Gene transfer to plants - Principles of Gene Manipulation - page 238

in a high frequency of cotransformation (Schocher
et al. 1986). Putative transformants are transferred
to selective medium, where surviving protoplasts
regenerate their cell walls and commence cell divi-
sion, producing a callus. Subsequent manipulation
of the culture conditions then makes it possible to
induce shoot and root development, culminating in
the recovery of fertile transgenic plants. The major
limitation of protoplast transformation is not the
gene-transfer process itself, but the ability of the host
species to regenerate from protoplasts. A general
observation is that dicots are more amenable than
monocots to this process. In species where regenera-
tion is possible, an advantage of the technique is that
protoplasts can be cryopreserved and retain their
regenerative potential (DiMaio & Shillito 1989).
The first transformation experiments concentrated
on species such as tobacco and petunia in which
protoplast-to-plant regeneration is well documented.
An early example is provided by Meyer et al. (1987),
who constructed a plasmid vector containing the
nptII marker gene, and a maize complementary DNA
(cDNA) encoding the enzyme dihydroquercetin 4-
reductase, which is involved in anthocyanin pig-
ment biosynthesis. The transgene was driven by the
strong and constitutive cauliflower mosaic virus
(CaMV) 35S promoter. Protoplasts of a mutant,
white-coloured petunia strain were transformed
with the recombinant plasmid by electroporation
and then selected on kanamycin-supplemented
medium. After a few days, surviving protoplasts had
given rise to microcalli, which could be induced to
regenerate into whole plants. The flowers produced
by these plants were brick-red instead of white,
showing that the maize cDNA had integrated into
the genome and was expressed.
After successful experiments using model dicots,
protoplast transformation was attempted in mono-
cots, for which no alternative gene-transfer system
was then available. In the first such experiments,
involving wheat (Lorz et al. 1985) and the Italian
ryegrass Lolium multiflorum (Potrykus et al. 1985b),
protoplast transformation was achieved and trans-
genic callus obtained, but it was not possible to
recover transgenic plants. The inability of most
monocots to regenerate from protoplasts may reflect
the loss of competence to respond to tissue-culture
conditions as the cells differentiate. In cereals and

grasses, this has been addressed to a certain extent
by using embryogenic suspension cultures as a source
of protoplasts. Additionally, since many monocot
species are naturally tolerant to kanamycin, the
nptII marker used in the initial experiments was
replaced with alternative markers conferring resist-
ance to hygromycin or phosphinothricin. With these
modifications, it has been possible to regenerate
transgenic plants representing certain varieties of rice
and maize with reasonable efficiency (Shimamoto
et al. 1988, Datta et al. 1990, Omirulleh et al. 1993).
However, the extended tissue-culture step is unfavour-
able, often resulting in sterility and other phenotypic
abnormalities in the regenerated plants. The trans-
formation of protoplasts derived from stomatal guard
cells has recently been identified as an efficient and
genotype-independent method for the production of
transgenic sugar beet (Hall et al. 1996).

Particle bombardment

An alternative procedure for plant transformation
was introduced in 1987, involving the use of a
modified shotgun to accelerate small (1- 4

m) metal
particles into plant cells at a velocity sufficient to
penetrate the cell wall (~250 m/s). In the initial
test system, intact onion epidermis was bombarded
with tungsten particles coated in tobacco mosaic
virus (TMV) RNA. Three days after bombardment,
approximately 40% of the onion cells that contained
particles also showed evidence of TMV replication
(Sanford et al. 1987). A plasmid containing the cat
reporter gene driven by the CaMV 35S promoter
was then tested to determine whether DNA could
be delivered by the same method. Analysis of the
epidermal tissue 3 days after bombardment revealed
high levels of transient chloramphenicol trans-
acetylase (CAT) activity (Klein et al. 1987).
The stable transformation of explants from
several plant species was achieved soon after these
initial experiments. Early reports included the trans-
formation of soybean (Christou et al. 1988), tobacco
(Klein et al. 1988b) and maize (Klein et al. 1988a). In
each case, the nptII gene was used as a selectable
marker and transformation was confirmed by the
survival of callus tissue on kanamycin-supplemented
medium. The ability to stably transform plant cells
by this method offered the exciting possibility of

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