Update on Plant Transformation
Arabidopsis in Planta Transformation. Uses,
Mechanisms, and Prospects for Transformation of
Other Species1
Andrew F. Bent*
Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706
The ability to move DNA into an organism and
thereby alter its phenotype is central to both basic
and applied molecular biology. Transformation is a
simple task with Escherichia coli or Saccharomyces cerevisiae, but is usually more difficult with multicellular
eukaryotes and can be particularly challenging with
some important plant species. However, for Arabidopsis, in planta transformation methods have been
developed that are incredibly simple. Attempts to
apply in planta transformation methods to other
plant species have often failed. This may be due in
part to a poor understanding of the mechanisms that
underlie the successful Arabidopsis transformation
method. Studies of Arabidopsis transformation have
accordingly been pursued, and three groups have
recently published relevant findings. Successful in
planta transformation of the legume Medicago truncatula was also reported recently, showing that the
method can be adapted to other species. The cellular
target for transformation of M. truncatula may differ
somewhat from the target in Arabidopsis. The above
findings may guide future efforts to improve transformation of other plant species.
This update opens by briefly reviewing transformation protocols that avoid tissue culture, and their
impressive utility. Recent findings concerning Arabidopsis and M. truncatula transformation are then described. The review closes by commenting on possible avenues for improvement of transformation in
other plant species.
BACKGROUND
Genetic transformation of plants occurs naturally
(Hooykaas and Schilperoort, 1992). Scientists have
been able to carry out controlled plant transformation with specific genes since the mid-1970s. The
most common methods for introduction of DNA into
plant cells use Agrobacterium tumefaciens bacteria or
rapidly propelled tungsten microprojectiles that have
been coated with DNA (Birch, 1997; Hansen and
1
Plant transformation research in the author’s laboratory was
supported by the North Central Soybean Research Program.
* E-mail [email protected]; fax 608 –263–2626.
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Wright, 1999). Other methods such as electroporation, microinjection, or delivery by virus have also
been exploited. To allow physiological selection of
cells that have been successfully transformed, the
DNA of interest is typically cloned adjacent to DNA
for a selectable marker gene such as nptII (encoding
kanamycin antibiotic resistance).
Genetic transformation can be transient or stable,
and transformed cells may or may not give rise to
gametes that pass genetic material on to subsequent
generations. Transformation of protoplasts, callus
culture cells, or other isolated plant cells is usually
straightforward and can be used for short-term studies of gene function (Gelvin and Schilperoort, 1998).
Transformation of leaf mesophyll cells or other cells
within intact plants may in some cases broaden the
utility of single-cell assays (e.g. Tang et al., 1996).
Exciting new approaches such as virus-induced gene
silencing may also be applicable for some studies
(Baulcombe, 1999). In the era of genomics these shortterm assays will become increasingly important.
However, in many cases it is desirable or necessary to
produce a uniformly transformed plant that carries
the transgene in the nuclear genome as a single Mendelian locus.
The generation of genetically homogeneous plants
carrying the same transformation event in all cells
has typically presented two separate hurdles: transformation of plant cells and regeneration of intact,
reproductively competent plants from those transformed cells (Birch, 1997; Hansen and Wright, 1999).
Although many successful plant regeneration methods have been developed, these methods often require a great deal of protocol refinement and the
focused effort of expert practitioners. It is unfortunate that plant regeneration from single transformed
cells often produces mutations ranging from single
base changes or small rearrangements to the loss of
entire chromosomes. In addition, significant epigenetic changes (for example, in DNA methylation) can
also occur (Phillips et al., 1994). It is often necessary
to generate and screen a dozen or more independent
plant lines transformed with the same construct to
find lines that have suffered minimal genetic damage
and that carry a simple insertion event (Birch, 1997;
Hansen and Wright, 1999). Transformation is feasible
Plant Physiology, December 2000, Vol. 124, pp. 1540–1547, www.plantphysiol.org © 2000 American Society of Plant Physiologists
Uses and Mechanisms of in Planta Transformation
in many plant species, but has required acceptance of
the above limitations.
