Transgenic Research (2005) 14:273–278 Ó Springer 2005

DOI 10.1007/s11248-004-8081-9

Chlorsulfuron resistant transgenic tobacco as a tool for broomrape control

S. Slavov*, V. Valkov**, R. Batchvarova, S. Atanassova, M. Alexandrova & A. Atanassov

AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria

Received 10 March 2004; accepted 8 December 2004

Key words: tobacco, broomrape, chlorsulfuron, genetic transformation

Abstract
Broomrape (Orobanche ramosa L.) is the most important parasitic plant that infests tobacco (Nicotiana
tabacum L.). Chemical treatment of the soil is not effective and crop rotation is not acceptable to solve
this problem because of the long viability period of Orobanche seeds in the soil. Application of systemic
herbicides in the field with herbicide resistant tobacco could be a successful tool for broomrape control.
Several tobacco cultivars were transformed with a mutant ahas3R gene for resistance to the herbicide
chlorsulfuron (GleanÒ, DuPont). Transformed plants were selfed and the segregation of resistance was
followed in the next generation. The efficiency of the herbicide was demonstrated in greenhouse and field
trials. An Orobanche/tobacco growth system was used in order to prove the lethal effect of the herbicide
to the attached broomrape plants.

Introduction low (Dhanapal et al., 1996). Cultivation of

Broomrapes (Orobanche spp.) are obligate
destructive holoparasitic plants infecting roots of
many economic crops (Parker, 1994). The
broomrape species Orobanche ramosa L. causes
severe damage and yield loss (up to 70%) in
tobacco (Lucas, 1975). The parasitic plant is dis-
tributed in many countries where tobacco is
grown. Orobanche seeds remain viable in soil for
a long period of time (15–20 years) and germi-
nate in the presence of the host plant.
Breeding of tobacco as a monocrop leads to
high infestation of the field with the parasitic
seeds (Lolas, 1994), which makes control of the
parasite extremely difficult. Several methods of
control including crop rotation, and chemical
and biological control have been investigated,
but the efficiency of most measures is relatively
*Author for correspondence
E-mail: sbslavov@abi.bg
**Present address: CNR-IGV, Institute of Plant Genetics,
Research Division Portici, Via Universita 133, 80055 Portici,
Italy
tobacco cultivars that are resistant to broomrape
is the only sustainable control of the parasite.
Thus far, no cultivars or species that are natu-
rally resistant to broomrape have been found in
the genus Nicotiana.
Several herbicides have been tested for their
effect on broomrape control with different crops
with varying success (Lolas, 1986; Foy et al.,
1989; Garcia-Torres et al., 1994; Khalaf et al.,
1994; Kogan & Ureta, 1996; Hershenhorn
et al., 1998a). In most cases, for effective control
of the parasite the herbicides have to be applied
in high dosages and they affect the crop plants
as well (Kotonla-Syka & Eleftherohorinos,
1991). There are several examples of observed
phytotoxicity in tobacco, when herbicides are
used for control of broomrape (Raju, 1996).
Even low doses of herbicides applied on tobacco
plants may decrease the yield and change the
leaf quality (Lolas, 1997).
Despite their effect on the host plant, sulfo-
nylurea and imidazolinone herbicides are rela-
tively effective when used against broomrape
274
(Joel et al., 1995). They are systemic herbicides
that are not metabolized by the plants. They pass
through the host plant reaching the roots and
kill the parasite in a very early stage of its devel-
opment (tubercule phase). Hence, obtaining
sulfonylurea and imidazolinone resistant tobacco
cultivars could be useful in applying those herbi-
cides as effective broomrape control.
