title: Screening for recombinants of Crambe abyssynica 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.
authors.affiliation: Jiangsu Key Laboratory for Bioresources of Saline Soils, Provincial Key Laboratory of Agrobiology, Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, People's Republic of China.|Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Weicong
authors.initials: W
authors.lastname: Qi
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Iris E M
authors.initials: IE
authors.lastname: Tinnenbroek-Capel
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Elma M J
authors.initials: EM
authors.lastname: Salentijn
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Jan G
authors.initials: JG
authors.lastname: Schaart
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Jihua
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Christel
authors.initials: C
authors.lastname: Denneboom
authors.affiliation: Department of Ornamental Horticulture, China Agricultural University, Beijing, 100193, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory for Bioresources of Saline Soils, Provincial Key Laboratory of Agrobiology, Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, People's Republic of China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory for Bioresources of Saline Soils, Provincial Key Laboratory of Agrobiology, Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, People's Republic of China.
authors.forename: Han
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Richard G F
authors.initials: RG
authors.lastname: Visser
authors.affiliation: College of Life Science, Hubei University, Zip code 430062, Wuhan City, China.
authors.forename: Bangquan
authors.initials: B
authors.lastname: Huang
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Eibertus N
authors.initials: EN
authors.lastname: Van Loo
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands.
authors.forename: Frans A
authors.initials: FA
authors.lastname: Krens
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics*; D018519:Meristem / Q000187:drug effects; D010641:Phenotype; D030821:Plants, Genetically Modified; D011995:Recombination, Genetic; D012038:Regeneration; D064028:Tracheophyta / Q000187:drug effects / Q000502:physiology*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D003907:Dexamethasone; D005472:Fluorouracil