TRANSFORMATION METHODS THAT AVOID
TISSUE CULTURE
A number of laboratories have pursued plant transformation methods that avoid tissue culture or regeneration. In many cases these methods have targeted
meristems or other tissues that will ultimately give
rise to gametes (Chee and Slighton, 1995; Birch,
1997). The same is true of popular tissue culturebased transformation methods for corn, rice, wheat,
and soybean, which target young apical meristems
for transformation (Birch, 1997). For those methods,
excised or partially disrupted meristems are transformed, subjected to antibiotic or herbicide selection,
and then carried through tissue culture to regenerate
shoots and roots from the transformed tissues. For
non-tissue culture approaches, Agrobacterium or
tungsten particles have been used in a number of
species to transform cells in or around the apical
meristems that are subsequently allowed to grow
into plants and produce seeds (Chee and Slighton,
1995; Birch, 1997). However, transformed sectors
have typically not persisted into gametes at reasonable frequencies, or the methods have been difficult
to reproduce (Birch, 1997). Injection of naked DNA
into ovaries has also been reported to produce transformed progeny (Zhou et al., 1983). Variations of this
method and “pollen tube pathway” delivery of DNA
are still practiced in China (Hu and Wang, 1999).
Electroporation-mediated gene transfer into intact
meristems in planta and a variety of pollen transformation procedures have also been reported (Chowrira et al., 1995; Touraev et al., 1997 and refs. therein).
However, most of these methods have been difficult
to reproduce and have not gained widespread
acceptance.
ARABIDOPSIS TRANSFORMATION WITHOUT
TISSUE CULTURE
Early stages of the revolution that transformed
Arabidopsis transformation were carried out by Ken
Feldmann and David Marks. They applied Agrobacterium to Arabidopsis seeds, grew plants to maturity
in the absence of any selection, then collected progeny seeds and germinated them on antibioticcontaining media to identify transformed plants
(Feldmann and Marks, 1987; Feldmann, 1992). Although the procedure was difficult to reproduce consistently, successful rounds produced transformants
at a high enough rate that thousands of transformed
lines were produced in a matter of a few years. These
“insertional mutagenesis” lines helped speed gene
cloning by the Arabidopsis community (AzpirozLeehan and Feldmann, 1997). The lines could be
Plant Physiol. Vol. 124, 2000
screened for mutant phenotypes of interest and the
mutated gene responsible for the phenotype could
often be identified by isolation of the Arabidopsis
chromosomal DNA flanking the previously known
T-DNA (transferred DNA from Agrobacterium).
Other laboratories later succeeded in generating
transformed Arabidopsis lines by “clip ‘n squirt”
methods (Chang et al., 1994; Katavic et al., 1994).
Reproductive inflorescences were clipped off,
Agrobacterium was applied to the center of the plant
rosette, new inflorescences formed a few days later
were again removed, Agrobacterium was re-applied,
and plants were then allowed to develop and set
seed. Transformants were obtained more reliably
than with the seed treatment method, but the methods were only marginally more productive than traditional tissue-culture approaches to Arabidopsis
transformation (e.g. Valvekens et al., 1988).
A third, crucial stage of the revolution in Arabidopsis transformation came when Georges Pelletier,
Nicole Bechtold, and Jeff Ellis reported success at
transformation by “vacuum infiltration” (Bechtold et
al., 1993). Arabidopsis plants at the early stages of
flowering were uprooted and placed en masse into a
bell jar in a solution of Agrobacterium. A vacuum was
applied and then released, causing air trapped within
the plant to bubble off and be replaced with the
Agrobacterium solution. Plants were transplanted
back to soil, grown to seed, and in the next generation stably transformed lines could be selected using
the antibiotic or herbicide appropriate for the selectable marker gene. Transformation rates often exceeded 1% of the seeds tested. Variations of this
extremely simple new method (Fig. 1) have been
widely adopted by Arabidopsis researchers. Tissue
culture and plant regeneration are no longer necessary and the associated high rates of mutation are
avoided.
THE UTILITY OF AN ACCESSIBLE
TRANSFORMATION METHOD
The impact of the vacuum infiltration method on
Arabidopsis research has been remarkable. Generation of transformed lines is simple and routine (Fig. 1;
Bechtold and Pelletier, 1998; Clough and Bent, 1998).
