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ChapterS
Direct Gene Transfer in Poplar1
Pierre J. Charest, Yvonne Devantier, Cathy Jones, Jim C. Sell mer,
Brent H. McCown, and David. D. Ellis
Introduction
Agrobacterium-mediated genetic transformation and regeneration of transgenic Populus has been achieved with selected
genotypes (Kim et al. this volume). Direct gene transfer
using electroporation and microprojectile-mediated DNA
delivery has also been accomplished (Chupeau et al. 1~94;
Devantier et al. 1993; McCown et al. 1991), with successful
regeneration of transgenic Populus from a few genotypes
(Chupeau et al. 1994; McCown et al. 1991). Microprojectile
DNA delivery has been also used to study transient expression of chimeric genes (Devantier et al. 1993).
Several differences exist between Agrobacterium-mediated DNA delivery and direct gene transfer. Using
Agrobacterium, only a fragment of the transformation vector within the T-DNA border of the TI plasmid is transferred to the host cell and, in most cases, only 1 copy or a
few copies of this fragment are integrated into the host
genome. With direct DNA delivery, the whole transformation vector is introduced into the host cell and often
several copies of the delivered DNA are integrated into
the host genome. Although Agrobacterium-mediated DNA
delivery is a more precise transformation method, direct
DNA delivery has two advantages; it is genotype independent, and it bypasses the long life cycle of tree species
by delivering DNA into a variety of plant tissues for gene
expression studies (Charest et al. 1993). For instance, somatic and zygotic embryos, embryonal masses, pollen,
germinated seedlings, flower organs, differentiating
wood, needles, and vegetative buds of coniferous trees
were used for transient gene expression using micro-
1
Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO:
U.S. Department of Agriculture, Forest Service, Rocky Mountain
Research Station. 326 p.
60
projectile-mediated DNA delivery (reviewed by Aronen
et al. 1994, 1995; Charest et al. 1993; Ellis 1995). Electroporation is less flexible because, as with protoplast culture, it is often genotype dependent and has a more
restricted range of tissues that respond to the procedure
(Adbudl-Baki et al. 1990; Dekeyser et al. 1990; D'Halluin
et al. 1992; Laursen et al. 1994; Luong et al. 1995; Songstad
et al. 1993; Xu and Li 1994; Yang et al. 1993). Both microprojectile-mediated DNA delivery and electroporation
could be useful alternatives to Agrobacterium-mediated
transformation for recalcitrant genotypes or transient gene
expression studies. Regeneration of transgenic poplar trees
following Agrobacterium-mediated transformation, which
required several months to obtain the transgenic plant
material, was used for gene expression studies (Leple et
al. 1992; Miranda Brasileiro et al. 1992; Strohm et al. 1995).
Similar results to evaluate the relative strength of promoters could have been obtained with microprojectile-mediated DNA delivery within a few days.
The most limiting factors to enhance foreign· DNA integration and/or the recovery of stable transformants for
direct DNA delivery are the host cell biology and defining
cellular factors that interact with the introduced DNA.
Experimental protoplast systems can be used for genetic
transformation but assessing cellular changes that interact with the integration of the introduced DNA is difficult.
Problems associated with protoplast systems include the
enzymatic cell treatment, gene transfer environment, and
need to transfer cells through a sequential series of different media. This difficulty is illustrated by the high mortality (50 percent) of poplar protoplasts following
electroporation (Sellmer 1991). In contrast, microprojectile
bombardment offers more potential for examining cellular factors involved in DNA incorporation because DNA
can be delivered into any plant cell or tissue. One drawback of this approach is the heterogeneity of the cell population used for bombardment; changes in 1 tissue may alter
a response by another tissue within an explant. For both
methods, regulation of the transiently expressed genes may
be less stringent than with transgenic plants because multiple copies of the gene can occur transiently, thus gene
expression is not necessarily controlled through chro~o-
Direct Gene Transfer in Poplar
some structure (Katagari and Chua 1992; Quail et al. 1987;
Vernet et al. 1982).
Electroporation
DNA transfer using electroporation is due to increased
cell wall and membrane permeability caused by a short electrical pulse applied to the target plant tissue, whether protoplasts or whole tissue explants (Van Wert and Saunders 1992).
