Plant Science 162 (2002) 181– 192
www.elsevier.com/locate/plantsci
Transgene silencing and reactivation in sorghum
Chandrakanth Emani, Ganesan Sunilkumar, Keerti S. Rathore *
Institute for Plant Genomics and Biotechnology, Department of Soil and Crop Sciences,
Norman E. Borlaug Center for Southern Crop Impro6ement, Texas A&M Uni6ersity, College Station, TX 77843 -2123, USA
Received 6 July 2001; received in revised form 7 September 2001; accepted 11 September 2001
Abstract
Amongst the important cereals, sorghum has been the most recalcitrant to genetic transformation. There are a few reports on
sorghum transformation and the majority of these have reported silencing of one of the transgenes. We regenerated plants from
two independent transgenic sorghum callus lines that were cotransformed with Ubi promoter:bar and Act1 -D promoter:gusA gene
constructs using the particle bombardment method. Southern analyses indicated integration of multiple copies of both the
transgenes. T0 plants were found to express the bar gene. The gusA gene, however, was silenced. It was possible to activate gusA
gene expression in T1 seedlings and in calli derived from immature T1 and T2 embryos by 5-azacytidine (azaC) treatment. In
certain cases, spontaneous expression of the gusA gene was observed in T1 and T2 immature embryo-derived calli. Expression of
the bar gene, as analyzed by Basta™ tolerance and Phosphinothricin acetyltransferase (PAT) assays, was detected in T0, T1 and
T2 plantlets; however, the expression was reduced in the T2 progeny obtained from a homozygous T1 parent. PAT activity was
also lower in the immature embryo-derived T2 calli from the same homozygous T1 parent. Again, culture on azaC increased the
level of PAT activity in these calli. Moreover, in a separate set of stable transformation experiments, it was possible to recover
a much higher than usual number of gusA gene expressing transgenic calli by growing the bombarded tissues in the presence of
azaC. Taken together, these results suggest that methylation-based silencing is frequent in sorghum and probably responsible for
several cases of transgene inactivation reported earlier for this crop. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: 5-Azacytidine; Biolistics; Gene silencing; Methylation; Sorghum; Transgenic
1. Introduction
Sorghum (Sorghum bicolor L., Moench) is one of the
most important grain and forage crops of the world,
ranking sixth among the most planted crops. It is an
important staple cereal of the semi-arid tropics of Asia
and Africa. In the US, it is usually grown primarily as
a livestock feed.
Improvement of sorghum for superior agronomic
traits has mostly been carried out using traditional
plant breeding methods. Improvements through genetic
engineering have been slow in sorghum. The main
reason for this has been its recalcitrance to genetic
transformation, even though sufficient advances have
been made in the area of tissue culture [1 – 6]. Research
* Corresponding author. Tel.: + 1-979-862-4795; fax: +1-979-8624790.
E-mail address: [email protected] (K.S. Rathore).
on sorghum transformation began a decade ago with
the first reports of DNA introduction into protoplasts
[7] and particle bombardment of a non-regenerable cell
suspension [8]. The first transgenic sorghum plants were
obtained by microprojectile bombardment of immature
embryos [9] and later using the same method with
immature inflorescence-derived calli [10]. There is also a
report on introduction of a rice chitinase gene into
sorghum by microprojectile bombardment [11]. Two
recent studies suggest that it is possible to genetically
engineer sorghum through Agrobacterium-mediated
transformation [12,13].
Success in generating improved sorghum cultivars by
genetic engineering requires efficient gene transfer, stable integration and predictable expression of the transgene. In all the reports mentioned earlier, only the
Agrobacterium method resulted in as high as 2.1%
transformation efficiency [13]. In contrast, the bombardment method yielded efficiencies of 0.2 [9,10] and
0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.
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C. Emani et al. / Plant Science 162 (2002) 181–192
0.5% [11]. Silencing of the introduced gusA gene was
observed in three different reports [9,10,12]. Zhu et al.
[11] also observed random silencing of the introduced
chitinase gene at different growth stages of the primary
transgenic plants and in some of their progeny. Though
gene silencing was cited as the prime reason for reduced
transformation efficiencies [9,10,12], the phenomenon
was not examined in detail. In light of these reports, it
is useful to carry out detailed analyses of silencing,
which may help explain the recovery of extremely low
number of transformants in sorghum due to the inactivation of selectable marker gene.
The present study is an attempt to examine the
involvement of methylation in silencing of transgenes in
sorghum. Cases of methylation-mediated transgene silencing have been reported in dicots [14] and in cereal
crops like rice [15–17] and wheat [18]. We made use of
the cytidine analog, 5-azacytidine (azaC), in reversing
the methylation-mediated transgene silencing as reported earlier for tobacco [19,20] and rice [15]. The
results presented in this study add to the existing evidence of methylation-based transgene silencing in another important cereal crop. The findings may aid
attempts being made to improve the efficiencies of
sorghum transformation.
2. Materials and methods
2.1. Plant material
S. bicolor (L.) Moench, genotype RT× 430 was used
in all experiments. Plants were grown in a greenhouse
in soil (Metromix 366) and watered on alternate days.