First and foremost, barriers to in planta testing of a
gene of interest have been dramatically lowered.
With minimal effort and in a matter of 3 to 6 months,
multiple transgenic plant lines can be constructed
and numerous DNA constructs can be tested.
A second example of this method’s utility can be
seen in positional cloning projects, in which a gene of
unknown structure is isolated based on its genetic
map position (e.g. Clough et al., 2000). Once a gene
has been mapped to a genetic interval of a few centiMorgans, an Arabidopsis chromosome walk can be
accelerated by the use of publicly available bacterial
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Bent
Figure 1. It’s really that simple. For floral dip transformation of
Arabidopsis, plants are grown to a stage when they have just started
to flower (A), plants are dipped briefly in a suspension of Agrobacterium, Suc, and surfactant (B), plants are maintained for a few more
weeks until mature and then progeny seeds are harvested (C), and
seeds are germinated on selective medium (e.g. containing kanamycin) to identify successfully transformed progeny (D).
artificial chromosome collections. Bacterial artificial
chromosome clones containing insert DNAs that
span the genomic region can be subcloned into a
transformation-competent binary vector, moved into
Agrobacterium, and used for “focused shotgun complementation” of a mutant plant line. With the genome
sequence available and anchored to genetic maps, researchers may even choose to target their effort to
specific candidate genes. The gene of interest is identified by screening sets of transformed plants for individuals that exhibit a corrected phenotype.
Another important use of simple, high-throughput
transformation returns to the insertional mutagenesis
methods pioneered by Feldmann and others. Collections of Arabidopsis containing tens of thousands of
independent transformed lines are now available for
screening (Azpiroz-Leehan and Feldmann, 1997;
Krysan et al., 1999). Researchers alternatively can
pursue insertional mutagenesis of a unique plant
line, for example to carry out screens for genetic
suppressors of a particular mutation. Activation1542
tagging T-DNAs can be used that enhance the expression level of genes near the T-DNA insert, or one
can use T-DNAs that place ␤-glucuronidase (GUS) or
other marker genes under the control of host promoter or enhancer elements that may flank the
T-DNA at the site of insertion (Weigel et al., 2000).
Forward genetic approaches study phenotype first
and then genotype, whereas reverse genetic strategies start with a DNA sequence and then seek a plant
line mutated in that gene. Efficient transformation
methods have facilitated reverse genetic screening in
plants. Public collections have been created that allow PCR-based screening of ordered pools of DNA
from thousands of transgenic lines (Krysan et al.,
1999). Once the subpool of DNA carrying an insert in
the gene of interest has been identified, progeny seed
corresponding to that pool can be requested. It is
important to note that many of these insertional mutagenesis methods can also be accomplished using
transposon mutagenesis (e.g. Tissier et al., 1999). A
goal for many T-DNA and transposon-mutagenized
seedbank collections is to obtain and compile in databases a small stretch of sequence data for the DNA
that flanks each insertion (Tissier et al., 1999). This
will allow researchers to simply request plant lines
that carry a mutation within any DNA sequence in
the genome.
The above strategies could be extremely useful in
research with other plant species. For some species,
the current transformation protocols are close to being sufficient (witness the recent production of more
than 18,000 fertile transgenic rice lines to form an
insertionally tagged population; Jeon et al., 2000). For
many important species, however, pursuit of the
above strategies would be greatly facilitated by the
availability of high-throughput/non-tissue culture
transformation methods. After the success of the Arabidopsis vacuum infiltration protocol, a number of
laboratories tried to use Agrobacterium vacuum infiltration with other plant species, but failed to obtain
transformants. Why? In the absence of a suitable
answer, study of the successful Arabidopsis methods
was a logical next step.
HOW DO IN PLANTA ARABIDOPSIS
TRANSFORMATION PROCEDURES WORK?
What is the cellular target of transformation? For
the Arabidopsis seed transformation and vacuum
infiltration methods, it was shown early on that most
primary transformants carry hemizygous T-DNA insertion events (Feldmann, 1992; Bechtold et al., 1993).
