J. Appl. Genet. 44(4), 2003, pp.

449-458

Agrobacterium-mediated transient GUS gene expression

in buffel grass (Cenchrus ciliaris L.)

Shweta BATRA1, Suresh KUMAR 2

1
Department of Microbiology, School of Life Sciences, Bundelkhand University, Jhansi - 284 128,
India
2
Biotechnology Section, Division of Crop Improvement, Indian Grassland and Fodder Research
Institute, Jhansi - 284 003, India

Abstract. The study was conducted to standardize a protocol for Agrobacterium-medi-

ated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli,
produced from one-year-old mature seeds of buffel grass, were used as target cells for
Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring
pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic
calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli
with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM
acetosyringone for 3 days was found to be optimum for maximum transformation effi-
ciency. Presence of acetosyringone during co-cultivation was found to be necessary for
transformation. Transient GUS (b-glucuronidase) gene expression was used to monitor
T-DNA delivery into the target cells. Significant genotypic variations in response to
transformation were observed among the tested genotypes. A very high frequency
(63.3%) of GUS gene expression was obtained following Agrobacterium-mediated
gene transfer into embryogenic calli. The standardized protocol would be useful for
Agrobacterium-mediated genetic transformation of buffel grass with genes of agro-
nomic importance.

Key words: Agrobacterium-mediated transformation, Cenchrus ciliaris, forage quality,

GUS gene.

Introduction

Buffel grass (Cenchrus ciliaris L.), also known as African foxtail and anjan grass,
is one of the most important forage crops of India. It is an apomictic, perennial,
polymorphic and polyploid warm-season forage grass suited to pastures, range-
Received: December 9, 2002. Revised: April 29, 2003. Accepted: September 22, 2003.
Correspondence: S. KUMAR, National Research Centre on Plant Biotechnology, I.A.R.I., New Delhi
110 012, India, e-mail: suresh_kumar33@rediffmail.com
450 S. Batra, S. Kumar

lands, tropics and sub-tropical regions of Australia, South Africa, and India
(CAVAYE 1991, HIGNIGHT et al. 1991). It can withstand heavy grazing and is ex-
tremely fire resistant (STARKER 1934). It is highly drought tolerant and well
adapted to arid and semi-arid areas. Though mainly used as a pasture grass for ru-
minants, it may also be used for hay or silage making. It is highly palatable to all
kinds of grazing animals, but the substantially high lignin content (3-5%) reduces
its digestibility (MINSON, BRAY 1986). Since it is an apomict, its genetic improve-
ment through conventional breeding methods is difficult, time consuming
and presently restricted to the selection of elite lines from natural variants.
The clone approach in apomictic species imposes a restriction on the genetic vari-
ability within a variety. A reduced genetic basis of any widely used crop repre-
sents a threat, giving pests and pathogens an opportunity of becoming virulent
with a single mutation. Once having become virulent, the pathogen is capable
of attacking all plants since all are equally susceptible. As expected, such an event
has occurred in the form of an epidemic of buffel grass blight caused by
Magnaporthe grisea (RODRIGUEZ et al. 1999). On the other hand, apomixis may
facilitate varietal improvement by genetic transformation, since no further breed-
ing is required to fix the transferred character (VIELLE CALZADA et al. 1996).
Hence, genetic transformation would be a powerful tool for improvement of this
pasture grass. An efficient protocol for genetic transformation of this grass, for ex-
ample genetic manipulation of lignification to increase forage digestibility, would
be very useful (CHERNEY et al. 1991).
Preliminary studies of transient expression of a reporter gene following parti-
cle bombardment have been reported in buffel grass (ROSS et al. 1995, BHAT et al.
2001). Plant transformation mediated by A. tumefaciens has become the most
commonly used method for introduction of foreign genes into plant cells and sub-
sequent regeneration of transgenic plants. Agrobacterium-mediated gene transfer
into monocotyledonous plants was not possible until recently, when reproducible
and efficient methods were established for many monocotyledons, like rice (HIEI
et al.1994, CHENG et al.1998), wheat (CHENG et al. 1997) and maize (ISHIDA et al.
1996). Transformation has now successfully been reported in several other spe-
cies, like barley (TINGAY et al. 1997), sugarcane (ARENCIBIA et al. 1998,
ENRIQUEZ-OBREGON et al. 1998), banana (MAY et al. 1995), Asparagus
officinalis (DELBREIL et al. 1993), Agapanthus praecox (SUZUKI et al. 2001).
The ease with which gene transfers are effected, higher predictability of gene
transfers and incorporation of fewer copies of the transgene, which is an important
factor in transgene preservation through subsequent generations, are some
of the advantages of the Agrobacterium-mediated transformation method over
the others. We report here, for the first time, an optimized protocol for
Agrobacterium-mediated transient GUS gene expression in embryogenic calli of
buffel grass. Once the transformation protocol is standardized, we envisage ge-
netic manipulation of buffel grass with genes of agronomic importance. Further,
there are prospects for genetic manipulation of the lignin biosynthesis pathway to
 Agrobacterium-mediated transformation of buffel grass 451

increase forage digestibility and identification/isolation of gene(s) responsible for

apomixis through T-DNA mutagenesis.