When plants reached the six-leaf stage, they were fertilized every week. Immature caryopses were collected
10–12 dpa and sterilized by treating with 70% ethanol
for 30 s followed by 40% commercial bleach with two
drops of Tween-20 for 30 min. Immature embryos were
dissected aseptically and cultured either for 1 week or
for 8 weeks before transformation.
2.2. Culture media and transformations
Immature embryos were pre-cultured for a week on
MSB medium (MS salts, modified B5 vitamins [9], 100
mg l − 1 myo-inositol, 1 g l − 1 asparagine, 1 g l − 1
proline, 2 mg l − 1 2,4-D, 30 g l − 1 sucrose, pH 5.7,
solidified with 2 g l − 1 Phytagel) before transformation.
MSB medium served as the callus-induction medium
and also as callus-maintenance medium in majority of
the experiments unless otherwise stated. Callus-induction, maintenance, and selection were carried out in
dark at 25 °C. In one set of experiments, immature
embryos were cultured on MSM medium (MSB with 30
g l − 1 maltose instead of sucrose). The callus from
scutellum that formed within 2 weeks was excised and
subcultured every 3 weeks onto fresh MSM media.
Eight-week-old embryogenic calli (white and compact)
were cultured for a week on fresh MSM medium before
bombardment. Prior to particle bombardment, both the
pre-cultured embryos and 9-week-old calli were transferred to high-osmoticum medium (MSB with 0.6 M
sucrose or MSM with 0.6 M maltose, final concentration) and incubated for 4 h.
Plasmids used in this study were pAHC20 [21], which
has the bar gene driven by the maize ubiquitin promoter and pAct1 -D [22], which has the gusA gene
driven by the rice actin promoter. A biolistic PDS-1000/
He (Bio-Rad) gene gun was used for transformations.
The plasmids were coated onto 1 mm gold particles
(Bio-Rad). Two milligram particles were washed first
with 70% ethanol and sterile water (twice) and then
resuspended by sonication in 220 ml of sterile water.
12.5 ml of 1 mg ml − 1 solutions of each plasmid DNA,
250 ml of 2.5 M CaCl2, and 50 ml of 0.1 M spermidine
were added sequentially to the particle suspension. After thorough mixing by short spurts of vortexing, the
particles were pelleted and washed in 70% ethanol and
finally resuspended in 40 ml of absolute ethanol. Ten
microliter of this suspension was spread onto macrocarrier discs for bombardment. Tissues were bombarded
twice at 900 psi. The distance between the target tissue
and rupture disc was 10 cm, with a 10 mm distance
between the rupture disk and macrocarrier and a 12
mm macrocarrier flying distance.
Following bombardment, the tissues were incubated
for 16 h on the same high-osmoticum medium before
transferring to the original medium. After 1-day culture
on this medium (approximately 48 h after bombardment), calli or immature embryos were cultured on
selection medium [MSB or MSM (excluding asparagine
and proline) supplemented with 1 mg l − 1 phosphinothricin (PPT; Crescent Chemical Co., Inc.)] for 2
weeks. Tissues were then cultured for two additional
rounds of 3 weeks each on MSB or MSM media
containing 2 mg l − 1 PPT and finally onto MSB or
MSM media containing 5 mg l − 1 PPT for 2 weeks.
Calli surviving the selection were transferred to pre-regeneration (PR) medium (MS salts, modified B5 vitamins [9], 0.25 mg l − 1 Kinetin, 0.5 mg l − 1 IAA, 30 g l − 1
sucrose containing 2 mg l − 1 PPT, pH 5.7, solidified
with 2 g l − 1 Phytagel) and cultured for 2 weeks (16 h
photoperiod, 90 mmol m − 2 s − 1) at 25 °C. All subsequent regeneration steps were carried out under similar
culture conditions. Green sectors of calli were transferred to regeneration medium (MS salts, modified B5
vitamins [9], 0.5 mg l − 1 Kinetin, 1 mg l − 1 IAA, 30 g
l − 1 sucrose, containing 2 mg l − 1 PPT, pH 5.7, solidified with 2 g l − 1 Phytagel) to induce shoot development. The regenerated shoots were finally transferred to
jars with medium for root development (0.5×MS
C. Emani et al. / Plant Science 162 (2002) 181–192
salts, modified B5 vitamins [9], 0.5 mg l − 1 NAA, 0.5
mg l − 1 IBA, 20 g l − 1 sucrose, containing 2 mg l − 1
PPT, pH 5.7, solidified with 2 g l − 1 phytagel).
2.3. GUS assay
Expression of gusA gene was examined histochemically in the calli, leaves, roots, and immature and
mature inflorescence of all the plants surviving selection
as described by Hiei et al. [23]. Fluorometric GUS
analysis was carried out according to the method described by Jefferson et al. [24].
Seeds collected from primary transgenic plants were
germinated in dark on MS medium, with (310 mM) or
without azaC. One-week-old seedlings were subjected to
histochemical gusA analysis. Fluorometric GUS assays
were conducted with 5– 6 mm individual coleoptilar
node segments from a set of 50 control seedlings germinated in dark on azaC-free medium, and 50 T1
seedlings each grown on medium with (310 mM) or
without azaC. Another tissue examined for GUS activity was callus grown from T1 immature embryos. Embryos 10–12 dpa from the primary transgenic plants
were cultured either on MSB medium with (20 mM) or
without azaC. After 4 weeks, a portion of the callus
developing from the embryos was dissected out and
subjected to histochemical analysis for gusA expression.