The presence of the T-DNA on only one of two
homologous chromosomes implies that productive
transformation occurs late in floral development,
after the divergence of male and female germ
lines (Arabidopsis self-pollinates within individual
flowers, and if transformation occurred earlier, selfPlant Physiol. Vol. 124, 2000
Uses and Mechanisms of in Planta Transformation
fertilization would be expected to give rise to some
homozygous transformants due to presence of the
same T-DNA insert in pollen and embryo sac cells).
The transformation target is further defined in that
transformants obtained from a given plant usually
carry independent T-DNA insertion events (Feldmann, 1992; Bechtold et al., 1993). This suggests that
transformation occurs after the divergence of individual pollen or egg cell lineages within a flower. A
developmental endpoint for the typical target of transformation can also be postulated. Although the result
is not as well established, typical primary transformants apparently carry the transgene in all parts of the
plant, suggesting that transformation occurred before
the cell divisions in a fertilized embryo that establish
independent meristems and other distinct adult plant
cell lineages. Hence, transformation seems to occur in
developing flowers after individual gametophyte cell
lineages form, but before extensive development of
the embryo. The next question was: Does transformation occur primarily in pollen, ovules, fertilized embryos, or any of the three?
It is an interesting historical sidelight that rather
than addressing this key question, Arabidopsis researchers in the mid-1990s focused on empirical
transformation protocol improvement. Practical motivation to proceed with the generation of transformants was understandably paramount, and overall satisfaction with the new transformation method
delayed efforts to understand how it worked. Nevertheless, protocol modifications, ideas, and anecdotal observations were shared widely through
meetings, word-of-mouth, and the Arabidopsis electronic newsgroup (http://www.bio.net/hypermail/
Arabidopsis/).
Significant findings resulting from this community
effort included the discoveries that (a) Plants did not
need to be uprooted, treated with Agrobacterium, and
re-planted. Transformants could be obtained by
treating only the protruding inflorescences; (b) inclusion of Silwet L-77, a strong surfactant that shows
relatively low toxicity to plants, often enhanced
transformation reliability; and (c) many different
Arabidopsis ecotypes were transformable and many
different Agrobacterium strains could be used, although notable differences in efficiency were observed. Most important, the popular name “vacuum
infiltration” was superceded when a number of
groups found that plants could be transformed when
dipped in Agrobacterium solution with no vacuum
infiltration. Some workers subsequently moved to
spray application of Agrobacterium rather than dipping. A number of other mechanistic clues and procedural tips were shared (see http://www.bio.net/
hypermail/Arabidopsis/; Clough and Bent, 1998).
A simplified protocol for “floral dip” transformation of Arabidopsis is available at http://plantpath.
wisc.edu/wisc.edu/⬃afb/protocol.html.
Plant Physiol. Vol. 124, 2000
OVULES ARE THE PRIMARY TARGET
FOR TRANSFORMATION
Returning to the question of the cellular target of
transformation, three research groups worked in parallel to address this issue and have now published
their results (Ye et al., 1999; Bechtold et al., 2000;
Desfeux et al., 2000). Given that transformation can
occur by mere dipping of flowers in Agrobacterium
solution and that anthers and pollen are exposed
whereas ovules are not, it seemed likely that the male
germ-line would be the target of transformation.
However, all three groups found that the female
germ-line is the primary target of transformation.
In one set of experiments, transformants were produced by outcrossing after Agro-inoculation of only
the pollen donor or pollen recipient. No transformants were observed among more than 14,000 seeds
produced following inoculation of the pollen donor,
but 71 transformants were recovered out of roughly
14,800 seeds produced following inoculation of the
pollen recipient (Fig. 2; Desfeux et al., 2000). Ye and
colleagues observed zero and 15 transformants, respectively, in a similar study (Ye et al., 1999). These
findings seemingly to rule out transformation of pollen as it develops within anthers, but do not preclude
the possibility that pollen is transformed after it ger-
Figure 2. Bar graph showing the number of Arabidopsis transformants obtained by crossing Agro-inoculated pollen recipients with
noninoculated pollen donors or vice versa. Breakdown by days prior
to anthesis at time of inoculation (days after inoculation) shows that
no transformants were obtained unless Agrobacterium was applied to
relatively young pollen-recipient flowers that were 5 or more d away
from anthesis (Desfeux et al., 2000).
