Gene Transfer to Rice Mediated by Agrobacterium tumefaciens:

Transient Expression of sgfp in Rice Calluses of indica cv. Fatmawati and

japonica cv. Nipponbare
Transfer gen pada padi dengan menggunakan mediasi Agrobacterium tumefaciens:
Ekspresi transient dari sgfp dalam kalus padi spesies indica cv. Fatmawati dan japonica cv.
Nipponbare

Tomohiko Yoshida1 and Nono Carsono2

Key words : Agrobacterium, genetic transformation, reporter gene, rice calluses,

synthetic green fluorescent protein, transient expression.
Kata kunci : Agrobacterium, transformasi genetic, gen pelacak, kalus padi, synthetic
green fluorescent protein, ekspresi pada waktu yang terbatas.

transgene were obtained from

Abstract Nipponbare and Fatmawati,
respectively.
Transient expression of synthetic green
fluorescent protein (sgfp) mediated by
Agrobacterium is rapid and useful Sari
approach for visual monitoring the
Ekspresi pada waktu yang terbatas dari
genetic transformation event in
synthetic green fluorescent protein (sgfp)
transformed cells/tissues of examined
yang dimediasi dengan Agrobacterium
genotype. The significant differences in
merupakan cara cepat dan berguna
transient expression of two genotypes
untuk memonitor secara visual peristiwa
(indica cv. Fatmawati and japonica cv.
transformasi genetik pada berbagai sel
Nipponbare) were found with regard to
atau jaringan suatu genotipe yang diuji.
the osmotic treatment (0.4 M mannitol),
Perbedaan yang nyata dalam hal ekspresi
solid-subcultured callus, and 10 min. air
dari sgfp pada kedua genotipe (indica
drying. While, no significant differences
kultivar Fatmawati dan japonica
in sgfp expression were observed in two
Nipponbare) ditemukan dalam
genotypes on without air-drying and 5
kaitannya dengan perlakuan osmotik
min. air-drying of calluses prior
(0,4 mannitol), perlakuan kalus yang
immersed in Agrobacterium suspension.
disubkultur dengan media padat dan
Surprisingly, the sgfp expression could
kalus yang dikering-anginkan selama 10
not be detected in suspension-
menit. Secara mengejutkan ekspresi sgfp
subcultured callus of both cultivars. The
tidak dapat dideteksi pada kalus yang
highest sgfp expression was achieved in
disubkultur dengan media cair (suspensi).
Nipponbare callus treated with 10 min.
Ekspresi sgfp yang tertinggi diperoleh
air drying. The level of green fluorescent
pada kalus kultivar Nipponbare yang
spots was higher in Nipponbare than
dikering-anginkan selama 10 menit. Level
that in Fatmawati, however, plants
ekspresi sgfp lebih tinggi pada
regenerated from Fatmawati were
Nipponbare dibandingkan dengan
considerably comparable with those of
Fatmawati, akan tetapi plantlet yang
Nipponbare. Seventeen and 16 putative
diregenerasikan dari Fatmawati relatif
transgenic rice plants expressing sgfp
sama dengan yang diperoleh dari
Nipponbare. Sebanyak 17 dan 16
1) Professor at Lab of Crop Science, Faculty of tanaman padi transgenik yang
Agriculture, Utsunomiya University, Japan. mengekspresikan sgfp diperoleh dari
2) Lecturer at Lab. of Plant Breeding, Faculty of masing-masing kultivar Nipponbare dan
Agriculture, Padjadjaran University, Campus Fatmawati.
Jatinangor 40600.