The remaining callus was removed from the azaC
medium and cultured on azaC-free medium for an
additional 4 weeks. After this culture, individual callus
lines were equally divided and one half was kept on
regeneration medium and the other half was again
subjected to histochemical analysis for GUS activity. A
separate set of control and transgenic calli, cultured
and maintained under similar conditions, were used for
fluorometric GUS analysis.
Immature embryos (T2) were also isolated from T1
homozygous plants and cultured on MSB medium for 2
weeks. The emerging calli were cultured on medium
with (20 mM) or without azaC for 4 weeks and then
subjected to histochemical and fluorometric GUS
analyses. The calli that were not used for GUS analyses
were transferred to azaC-free medium. Three weeks
after the transfer, the calli were subjected to flourometric GUS analysis.
183
ual T1 plants were germinated in soil in separate trays.
The resulting T2 seedlings were sprayed with 0.25%
Basta™ at the three-leaf stage.
PAT assays were performed on leaf tissues from
young plantlets as described by Rathore et al. [25]. For
T1 and T2 plants, leaf tissues used for PAT assays were
obtained from 8-day-old soil-grown seedlings. For T0
generation, the leaf tissue was obtained from small
tillers arising from the base of the original T0 plants
from experiment c 8 (Fig. 2, lanes 5 and 6). These
tillers were about the same size as T1 and T2 seedlings
that were used for PAT assays. The T0 plantlets from
experiment c 11 were in Magenta boxes growing on
PPT-supplemented medium at the time of PAT assays
(Fig. 2, lanes 7 and 8). Care was taken to ensure PAT
reaction conditions were same for all three generations.
X-ray films were exposed to the TLC plates for the
same length of time and they were developed at the
same time. PAT activity was also determined for the
callus tissues obtained from immature T2 embryos of a
homozygous T1 parent cultured on medium with (20
mM) or without azaC for 6 weeks.
2.5. Molecular analyses
Genomic DNA was isolated [26] from the PCR-positive plants. Ten microgram of DNA was digested with
HindIII and transferred to nylon membrane (Hybond,
Amersham). Southern blot analysis [27] was conducted
to confirm integration of both the bar and gusA genes.
Details for each blot are described in the figure legends.
2.6. Selection of transformants in the presence of azaC
Two additional bombardment experiments were carried out with 100 (Experiment c 11) and 200 (Experiment c 12) immature embryos (see Section 3.4).
During selection, the embryos were equally divided and
cultured on selection medium with (20 mM) or without
azaC for 4 weeks. The embryos were then transferred to
azaC-free selection medium and cultured for two additional weeks. The calli emerging from these embryos
were subjected to histochemical analysis for GUS
activity.
2.4. PAT assays and basta tolerance tests
3. Results
Twenty-five T1 plants were grown from seeds obtained from one of the Southern-positive primary transgenic plants. Two-week-old plants were examined for
the expression of the bar gene by dipping 4 –5 cm
portions of tips of young leaves into a 0.25% Basta™
(500 mg l − 1 PPT) solution. Following the herbicide
resistance test, these T1 plants were grown to maturity
in the greenhouse. Seeds obtained from several individ-
3.1. Regeneration of transgenic plants and transgene
acti6ity
In nine different experiments, 2160 immature embryos were used for co-transformation with the
pFF19H (CaMV 35S:hph, [28]) and pAct1 -D plasmids.
However, no transgenic plants were recovered following
selection on hygromycin.
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C. Emani et al. / Plant Science 162 (2002) 181–192
Transgenic sorghum plants were recovered only when
transformations were carried out with the pAHC20 and
pAct1 -D plasmids. In seven separate experiments, a
total of 1100 immature embryos pre-cultured on MSB
medium were subjected to particle bombardment-mediated transformation. In three other experiments, 150
calli (white, compact and embryogenic callus pieces
from 9-week-old callus lines) cultured on the MSM
medium were used for transformation. Most of the calli
did not survive the selection process, except from two
experiments. In experiment c4 where 150 pre-cultured,
immature embryos were bombarded and then cultured
on MSB medium, one callus line survived selection
from which eight plants were regenerated. As described
later, PCR analyses revealed that only four of these
were positive for the bar gene and the rest were escapes.
The transgenic plants recovered from this experiment
were, however, stunted and infertile. In experiment
c 8, where 50 individual callus pieces grown on MSM
medium were used as target tissues, again, only one
callus line survived selection from which 15 plants were
regenerated. Only six of the regenerated plants from
this experiment were transgenic and nine were escapes.
All six transformed plants from experiment c 8 grew
normally and set seeds in the greenhouse with no
apparent morphological differences from the control
plants. The results presented for GUS and PAT assays
are on T0, T1 and T2 generations derived from this
callus line.
