Download Direct DNA transfer using electric discharge particle acceleration

Plant Cell, Tissue and Organ Culture 33: 227-236, 1993.
© 1993KluwerAcademic Publishers. Printedin the Netherlands.
Special section on particle bombardment
Direct DNA transfer using electric discharge particle acceleration
(ACCELL T M technology)
Dennis McCabe & Paul Christou*
Agracetus, Inc., 8520 University Green, Middleton, WI 53562, USA (*requests for offprints)
Key words: dicot transformation, monocot transformation, particle bombardment, transgenic cotton
Gossypium hirsutum, transgenic rice Oryza sativa, transgenic soybean Glycine max
Abstract
Direct D N A transfer methods based on particle bombardment have revolutionized plant genetic
engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been
engineered using variations of this technology. In many cases variety-independent and efficient
transformation methods have been developed enabling application of molecular biology techniques to
crop improvement. The focus of this article is the development and performance of electric discharge
particle bombardment (ACCELL TM) technology. Unique advantages of this methodology compared to
alternative propulsion technologies are discussed in terms of the range of species and genotypes that
have been engineered, and the high transformation frequencies for major agronomic crops that enabled
the technology to move from the R&D phase to commercialization.
Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the
development of variety-independent and efficient gene transfer methods for most of the major
agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment
has resulted in variety-independent and practical generation of large numbers of independently-derived
crop plants. ACCELL TM technology is currently being utilized for the routine transfer of valuable genes
into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.
Introduction
In order to develop a practical transformation
system for any crop, a number of criteria need to
be fulfilled to make engineering of the crop
useful:
- transformation systems have to be independent
of genotype or cultivar in order to facilitate
introduction of useful genes into elite varieties;
-large numbers of transgenic plants should be
recovered in order to assess useful levels of
gene expression; and
-extensive tissue culture manipulations involving time-consuming and labor-intensive operations such as protoplast and embryogenic
suspension cultures should be minimized or if
possible eliminated in order to avoid cultureinduced mutations and somaclonal variation.
It is apparent that promising reported gene
transfer methods for many important crops suffer from some or all of the above drawbacks. An
example is the engineering of maize by biolistic
particle bombardment which requires development of regenerable embryogenic suspension
cultures (Gordon-Kamm et al. 1990; Fromm et
al. 1990) amenable to transformation, a not so
trivial task. The requirement for developing such
cultures compatible with current transformation
technologies make practical engineering of elite
cultivars of maize elusive, and represents an
unfortunate under-utilization of the potential of
particle bombardment-based technologies.
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Particle bombardment employs high velocity
metal particles to deliver biologically active
D N A into plant cells. The concept has been
described in detail by Sanford (Sanford 1988,
1990). Christou et al. (1988) demonstrated that
the process could be used to deliver biologically
active D N A into living cells and result in the
recovery of stable transformants. The ability to
deliver foreign D N A into regenerable cells,
tissues or organs, appears to provide the best
method, at present, for achieving truly genotypeindependent transformation in many agronomic
crops bypassing Agrobacterium host-specificity
and tissue-culture related regeneration difficulties. Due to the physical nature of the technique
there is no biological limitation to the actual
D N A delivery process thus genotype should not
be a limiting factor. Combining the relative ease
of D N A introduction into plant cells with an
efficient regeneration protocol avoiding protoplast or suspension culture, we appear to have the
most optimum system in place for transformation. Important advancements and refinements
in the process described subsequently, using
soybean.(McCabe et al. 1988; Christou et al.
1990), cotton (McCabe & Martinell 1991), and
rice (Christou et al. 1991, 1992) as models for
dicots and monocots demonstrated the power of
the technique.
A number of advantages make microprojectile
bombardment the method of choice for engineering various crops.
ferent species. Even though recovery of transgenic plants has not been reported from a
number of important agronomic crops, this deficit is more due to the lack of a favorable tissue
culture response than due to the D N A delivery
method.