8 Zuriat, Vol. 19, No. 1, Januari-Juni 2008

 Introduction occurs almost immediately after gene
transfer, it does not require the
Genetic transformation mediated by regeneration of whole plants, and it
Agrobacterium tumefaciens, a soil plant occurs at much higher frequency than
pathogenic bacterium, is one of the stable integration (Altpeter et al., 2005).
methods for the introduction of the Thus, in this paper, the author applied a
foreign genes into plant cells. This is sgfp gene as reporter and non-
due to Agrobacterium has the destructive markers to visualize the
exceptional ability to transfer a genetic transformation event mediated
particular DNA segment (T-DNA) of by Agrobacterium in transformed callus
the tumor-inducing (Ti) plasmid into the cells of cvs. Fatmawati and Nipponbare.
nucleus of infected cells where it is then These two genotypes were selected
stably integrated into the host genome based on their high capacity in both
(Binns and Thomashow, 1988). The callus induction and plant regeneration
initial studies on T-DNA transfer (Carsono and Yoshida, 2006 a,b).
process to plant cells demonstrate three
important facts for the practical use of
this process for plant transformation. Materials and Methods
Firstly, the tumor formation is a
A. tumefaciens strain EHA105
transformation process of plant cells
harboring plasmid pIG001 was used.
resulted from transfer and integration of
Plasmid pIG001 was a binary vector
T-DNA and the subsequent expression
that contains a neomycin
of T-DNA genes. Secondly, the T-DNA
phosphotransferase (npt II) gene, a
genes are transcribed only in plant cells
hygromycin-resistance gene (hpt) and
and do not play any role during the
sgfp gene (Fig. 1). Agrobacterium was
transfer process. Thirdly, any foreign
grown in LB medium (Tabel 1) at 270C
DNA placed between the T-DNA
in a gyratory shaker for 3 days.
borders can be transferred to plant cells,
Agrobacterium cells were collected and
no matter it comes from. These well-
resuspended in NB5 liquid medium with
established facts allowed the
construction of the first vector and 200 µM acetosyringone. Six weeks old
bacterial strain systems for plant genetic calluses, induced from mature seeds of
transformation (Torisky et al., 1997). Fatmawati and Nipponbare, were placed
in bacterial suspension for 10 min. After
Agrobacterium-mediated transformation briefly draining the calluses on
has been established for many years in sterilized paper filter, calluses were then
dicotyledonous plants, and recently has transferred to NB5 solid medium with
been adapted to monocotyledonous 200 µM acetosyringone and co-
plants including rice (Hiei et al., 1994; cultivated at 270C in the dark for 3 days.
Rashid et al., 1996). In order to The co-cultivated calluses were washed
visualize the genetic transformation three times with sterile water containing
event mediated by Agrobacterium, 250 mgL-1 cefotaxime and 250 mgL-1
transient expression of reporter gene i.e. carbenicilin to kill Agrobacterium.
synthetic green fluorescent protein
(sgfp) is rapid and useful method for
this purpose. Transient expression

Gene Transfer to Rice Mediated by Agrobacterium tumefaciens 9

 RB nptII sgfp hpt LB

Pnos Tnos PUbi Tnos Tnos P35S

Figure 1. Schematic map of plasmid pIG001. hpt, hygromycin phosphotransferase

gene, conferring resistance to hygromycin B; npt II, neomycin phosphotransferase II
gene, harboring resistance to kanamycin; sgfp, synthetic green fluorescent protein
gene; PUbi, promoter of a maize Ubiquitin-1; Pnos, promoter of nos (nopaline
synthase) gene; P35S, the 35S promoter of cauliflower mosaic virus; Tnos, terminator
of nos; RB, right border; LB, left border.
Table 1. Medium used for bacterial culture, tissue culture and Agrobacterium-
mediated transformation.
Culture media Composition
Callus induction MS* medium, 3 mgL-1 2,4-D, 30 gL-1 sucrose, 2.5 gL-1
Phytagel, pH 5.8
Callus subculture suspension: NB5** medium, 3 mgL-1 2,4-D, 500 mgL-1 l-
proline, 500 mgL-1 l-glutamine, 30 gL-1 maltose, 2.5 gL-1
Phytagel, pH 5.8
solid: NB5 medium, 3 mgL-1 2,4-D, 500 mgL-1 l-proline, 500
mgL-1 l-glutamine, 30 gL-1 maltose, pH 5.8
Agrobacterium culture LB medium, 10 gL-1 glucose, 50 mgL-1 streptomycin and 50
mgL-1 hygromycin
Co-cultivation liquid: NB5 medium, 10 gL-1 glucose, 200 µM
acetosyringone, pH 5.4
solid: NB5 medium, 10 gL-1 glucose, 4 gL-1 phytagel, 200
µM acetosyringone, pH 5.4
Callus selection NB5 medium, 50 mgL-1 hygromycin, 500 mgL-1 l-proline,
500 mgL-1 l-glutamine, 250 mgL-1 carbenicillin, 250 mg L-1
cefotaxime, pH 5.8
Plant regeneration pre-regeneration (1 wk): NB5 medium, 3 mgL-1 Kinetin, 3
mg L-1 BA, 10 mgL-1 ABA, 0.3 mgL-1 IAA, 0.3 mgL-1 NAA,
500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1
casein hydrolysate, 30 gL-1 maltose, 50mgL-1 hygromycin, 8
gL-1 agarose, pH 5.9
regeneration: NB5 medium, 3 mgL-1 Kinetin, 3 mgL-1 BA,
0.3 mgL-1 IAA, 0.3 mgL-1 NAA, 500 mgL-1 l-proline, 500
mgL-1 l-glutamine, 800 mgL-1 casein hydrolysate, 30 gL-1
maltose, 50mgL-1 hygromycin, 8 gL-1 agarose, pH 5.9
Plantlet rooting Half strength of MS medium, 50mgL-1 hygromycin, 2 gL-1
Phytagel, pH 5.9
Note: *Medium MS from Murashige and Skoog, (1962); ** Medium NB5: adapted from
Chu et al. (1975) for macronutrient, and Gamborg et al. (1968) for micronutrient and
organic components

