Screening for recombinants of Crambe abyssinica after transformation by the PMF1 marker-free vector based on chemical selection and meristematic regeneration

W. Qi, I.E.M. Tinnenbroek-Capel, E.M.J. Salentijn, J.G. Schaart, J. Cheng, C. Denneboom, Z. Zhang, X. Zhang, H. Zhao, R.G.F. Visser, Huang Bangquan, E.N. van Loo, F.A. Krens

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Abstract

The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.
Original languageEnglish
Article number14033
Number of pages15
JournalScientific Reports
Volume5
DOIs
Publication statusPublished - 2015

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Crambe abyssinica
dexamethasone
screening
gene fusion
chimerism
fluorouracil
DNA
hexaploidy
tissue repair
meristems
germplasm
cells

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@article{fd0e412a2afc4a15ad23ec2b9912a640,
title = "Screening for recombinants of Crambe abyssinica after transformation by the PMF1 marker-free vector based on chemical selection and meristematic regeneration",
abstract = "The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.",
author = "W. Qi and I.E.M. Tinnenbroek-Capel and E.M.J. Salentijn and J.G. Schaart and J. Cheng and C. Denneboom and Z. Zhang and X. Zhang and H. Zhao and R.G.F. Visser and {Huang Bangquan} and {van Loo}, E.N. and F.A. Krens",
year = "2015",
doi = "10.1038/srep14033",
language = "English",
volume = "5",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - Screening for recombinants of Crambe abyssinica after transformation by the PMF1 marker-free vector based on chemical selection and meristematic regeneration

AU - Qi, W.

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

AU - Salentijn, E.M.J.

AU - Schaart, J.G.

AU - Cheng, J.

AU - Denneboom, C.

AU - Zhang, Z.

AU - Zhang, X.

AU - Zhao, H.

AU - Visser, R.G.F.

AU - Huang Bangquan, null

AU - van Loo, E.N.

AU - Krens, F.A.

PY - 2015

Y1 - 2015

N2 - The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

AB - The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

U2 - 10.1038/srep14033

DO - 10.1038/srep14033

M3 - Article

VL - 5

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 14033

ER -