Transformation of recalcitrant species
Most of the important agronomic crops such as
soybean, cotton, maize etc., cannot be engineered using conventional methods. In the case
of soybean (Glycine max Merril.), Agrobacterium host-specificity restricts utilization of this
method to one specific variety which is of no
commercial importance (Hinchee et al. 1988)
whereas for cotton (Gossypium hirsutum L.),
tissue culture limitations only allow engineering
of a specific variety (Umbeck et al. 1987). For
maize (Zea mays L.), transformation using electroporation and protoplasts resulted in the recovery of infertile plants (Rhodes et al. 1988).
Study of basic plant developmental processes
By utilizing chromogenic markers it is possible to
study developmental processes and also clarify
the origin of germline in regenerated plants
(Christou & McCabe 1992). Chimeras can be
created very easily (Christou 1990) and contribution of the various cell layers in the apical
meristem to the ontogeny of plants can now be
studied.
Transformation of organized tissue
The ability to engineer organized and potentially
regenerable tissue permits introduction of
foreign genes into elite germplasm. Consequently, backcrossing is not required to restore the
original line as compared to other transformation
methods limited by genotype and host-specificity. Recovery of transformed R1 seed is considerably shortened and this is of paramount
importance in commercial programs where timing is vital in bringing products to market.
Universal delivery system
Transient gene expression has been demonstrated in numerous tissues representing many dif-
Critical variables
A number of parameters have been identified
and need to be considered very carefully in
experiments involving transformation through
particle bombardment. These can be classified in
three general categories:
- physical parameters,
-environmental factors and
- biological factors.
Physical parameters include: the nature,
chemical and physical properties of the metal
particles utilized to carry the foreign DNA;
nature, preparation and binding of D N A onto
the particles; target tissue. Particles should be of
229
high enough mass in order to possess adequate
momentum to penetrate into the appropriate
tissue. Suitable metal particles include gold,
tungsten, palladium, rhodium, platinum, iridium
and possibly other second and third row transition metals. Metals should be chemically inert to
prevent adverse reactions with the DNA or cell
components and they should also be able to form
organometallic complexes with the DNA possessing the correct stereochemistry that will allow
optimal dissociation of the metal-DNA entity
once the coated particle enters the target cell.
Additional desirable properties for the metal
include size and shape as well as agglomeration
and dispersion properties. The nature, form and
concentration of the DNA need also be carefully
considered. In the process of coating the metal
particles with exogenous DNA, certain additives
such as spermidine and calcium chloride appear
to be useful. The nature of the DNA, e.g. single
versus double stranded may also be important
under some conditions, even though this was
shown not to be a significant variable in specific
cases. Finally, it is very important to target the
appropriate cells that are competent for both
transformation and regeneration. It is apparent
that different tissues have different requirements; extensive histology needs to be performed in order to ascertain the origin of regenerating tissue in a particular transformation
study. Depth of penetration thus becomes one of
the most important variables and the ability to
tune a system to achieve particle delivery to
specific cell layers may be the difference between
success and failure in recovering transgenic
plants from a given tissues.
Environmental variables include such parameters as temperature, photoperiod and humidity
of donor plants, explants and bombarded tissues.
These parameters have a direct effect on the
physiology of tissues and this is also an important
variable. Such factors will influence receptiveness of target tissue to foreign DNA delivery and
also affect its susceptibility to damage and injury
that may adversely affect the outcome of the
transformation process. Some explants may require a 'healing' period after bombardment
under special regimens of light, temperature and
humidity.
Biological factors include choice and nature of
explant, pre- and post-bombardment culture
conditions etc. In addition, explants derived
from plants that are under stress, e.g. infected
with bacteria or fungi, will provide inferior
material for bombardment experiments. Interaction between introduced DNA and cytoplasmic or nuclear components need also be examined.
Particle bombardment using ACCELL T M
technology hardware and operating principles
ACCELL T M technology utilizes an instrument
(Fig. 1) to accelerate DNA coated gold beads to
any desired velocity by varying the input voltage.
Conceptually, it can be viewed as a shock wave
generator. Early in the development of the
device it became apparent that the driving force
Fig. 1. ACCELLT M device developed by Agracetus for
electric dischargeparticle acceleration.