Histochemical GUS activity was not observed in
several batches of calli tested during the selection process. GUS-dependent histochemical staining was also
not observed in the leaves, roots, and immature and
mature inflorescence of regenerated plants. Results of
fluorometric analysis performed on the leaves and seeds
of regenerated plants were also negative for GUS-specific activity (data not shown).
Twenty-five T1 plants from one of the primary,
fertile T0 plant were tested for herbicide tolerance by
treating a portion of their leaves with Basta™. Leaves
of 16 plants showed resistance to Basta™ application
with little or no damage. The treated leaf portions of
the remaining nine plants were susceptible to the herbicide and showed complete yellowish to brown coloration in the leaves indicative of substantial necrosis.
The Basta™ spray test was conducted on young T2
seedlings obtained from 12 different herbicide tolerant
T1 plants and five different herbicide susceptible T1
(possible null segregants) plants. T2 progeny from the
five susceptible T1 plants were all damaged and killed
by herbicide spray (Fig. 1J) confirming that these five
T1 plants were indeed null segregants. All of the T2
seedlings from three different resistant T1 plants were
herbicide-tolerant (Fig. 1K) indicating that these were
homozygous for the bar gene. T2 seedlings from the
remaining nine resistant T1 plants segregated for herbi-
cide resistance, indicating that the parent T1 plants
were hemizygous for the transgene (results not shown).
We also carried out PAT assay on the leaf tissues
from T0, T1, and T2 seedlings. The results from this
analysis are shown in Fig. 2. The results show the
expression of the bar gene in all three generations. PAT
activity in the leaves of T0 plants were similar to the
activity levels detected in bar-gene expressing rice leaves
(kindly provided by Dr Roberta Smith) (Fig. 2, panel
1). Segregation of the PAT activity was evident in T1
generation. It is interesting that three out of 12 positive
plants showed a lower level of PAT activity (Fig. 2,
panels 2 and 3). A PAT assay on a second set of 16 T1
seedlings also showed a lower level of PAT activity in
five out of 11 PAT-positive plants (data not shown).
Thus, the PAT phenotype appeared to be segregating as
eight with low-level activity: 15 with high-level activity:
nine with no activity. PAT assay on T2 seedlings obtained from a homozygous T1 parent showed that the
bar gene was being expressed in all the seedlings tested;
however, at a considerably lower level compared to T0
and many of the T1 seedlings (Fig. 2, panel 4). This low
level of bar gene expression was nonetheless sufficient
to confer resistance to the herbicide treatment (Fig.
1K).
3.2. Molecular analyses
Amongst the regenerated plants from the transformations involving the bar gene, four plants from experiment c 4 and six plants from experiment c 8 showed
the expected PCR products for the bar and gusA genes
(results not shown). These plants were also tested for
the integration of the bar and gusA genes by Southern
blotting. In each of the two plasmids used for transformation, there is a single HindIII site just outside the
expression unit. In the Southern blot with HindIII
digested DNA, multiple copies of both the bar (Fig.
3A) and gusA (Fig. 3B) genes were observed in all the
plants. There are PstI sites on either side of the bar
gene in the plasmid, pAHC20. In the blot with PstI
digested DNA, the expected fragment for the bar gene
insert ( 570 bp) was seen in all the plants (results not
shown). Presence of the bar gene in the Basta™-resistant progeny plants was confirmed by PCR, where all
of the 16 resistant T1 plants showed the expected PCR
product (results not shown).
3.3. Reacti6ation of transgenes in the progeny
In order to ascertain if methylation of the transgene
was responsible for gusA gene silencing, we carried out
a series of experiments where seedlings or callus tissues
from T1 and T2 progeny were treated with azaC. Of
the 25 T1 seedlings grown on 310 mM azaC-supplemented medium, low-level blue staining confirming the
C. Emani et al. / Plant Science 162 (2002) 181–192
reactivation of the silenced gusA gene was observed in
the coleoptilar node regions (measuring up to 5– 6 mm)
in five seedlings (Fig. 1D and DD). The fluorometric
GUS assay was carried out with coleoptilar node segments from 50 individual T1 seedlings grown on media
with and without azaC. Results showed an activation of
gusA gene leading to increased level of GUS activity in
several seedlings treated with azaC (Fig. 4). However,
185
this GUS reactivation was low, thus confirming the
results obtained from histochemical analyses.
