs
Publication statusPublished - 2014

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Crambe abyssinica
Crambe (Brassicaceae)
explants
kanamycin
buds
cotyledons
Agrobacterium
shoots
industrial oils
erucic acid
chemical industry
oilseed crops
seedlings
DNA
genetic transformation
selection methods
petioles
feedstocks
genetic engineering
seed oils

Keywords

  • gene
  • agrobacterium
  • tissue
  • plants

Cite this

@article{50be9346a3e24f5a838c9cf68119fc4d,
title = "Regeneration and transformation of Crambe abyssinica",
abstract = "Background: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. Results: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10{\%} transformation frequency, and cotyledons 4{\%}, while with the consistent method (25 mg · L-1) lower frequencies were found, 1{\%} for cotyledonary nodes and 0{\%} for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2{\%}. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4{\%}. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50{\%} of the transgenic lines had a single-copy insertion. Conclusions: Present research revealed the potential of using crambe meristematic tissue for genetic transformation andin vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.",
keywords = "gene, agrobacterium, tissue, plants",
author = "W. Qi and I.E.M. Tinnenbroek-Capel and J. Schaart and {Huang Bangquan} and J. Cheng and R.G.F. Visser and {van Loo}, E.N. and F.A. Krens",
year = "2014",
doi = "10.1186/s12870-014-0235-1",
language = "English",
volume = "14",
journal = "BMC Plant Biology",
issn = "1471-2229",
publisher = "Springer Verlag",

}

Regeneration and transformation of Crambe abyssinica. / Qi, W.; Tinnenbroek-Capel, I.E.M.; Schaart, J.; Huang Bangquan; Cheng, J.; Visser, R.G.F.; van Loo, E.N.; Krens, F.A.

In: BMC Plant Biology, Vol. 14, 235, 2014.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Regeneration and transformation of Crambe abyssinica

AU - Qi, W.

AU - Tinnenbroek-Capel, I.E.M.

AU - Schaart, J.

AU - Huang Bangquan, null

AU - Cheng, J.

AU - Visser, R.G.F.

AU - van Loo, E.N.

AU - Krens, F.A.

PY - 2014

Y1 - 2014

N2 - Background: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. Results: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L-1) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. Conclusions: Present research revealed the potential of using crambe meristematic tissue for genetic transformation andin vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.

AB - Background: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. Results: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L-1) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. Conclusions: Present research revealed the potential of using crambe meristematic tissue for genetic transformation andin vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.

KW - gene

KW - agrobacterium

KW - tissue

KW - plants

U2 - 10.1186/s12870-014-0235-1

DO - 10.1186/s12870-014-0235-1

M3 - Article

VL - 14

JO - BMC Plant Biology

JF - BMC Plant Biology

SN - 1471-2229

M1 - 235

ER -