230
for the projectiles was not the expansion of hot
gases as in a c a n n o n or rifle. T h e shock wave
p r o d u c e d by the initial electrical discharge app e a r s to be responsible for the particle acceleration. This realization led to new designs that no
l o n g e r r e s e m b l e 'guns'. In the current design the
accelerating force is g e n e r a t e d in a two c h a m b e r
e n c l o s u r e s e p a r a t e d by a partition (Fig. 2). O n e
side contains two steel arc points which provide
the initial m o t i v e force. T h e other side acts as a
reflection c h a m b e r . T h e expanding shock wave
reflects back into the c h a m b e r as it hits the
deflector and the bulk of the pressure wave is
r e l e a s e d as the deflector is blown away. T h e
s h o c k f r o n t is p r e v e n t e d f r o m directly interacting
with the carrier sheet by an obstructing barrier.
A f t e r o n e or m o r e reflections f r o m the floor and
Fig. 2. The blast chamber consists of a PVC block milled to
form a rectangular cavity divided into two chambers by a
partial wall. In one of these chambers are two arc points (a)
spaced equidistant from the sides and rear wall. The points
are set 0.5 mm apart and bridged by a 10 ixl drop of water
prior to each discharge. The spark chamber is covered by a
wafer of PVC (b) which reflects the primary shock wave back
into the chamber avoiding energy loss. The cavity is partially
obstructed by a partition which separates the spark chamber
from the reflection chamber (c). The partition serves to
prevent the primary shock wave from interacting directly
with the carrier sheet. As secondary waves are formed by
reflection from the chamber walls, their combined fronts
distribute their force evenly over the carrier sheet. This sheet
which forms the roof of the reflection chamber, is accelerated
upward until it encounters the retaining screen (d). The
sheet, and 18 mm square of 0.5 mil metalized mylar (Dupont) is held by the screen (100 mesh stainless steel) as the
gold beads on its surface proceed to the target tissue. The
energy for the arc is provided by a 25 Kv 2 ixF capacitor
charged from a 25 Kv DC variable power supply. The
capacitor is discharged through the arc gap by activating a
solenoid-actuated double-pole double-throw switch.
Fig. 3. Diagram of the acceleration mechanism, illustrating
spark chamber with arc points, film carrying gold particle
with DNA, retaining screen and target tissue.
walls, the c o m b i n e d wave fronts reach the overlying carrier sheet which is accelerated upwards.
A n y particles on its surface will be passively
accelerated as well. F o r this p u r p o s e , a lightweight sheet of metalized m y l a r ( D u p o n t 50
M M C ) is used as an intermediate carrier for the
small beads. The sheet along with the beads is
accelerated as it absorbs e n e r g y f r o m the s h o c k
wave. It is then s t o p p e d by a retaining screen to
allow the beads to p r o c e e d to the target alone.
Since the shock wave carries no hot exhaust
gases or debris it is possible tO place the target
tissue relatively close to the gun w i t h o u t fear of
incineration. T h e entire assembly m a y be partially e v a c u a t e d to r e d u c e drag and tissue d a m a g e ,
h o w e v e r , too high a v a c u u m (less than 200
millibars) will cause the tissue to lose m o i s t u r e
rapidly with subsequent reduction in cell viability. A schematic of the acceleration m e c h a n i s m is
shown in Fig. 3.
Bombardment protocol
D N A is typically l o a d e d o n t o 1 . 5 - 3 ~ m gold
beads ( A l p h a Chemicals Inc.) at a rate of up to
40 Ixg D N A / m g of gold using CaC12 and spermidine (Klein et al. 1987) to precipitate the
D N A o n t o the gold. T h e c o a t e d beads are gently
centrifuged and r e s u s p e n d e d in 100% ethanol,
then p i p e t t e d o n t o the carrier sheets ( 1 8 x 1 8 m m
squares o f 1/2 mil metalized mylar; D u p o n t
5 0 M M C ) . A f t e r a brief period of settling, the
231
ethanol is drained away and the sheet dried. The
'gun' is loaded by placing a 10 Ixl drop of water
between the points and covering the spark
chamber with the reflecting cap. The carrier
sheet is laid over the top of the reflection
chamber and the retaining screen put in place.
