Plant Molecular Biology 42: 819–832, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
pGreen: a versatile and ﬂexible binary Ti vector for
Agrobacterium-mediated plant transformation
Roger P. Hellens
, E. Anne Edwards, Nicola R. Leyland, Samantha Bean and Philip M.
John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK (
author for correspondence;e-mail:
firstname.lastname@example.org; fax: +44 1603 456844)
Received 8 June 1999; accepted in revised form 30 January 2000
Key words: Agrobacterium, binary vectors, plant transformation, reporter genes, selectable marker genes, Ti vector
Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols.
The pGreen series of binary Ti vectors are conﬁgured for ease-of-use and to meet the demands of a wide range
of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable
marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites
for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its
size and copy number in Escherichiacoli offers increased efﬁcienciesin routine in vitro recombinationprocedures.
pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup
provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size
of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with
the same efﬁciencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an
Internet site (http://www.pgreen.ac.uk)through which comprehensive information, protocols, order forms and lists
of different pGreen marker gene permutations can be found.
Stable plant transformation is commonly achieved by
Agrobacterium tumefaciens-mediated procedures (El-
lis, 1993). Agrobacterium is a plant pathogen which
causes the formation of crown galls or tumours in
tissues infected by the bacterium (see Sheng and
Citovsky, 1996, and Gheysen et al., 1998, for a
detailed review). Brieﬂy, the determinants for estab-
lishing and sustaining tumours are located mostly on
large (>200 kb) Ti (tumour-inducing) plasmids. The
T-DNA and the virulence (vir) region are two dis-
tinct regions of all Ti plasmids which are essential for
Agrobacterium-mediated plant transformation. The T-
DNA is a discrete section of the Ti plasmid bounded
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number AJ007829 (pGreen0000).
by 25 bp imperfect repeats termed the right (RB) and
left borders (LB). The T-DNA is transferred to, and
integrated in, the host cell nuclear genome at the on-
set of infection. The processing of the T-DNA and its
transfer to the host plant cell nucleus is achieved pri-
marily by the concerted action of about 20 vir gene
products. All the plasmid-encoded vir genes reside in
a region of the Ti plasmid. Ti plasmid-encoded vir
genes can function in trans to promote the transfer of
T-DNAs from co-resident plasmids to recipient plant
cells (Hoekema et al., 1983). Such T-DNA-containing
plasmids are termed Ti vectors (Bevan, 1984; Guer-
ineau and Mullineaux, 1993). Genes and sequences to
be transformedinto plants are insertedbetween the LB
and RB of the Ti vector T-DNA.
Binary Ti vectors are able to replicate in Es-
cherichia coli and Agrobacterium species. In such
plasmids, a broad-host-rangereplication locus is often