On the basis of these results on the reactivation of
gusA gene following short-term treatment of seedlings
with azaC, we wanted to evaluate the effects of more
intimate and long-term exposure to azaC. The experiment to test this was carried out by isolating immature
T1 embryos from primary transgenic plants, and then
Fig. 1. Transgene activity in sorghum. (A) Spontaneous reactivation of gusA gene expression in T1 immature embryo-derived callus after an
8-week culture. (B) Reactivation of gusA gene expression in T1 immature embryo-derived callus after a 4-week culture on 20 mM azaC-supplemented medium. (C) GUS activity in T1 immature embryo-derived callus after an 8-week culture (4 weeks on azaC medium and 4 weeks on
azaC-free medium). (D) Reactivation of gusA gene expression in T1 seedling after 1 week-long germination on medium with 310 mM azaC. Note
that the expression was detected only in the coleoptilar node region. (DD) A higher magnification image of the coleoptilar node region of the same
seedling. (E) Spontaneous reactivation of gusA gene expression in T2 immature embryo-derived callus after a 6-week culture. (F – I) Reactivation
of gusA gene expression in T2 immature embryo-derived calli after a 6 week culture (2 weeks on MSB medium followed by 4 weeks on 20 mM
azaC-supplemented medium). All the calli were derived from immature embryos that were obtained from a single homozygous T1 parent. (J)
Basta™ (500 mg l − 1 PPT) spray test on young T2 seedlings derived from a T1 null segregant. Note that all of the seedlings were damaged and
eventually killed. (K) Basta™ spray test on young T2 seedlings derived from a homozygous T1 parent. Note that all the seedlings were herbicide
tolerant. (L) One of the rare cases of GUS activity seen in a T0 callus line after 4 weeks of PPT selection following transformation. (LL) magnified
image of the part of the same callus showing GUS activity. (M – O) GUS activity in three different T0 calli that were cultured following
bombardment on PPT selection medium containing 20 mM azaC. Bar = 1 mm.
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C. Emani et al. / Plant Science 162 (2002) 181–192
Fig. 2. PAT activity in leaf extracts from control and three generations of transgenic sorghum plants. First panel — lane 1: empty; lanes 2 and 3:
two untransformed sorghum plants; lane 4: rice plant expressing the bar gene; lanes 5 and 6: two T0 transgenic sorghum plants obtained from
a single line in experiment c8; lanes 7 and 8: two T0 transgenic sorghum plants obtained from two different lines in experiment c 11. Second
and third panels —16 individual T1 seedlings obtained from one of the T0 transformant from experiment c 8. Fourth panel — Eight individual
T2 seedlings obtained from a T1 homozygous plant. The arrows indicate the position of acetylated PPT.
culturing them in the presence of azaC. AzaC at the
concentration that was used for seedlings (310 mM)
proved to be toxic to calli. Therefore, a lower concentration (20 mM) of this chemical was employed in the
reactivation studies in calli. In the histochemical analysis for gusA expression conducted on the resulting T1
calli after 4 weeks of culture, 20 calli out of the 25
tested showed large sectors of intense blue staining
(Fig. 1B; Table 1). In the control treatment where the
T1 calli from immature embryos were cultured on MSB
medium, only three out of the 25 calli tested showed
blue staining (Table 1). The remaining calli (from azaC
treatment) that were not used for analysis at this time
were transferred to azaC-free medium for an additional
4-week culture and then subjected to histochemical
analysis. Amongst these calli, 41 out of 57 tested still
showed the blue staining (Table 1; Fig. 1C). Since these
calli represent the T1 generation, they will be expected
to segregate for the transgene and therefore transgene
expression. Amongst the calli that were grown on azaCfree medium throughout the 8-week culture period,
only 12 out of the 43 tested showed blue staining (Table
1; Fig. 1A). However, these calli had weaker staining
compared to the calli cultured on azaC medium. Quantitative, fluorometric GUS analyses carried out on a
separate set of T1 calli, showed an overall 46-fold
increase in GUS activity in the callus tissues that were
grown on azaC-supplemented medium for 4 weeks (Fig.
5). Null segregants were quite obvious with activity
levels similar to non-transgenic controls. The remaining
calli had varying degrees of GUS activity. We were able
to regenerate some plants from the remaining portions
of calli that tested GUS positive. However, none of the
regenerated plants exhibited any GUS activity indicating that the gusA gene that was reactivated in the callus
tissue was once again silenced (data not shown).
Reactivation of the gusA gene expression as tested by
histochemical analysis was also observed in the immature embryo-derived T2 calli cultured on azaC-supplemented media (Table 1). These T2 calli were obtained
from the embryos from three different T1 plants that
had been identified as homozygous for the bar gene
using the herbicide spray test on their progeny as
described earlier. It is interesting that 100% of the T2
calli from the azaC treatment showed GUS reactivation
(Fig. 1F–I; Table 1). Spontaneous reactivation of gusA
gene expression was observed in some of the T2 calli
cultured on azaC-free medium in all three samples of
calli from the homozygous parents (Fig. 1E; Table 1).
Again, these calli showed a much lower intensity of
GUS staining. The fluorometric analysis carried out on
individual T2 calli from one of the homozygous T1
parents (24 calli each from media with or without azaC)
revealed an overall 4.6-fold increase in GUS activity in
C. Emani et al. / Plant Science 162 (2002) 181–192
calli cultured on azaC-supplemented medium (Fig. 6A).
The figure also shows that some of the calli in the
azaC-free medium showed spontaneous reactivation of
GUS activity. However, with azaC treatment, 23 out of
24 calli tested were positive for GUS activity and the
range of reactivated GUS activity in these calli was
much higher. Again, as observed in T1 calli, the reactivation of gusA expression did not occur throughout the
callus (Fig. 1F–I). Fluorometric analysis was repeated
on the remaining T2 calli from the above experiment
(see Fig. 6A) after an additional 3-week culture on
187
azaC-free medium. As shown in Fig. 6B, the previously
azaC-treated calli showed GUS activity, however, there
was a decrease in the activity suggesting that the reactivated gusA gene was being silenced.