The target is prepared in a way that allows the
desired area to be exposed as it is inverted above
the retaining screen. The assembly is then
evacuated to 600 millibars before the discharge is
activated.
Bombardment parameters
As the gun is being refined and tested under the
particular conditions required for a given system,
it is important to be able to monitor its performance. Penetration of the gold beads into 1%
w/v agar provides a direct measure of the effects
of bombardment conditions on the purely physical, non-biological, behavior of the accelerated
beads. Figure 4 illustrates the effect of the
charging voltage on penetration into agar. Similar tests may also be performed directly on the
tissue of choice. The progressively increasing
penetration of the beads into soybean hypocotyl
tissue as a function of voltage can be seen in Fig.
5A. It is also possible to evaluate the effectiveness of DNA delivery into the different cell
layers (Fig. 5B) by using transient expression of
the ¢3-glucuronidase (gus) gene (Jefferson et al.
1987). The number of cells expressing GUS in
O,.
Transient expression studies
Transient expression studies should only be used
as a guide to develop systems for the stable
transformation of a given species. Routinely,
many investigators have overstressed the importance and significance of transient expression
data. In some cases exhaustive experiments were
performed using transient expression data in an
attempt to achieve complete protocol optimization for the recovery of stable transformants.
This, however, may be unwise as optimization or
maximization of transient activity does not
necessarily result in optimal or any stable transformation. Therefore, studies involving numbers
of transiently expressing cells and loci per unit
mass or volume of recipient cells is in our
opinion meaningless and in many cases irrelevant
to the final outcome particularly when the objective is recovery of transgenic plants. We would
strongly urge utilizing data from stable transformation experiments to draw conclusions pertaining to stable transformation.
Stable transformation of agronomic
crops - dicotyledonous species
600
A
each cell layer of the soybean hypocotyl shows a
more complicated relationship to voltage reflecting the more critical nature of the interaction
between bead delivery and viability of the cells.
Such studies combined with the tunable nature
of the device are invaluable when designing
transformation protocols using this approach.
Transgenic soybean as a model
500
1
400
300
0
10
20
30
Voltage (Kv)
Fig. 4. Penetration of 1.5-3.0 txm gold beads into 1% agar.
A 1 m m cross section of the b o m b a r d e d agar was examined
to d e t e r m i n e depth of penetration at each voltage.
The first example of the versatility and usefulness of particle bombardment is exemplified by
the development of a genotype-independent
transformation protocol for soybean (McCabe et
al. 1988; Christou et al. 1990). Starting with
isolated immature embryonic axes we were able
to develop a simple protocol that permitted
recovery of clonal plants from every elite variety
we attempted to transform (approximately 30
varieties to date covering all maturity groups).
The overall transformation frequency can be as
232
% Expressing Cells
B
20-
% Cells with Beads
Soybean Penetration vs. Voltage
Soybean Penetration vs. Expression
181614-
60-
12-
501040-
8-
30-
6-
20-
4-
10-
2-
ss
ss
4
6
8
10
12
14
Voltage (KV)
4
6
8
10
12
14
Voltage (KV)
Fig. 5. Soybean hypocotyls were prepared by pre-germinating sterilized seed in petri dishes containing MS salts with 1% agar for
24 h. Cotyledons were then dissected away and axes replated on OR medium (McCabe et al. 1988) for an additional 24 h. The
axes were then oriented on their side with the flattened face of the hypocotyl to be bombarded; 1-3 Ixm gold beads (Alpha) were
coated with DNA at a ratio of 0.1 Ixg mg 1 of gold containing the [3-glucuronidase gene (gus) by precipitation with CaC12 and
spermidine. Carrier sheets were loaded with beads at 0.075 mg cm--'. The assembly was evacuated to 600 millibars prior to each
blast. Following bombardment the hypocotyls were replated on OR medium and incubated for 1.5-4h then fixed in 5%
formaldehyde in 25 mM KH2PO 4 + 1% Triton X 100 at room temperature for 5 min. Fixation was necessary to reduce diffusion of
the GUS protein into adjacent cells. Following fixation tissues were incubated in substrate at 37°C overnight. The hypocotyls were
then cut into 35 Ixm sections with a freezing microtome (Rheikert), cleared and examined under a microscope. Cells were scored
for the presence or absence of a bead (A) as well as GUS activity (B). Only cells containing a bead visibly in the nucleus and
showing precipitated dye were scored as cells expressing the gus gene. These criteria were imposed to reduce the chance of
scoring false positives.
