Gene transfer to plants - Principles of Gene Manipulation - page 240

the way in the development of so-called in planta
transformation techniques, where the need for
tissue culture is minimized or eliminated altogether.
Such methods involve the introduction of DNA,
either by Agrobacterium or by direct transfer, into
intact plants. The procedure is carried out at an
appropriate time in the plant's life cycle, so that
the DNA becomes incorporated into cells that will
contribute to the germ line, directly into the germ
cells themselves (often at around the time of fertiliza-
tion) or into the very early plant embryo. Generally,
in planta transformation methods have a very low
efficiency, so the small size of Arabidopsis and its
ability to produce over 10 000 seeds per plant is
advantageous. This limitation has so far prevented
in planta techniques from being widely adopted for
other plant species.
The first in planta transformation system involved
imbibing Arabidopsis seeds overnight in an Agrobac-
terium culture, followed by germination (Feldmann
& Marks 1987). A large number of transgenic plants
containing T-DNA insertions were recovered, but in
general this technique has a low reproducibility.
Bechtold et al. (1993) has described a more reliable
method, in which the bacteria are vacuum-
infiltrated into Arabidopsis flowers. An even simpler
technique called floral dip has become widely used
(Clough & Bent 1998). This involves simply dipping
Arabidopsis flowers into a bacterial suspension at
the time of fertilization. In both these methods, the
transformed plants are chimeric, but give rise to a
small number of transgenic progeny (typically
about 10 per plant). Similar approaches using direct
DNA transfer have been tried in other species,
but germ-line transformation has not been repro-
ducible. For example, naked DNA has been injected
into the floral tillers of rye plants (De La Pena et al.
1987) and post-fertilization cotton flowers (Zhou
et al. 1983), resulting in the recovery of some trans-
genic plants. Transgenic tobacco has been produced
following particle bombardment of pollen (Touraev
et al. 1997).
An alternative to the direct transformation of germ-
line tissue is the introduction of DNA into meristems
in planta , followed by the growth of transgenic
shoots. In Arabidopsis , this has been achieved simply
by severing apical shoots at their bases and inoculat-
ing the cut tissue with Agrobacterium suspension

(Chang et al. 1994). Using this procedure, trans-
genic plants were recovered from the transformed
shoots at a frequency of about 5%. In rice, explanted
meristem tissue has been transformed using Agro-
bacterium and particle bombardment, resulting in the
proliferation of shoots that can be regenerated into
transgenic plants (Park et al. 1996). Such proced-
ures require only a limited amount of tissue culture.

Chloroplast transformation

So far, we have exclusively considered DNA transfer
to the plant's nuclear genome. However, the chloro-
plast is also a useful target for genetic manipulation,
because thousands of chloroplasts may be present in
photosynthetic cells and this can result in levels of
transgene expression up to 50 times higher than
possible using nuclear transformation. Furthermore,
transgenes integrated into chloroplast DNA do not
appear to undergo silencing or suffer from position
effects that can influence the expression levels of
transgenes in the nuclear DNA (see Boxes 11.1 and
13.2). Chloroplast transformation also provides a
natural containment method for transgenic plants,
since the transgene cannot be transmitted through
pollen (reviewed by Maliga 1993).
The first reports of chloroplast transformation were
serendipitous, and the integration events were found
to be unstable. For example, an early experiment
in which tobacco protoplasts were cocultivated
with Agrobacterium resulted in the recovery of one
transgenic plant line, in which the transgene was
transmitted maternally. Southern-blot analysis of
chloroplast DNA showed directly that the foreign DNA
had become integrated into the chloroplast genome
(De Block et al. 1985). However, Agrobacterium does
not appear to be an optimal system for chloroplast
transformation, probably because the T-DNA com-
plex is targeted to the nucleus. Therefore, direct
DNA transfer has been explored as an alternative
strategy. Stable chloroplast transformation was
first achieved in the alga Chlamydomonas reinhardtii ,
which has a single large chloroplast occupying most
of the volume of the cell (Boynton et al. 1988).
Particle bombardment was used in this experiment.
The principles established using this simple organ-
ism were extended to tobacco, allowing the recovery
of stable transplastomic tobacco plants (Svab et al.