Genetic engineering for herbicide tolerance
has been based on inserting genes to express pro-
teins which confer herbicide tolerance by target
site modification, target site overproduction or
metabolic inactivation of the herbicide (Hinchee
et al., 1993). Target site modification has been
used in developing tolerance to sulfonylureas,
imidazolinones, glyphosate and atrazine.
The sulfonylurea herbicides inhibit acetolac-
tate synthase (ALS – EC 4.1.3.18) or acetohydr-
oxy acid synthase (AHAS), the first enzymes in
the biosynthetic pathway for the branched-chain
amino acids valine, leucine and isoleucine
(Chaleff & Mauvais, 1984). Several genes for
resistance to sulfonylurea were cloned (Mazur
et al., 1987; Hattori et al., 1992). In most cases
resistance is due to a single amino acid change
within a conserved region of AHAS, which com-
prises a single site essential for the binding of the
herbicide. In this work a mutant ahas3R gene
isolated from a sulfonylurea resistant Brassica
napus cell line (Ouellet et al., 1994) was used.
In the present study, we report that transgenic
tobacco plants harboring a Brassica mutant
ahas3R gene expressed high resistance to chlor-
sulfuron, a synthetic sulfonylurea herbicide, con-
ferring efficient broomrape control.
Materials and methods
Plant material and Agrobacterium mediated
transformation
Three Bulgarian oriental tobacco cultivars ‘Khan
Tervel 39’, ‘Krumovgrad 90’ and ‘Nevrokop
1146’ were selected for transformation in order
to give resistance to the herbicide chlorsulfuron
(GleanÒ, 75% a. i., DuPont). Vector pYWMC-2H
carrying the chlorsulfuron-resistant ahas3R gene
from mutant Brassica napus under the 35S
promoter and containing a chimeric 35S-nptII-nos
gene for selection on kanamycin, was kindly pro-
vided by Dr Brian Miki from Plant Research Cen-
tre, Ottawa, Canada. The foreign gene was
transferred to Agrobacterium tumefaciens via tripa-
rental mating and introduced into tobacco plants
by Agrobacterium mediated transformation follow-
ing the leaf disc method (Horsch et al., 1985).
After co-cultivation with Agrobacterium, leaf
explants were selected on MS (Murashige & Sko-
og, 1962) medium for direct organogenesis supple-
mented with 500 mg l)1 cefotaxime and
100 mg l)1 kanamycin. Developed plantlets were
rooted on MS basal medium containing
100 mg l)1 kanamycin. The efficiency of transfor-
mation was calculated as a percent of rooted
regenerants per number of explants.
Molecular analyses
Total DNA was isolated according to Dellaporta
et al. (1983). PCR (Polymerase Chain Reaction)
was carried out under the following conditions: 1
cycle of 94°C 3 min and 30 cycles of 94°C 1 min,
62°C 1 min, 72°C 1 min. Two specific primers
for the mutant ahas3R gene were used to prove
successful transformation:
50 -ACG ATG AGT TGT CCC TGC AG-30
50 -AGA TCT CGT TCT CCC TTG CC-30
Products were analyzed on 0.8% agarose gel.
Southern hybridization was done following
standard procedures according to Sambrook et al.
(1989). DNA probes were labeled with an Amer-
shamPharmaciaBiotech multiprime labeling kit.
Tests for resistance to chlorsulfuron
Nontransformed tobacco plants were sprayed
with different concentrations (from 3.75 to
37.5 g a.i. ha)1) of the herbicide GleanÒ to estab-
lish the lethal dose for tobacco. Transformants
carrying the foreign ahas3R gene were trans-
ferred in soil and grown to maturity in the green-
house. In vivo grown transgenic plants were
treated with chlorsulfuron at the four-leaf stage
with concentrations from 37.5 to 112.5 g a.i. ha)1
(three times higher than doses applied under nor-
mal agricultural practices). Plants were sprayed
twice with 15 days between treatments. Resistant
plants were recorded two weeks after the second
treatment. They were selfed and grown for