Material and methods

Plant material and callus induction

Three genotypes of buffel grass, viz. IG-3108, IG-9757 and IG-97101, obtained
from the Central Research Farm, I.G.F.R.I., Jhansi, were used in the study.
One-year-old seeds of the selected genotypes were used for in vitro callus induc-
tion. Mature seeds were dehusked manually, surface-sterilized in 70% ethanol for
1 minute, followed by 0.1% mercuric chloride with a drop of Tween-20 for 5 min-
utes, and then rinsed several times with sterile distilled water. Seeds were then
placed on an MS medium (MURASHIGE, SKOOG 1962) solidified by 0.7% agar,
supplemented with 13.5 mM 2,4-D and 2.2 mM BAP (CLM medium), and incu-
bated at 25 2C in the dark. Embryogenic calli obtained 21 days after incubation,
were subcultured on a freshly prepared CLM medium at 2-3-week intervals for
3-4 passages. In vitro culture of embryogenic calli was performed as reported ear-
lier by the authors (BATRA, KUMAR 2002).

Agrobacterium strain and plasmid vectors

A. tumefaciens strain LBA4404, harbouring binary vector pCAMBIA-1301 or

pCAMBIA-2301 (ROBERTS et al. 1997), was used in the transformation experi-
ments. The constructs contain the uidA gene (interrupted by catalase intron), en-
coding the enzyme b-glucuronidase, under the control of the CaMV35S promoter
and the nos terminator. Plasmid pCAMBIA-1301 carries the hygromycin
phosphotransferase (hpt) gene, while pCAMBIA-2301 has the neomycin
phosphotransferase II (npt II) gene, both of them driven by the CaMV35S pro-
moter and terminated by the CaMV35S polyA sequence. Bacterial cultures were
grown in the YEM medium (VINCENT 1970) supplemented with kanamycin
monosulphate (Sigma Chemical Co., USA) 100 mg L1 and rifampicin 10 mg L1
for 2-3 days at 28 2C (150 rpm).

Co-culture and co-cultivation of embryogenic calli

The transformation protocol described by HIEI et al. (1994) was followed with mi-
nor modifications. Cultured bacterial cells were pelleted by centrifugation at
5000 rpm for 5 minutes and resuspended in the YEM medium at a density of
3-5 109 cells mL1, considering the optical density of bacterial culture at 600 nm.
White compact embryogenic calli (5-6 mm in diameter) were used as explants
in co-cultivation experiments. The selected calli were co-cultured with
452 S. Batra, S. Kumar

Agrobacterium for varying periods of time (10-60 minutes) by immerging them

into the bacterial suspension. The excessive bacterial cells on the surface of calli
were blot dried with sterile filter paper. The agroinfected calli were then trans-
ferred onto the CLM medium with or without 0.1 mM acetosyringone
(3, 5-dimethoxy-4-hydroxy-acetophenone; Aldrich Chemical Co.) for co-culti-
vation in the dark at 28 2C for 1-4 days. After co-cultivation, the calli were
washed thoroughly with cefotaxime (Melford Laboratories Ltd., England)
250 mg L1 in sterile distilled water and transferred onto the CLM medium con-
taining hygromycin B (Sigma Chemical Co., USA) or kanamycin 50 mg L1
along with cefotaxime 250 mg L1 and incubated in the dark at 25 2C for
21 days.

GUS histochemical assay

The GUS staining solution was prepared by taking 1 mM X-Gluc (5-bromo,

4-chloro, 3-indolyl-b-D-glucuronide: Cyclohexylammonium salt (X-GlcA);
Melford Laboratories Ltd., England), from a 20 mM stock made in
dimethylformamide, 100 mM sodium dihydrogen phosphate dihydrate,
and 0.05% Tween-20 (GALLAGHER 1992). The pH of the solution was adjusted
to 7.0 with 1N NaOH. The histochemical GUS assay was performed as described
by JEFFERSON (1987) to monitor GUS gene expression in putative transgenic
calli. The GUS assay was carried out on 25% of the randomly selected calli imme-
diately after co-cultivation as well as 21 days after co-cultivation. Calli were im-
mersed in GUS assay solution and incubated at 37C overnight. GUS gene
expression was observed and photographed by using a Nikon-SMZ-2T binocular
stereozoom microscope.