10 Zuriat, Vol. 19, No. 1, Januari-Juni 2008

The transformed calluses were then recalcitrant to Agrobacterium. The
incubated in the selection medium studies on genetic transformation of rice
containing hygromycin B 50 mgL-1, suggest that numerous factors including
cefotaxime 250 mgL-1 and carbenicilin Agrobacterium strain, vectors, plant
250 mgL-1 for 2-3 weeks. Small, good- genotype, explant, selection marker and
looking, hygromycin resistant calluses selective agent, various conditions of
were obtained, and then transferred to culture media, are of importance (Hiei
pre-regeneration medium for one wk et al., 1997). The results, here,
and subsequently transferred to fresh demonstrate that transient expression of
regeneration medium (Table 1). sgfp was significantly different in two
genotypes with regard to osmotic
Monitoring sgfp expression was
treatment (0.4 M mannitol) (Table 2).
performed according to Carsono and
Yoshida (2008). A completely Osmotic treatment, provided by 0.4 M
randomized design with three mannitol, could act to protect the target
replications, each with 10 calluses, was tissue to less extrude their protoplasm
used in this study. Transformation following Agrobacterium infection, due
frequency was evaluated on day 2 after to a plasmolysis effect (Vain et al.,
co-cultivation as the total number of 1993) and reducing cell turgidity.
green fluorescent spots (transformation Osmotic treatment for enhancement of
events) per explant. Data were then Agrobacterium-mediated transformation
analyzed statistically by analysis of is largely dependent on genotype as
variance and the differences between found in this study. It is likely both
means of two genotypes in sgfp genotypes had discrepancy of response
expressions/spots were evaluated by to osmotic treatment, in which
Duncan’s multiple range test (DMRT) Nipponbare was the most susceptible by
Agrobacterium infection since we
Results and Discussion obtained higher expression of sgfp in
japonica Nipponbare than in indica
Two days after co-cultivation of rice Fatmawati (Table 3). Indica varieties
calluses with Agrobacterium are generally recalcitrant in both tissue
suspension, green fluorescent spots culture and plant transformation, and
corresponding to transformation events are poor hosts for Agrobacterium (Hiei
were observed in both cultivars (Fig. 2a, et al. 1997). A recalcitrant of
b). These sectors were clearly monocotyledonous plants to
distinguishable from untransformed Agrobacterium-mediated transformation
parts. Around 2 to 90 sgfp-expressing may be due to inappropriate process in
cells were observed per explant on back the following one or more steps
and upper of Petri dish. However, the involved in transformation: chemotaxis
fluorescent was limited only in small Agrobacterium towards wounded plant
parts of the callus cells. It is apparent cells (Parke et al., 1987), binding of
that the sgfp gene driven by ubiquitin-1 Agrobacterium to plant cells (Douglas
promoter isolated from maize et al., 1982), inducing of vir genes by
(Christensen dan Quail, 1996) was plant signal molecules (Stachel et al.,
working well in rice callus. Fluorescent 1985; Vijayachandra et al., 1995),
was not observed in the callus of the generation of T-DNA transfer
control (immersed in water). intermediates (Veluthambi et al., 1987),
The genetic transformation mediated by transfer of T-DNA transfer
Agrobacterium has been adapted for intermediates to plant cells and
various plant species, including rice, integration of T-DNA into plant genome
which was formerly considered to be (Simpson et al., 1982).

Gene Transfer to Rice Mediated by Agrobacterium tumefaciens 11

 a b

c d

Fig. 2. The expression of sgfp in rice calluses cv. Fatmawati (a, 80X) from solid-grown-
derived callus, and Nipponbare (b, 64X) treated with 10 min. air drying. The
transformed callus and regenerating shoots expressing sgfp cv. Nipponbare (d),
and leaves of control (cv. Nipponbare, non-transformant). Bars on a) and b) are
50µm, while c) and d) are 0.2 mm.
Tabel 2. Analysis of variance for parameters tested on sgfp expression of two
cultivars.

Source of df Osmoti Solid-grown No-air 5 min. air 10 min. air

variation c (0.4 derived callus drying drying drying
M)
Genotype 1 421.4** 3212.0** 3.8ns 156.8 ns 1041.7*
Error 58 56.5 225.0 178.1 129.8 234.6
Total 59

Data show mean square values. ns: nonsignificant, *: significant at p = 0.05, **:
significant at p = 0.01, respectively.