subsequent progeny. The segregation for the
resistance to chlorsulfuron was followed in the
R1 generation of one line from cv. Krumovgrad
90 and one line of cv. Khan Tervel 39.
Broomrape control
Plants from R2 progeny were planted in pots
with O. ramosa infected soil (200 mg seeds per
1 kg soil) and in the field infested with broom-
rape seeds. In total sixty transgenic plants from
line 1 of cv. Krumovgrad 90, were grown in a
restricted field infested with Orobanche seeds.
Half of the plants were treated twice with
chlorsulfuron 37.5 g and 60 g a.i. ha)1 to inves-
tigate the efficiency of the herbicide against the
parasite. The herbicide was applied on tobacco
leaves before appearance of the parasite above
the soil. The numbers of infected and noninfect-
ed tobacco plants were recorded at the end of
the vegetation period and the results were com-
pared with those from control, nonsprayed,
transgenic plants.
A system with artificial inoculation of host
plants in vitro was performed to prove exactly
that the herbicide treatment kills the parasite
attached to the tobacco roots. Transgenic tobacco
plants of cv. Krumovgrad 90 were grown in an
artificial hydroponic system and 7 days later were
transferred in another artificial growth system
according to Lane et al. (1991). Seven days after
transferring, the roots of the plants were artifi-
cially inoculated with germinated broomrape
seeds, stimulated with GR24 (Mangnus & Zwan-
enburg, 1989). After the attachment of the para-
site, tobacco plants were treated twice with
chlorsulfuron at a low concentration
(20 g a.i. ha)1) with a difference of one week
between treatments. The development of the par-
asite was observed under the microscope two
weeks after the second treatment.
Table 1. Efficiency of Agrobacterium mediated transformation of tobacco cultivars
Cultivar Number of co-cultivated
explants
Khan Tervel 39 200
Krumovgrad 90 200
Nevrokop 1146 200
275
Results and discussion
Three tobacco cultivars were successfully trans-
formed by Agrobacterium tumefaciens strain car-
rying ahas3R gene. All the regenerants grew
vigorously on a selective medium containing
100 mg l)1 kanamycin. Efficiency of transforma-
tion was higher with leaf explants from cv. Khan
Tervel 39–20% and lower in the other two culti-
vars (Table 1).
Presence of the gene for resistance into
putative transformants was verified by PCR anal-
yses. All regenerants that were resistant to kana-
mycin gave PCR positive reactions and the
fragments with the predicted sizes for the ahas3R
and nptII gene were observed. In these transfor-
mants the mutant ahas3R gene gives the expected
product of 730 bp (base pairs). No signal was
detected in the corresponding negative control
DNA samples (Figure 1). Specificity of the PCR
product was further proved by Southern blot
analyses. Successfully transformed plants showed
a strong hybridization signal demonstrating that
the foreign ahas3R gene was integrated in the
genome of putative transgenic plants (Figure 2).
No signals were detected when DNA from con-
trol, nontransformed, plants was hybridized, con-
firming that the hybridization patterns were the
result of the integration of the transgene.
After treatment of nontransformed (control)
tobacco plants with 3.75 g a.i. ha)1 chlorsulfuron
in the greenhouse, the upper leaves of the plants
turned yellow and were stunted within 2 weeks.
Later they overcame the herbicide effect, but
grew relatively slowly. Doses of 7.5 g a.i. ha)1
and higher killed all of the treated control plants.
A five times higher dose (37.5 g a.i. ha)1) was
applied for the in vivo selection of transformants
for resistance to chlorsulfuron under greenhouse
conditions. The treated transgenic plants were
fully resistant to the applied dose of the herbicide
Number of regenerants rooted Efficiency of
on a selective MS medium transformation (%)
40 20
13 6.5
17 8.5
276
Figure 1. PCR analyses of transformed plants. All
transformants show positive signal for the mutant ahas3R
gene. From left to right: from 1 to 15 – PCR product of
transgenic plants with primers for the mutant ahas3R gene;
16 – negative control (non transgenic plant); 17 – DNA
marker k/HindIII.
Figure 2. Southern blot analyses of PCR product from
transformed tobacco plants. Hybridization probe is ahas3R
gene, isolated by restriction with NcoI and SmaI from
plasmid pYWMC-2H. (1 – negative control; from 2 to
10 – transformed tobacco plants).
(Figure 3). They were fertile and produced
sufficient and viable seeds for analysis of the
inheritance of the foreign genes in the next gener-
ation. Some overcame even the treatment with an
extremely high dose of 112.5 g a.i. ha)1 chlorsul-
furon, which showed a very high expression and
efficiency of the introduced ahas3R gene. The
percent of resistant transformed plants differs
between the cultivars used (Table 2). This is
probably due to the cultivar specificity of
tobacco/Agrobacterium interactions. Seeds from
transgenic plants were collected and subsequent
generation progeny were tested for segregation of
resistance. Transformants that survived treatment
Table 2. Resistance of transformed tobacco plants to the herbicide chlorsulfuron
Cultivar Number of treated kanamycin
resistant regenerated plants
Khan Tervel 39 20
Krumovgrad 90 8 6
Nevrokop 1146 14
Figure 3. Transgenic (right) and control nontransgenic (left)
tobacco plants of cultivar Nevrokop 1146 treated with
chlorsulfuron (GleanÒ) at concentration 37.5 g a.i. ha)1.
with the herbicide were evaluated for the typical
phenotype of the respective cultivar and one line
of cv. Krumovgrad 90 (line 1) and one of cv.
Khan Tervel 39 (line 4) were further investigated
(Table 3). The observed segregation ratio for
resistance was 5.3:1 for cultivar Krumovgrad 90-
line 1 and 5.6:1 for cultivar Khan Tervel 39-line
4 corresponding to the integration of one copy of
the gene into the plant genome.
The efficiency of chlorsulfuron for broomrape
control was investigated under field and green-
house conditions. Transgenic tobacco plants
from both cultivars were planted in pots infected
with Orobanche seeds. Half of the plants were
sprayed twice with the herbicide at the same con-
centration applied for selection. No broomrape
plant was observed in any pots with herbicide
treated plants. Some of the non-treated trans-
genic plants were heavily infested with the para-
site. At least 10 parasitic plants per infested
tobacco plant were counted. This showed that
resistance to broomrape is due to the herbicide
application and not on the transformation event.
The results obtained in the greenhouse were con-
firmed in the field experiments. Two applications
Number of plants Resistant R0 plants in %
resistant to chlorsulfuron
9 45
75
3 22
 277