Results presented in Fig. 2 suggested that the bar
gene expression had also declined in the T2 generation.
Our observations that the gusA gene can be reactivated
in the calli in response to azaC treatment suggested the
possibility that the bar gene may also show an increase
in expression as a result of long-term azaC treatment.
In order to explore this possibility, we carried out PAT
Fig. 3. (A) Southern blot analysis of genomic DNA from leaves of a control and ten transgenic sorghum plants. Genomic DNA was digested with
HindIII and bar-coding region was used as the probe. Lane 1: HindIII-linearized pAHC20 plasmid. Lane C: DNA from a control plant. Lanes
M1– M6: DNA from six plants regenerated from a transgenic line obtained in experiment c 8. Lanes S1 – S4: DNA from four plants regenerated
from the second transgenic line obtained in experiment c 4. (B) Southern blot analysis of genomic DNA from leaves of a control sorghum plant,
a gusA transformed rice plant and 10 transgenic sorghum plants. Genomic DNA was digested with HindIII and gusA-coding region was used
as the probe. Lane 1: HindIII-linearized pAct1 -D plasmid. Lane R: DNA from a gusA transformed rice plant. Lane C: DNA from a control
plant. Lanes M1 –M6: DNA from six plants regenerated from a transgenic line obtained in experiment c 8. Lanes S1 – S4: DNA from four plants
regenerated from the second transgenic line obtained in experiment c4.
C. Emani et al. / Plant Science 162 (2002) 181–192
188
Fig. 4. Effect of 5-azacytidine on gusA reactivation in coleoptilar node segments from transgenic sorghum seedlings. GUS-specific activity in node
segments from 50 individual seedlings each from an untransformed control, T1 seedlings germinated on azaC-free medium, and T1 seedlings
germinated on azaC-supplemented medium. Mean GUS activity ( 9 SE) of 50 individual seedlings is shown in the graph.
Table 1
Effect of 5-azacytidine on reactivation of gusA gene expression in individual callus lines derived from immature T1 and T2 embryos
Callus line
T1
T2
a
b
Duration of culturea (weeks)
Assay method
No. Showing GUS activity/No. tested (percent calli expressing
gusA gene)
−azaC
+azaC
4
Histochemical
Fluorometric
3/25 (12%)
0/50 (0%)
20/25 (80%)
42/50 (84%)
4+4
Histochemical
12/43 (27.9%)
41/57 (71.9%)
2+4
Histochemicalb
3/34 (8.8%)
9/25 (36%)
11/25 (44%)
42/42 (100%)
25/25 (100%)
25/25 (100%)
Fluorometric
5/24 (20.8%)
23/24 (95.8%)
The numbers in bold represent duration of culture period on azaC-supplemented medium.
Results from three sets of T2 calli obtained from immature embryos from three different homozygous T1 parents.
assays on individual T2 calli (obtained from a homozygous T1 plant) that were grown either in the
presence or absence of azaC for 6 weeks. The results
from this analysis, presented in Fig. 7, showed a substantial increase in bar gene expression as a result of
azaC treatment. These results suggest that bar gene
expression in the T2 calli, without azaC, was only
partial and it was possible to further enhance its expression by demethylation.
3.4. Impro6ed reco6ery of GUS expressing calli by
azaC
The results described thus far and the results from
other studies [9–12] suggest that transgene silencing is a
serious problem in sorghum. However, our results also
indicated that azaC treatment of transformed tissues
might increase the recovery of stable transformation
events. In order to explore this possibility, we carried
out two experiments (c 11 and c 12) where following
bombardment, the tissues were selected in the presence
of azaC. In experiment c 11, where tissues were cultured on azaC-supplemented medium after bombardment, four out of the 35 calli tested after 4 weeks of
selection showed sectors of intense blue staining and
three showed a single blue spot observable under a
microscope (Table 2). No blue staining was observed in
an equal number of calli, which were grown on azaCfree selection medium. In experiment c 12, 21 out of
the 43 calli cultured on azaC-supplemented medium
showed sectors of intense blue staining (Fig. 1M– O;
Table 2). In the same experiment, only one callus out of
45 tested, that were selected on azaC-free medium,
showed a few blue spots (Fig. 1L and LL; Table 2). The
percentage of GUS expressing calli with azaC treatment
in the two experiments was thus 20 and 49%, respec-
C. Emani et al. / Plant Science 162 (2002) 181–192
tively (Table 2). These results indicate clearly that the
reason for the absence of GUS activity in the transformed callus under normal culture conditions, as
noted in the early stages of this investigation, was
methylation-based silencing of the gusA gene.
4. Discussion
Genetic engineering relies on stable integration, desired level of expression, and predictable inheritance of
the introduced transgenes. Although transgene silencing
phenomenon has been observed in both dicots [14] and
monocots [29], it appears to be a major obstacle in the
transformation of sorghum. In most of the reports on
sorghum transformation, it was observed that the introduced gusA gene expression was either very poor or
totally absent. Hagio et al. [8] observed that GUS
enzyme activities were very low in sorghum cells compared to other gusA gene-transformed monocot cells.