Statistical analysis

All the experiments were replicated three times. Experimental data were analysed
using the analysis of variance. Duncans multiple range test was used to find sig-
nificant differences among treatment means at 5% level of significance.

Results

Various parameters were analysed for optimizing Agrobacterium-mediated

T-DNA delivery into embryogenic calli of buffel grass. Transient GUS gene ex-
pression frequency, ranging from 62.3% to 86.7%, was obtained in the tested ge-
notypes (Figure 2). Co-cultivation of white, compact, embryogenic calli induced
from mature seeds of C. ciliaris cv. IG-97101 with A. tumefaciens
(LBA4404/pCAMBIA-1301) gave the maximum transient GUS gene expression
frequency (86.7%). Gene transfer frequency was monitored through transient
 Figure 1. Agrobacterium-mediated transient GUS gene expression in buffel grass
(a) Transient GUS gene expression in embryogenic calli (C. ciliaris cv. IG-9757) immediately after
co-cultivation with A. tumefaciens strain LBA4404/pCAMBIA-1301 (T). No GUS gene expression
in control (C). (b) GUS gene expression in putative transgenic calli (C. ciliaris cv. IG-97101) growing on
CLM medium containing hygromycin (50 mg L1), 21 days after co-cultivation.

pCAMBIA-1301 pCAMBIA-2301
100
Transient GUS expression frequency (%)

90 86.7

80 76.7 76.7
73.38
70
63.3 62.3
60

50

40

30

20

10

0
IG-3108 IG-9757 IG-97101

Genotype

Figure 2. Variable response to Agrobacterium-mediated genetic transformation

of embryogenic calli of three genotypes of buffel grass using two different constructs
454 S. Batra, S. Kumar

Table 1. Effect of duration of co-culture on A. tumefaciens (LBA4404/pCAMBIA-1301) -

mediated transformation of embryogenic calli of buffel grass
Co-culture of embryogenic calli in bacterial suspension (min.)
Genotype
10 20 30 40 50 60
a a b b b
IG-3108 63.3 66.8 76.7 73.3 73.3 76.7 b
a b b b b
IG-9757 63.3 73.3 76.7 73.3 76.7 73.3 b
IG-97101 73.3 a 76.7 a 86.7 b 86.7 b 83.3 b 83.3 b
* Co-culture was followed by three days of co-cultivation. Figures are means of three replications showing per-
centage of calli with GUS positive response. In a row, means followed by the same letter are not significantly dif-
ferent at 5% level of significance.

Table 2. Effect of duration of co-cultivation of agroinfected calli of buffel grass

on a medium with 0.1 mM acetosyringone
Co-cultivation of agroinfected calli with acetosyringone (days)
Genotype
1 2 3 4
a b c
IG-3108 0.0 30.0 76.7 73.3 c
IG-9757 0.0 a 36.7 b 73.3 c 73.3 c
IG-97101 0.0 a 33.3 b 86.7 c 83.3 c
* Co-cultivation was preceded by 30 minutes of co-culture. Figures are means of three replications showing per-
centage of calli with GUS positive response. In a row, means followed by the same letter are not significantly dif-
ferent at 5% level of significance.

GUS gene expression in co-cultivated embryogenic calli and antibiotic selection.

GUS gene expression was not observed in control, nonco-cultivated calli
(Figure 1a). Significant differences in GUS gene expression frequency were ob-
served between IG 97101 IG 9757 and IG 97101 IG 3108, while the difference
was insignificant for IG 9757 IG 3108 genotypes (Figure 2). Varying periods
of co-culture (leading to attachment of bacterial cells on the surface of calli)
and co-cultivation (leading to T-DNA delivery into the cells) of embryogenic calli
with Agrobacterium, were effective for gene transfer and expression in buffel
grass. Co-culturing for 30 minutes and co-cultivation for 3 days, was found to be
optimum. Longer periods of co-culture and co-cultivation did not significantly
change the gene transfer frequency (Tables 1 and 2). Moreover, co-cultivation
of embryogenic calli with Agrobacterium for 3 days on the CLM medium contain-
ing 0.1 mM acetosyringone, was found to be the most effective for the gene trans-
fer from bacteria to plant cells. No GUS gene expression was observed in calli
co-cultivated on CLM medium without acetosyringone. GUS gene expression
was observed 21 days after co-cultivation in 57 out of 90 (63.3%) randomly se-
lected putative transgenic calli of genotype IG 97101 proliferating on CLM me-
dium containing 50 mg L1 hygromycin and 250 mg L1 cefotaxime (Figure 1b).
 Agrobacterium-mediated transformation of buffel grass 455