Table 3. Segregation of R1 generation of transgenic plants resistant to chlorsulfuron

Cultivar/Line Number of Number of Number of Ratio resistant to v2

treated plants resistant plants susceptible plants susceptible plants

Krumovgrad 90, line 1 57 48 9 5.3:1 2.36

Khan Tervel 39, line 4 20 17 3 5.6:1 1.6

p < 0.05

of the herbicide over half of the total number of plants was observed (Figure 4). The herbicide
the plants contributed to complete broomrape kills the parasitic plants at tubercular phase, a
control. The parasite did not grow on (or affect) very early stage of development, when
the treated tobacco plants, in contrast to the penetration of host roots is established. In our
nontreated controls, which were even more infested investigation, treatment with the herbicide chlor-
than the plants in the greenhouse (Table 4). sulfuron of transgenic sulfonylurea resistant
Hershenhorn et al. (1998b) observed the effect tobacco is a very promising approach for effec-
of sulfonylurea herbicides on early development tive broomrape control. The herbicide sprayed
of Egyptian broomrape (O. aegyptiaca) on on plant leaves is translocated through the whole
tomato. We demonstrated a similar effect on plant to the root system and kills the attached
O. ramosa infected tobacco plants. After herbi-
cide treatment only a very low percentage (from
0.1 to 4%) of its active ingredient reaches the
plant roots (Kotonla-Syka & Eleftherohorinos,
1991). In our experiments, after two treatments
this presumed amount of the herbicide passing
(or transferred) through the plant was sufficient
to kill the parasite at the early stage of its devel-
opment. After a single treatment with the herbi-
cide, some of the plants were infected with
broomrape. These parasitic plants appeared on
the late stage of tobacco development and had
no significant effect on the host plants. The via-
bility of the seeds of these parasitic plants was
very low (data not shown). In this way even a
single herbicide application is effective for Figure 4. Necrosis of broomrape plants attached to the roots
of transgenic tobacco plants of cv. Krumovgrad 90 treated
broomrape control and decreases the infestation with chlorsulfuron (GleanÒ, 75% a.i.). Left –transgenic
of soil with broomrape seeds. tobacco plants not treated with chlorsulfuron. Broomrape
The effect of chlorsulfuron on broomrape was plants developed on host plant roots (indicated with arrows).
Middle and right –transgenic tobacco plants treated with
clearly demonstrated in a hydroponic system.
chlorsulfuron, two and four weeks after treatment respec-
Two weeks after applying the low dose of herbi- tively. All of the broomrape plants attached to roots are
cide, necrosis and death of the attached parasitic necrotic and dead. No parasitic plants developed.

Table 4. Number of tobacco plants infected with broomrape after treatment with the herbicide chlorsulfuron

Under greenhouse conditions Under field conditions

Total Number Number of infected Total Number of Number of infected

of the plants plants by the parasite the plants plants by the parasite

Treated 20 0 30 0
Non treated 10 4 30 19
278
broomrapes. Thus in addition to protection of
tobacco crop, cleaning of the field from O. ramo-
sa seeds is another achievement for practical
tobacco breeding.
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