189
In addition, the RNA blot analysis revealed accumulation of aberrant transcripts, which the authors attributed to transgene rearrangements and in vivo
degradation of the transcripts. Battraw and Hall [7]
observed that the majority of gusA-transformed cells
did not stain blue upon incubation with histochemical
substrate x-gluc. Casas et al. [9,10] noted that GUS
activity that was high in transient assays could not be
detected later than 3 weeks after bombardment. They
suggested that transgene methylation might have occurred in sorghum cells that inhibits expression of the
reporter gene. In none of these reports was GUS activity detected in the transgenic plants despite the fact that
Southern analyses clearly indicated integration of the
gusA gene.
We introduced the bar gene under the control of
maize ubiquitin promoter and the gusA gene under the
control of rice actin promoter into sorghum. Molecular
analyses confirmed that multiple copies of bar as well as
gusA genes had integrated into the sorghum genome.
Fig. 5. Effect of 5-azacytidine on gusA reactivation in callus tissues obtained from immature T1 embryos. GUS-specific activity in 10
untransformed control calli each grown on medium without (C) or with azaC (C + azaC), and 50 T1 calli each grown on medium without (T1)
or with azaC (T1 +azaC). Mean GUS activity ( 9 SE) for each group is shown in the graph.
Fig. 6. Effect of 5-azacytidine on gusA reactivation in callus tissues obtained from immature T2 embryos. (A) GUS-specific activity in 24 T2 calli
each grown on medium without (T2) or with azaC (T2 + azaC) for 4 weeks. (B) GUS-specific activity in 24 T2 calli grown on medium without
azaC (T2) for 7 weeks, and 24 T2 calli grown on azaC-supplemented medium for the first 4 weeks and then for 3 weeks on azaC-free medium
(T2+ azaC). Note the difference in scale between the two figures. Mean GUS activity ( 9 SE) for each group is shown in the graphs.
190
C. Emani et al. / Plant Science 162 (2002) 181–192
Fig. 7. Effect of 5-azacytidine on bar gene expression in callus tissues derived from T2 immature embryos that were obtained from a homozygous
T1 parent. PAT activity in 16 individual calli each grown on medium with or without azaC for 6 weeks. Each panel represents a single TLC plate,
where the first four samples loaded were from calli grown on azaC-free medium and the next four samples loaded were from calli grown on
azaC-supplemented medium. The arrows indicate the position of acetylated PPT.
Expression of the bar gene was observed in T0, T1 and
T2 generations as confirmed by growth of the plantlets
on PPT-supplemented medium, PAT assays or herbicide tolerance. The plasmid pAct1 -D had been successfully used in other monocots like rice [22,25] and it also
proved effective in transient expression studies in sorghum during the early stages of this investigation.
However, histochemical GUS activity was absent in all
tissues tested from regenerated T0 plants obtained from
both lines investigated in this study. In fact, no GUS
activity was observed in the leaf tissues of T0, T1 and
T2 generations. One possible reason for this may be
that both the transgenic lines obtained in this investigation had a large number of gusA gene copies integrated
with possible concatameric arrangements. There are
several reports [15,25,30] demonstrating transgene inactivation as a result of high copy number integration.
However, results from a number of other studies suggest that transgenes with even low copy number integration can become silenced [9,10,12,16,17,29,31]. Thus,
lack of GUS activity noted in the majority of stably
transformed calli in this investigation (Table 2, and
results from earlier stages of this investigation) and in
other sorghum transformation studies [9,10,12] cannot
be attributed entirely to high copy number integrations.
As shown in Fig. 1L, LL and Table 2, gusA-expressing
stable transformants were extremely rare events in sor-
ghum. It is also possible that the rice actin promoter
was particularly prone to silencing in sorghum, thus
accounting for the extremely poor, stable gusA gene
expression noted in this study. This notion is supported
by the fact that the bar gene that was driven by the
ubiqutin promoter was expressed well in T0 and T1
generations despite the fact that several copies of this
gene had integrated into the genome of the same line
showing no GUS activity.
It is interesting that during the culture of T1 and T2
embryos there was spontaneous reactivation of gusA
gene expression in a small number of calli. There are at
least two other studies on sorghum reporting silencing/
reactivation of transgenes at various stages of plant
growth and development. As stated earlier, Zhu et al.
Table 2
Improved recovery of gusA-expressing transformants by 5-azacytidine treatment (20 mM)
Experiment No.
11
12
No. showing blue staining/No.
tested (percent calli expressing
gusA gene)
−azaC
0/35 (0%)
1/45 (2.2%)
+azaC
7/35 (20%)
21/43 (49%)
The treatment was applied for 4 weeks to immature embryo-derived
calli following bombardment during the selection process.