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Bent
minates on the stigmatic surface of the pollen
recipient.
Ovule transformation was convincingly demonstrated when constructs containing a GUS marker
gene were used to document sites of delivery of
T-DNA (Fig. 3; Ye et al., 1999; Desfeux et al., 2000).
35S and other standard promoters are poorly expressed in gametophyte tissues, so additional promoters used for GUS fusions were Arabidopsis
ACT11 (Desfeux et al., 2000), an oilseed rape Skp1like promoter (Bechtold et al., 2000), or a Figwort
mosaic virus promoter (Ye et al., 1999). Staining was
observed in ovules in mature flowers and in younger
flowers that had not yet reached pollination (Ye et al.,
1999; Desfeux et al., 2000). Desfeux et al. (2000) and
Bechtold et al. (2000) did not observe GUS staining of
anthers or pollen (except in stably transformed positive controls), providing another line of evidence
that pollen transformation is not common. Ye et al.
(1999) reported frequent GUS staining of ovules and
pollen, but also concluded that ovules are the primary target for transformation. It is curious that
Bechtold et al. (2000) did not observe staining of
ovules or embryos in their work.
A third, very different line of evidence also points
to ovules as the primary site of productive transformation. Genetic linkage analysis with a marked chromosome demonstrated that most transformants (25
of 26 tested) carry T-DNA on the maternally derived
chromosome set (Bechtold et al., 2000). For the one of
26 events associated with the paternal chromosome
set, the most likely origin was pollen transformation
or integration of T-DNA within the diploid genome
of a fertilized embryo. However, the aggregate message from the efforts of these three labs seems convincing: developing ovules are the primary target of
productive transformation in the Arabidopsis floral
dip or vacuum infiltration transformation procedures
Figure 3. ACT11-GUS staining of Arabidopsis ovules, embryo sacs,
and entire locules due to inoculation with Agrobacterium. A, View of
an entire flower, with staining present in multiple ovules. B, Ovules
in a segment of a dissected flower, showing no staining, localized
staining, or complete staining of individual ovules. C, Ovules with
staining of embryo/embryo sac rather than entire ovule. D, Staining
of an entire locule cavity due to a bacterially expressed GUS construct within Agrobacterium that has colonized the locule interior.
Some panels from Desfeux et al. (2000).
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(Ye et al., 1999; Bechtold et al., 2000; Desfeux et al.,
2000).
GUS STAINING AND TRANSFORMANT
GENERATION: SALIENT DETAILS
In the above experiments GUS staining was often
observed only within the embryo sac of ovules, indicating a time of transformation late in megagametophyte development (Fig. 3c; Ye et al., 1999; Desfeux et
al., 2000). Late transformants were also obtained,
albeit at a very low frequency, from inoculation of
flowers that were sufficiently developed to contain
trinucleate pollen and embryo sacs at the four-nuclei
or mature stage of development (Bechtold et al.,
2000). Uniform blue staining of entire ovules was
sometimes observed, suggesting that transformation
can also occur earlier in the formation of the megagametophyte cell lineage (Fig. 3b). However, earlier
transformation events that give rise to larger transformed sectors encompassing multiple ovules seem
unlikely. Arabidopsis transformants from the same
plant (Bechtold et al., 1993; Ye et al., 1999) or even
from the same silique (seed pod; Desfeux et al., 2000)
are usually independent. This latter point contrasts
with recent results from the legume M. truncatula
(discussed below).
Desfeux and colleagues tracked the presence of
Agrobacterium by using GUS constructs that are expressed within Agrobacterium (Desfeux et al., 2000).
Floral dip inoculation produced staining along the
stigmatic surface and in various crevices of the
flower (not shown), but also produced examples in
which closed locules were filled with blue stain (Fig.
3d), suggesting that locules can harbor substantial
colonies of Agrobacterium.
