Gene transfer to plants - Principles of Gene Manipulation - page 235

Surface-sterilize Inoculation

Culture overnight
in liquid with
Agrobacterium to
infect cut edg es of disc

Small discs
punched
from leaf

Blot disc
dry

Leaf disc

Feeder plate

Filter paper

Shoot-inducing solid
medium + kanamycin
+ carbenicillin
Culture for about
20 days

A layer of feeder
cells, previously
grown in
suspension

Shoot-inducing
solid medium
(high in cytokinin)
Culture for 2 days

Shoot
formation

Root-inducing solid
medium (high in
auxin) + kanamycin +
carbenic illin

Excise
shooted callus,
transfer to
root-inducing
medium

Transfer plantlets to
soil as soon as roots
appear 4-7 weeks
after inoculation

Fig. 12.14 Leaf-disc transformation by
Agrobacterium tumefaciens .

Agrobacterium (De Block et al. 1984, Horsch et al.
1984). Contemporary protocols for the Agrobacterium -
mediated transformation of many solanaceous
plants are variations on the theme of the leaf-disc
protocol, although the optimal explant must be
determined for each species. Alternative procedures
are required for the transformation of monocots, as
discussed below.

that some monocots might be susceptible to Agro-
bacterium infection (see, for example, the discussion
of agroinfection with maize streak virus DNA on
p. 243). However, in most cases there was no
convincing evidence for T-DNA integration into the
plant genome. In the laboratory, it proved possible
to induce tumours in certain monocot species, such
as asparagus (Hernalsteens et al. 1984) and yam
(Schafer et al. 1987). In the latter case, an important
factor in the success of the experiment was pretreat-
ment of the Agrobacterium suspension with wound
exudate from potato tubers. It has been argued that
Agrobacterium infection of monocots is inefficient
because wounded monocot tissues do not produce
phenolics, such as acetosyringone, at sufficient
levels to induce vir gene expression.

Agrobacterium and monocots

Until recently, most monocotyledonous plants (mono-
cots) were thought to be outside the host range of
Agrobacterium , prompting research into alternative
transformation methods, as discussed below. During
the 1980s, limited evidence accumulated showing