high as 15% with germline transformation frequencies in the range of 0.05-0.5% based on the
number of bombarded explants. The ~-glucuronidase gene (gus) from Escherichia coli
proved to be a useful tool in these experiments.
The meristematic regions were exposed and gold
particles coated with plasmid DNA were introduced into the general area of the meristem as
described elsewhere. Plant regeneration proceeded through organogenesis using high levels
of cytokinin to induce multiple shoots from the
general area of both the primary and axillary
meristems (McCabe et al. 1988). On average,
eight to ten shoots were recovered per explant.
Regenerated shoots were harvested and transferred to the greenhouse after an appropriate
acclimation period. Conversion of shoots to
plants was greater than 95%. Expression of the
gus gene was originally used to visualize transformed plant tissues. Due to the nature of the
transformation process most of the plants recovered were chimeric; however, clonal plants
233
were also obtained at a significant frequency
(Christou et al. 1989, 1990). These plants were
found to express the gene in essentially all their
tissues and usually passed the transgene to their
progeny in a manner consistent with mendelian
inheritance of a single dominant locus. Though
germline transformation frequency appears to be
somewhat low, it is relatively straightforward to
identify transformed plants due to the ease of the
histochemical GUS assay. A procedure for the
early identification of germline transformants has
been described recently (Christou & McCabe
1992). Consequently, production of reasonably
large numbers of transformed plants is practical
by this method which does not utilize selection to
identify transgenic plants.
In experiments in which we evaluated transformation frequencies using two genes, either
linked on the same plasmid or unlinked on
separate plasmids, we found that in the former
case cotransformation frequencies between the
screenable and the agronomic or non-selected
genes exceeded 95%. In all cotransformed
families we found that progeny plants expressing
one gene also expressed the other indicating that
the two genes were genetically linked even when
they were introduced into the explant on separate plasmids (Christou & Swain 1990). Both
chimeric and clonal plants gave rise to transformed progeny and most of the transgenic
families segregated in a mendelian fashion in the
R1 and R2 generations (Christou et al. 1990).
Hundreds of independently-derived soybean
plants transformed by this method have maintained the foreign genes for many generations.
Elite soybean varieties expressing resistance to
the herbicides Basta TM and Roundup TM, engineered through ACCELL TM technology are
currently undergoing large scale field evaluation
with projected commercialization dates well before the end of the decade. Additional experiments involving modification of protein and oil
composition are currently in progress.
Variety-independent gene transfer into cotton
The technique originally developed to engineer
cotton utilized Agrobacterium tumefaciens vectors and was applicable to only a few varieties
(Umbeck et al. 1987; Firoozabady et al. 1987).
Finer & McMullen (1990) reported recovery of
transgenic cotton plants from the same cultivar
utilizing bombardment of embryogenic suspension cultures. Most varieties of commercial interest proved difficult or impossible to regenerate
into plants from the obligatory callus phase.
Traits introduced into regenerable varieties
could be bred into other lines but the process
was lengthy and prone to somaclonal variation.
Recently, McCabe & Martinell (1991) developed
a procedure, based on ACCELL TM technology,
to deliver foreign genes directly into the meristematic tissue of excised embryonic axes of
cotton, that resulted in the recovery of transgenic plants in a variety-independent fashion.