Transformants have been obtained from siliques
located at multiple sites across the inflorescence
(Bechtold et al., 1993; Ye et al., 1999). However, transformants in one study were not randomly (Poisson)
distributed on a per silique or per plant basis (Bechtold et al., 2000). In another study roughly one-half of
the transformant-bearing siliques contained more
than one and up to seven transformants (Desfeux et
al., 2000). In addition, although most siliques on inoculated plants showed no GUS staining, a few siliques showed multiple stained embryos (Desfeux et
al., 2000). It is apparent that some flowers are particularly amenable to transformation.
TIME OF INOCULATION RELATIVE TO FLOWER
DEVELOPMENT IS CRUCIAL
If productive transformation events occur in the
female germ-line, one is forced to wonder how
Agrobacterium gains access to ovules that develop
within enclosed locules. However, Arabidopsis locules are not always closed. The ovary develops as a
ring of cells that protrude from the floral meristem,
Plant Physiol. Vol. 124, 2000
Uses and Mechanisms of in Planta Transformation
extending to form a vase-shaped structure that is
open at the top. It is only late in floral development,
roughly 3 d prior to anthesis, that locules become
sealed by formation of the stigma as a cap at the top
of this vase. This timeline correlates strikingly with
the outcrossing study (Fig. 2; Desfeux et al., 2000) in
which siliques that gave transformants were all from
flowers inoculated 5 to 10 d prior to anthesis, a time
when the locule is open. No transformants were obtained from flowers that were 4 d or fewer away from
anthesis at the time of Agro-inoculation. Furthermore, GUS staining of ovules occurred only in flowers that were inoculated 5 or more d prior to anthesis
(Desfeux et al., 2000). Agrobacterium apparently enters Arabidopsis locules prior to their closure. Alternative interpretations of these timing-of-inoculation
results are that it takes Agrobacterium a number of
days to build up sufficient numbers and/or to adapt
to the plant and activate transformation. In addition,
the finding of Bechtold and colleagues (2000) that
transformants can be obtained at a low frequency
from inoculation of relatively mature flowers suggests that some transformation events may arise from
Agrobacterium delivered by other means; for example,
via pollen tubes. On the whole, however, it is clear
that Agrobacterium must gain access to ovules or to
ovule-progenitor tissue for the high-efficiency Arabidopsis transformation procedures to succeed.
M. TRUNCATULA: SOMETIMES A DIFFERENT
TRANSFORMATION TARGET?
In a finding that suggests that improved transformation of many plant species is within reach, successful transformation of M. truncatula by Agrobacterium vacuum infiltration has been reported (Trieu et
al., 2000). As with Arabidopsis, the investigators had
success inoculating flowering plants or younger
seedlings. However, amazing transformation rates
were reported for M. truncatula, ranging from 2.9% to
76% of all seeds tested (Trieu et al., 2000). It is intriguing that when flowering M. truncatula plants
were inoculated, transformants homozygous for the
transgene were observed and the majority of transformants from a given plant were siblings derived
from the same T-DNA integration event. M. truncatula transformation can apparently occur at earlier
stages of plant development than in Arabidopsis.
However, five of nine and six of seven transformants
examined in two seedling inoculation experiments
were judged to be independent, showing that multiple transformation events can arise on a plant even if
it is inoculated as a seedling (Trieu et al., 2000). The
cellular targets of these transformation events are not
known, and determination of these targets should be
a high priority for future research.
In another intriguing, but unexplained result, Trieu
and colleagues (2000) obtained transformants only if
seedlings were subjected to a 4°C/14 d vernalization
treatment that induced earlier flowering.
Plant Physiol. Vol. 124, 2000
The occurrence of sibling transformants is not desirable for applications such as insertional mutagenesis, but the overall high rates of transformation obtained (Trieu et al., 2000) indicate that this method
will be of tremendous utility to researchers who
study M. truncatula.
WILL THE ABOVE INFORMATION HELP WITH
TRANSFORMATION OF OTHER SPECIES?
Transformation by infiltration of adult plants with
Agrobacterium has also been reported for pakchoi (Liu
et al., 1998) and has been informally reported for
other Brassicaceae beyond pakchoi and Arabidopsis.
