Arabidopsis plants were transformed as described previously [ 19, 56] with Agrobacterium tumefaciens strains GV3101 pMP90 [ 101] or LBA4404 [ 102] containing the GFP binary vectors using the standard floral dip method [ 103].
The generated 126bp-fragments were cloned into the binary vector using HindIII and PstI restriction sites, 5' of the minimal promoter in pGreen- m35S::GFP.
After confirmation of the mutations by DNA sequencing, the precursor was placed downstream of the UBQ10 promoter in a binary vector using standard molecular biology procedures.
The GUSPlus gene was PCR amplified from pCambia 1305.1 binary vector using high fidelity Platinum TAQ DNA polymerase (Invitrogen life technologies, Carlsbad, CA) and gene specific primers complementary to the GUSPlus gene.
cDNAs from expressed sequenced apple libraries [ 58] were cloned into pART27 binary vector using restriction enzymes or pHEX2 using Gateway cloning.
Each cDNA molecule was flanked at the 5′ and 3′ termini by a short sequence providing the required homology to the 5′ and 3′ termini of the linearized binary vector used for cloning of the cDNA molecules.
The T-DNA of the binary vector used has unique restriction sites for EcoRI and DraI at the left and right borders of the sequence, respectively, and digestion of the DNA with either of these enzymes generated unique fragments between the T-DNA and plant DNA.
A. thaliana was transformed with a CaMV 35S binary expression vector using the floral dip method, and events containing a single T-DNA insertion selected.
The resulting ST111 binary vector used for soybean transformation contained the 7S β-conglycinin promoter, Tobacco Etch Virus TEVV) translational enhancer, glycinin signal peptide, hMBP-Sigma fusion protein and the 35 s terminator.
In addition, the T-DNA region of the binary vector (used for rice transformation) contained a selectable marker gene bar for the herbicide bialaphos resistance (Fig. 1A).