This method has been used to study transient gene expression and produce transgenic tissues and plants (Chupeau et
al. 1993; Russell and McCown 1986; Wang et al. 1991). A prerequisite is the availability of tissue cultures amenable to
electroporation. For poplar, protoplast culture and tree regeneration were achieved (Chupeau et al. 1993; Russell and
McCown 1986; Wang et al. 1991) that provided a basis for
gene transfer via electroporation. Transgenic P. tremula x P.
alba trees were produced following electroporation of protoplasts with a decay-wave electroporation device (Chupeau
et al. 1994). Transformation selection was achieved using
phosphinotricin with the phosphinotricin acetyl transferase
(PAT) gene, and chlorsulfuron with the acetolactate synthase
(ALS) gene. The transformation frequency was from 1.3 to
7.5 x 10-6, and 56 independent transgenic trees were regenerated. An interesting finding of this study was that the introduced genes were integrated in a relatively simple and
straightforward pattern.
Microprojectile-mediated DNA
Delivery
In poplar, the propulsion of microprojectiles coated with
DNA was accomplished using 3 different mechanisms: electric discharge, gunpowder, and helium gas (Devan tier 1992;
Devantier et al. 1993; McCown et al. 1991; Sellmer 1991).
These methods were optimized primarily using cell suspensions and were not directly compared. Transgenic trees were
obtained using the electric discharge method (McCown et
al. 1991), while only transgenic. calli were obtained with the
gunpowder method (Devantier 1992; Devantier et al. 1993).
Electric Discharge Propulsion
Optimization with Cell Suspension
With the electric discharge method, optimization for
transient expression was performed using a cell suspension of P. alba x P. grandidentata cl. 'NC5339' (cv. 'Crandon')
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
from leaf callus. The experiment included an evaluation
the effects of particle size, discharge voltage, and particle load (Sellmer 1991). Although no strong correlation
between transient expression and stable expressi.on of
transgenes has been reported, maximizing transient expre~sion in the cell suspension provides a uniform, reproduCible system to evaluate biological parameters that affect
transgene expression.
The first step toward developing a reproducible method
for gene transfer is characterization of the target cells. Cell
density (initial volume of cells/ volume of tissue culture
medium) on the target plate did not affect transient gene
expression 2 days after bombardment for cell concentrations of 5 percent, 10 percent, and 15 percent; however, 50
~ercent yielded a significant increase in transient expressiOn. Interestingly, no differences in f3-glucuronidase (GUS)
gene expression were observed among the 4 concentrations 1 week after bombardment. Cell culture age significantly affected transient expression levels of the GUS gene.
Based on weekly subculturing (5 ml packed cells into 50
ml fresh medium), 3-day-old cell suspensions had significantly higher GUS gene expression compared to cells harves.ted at other times in the culture cycle. Based on dry
weight, the cell suspension culture was in a lag phase
through day 4, underwent a rapid growth phase during
the next 4 days, and were in a stationary phase at day 9.
Maximum transient gene expression corresponded to the
mid-lag phase. During this 3-day period, growth was not
noticeable, but a profound change in cellular morphology
and the initiation of active cell division occurred. Cytologically, 32 percent of the cells on day 3 contained a division plate, in contrast with only 13 percent on day 1 and 9
percent on day 5. Furthermore, cells from 3-day-old cultures had almost twice as much 3H-thymidine incorporation compared with cells from other stages. Cell size was
also the smallest (less than 100 11m) from 3-day-old cultures. Because of this smaller size, more cells per volume
were used causing a bias in transient GUS expression.
When adjusted for cell number, cells from 1-day-old and
3-day-old cultures exhibited similar expression levels, but
both produced higher GUS expression than those at other
stages. However, this difference was diminished in GUS
expression assays 21 days after bombardment, and no difference was detectable 56 days after bombardment. This
long-term pattern of gene expression was independent of
transformation vector size (Sellmer 1991 ).
Later experiments focused on factors that could promote
cell competence for transient gene expression and stable
transgene incorporation. These factors were associated
with diverse approaches including preculturing cells with
growth regulators, cell synchronization, and DNA breakage experiments. To test the effect of cell division (rate and
synchronization) on transient GUS gene expression, cultures were grown on various hormone regimes. Experiments were inspired by previous cell-age experiments and
~f
61
Section II Transformation and Foreign Gene Expression
other reports that preculturing of tissue culture materials
can enhance gene transfer and transient gene expression
by promoting axillary and adventitious meristem development before bombardment (Ellis et al. 1991; McCown et
al. 1991; Serres et al. 1992). In 1 set of experiments, 2,4dichlorophenoxyacetic acid (2,4-D) was required for cell
growth. Higher cytokinin levels or the inclusion of
thidiazuron (TDZ) did not significantly change the growth
rate of the cultures. Although growth rates were unchanged,
incorporation of 3H-thymidine was the highest with the
highest concentration of cytokinin tested (1.0 ~-tM benzyladenine). However, transient GUS gene expression did not
change (Sellmer 1991). These results suggest that tissue cultures responded toward differentiation and meristematic
development, but transient gene expression was unaffected.
