Gene transfer to plants - Principles of Gene Manipulation - page 234

Bsp H I (3031)

pSa-ORI

Bgl II (536)
Hpa I (568)

Asp 7181 (783)
Apa I (793)
Xho I (798)
Sal I (804)
Cla I (814)
Eco RV (827)
Eco R I (831)
Pst I (841)
Sma I (845)
Bam H I (849)
Spe I (855)
Xba I (861)
Not I (868)
Sac II (880)
Sac I (889)

LB

npt I

lacZ

pGreen II 0000
3304 bp

RB

Fig. 12.13 The small and versatile
binary vector pGreen, reproduced with
permission of Roger Hellens and Phil
Mullineaux.

Bsp H I (2070)

ColE I ori

Stu I (1292)
Bgl II (1345)

on recombination, and the binary vector's copy
number is not determined by the Ti plasmid, making
the identification of transformants much easier.
All the conveniences of bacterial cloning plasmids
have been incorporated into binary vectors, such as
multiple unique restriction sites in the T-DNA region
to facilitate subcloning, the lacZ gene for blue -white
screening (McBride & Summerfelt 1990) and a

A simple experimental procedure for
Agrobacterium -mediated
transformation

Once the principle of selectable, disarmed T-DNA
vectors was established, there followed an explosion
in the number of experiments involving DNA trans-
fer to plants. Variations on the simple general
protocol of Horsch et al. (1985) have been widely
used for dicot plants (Fig. 12.14). In the original
report, small discs (a few millimetres in diameter)
were punched from leaves, surface-sterilized and
inoculated in a medium containing A. tumefaciens
transformed with the recombinant disarmed T-DNA
(as a cointegrate or binary vector). The foreign DNA
contained a chimeric neo gene conferring resistance
to the antibiotic kanamycin. The discs were cultured
for 2 days and transferred to medium containing
kanamycin, to select for the transferred neo gene,
and carbenicillin, to kill the Agrobacterium. After
2- 4 weeks, developing shoots were excised from the
callus and transplanted to root-induction medium.
Rooted plantlets were subsequently transplanted
to soil, about 4 -7 weeks after the inoculation step.
This method has the advantage of being simple
and relatively rapid. It is superior to previous
methods, in which transformed plants were regener-
ated from protoplast-derived callus, the protoplasts
having been transformed by cocultivation with the

cos
site for preparing cosmid libraries (Lazo et al. 1991,
Ma et al. 1992). A current binary vector, pGreen,
is shown in Fig. 12.13 (Hellens et al. 2000b). This
plasmid is less than 5 kbp in size and has 18 unique
restriction sites in the T-DNA, because the T-DNA is
entirely synthetic. It has a lacZ gene for blue -white
selection of recombinants, and a selectable marker
that can be used both in bacteria and in the trans-
formed plants. The progressive reduction in size has
been made possible by removing essential genes
required for replication in Agrobacterium and trans-
ferring these genes to the bacterium's genome or
on to a helper plasmid. The pGreen plasmid, for
example, contains the Sa origin of replication, which
is much smaller than the more traditional Ri and
RK2 regions. Furthermore, an essential replicase gene
is housed on a second plasmid, called pSoup, resident
within the bacterium. All conjugation functions
have also been removed, so this plasmid can only
be introduced into Agrobacterium by transformation
(Hellens et al. 2000b).

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