Discussion

In the present study, we report an optimized protocol for Agrobacterium-mediated

gene delivery into buffel grass. Although transient GUS gene expression has al-
ready been reported in Cenchrus ciliaris following particle bombardment (ROSS
et al. 1995, BHAT et al. 2001), to the best of our knowledge, this is the first report
to testify the possibility of Agrobacterium-mediated genetic transformation of
buffel grass.
We opted for one-year-old mature seeds of certain genotypes that initially re-
sponded better to in vitro culture, as a more readily available source of tissues for
embryogenic callus production. We studied the effects of various factors,
viz. genotypic difference, duration of co-culture and co-cultivation and presence
of acetosyringone during co-cultivation, on Agrobacterium-mediated gene trans-
fer frequency in buffel grass. Significant differences in genotypic responses for
in vitro culture (BATRA, KUMAR 2002) and transformation efficiency were ob-
served between IG-97101 and IG-9757 or IG-3108. The effects of co-culture
and co-cultivation periods on transformation efficiency have been reported
in a number of plant species. Several minutes of co-culture and 2-3 days of
co-cultivation have generally been recommended for best results (HIEI et al. 1994,
HOLFORD et al. 1992, MUTHUKUMAR et al. 1996). We obtained the optimum re-
sults with 30 minutes of co-culture and 3 days of co-cultivation. Prolonged
co-culture and co-cultivation did not increase gene transfer frequency. While lon-
ger co-culture periods have been reported to provide a better chance for
Agrobacterium to attach to plant cells for infection (POTRYKUS 1990, DONG,
MCHUGHEN 1991), plant tissue damage caused by agroinfection has also been re-
ported (PU, GOODMAN 1992, HANSEN 2000, ZHAO et al. 2000).
Among various factors analysed, addition of acetosyringone to the co-culti-
vation medium was found to be critical for the gene transfer. No GUS gene expres-
sion was observed in the calli co-cultivated on the CLM medium without
acetosyringone. Therefore, addition of acetosyringone seems necessary for
the gene transfer. Presence of acetosyringone during co-cultivation has been re-
ported to be essential for certain monocotyledons, like Oryza sativa (HIEI et al.
1994), Zea mays (ISHIDA et al. 1996), Agapanthus praecox (SUZUKI et al. (2001),
and having general enhancement in transformation efficiency even for certain di-
cotyledons, like Arabidopsis (SHEIKHOLESLAM, WEEKS 1987). Phenolic com-
pounds, like acetosyringone, are needed in sufficient amounts for activation of vir
genes to initiate T-DNA transfer (STACHEL et al. 1985). These compounds are re-
ported not to be produced by many of the monocotyledons (USAMI et al. 1987).
Contrary to this, acetosyringone has not proved to be necessary for
Agrobacterium-mediated transformation of certain monocotyledons, like
Triticum aestivum (CHENG et al. 1997), Hordeum vulgare (TINGAY et al. 1997)
and Asparagus officinalis (DELBREIL et al. 1993). Therefore our results suggest
that the embryogenic calli of buffel grass used in the present study do not produce
456 S. Batra, S. Kumar

sufficient amounts of phenolic compounds for activating vir genes of

Agrobacterium, hence the external supply of acetosyringone during co-cultivation
is essential for the gene transfer to take place.
Thus, we have optimized the protocol for transient GUS gene expression
in the embryogenic calli of buffel grass following the A. tumefaciens-mediated
gene transfer method. The study has provided a useful basis for standardization of
the transformation protocol for genetic manipulation of this pasture grass with de-
sired/modified genes, such as genes for forage quality improvement. Although
GUS gene expression was obtained in 63.3% of the randomly selected putative
transgenic calli growing on the CLM medium containing hygromycin, further at-
tempts will be needed to regenerate transgenic plants and to study stable inheri-
tance of the transgene in T0 plants and their progenies.
Acknowledgements. The generous gift of plasmid constructs from CAMBIA,
Australia, is gratefully acknowledged. We are grateful to Dr. Malathi LAKSMI-
KUMARAN, Tata Energy Research Institute, New Delhi, and Dr. M.V. RAJAM, Uni-
versity of Delhi-South Campus, New Delhi, for providing the constructs
in Agrobacterium. Thanks are also due to Dr. P. S. PATHAK, Director and Dr.
R.N. CHOUBEY, Head of the Crop Improvement Division, I.G.F.R.I., Jhansi, for pro-
viding necessary facilities and encouragement.

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