Thus multiple plant species have now been successfully transformed using Agrobacterium in planta approaches. In addition, although the methods have not
been widely reproduced or adapted, Agrobacteriummediated shoot apex transformation and related
methods that minimize tissue culture have been reported for a number of other plant species (Chee and
Slighton, 1995 and refs. therein). Development of
robust in planta transformation protocols for other
plant species should be within reach.
Transformation technology development has been
regarded by some as art as much as science, but
success is most likely to come from efforts informed
by the scientific literature and past experiences (e.g.
see the reviews of Hansen and Wright, 1999 and
Birch, 1997). This must be coupled with a willingness
to try different approaches and to tolerate failures
along the way.
It is very probable that success will be easier to
achieve with some species and than with others, but
what are some of the criteria that may contribute to
success? Arabidopsis and M. truncatula are relatively
small plants with rapid generation times, but that
may enhance ease of effort more than ultimate success at transformation. A high seed set is also likely
to help, but is not an ultimate determinant: although
single Arabidopsis plants commonly produce 5,000
to 10,000 seeds, only 33 or fewer seeds per Agroinoculated plant were collected in the successful
transformation of M. truncatula (Trieu et al., 2000).
Aspects of experimental design such as planting configuration and mode of Agro-inoculation (i.e. inoculating seedlings as opposed to flowering plants) can
allow dramatic shifts in the number of specimens
processed. Large numbers can be important if rates of
success are low, but can also be a trap if quality of
treatment is more important than quantity. Prime
examples of this are the low success rates and/or
poor reproducibility that were achieved with Arabidopsis in planta transformation procedures until inoculation of plants in full flower was attempted. The
need for vernalization of M. truncatula to achieve
efficient transformation offers another example. Trying a larger variety of approaches may be more productive than trying very large-scale attempts with a
narrow set of methods.
1545
Bent
The discovery of the ovule as the site of productive
Arabidopsis transformation produces specific suggestions for floral transformation efforts with other
species. Application of Agrobacterium to flowering
tissues very early in their development and prior to
locule closure is likely to be important. In an alternate
manner, with some species it may be possible to
deliver Agrobacterium by microinjection of ovaries, or
by shooting Agrobacterium into flowers using microprojectiles or high-pressure air guns (e.g. see U.S.
patent no. 5,994,624). As a further alternative, plant
lines such as the Arabidopsis CRABS-CLAW mutants
that bear a more accessible locule may provide an
improved target for transformation (Desfeux et al.,
2000). Work with M. truncatula and Arabidopsis suggests that younger plants can also be treated, although onset of flowering soon after Agroinoculation appears to be preferable.
In numerous plant transformation systems, the
choice of host genotype and/or Agrobacterium genotype has been an important parameter (Birch, 1997).
If recent findings with Arabidopsis are any indication, surveys for compatible host and bacterial genotypes might best be focused on assays that monitor
transformation of ovules or ovule progenitor tissues.
With some plant species the use of anti-oxidants or
other necrosis-reducing approaches has improved
transformation rates, and many other modifications
can be considered (Birch, 1997; Hansen and Wright,
1999). A better understanding of T-DNA transfer and
other aspects of Agrobacterium/plant interactions
(e.g. Hooykaas and Schilperoort, 1992; Mysore et al.,
2000) may also allow engineering of better host/
bacteria combinations.
Other substantially different transformation methods also must be kept in mind (e.g. Chowrira et al.,
1995; Chen et al., 1998). Who would have dreamed,
20 years ago, that coating DNA on to little metal
particles and then shooting it into plants (Klein et al.,
1987) could be so successful?
Agrobacterium floral transformation procedures
have been a tremendous success with Arabidopsis,
and similar success now seems likely for M. truncatula. Such successes, along with the recent information about the targets for Arabidopsis transformation, should inspire a renewal of efforts to adapt
these methods to the transformation of other plant
species. The benefits are clear: transformation without tissue culture can provide a high throughput
method that requires minimal labor, expense, and
expertise. Rates of unintended mutagenesis are reduced. More important, simplified transformation
protocols facilitate positional cloning, insertional mutagenesis, and other transformation-intensive procedures, reducing the effort required to test any given
DNA construct within plants.
Received September 5, 2000; accepted September 21, 2000.
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