When bombarded explants had developed 2-3
leaves (2-3 weeks post bombardment) a segment
of each leaf was sampled for GUS activity.
Plants exhibiting activity were then mapped to
identify nodes or axillary buds subtending the
transformed leaves. These plants were transferred to the greenhouse and pruned to force
growth of the appropriate bud. Once selective
pruning produced a plant in which every leaf
exhibited GUS activity the potential for germline
transformation could be evaluated. Characterization of the transformants for this purpose relied
on the observations of Christou & McCabe
(1992). They had observed that in all cases in
which any part of the vascular system of a
soybean shoot produced from meristem bombardment expressed the reporter gene it usually
was able to pass that gene to its progeny. Cross
sections of either a leaf or petiole from the
forced cotton shoots were therefore stained to
identify the specific tissue types expressing GUS.
R 1 seeds recovered from pollen-positive R 0
plants were germinated and grown in the greenhouse. Leaf punches were taken from each plant
and analyzed for GUS activity to determine
segregation patterns. Subsequently pollen staining for GUS allowed selection of homozygous
individuals for the production of R 2 plants for
further analysis. Southern blot analyses of transgenic families showed the introduced gene migrating as high molecular weight DNA when
uncut and unit length when cut at restriction sites
at each end of the gene. Segregation data were
consistent with the expected 3:1 pattern for
mendelian segregation of a single dominant
234
marker gene. Southern blot and enzyme analyses
of R2 and R3 plants confirmed inheritance of
foreign genes into successive generations.
Stable transformation of agronomic cropsmonocotyledonous plants
Monocotyledonous plants which include some of
the world's most important food crops e.g.
cereals such as wheat (Triticum aestivum L.), rice
(Oryza sativa L.), maize, barley (Hordeum vulgare L.), etc. had until recently been extremely
recalcitrant to genetic manipulation in vitro.
Particle bombardment resulted in the recovery of
transgenic plants and progeny for such important
crops as maize (Gordon-Kamm et al. 1990;
Fromm et al. 1990), wheat (Vasil et al. 1992),
and rice (Christou et al. 1991, 1992).
In an attempt to explore methods to overcome
problems in cereal transformation we focussed
on rice as a model to address fundamental
problems which touch on many key areas of
plant sciences (Potrykus 1990). Until recently,
recovery of transgenic rice plants was only possible using direct D N A transfer methods such as
electroporation (Toriyama et al. 1988; Zhang et
al. 1988; Shimamoto et al. 1989; Tada et al.
1990)
or
PEG-mediated
transformation
(Hayashimoto et al. 1990; Datta et al. 1990) of
protoplasts.
Rice in vitro culture has a very strong genotype- and culture-dependent component. Even
though genetic engineering of rice has been
reported, only cultivars from japonica varieties
can be transformed using protoplast-dependent
methods; recently, transformation and subsequent regeneration of transgenic indica rice
plants, from specific cultivars, was reported
utilizing protoplasts (Datta et al. 1990, 1992).
Indica rice varieties provide the staple food for
more than two billion people worldwide, including Indochina and the Indian sub-continent.
Eighty percent of cultivated rice worldwide is of
the indica type (Swaminathan 1982; Wu et al.
1990).
Twelve to fifteen-day old rice immature embryos were isolated from greenhouse grown
plants and subjected to electric discharge particle-mediated transformation (Christou et al.
1991). The scutellar region of the embryo was
bombarded under previously described conditions. Bombarded tissue was then plated on
regeneration media supplemented with appropriate selective agents. Continuous selection of the
proliferating tissue resulted in the appearance of
transformed embryogenic callus. However,
transformed embryogenic callus and somatic
embryos also appeared in the absence of any
selection pressure. Subsequent transfer of this
embryogenic callus to appropriate media resulted in the development of plantlets and plants
expressing marker genes in addition to antibiotic-, herbicide-, and insect-resistance genes.