12 Zuriat, Vol. 19, No. 1, Januari-Juni 2008

Table 3. Comparison for cultivar on sgfp expression.
No Tested parameters Fatmawati Nipponbare
1. Osmotic medium (0.4 M 0.83 ± 2.33 A 6.13 ± 5.68 B
mannitol)
2. Solid-subcultured callus 1.50 ± 3.01 A 16.13 ± 9.31 B
3. Liquid-subcultured callus 0.00 ± 0.00 A 0.00 ± 0.00 A
4. No-air drying 5.07 ± 8.93 A 4.57 ± 6.24 A
5. 5 min. air drying 0.23 ± 0.74 A 3.47 ± 9.27 A
6. 10 min. air drying 0.17 ± 0.43 A 8.50 ± 2.50 B
Data show means with standard error of the means. The differences were analyzed by
DMRT.
In both cultivars, the sgfp expression Agrobacterium suspension, but not
was significantly influenced by callus significantly different when rice callus
that was subcultured in solid-agar media cells were treated with no air drying and
(Table 2). Such expression was lower in five min. air drying (Table 2 and Table
Fatmawati than in Nipponbare (Table 3). Ten min. air drying apparently
3), indicating Nipponbare is more improved the sgfp expression in
susceptible by Agrobacterium infection Nipponbare, but not in Fatmawati
than Fatmawati. But, surprisingly, no (Table 3), suggesting the difference in
sgfp spots were detected in the cultivar’s response to prolonged air-
suspension-subcultured callus of both drying treatment. Urushibara et al.
cultivars (Table 3), suggesting the (2001) reported that 10 to 15 min. air-
suspension-subcultured callus is drying treatment of Nipponbare
inefficient for Agrobacterium-mediated suspension culture enhanced the
transformation. This may be explained transformation efficiency 10-fold or
by the fact that, in many more as compared to the control without
monocotyledonous, the cells (at wound air drying (solid-subcultured callus).
site) tend to be lignified or sclerified
In this experiment, overgrowth
(Hiei et al., 1997), which may inhibit
of Agrobacterium was frequently found
the Agrobacterium to directly infect the
in calluses of Nipponbare. At the end of
rice cells. Another explanation is that
the course of Agrobacterium-mediated
inclusion of 200 µM acetosyringone
transformation, the author obtained
was not sufficient for inducing vir genes
around 58 calluses resistant to
for suspension-subcultured cells.
hygromycin in Nipponbare, while only
However, transgenic rice plants had
7 calluses in Fatmawati. However,
been obtained via Agrobacterium using
plants regenerated from Fatmawati were
a callus suspension-subcultured cells
considerably comparable with those of
(Urushibara et al., 2001; Deng et al.,
Nipponbare. Seventeen and 16
2003), without clearly stated the amount
transgenic rice plants expressing sgfp
of acetosyringone used in their
transgene (as screened by a fluorescent
transformation experiments.
microscope shown in Fig. 2c) were
A significant factor that enhances obtained from Nipponbare and
transformation of crop species is Fatmawati, respectively.
desiccation of explants prior to or after
Agrobacterium infection. Ten min. air
drying was found to be significant in
Concluding Remarks
obtaining the sgfp expression of two Gene transfer mediated by
cultivars prior immersed in Agrobacterium offers a valuable tool for

Gene Transfer to Rice Mediated by Agrobacterium tumefaciens 13

plant improvement programs, especially calluses derived from mature seed of
for application in developing country. five Indonesia rice genotypes. Plant
Transient sgfp gene expression will be Prod Sci 9: 71-77.
of greatly helpful in testing the
Carsono, N. and T. Yoshida. 2008.
expression vector, optimizing some
Transient expression of green
parameters for Agrobacterium-mediated
fluorescent protein in rice calluses:
transformation and selecting the suitable
optimization of parameters for
genotype for Agrobacterium-mediated
Helios gene gun device. Plant Prod
transformation, thus, in turn, it will
Sci 11: 88-95.
improve the efficiency of genetic
transformation mediated by Christensen, A.H. and P.H. Quail. 1996.
Agrobacterium, which is to date still far Ubiquitin promoter-based vectors for
from efficient for some rice genotypes. high level expression of selectable
and/or screenable marker genes in
monocotyledonous plants.
Acknowledgment Transgenic Res 5: 213-218.
We greatly acknowledge Dr. Hiroyuki Chu, C.C., C.C. Wang, C.S. Sun, C.
Anzai, Gene Research Center, Ibaraki Hsu, K.C. Yin, C.Y. Chu and F.Y.
University, Japan for kindly providing Bi. 1975. Establishment of an
Agrobacterium tumefaciens strain efficient medium for anther culture
EHA105 harboring plasmid pIG001. of rice through comparative
experiments on the nitrogen sources.
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Gene Transfer to Rice Mediated by Agrobacterium tumefaciens 15