C. Emani et al. / Plant Science 162 (2002) 181–192
[11] had observed random occurrence of silencing/reactivation of the introduced chitinase genes at different
growth stages of primary transgenic plants and in some
of their progeny. Carvalho [12] observed high level of
expression of the gusA gene only during certain stages
of development in only one of the five transgenic lines,
i.e. in immature inflorescences, florets and developing
seeds. The reasons for these random occurrences of
reactivation of silenced transgenes are not clear. Spontaneous reactivation of silenced genes was also observed in rice [17] and oats [31]. High auxin
concentrations have been proposed to cause demethylation via induction of the stress hormone ethylene with a
consequent depletion of the s-adenosyl-methionine
pool, which affects the level of maintenance methylation as s-adenosyl-methionine is the source of the
methyl group transferred to the cytosine residues [32].
The fact that the T1 and T2 embryos were cultured on
callus-induction medium with the auxin, 2,4-D, may
thus provide a possible explanation for the spontaneous
reactivation of the gusA gene in the resulting calli. This,
however, does not explain the lack of GUS activity in
the primary (T0) transgenic calli.
The results from experiments with azaC showed
clearly that this chemical, known to result in hypomethylation of DNA, was able to reactivate expression of silenced transgenes. 5-Azacytidine is a cytidine
analog, which integrates into DNA during replication
or repair and prevents methylation by inhibiting DNA
methyl transferase [33,34]. The reactivation of the GUS
activity observed in the azaC-treated tissues suggests
that the silencing of the gusA gene observed in this
investigation resulted from cytosine methylation-mediated silencing. This reactivation occurred both in the
seedlings as well as in calli grown on azaC-supplemented medium. Results from histochemical analyses
revealed that reactivation did not occur throughout the
tissues. Fluorometric analysis on these tissues showed
that the reactivation of the gusA gene was much
stronger in the callus tissue compared to the coleoptilar
node portion of the seedling. This may be due to the
fact that it was possible to keep the embryo-derived
calli in more intimate contact with azaC for much
longer periods of time. Weber et al. [32] compared GUS
activities after azaC treatment between tobacco protoplasts and callus tissue and found that azaC treatment
resulted in higher levels of reactivation in the calli. They
attributed this difference to the fact that the cell division activities were higher in callus tissues resulting in
faster rate of DNA synthesis and repair accounting for
hypomethylated DNA. A similar explanation may account for the results showing a higher degree of reporter gene reactivation in the callus tissue in the
present study.
Even in the callus tissues, the GUS activity was not
uniform. Individual tissues showed certain areas of
191
intense staining. Amongst a group of callus tissues
exhibiting reactivation of gusA gene, fluorometric analysis revealed a range of activities. The reasons for the
non-uniform reactivation are not clear, however, similar observations have been made in other studies.
Bochardt et al. [19], working with azaC-mediated reactivation of gusA gene expression in transformed tobacco suspension cultures, hypothesized that azaC will
be incorporated in a small population of rapidly dividing cells, resulting in severe DNA demethylation in a
fraction of progenitor cells. This suggestion may also
help explain results obtained in the present study.
As stated earlier, the bar gene expression, as tested by
herbicide-resistance and PAT assays, was observed in
three generations. It is interesting that PAT activities in
the T2 seedlings that were obtained from a homozygous
T1 parent were substantially lower compared to the
activities detected in T0 and many of the T1 seedlings.
A possible explanation for this reduction is the doubling of the already high copy number of the bar genes
in homozygous T1 progeny. This in turn could suppress
gene expression. It has been a general observation in
homology-mediated silencing that this phenomenon is
more readily observed in homozygotes compared to
heterozygotes. In this regard, it is interesting that at
least eight out of 23 PAT-positive T1 seedlings had
considerably lower PAT activities, perhaps suggesting
their homozygous status. In order to determine if the
reduction in the bar gene expression in the homozygous
T2 progeny was a result of methylation, we examined
the effect of azaC on bar gene expression in immature
embryo-derived T2 (homozygous) calli. The results
showing a substantial increase in PAT activity (Fig. 7)
in the cultures treated with azaC suggest that partial
methylation may have reduced the expression of the bar
gene in the homozygous T2 generation.
In the light of results described thus far, it is interesting but not surprising that azaC treatment during the
selection process following the transformation resulted
in increased number of gusA expressing calli. This
suggests that during the early stages of this investigation the reason for our inability to observe stable gusA
gene expression was transgene silencing due to methylation. Treatment of the cell cultures with azaC proved to
be an efficient way to obtain an increased number of
reporter gene-expressing sorghum cells. However, the
feasibility of such a treatment in enhancing the expression of selectable marker gene resulting in the recovery
of higher numbers of transgenic sorghum plants which
continue to express the transgenes needs to be investigated further.
The present study clearly demonstrates that methylation-based transgene silencing is a serious problem in
sorghum. It may also help explain the very poor transformation efficiencies obtained in previous sorghum
transformation studies. The results presented in this
192
C. Emani et al. / Plant Science 162 (2002) 181–192
report may also aid in designing more effective strategies
to increase the efficiency of sorghum transformation.
Acknowledgements
We thank LeAnne Mohr for her assistance with the
GUS assays. We also thank Drs William L. Rooney and
Robert Klein for providing sorghum seeds used in this
investigation and Dr Thomas Hodges and Dr Carol
Loopstra for their critical reading of this manuscript and
for their valuable comments. This research was supported by funds from the Rockefeller Foundation,
USDA-ARS and Texas Agriculture Experiment Station.
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