Continuing this rationale of enhancing cell competence
and GUS gene expression, experiments were conducted
to synchronize poplar cell suspension to potentially increase transient gene expression and stable incorporation
of genes. Some success was achieved with aphidicolin, a
tetracyclic diterpenoid that reversibly blocks cell division
before DNA strand synthesis. Aphidicolin was used to halt
cell division and was effective at blocking DNA synthesis
based on 3H-thymidine incorporation in early exponential
(3-day-old) and stationary (8-day-old) cultures. Effects of the
block apparently remained for 12 to 24 h after release from
the aphidicolin because little 3H-thymidine incorporation
was observed. In both cell ages, a sharp peak of 3H-thymidine incorporation was observed after 24 h. In the cells from
3-day-old cultures, this peak dropped back to the baseline
level suggesting synchronization of DNA synthesis. The
highest level of transient GUS gene expression was observed
at the 3-day-old stage although the ~evel was not significantly
different from cell suspensions at other times following
aphidicolin release. Differences were not apparent in GUS
gene expression, nor in the level of stably transformed calli
recovered from the different time points (Sellmer 1991).
In a complementary study, ionizing radiation (gamma
rays) was tested alone and in combination with aphidicolin
to evaluate if DNA repair in conjunction with synchronized
division could increase stable transformation. Radiation
levels up to 3,000 rads did not affect cell growth, although
no difference on the transient or long-term expression of
GUS was observed, even at higher radiation levels. Also,
no difference in GUS gene expression was evident when
this treatment was combined with the aphidicolin treatment (Sellmer 1991).
Gene Delivery into Protoplast-Derived Cells and
Nodules
Electric discharge propulsion was used to deliver
microprojectiles in cells from protoplasts and nodule cultu.res of P. alba x P. grandidentata cl. 'NC5339' and P. nigra x P.
trichocarpa cl. 'NC5331,' and to obtain transgenic trees con-
62
taining a chimeric Bacillus thuringiensis 8-endotoxin gene
conferring resistance to some lepidopteran pests (McCown
et al. 1991). Transient GUS gene expression was obtained
with 2 cell types (cell microcalli and nodules) and in stem
sections. Bombardment intensity, particle load, and target
tissue type were studied. Transgenic nodules resistant to
kanamycin were obtained with both genotypes, but only
nodules of P. alba x P. grandidentata regenerated transgenic
trees. Transgenic tree tissues were tested for feeding toxicity
against forest tent caterpillar and gypsy moth larvae. Reduced survival was observed for the forest tent caterpillar
only, but both insect species manifested reduced growth.
Gene Delivery into Plantlet Tissues
As with poplar cell suspensions, the developmental and
physiological stage of the poplar explant influences transient
expression following particle bombardment. With poplar
hybrid clones 'NC5339' (P. alba x P. grandidentata cv.
'Crandon') and 'NM6' (P. nigra x P. maximowiczii), explant
pretreatment on shoot inducing media significantly increased
transient GUS gene expression, driven by an enhanced
CaMV 35S promoter, in nodules, petioles, internodes, and
leaf explants (Sellmer 1991). Pretreatment of 0.5 to 1 em petiole segments in a liquid Woody Plant Medium (WPM) (Lloyd
and McCowm 1980) supplemented with 0.1 ~-tM each
benzyladenine (BA), naphthaleneacetic acid (NAA), and
thidiazuron (TDZ) progressively increased the number of
cells histochemically staining for GUS activity for up to 7
days (Wraith et al. 1994). Although proliferation at the cut
end of the petiole explant had initiated, cells ·were not yet
sloughing off. Interestingly, pretreatment on callus induction media did not markedly increase transient gene expression in petiole segments, despite the similar morphological
appearance of the explants (Wraith et al. 1994).
To increase the number of GUS-expressing cells, pretreatment must either increase the number of cells impacted or
alter cell competency for transiently expressing inserted
DNA. Explant pretreatment with a bud-induction medium
may induce differentiation of cells capable of forming
shoots or meristematic cells. These pretreated cells may
be similar to the cells of the late-lag-phase cell suspensions.