Southern blot analyses of R0 plants and their
progeny demonstrated stable integration of exogenous genes into the rice genome. Molecular
analysis of a number of independently-derived
transgenic rice plants indicated that the D N A
profile and integration patterns in rice were very
similar to those of soybean and cotton, which we
analyzed extensively in the past (Christou et al.
1989; McCabe & Martinell, in preparation).
When progeny from transgenic rice plants
carrying the bar gene were sprayed with the
herbicide bialaphos they were shown to express
total resistance to the herbicide at levels of 2000
ppm. Control plants died when sprayed with 500
ppm of the herbicide.
Advantages of ACCELL T M gene transfer
A number of features make electric discharge
particle acceleration unique as compared to
other bombardment procedures that utilize alternative acceleration principles:
-Transformation of organized tissue. There is
no need for the development of embryogenic
suspension cultures and other complex tissue
culture systems that under-utilize the potential
of particle bombardment. This is illustrate by
the published transformation systems for
maize, wheat, cotton etc. which are restricted
to one or two varieties that can be regenerated
from tissue culture.
-Variety-independent transformation into elite
commercial varieties, negating the need for
going through extensive backcrossing programs
to introgress foreign genes into varieties that
235
are not amenable to alternative b o m b a r d m e n t
procedures. Only A C C E L L T M technology has
resulted in variety-independent and practical
gene transfer procedures for such important
crops as soybean, cotton and rice.
-Minimal tissue culture operations with subsequent reduction or elimination of mutation
or culture-induced variation. In the case of
dicotyledonous species we have b e e n able to
bypass the o b l i g a t o r y callus phase that often
results in somaclonal variation and sterility in
r e g e n e r a t e d transgenic plants. In addition by
either utilizing de n o v o organogenesis or simple germination of b o m b a r d e d explants we
bypass most of the problems that impose
severe limitations on alternative acceleration
mechanisms.
- F a s t e r cycle times in breeding programs and
field testing as a result of engineering elite
cultivars, bypassing the need for extensive back
crossing.
- Precise control of particle penetration into cells
that are c o m p e t e n t for both transformation and
regeneration. This is the key feature of
A C C E L L T M that m a k e s the technology suitable for variety-independent gene transfer.
-Minimal d a m a g e to recipient tissue due to the
ability to modulate the accelerating force effectively. As a result, regenerable tissue which
would not survive alternative acceleration
mechanisms can be targeted and transgenic
plants can be recovered routinely.
Remaining problems
Until recently the key barrier in achieving effective transformation of agronomically important
species was the D N A delivery method. Microprojectile b o m b a r d m e n t and A C C E L L T M technology in particular has had a tremendous impact
on this limitation. The challenge now is shifting
b a c k to the biology of the explant used in
b o m b a r d m e n t experiments. It is apparent that
the conversion frequency of transient to stable
transformation events is low. This does not
m e a n , however, that transgenic plants from all of
the crops that have been engineered cannot be
obtained at high enough frequencies to m a k e the
process commercially useful and economical.
More attention needs to be paid to the biology of
explants prior to, and following b o m b a r d m e n t .
We need to identify how m o r e cells can be
induced to b e c o m e c o m p e t e n t for stable D N A
uptake and regeneration. Optimization of biological interactions between physical p a r a m e t e r s
and target tissue needs to be better studied and
understood. N o t much is known about the fate
of D N A from the time particles are introduced
into plant cells. Recipient tissue variation and
variability due to b o m b a r d m e n t conditions complicate the picture even further. Additional issues such as irregular particle size and uniformity
as well as i m p r o v e m e n t s in hardware design need
also be addressed.
References
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Plant Physiol. 87:671-674
Christou P, Swain WF, Yang N-S & McCabe DE (1989)
Inheritance and expression of foreign genes in transgenic
soybean plants. Proc. Natl. Acad. Sci. USA 86:7500-7504
Christou P (1990) Morphological description of transgenic
soybean chimeras created by the delivery, integration and
expression of foreign DNA using electric discharge particle
acceleration. Annals of Botany 66:379-386
Christou P & Swain WF (1990) Cotransformation frequencies
of foreign genes in soybean cell cultures. Theor. Appl.
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