For stable integration of delivered genes, a callus induction phase before culture on a bud-induction medium may
improve the recovery of transgenic tissues after bombardment. Although this phase is not always required when
no selection is applied, it is necessary when kanamycin
selection is performed after bombardment of the hybrid
poplar l~ne 'NM6' (Wraith et al. 1994). There was no strong
correlation between level of transient gene expression and
number of stably transformed tissues recovered. Eight
weeks after particle bombardment, the low number of
GUS-expressing cells was constant regardless of gene construct, level of transient gene expression, and prebombardment treatments.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Direct Gene Transfer in Poplar
Gunpowder and Helium Propulsions
Optimization With Cell Suspension
Similar to work with the electric discharge method, optimization of gene delivery conditions with the gunpowder and the helium propulsion methods (Biolistic
PDS-1000, Biolistic PDS-1000/He) was done with a cell
suspension of P. nigra x P. maximowiczii cl. 'NM6'
(Devantier 1992; Devantier et al. 1993). Evaluations were
conducted on the effects of propulsion mechanism type,
DNA precipitation method, DNA amount, bombardment number, microprojectile type and size, use of
screens on top of the target tissues, target sample position in the bombardment chamber, and transient gene
expression assay time. All these factors influenced gene
expression levels. In addition, the helium propulsion
device, using gold particles on which DNA was precipitated with CaC1 2 (calcium chloride), produced a
higher level of transient gene expression. The optimum
cell suspension age was between 7 and 9 days after subculturing, and the maximum transient gene expression
was 1 day after bombardment. No study of growth rate
or cell division was conduct~d. Following cell suspension bombardment, subcellular localization of
microprojectiles revealed that most GUS-expressing
cells contained microprojectiles in their cytoplasm suggesting that a mechanism may be available to transport
the DNA to the nucleus.
In this study, microprojectile-mediated DNA delivery in a cell suspension of 4 different poplar genotypes
(P. nigra x P. maximowiczii cl. 'NM1' and cl. 'NM6,' P.
deltoides x P. nigra cl. 'DN106,' and P. tremula x P. alba cl.
'7171-84') was used to evaluate the strength of different chimeric promoters (Devantier et al. 1993). A genotype effect was observed, with 'NM1' yielding the
highest level of transient gene expression by a vector
containing the GUS gene linked to a double 35S promoter with an alfalfa mosaic virus translational enhancer. This effect was perhaps associated with different
morphologies of the cell suspensions for each genotype.
Lower expression levels were obtained with chimeric
promoters containing only the single 35S promoter with
the alfalfa mosaic virus promoter, the double 35S promoter, and the single 35S promoter (in order of decreasing strength). Similar results were obtained with the
single and double 35S promoter in transgenic poplar
(Leple et al. 1992).
Stable integration of the introduced genes was obtained
following selection on 500 mg/1 kanamycin. Expression
of the introduced neomycin phosphotransferase II (NPTII)
gene was detected using a radioactive assay. However,
because· the cell suspension was over 5 years old, no
transgenic plants were regenerated (Devantier 1992;
Devantier et al. 1993).
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Gene Delivery Into Plantlet Tissues
As with the electric discharge method, explants were used
to attempt recovery of transgenic poplar trees (Devantier
1992; Devantier et al. 1993). Three genotypes (P. nigra x P.
maximowiczii cl.'NM1' and cl. 'NM6,' and P. tremula x P. alba
cl. '7141-84') were tested to optimize transient gene expression. The Biolistic Helium device using gold particles was
superior, and 6-day-old explants gave higher transient gene
expression. Different hormone treatments, a callusing phase,
bombardment time after excision, different chimeric promoter strengths, and the timing of kanamycin selection were
evaluated for stable genetic transformation; however, no
transgenic tissues were obtained Uones and Charest, unpublished). Furthermore, selection with hygromycin and methotrexate was attempted without success. A current approach
is to obtain cell suspensions from in vitro grown plantlets
and use these cells to produce transgenic calli from which
trees will be regenerated.
Conclusions
Direct gene transfer by electroporation.or microprojectile-mediated DNA delivery was achieved in poplar, and transgenic trees were obtained. These methods can
also be used for fast experiments on transient gene expression. However, only a few genotypes were useful for regenerating transgenic trees, despite several attempts to
extend these methods to a wider range of genotypes. Several biol.ogical parameters need exploring, and a better
understanding of the mechanisms of DNA integration into
plant cell genomes is required to improve currently used
protocols. Once DNA delivery parameters are established,
careful consideration must be given to the: 1) target cell
response to the delivery process; 2) cell competence for
DNA uptake, transient gene expression, and stable expression of the delivered DNA; and 3) interactions between
the delivered DNA and the target cell machinery.
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