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A protocol for Agrobacterium-mediated transformation in rice

Article  in  Nature Protocols · February 2006

DOI: 10.1038/nprot.2006.469 · Source: PubMed

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 PROTOCOL

A protocol for Agrobacterium-mediated transformation

in rice
Asuka Nishimura1, Ikuko Aichi2 & Makoto Matsuoka2
1Honda Research Institute Japan Co. Ltd, 2-1-4 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan. 2Bioscience and Biotechnology Center, Nagoya University, Nagoya
464-8601, Japan. Correspondence should be addressed to A.N. (asuka.nishimura@jp.honda-ri.com).

Published online 18 January 2007; doi:10.1038/nprot.2006.469

Agrobacterium-mediated transformation of rice is an important method that has been widely adopted by many laboratories. However,
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those
varieties with higher regeneration potential. Some very efficient methods have been developed for japonica varieties that enable
high-throughput functional analysis in rice; however, many elite japonica, and most indica, varieties are difficult to regenerate,
leading to low transformation efficiencies. Much effort has been devoted to improving transformation efficiency for all rice
genotypes. Here, we describe an Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in
addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in
about 2–3 months with a transformation frequency of 30–50%, both in easily cultured varieties and recalcitrant elite japonica rice.

INTRODUCTION
Agrobacterium tumefaciens-mediated transformation is a general
method for genetic modification in many plant species because it
allows efficient insertion of stable, unrearranged, single-copy sequen-
ces into plant genomes. In rice, an efficient method for Agrobacter-
ium-mediated transformation was developed more than 10 years
ago,1 and is now used as a routine protocol in many laboratories.
Establishment of a rice transformation method mediated by
Agrobacterium
In 1994, Hiei et al.1 reported the first efficient Agrobacterium-
mediated transformation method for japonica rice. These investi-
gators obtained a number of transgenic rice plants with a trans-
formation efficiency (the number of transgenic lines obtained per
co-cultivated callus) of 10–30% by using a method consisting of
seven steps requiring about 4 months. Two critical points for
successful transformation were indicated: the use of actively divid-
ing, embryonic callus cells derived from the scutella of mature seeds
as the starting material; and addition of a phenolic compound,
acetosyringone, in the co-cultivation steps1,2. On the basis of this
method, Agrobacterium-mediated transformation methods have
been developed with minor modifications for many rice varieties,
not only japonica3–5 but also indica6–8 and tropical japonica9,10.
Here, we refer to this method and the modified methods as
‘‘conventional’’ transformation methods. Experience has also
demonstrated that transformation efficiencies depend on the
culture response of each variety. Therefore, optimization of tissue
culture conditions is necessary for varieties that are more difficult
to regenerate1,2. Here, we refer to varieties that can be cultured
easily as ‘‘high-regeneration’’ varieties, those that are recalcitrant to
cell culture are referred to as ‘‘low-regeneration’’ varieties.
Development of a rapid transformation method for japonica rice
To improve transformation efficiencies in rice, Toki established a
rapid transformation procedure by modifying the method of
Hiei et al.3 In short, two culture conditions were modified: the
media composition for growing calli, and the light conditions for
all culture stages except during the co-culture period. By using a
culture system that accelerated the growth of calli, the time required
from callus induction to regeneration was reduced to only 2
months and could be carried out in five or six steps. As this method
was so simple, special techniques were unnecessary. This optimized
protocol has greatly facilitated routine transformation in japonica
rice; however, this method is based on a culture system optimized
for japonica rice, especially for varieties with higher regeneration
potential. Such rapid transformation has not yet been reported for
varieties of indica, tropical japonica or japonica genotypes with
poor regeneration potential.
Improvement of transformation efficiency for large-scale
transformation of japonica rice
Establishment of an efficient rice transformation method mediated
by Agrobacterium and completion of the rice genome sequence has
enabled rice to become a model organism for the plant sciences.
Many useful tools for functional analyses have been developed
using the transformation method, such as activation or silencing
and disruption of targeted/random gene(s). In fact, for functional
analyses covering the entire rice genome, it is essential to generate a
large number of transgenic plants. So far, such transgenic rice
populations have been produced by many investigators using the
conventional transformation methods with 10–50% transforma-
tion efficiency11–14. To improve the efficiency appropriate for
production of large populations of transgenic rice, Sallaud et al.15
developed a modified transformation method of japonica rice,
which permitted the generation of transgenic rice with high
transformation efficiency (75–95%). Their method relies on the
careful choice of the target callus source and a modification of the
co-culture and selection procedure, which consists of nine or ten
steps in total. Although the transformation efficiency is very high,
special techniques for selection of calli and long periods of time
(4 or 5 months) are required for this protocol.
Modified methods for culturing recalcitrant rice varieties
Transformation techniques currently in use still present significant
obstacles for the manipulation of rice because the tissue culture

2796 | VOL.1 NO.6 | 2006 | NATURE PROTOCOLS


response is genotype-dependent. In fact, the Agrobacterium-
mediated transformation frequency is quite low for many elite
rice varieties used in food production, including high-quality grain
and high-yielding cultivars. To break through this low transforma-
tion frequency barrier, additional modifications to the conventional
methods have been developed.
(1) Japonica varieties: Japonica varieties have relatively high
applicability for tissue culture, for example ‘‘Nipponbare’’ and
‘‘Taichung 65’’ show vigorous cell proliferation and high regenera-
tion, traits that facilitate transformation by conventional methods.
In contrast, many elite japonica varieties respond poorly to con-
ventional culture using mature seeds on MS16 or N6 medium (see
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
ref. 16 or 17) medium making it difficult to obtain transgenic rice
plants. Nishimura et al.18 have recently identified a key gene
controlling regeneration frequency and have developed an alter-
native transformation method for the elite japonica variety, ‘‘Koshi-
hikari.’’ The low-regeneration frequency of ‘‘Koshihikari’’ is due to
the low activity of ferredoxin-nitrite reductase (NiR), an enzyme
that catalyzes the reduction of nitrite to ammonium leading to the
accumulation of toxic nitrite in culture media. To test directly this
hypothesis, an NiR gene encoding high enzymatic activity was
introduced into ‘‘Koshihikari’’ and the regeneration ability of this
variety improved. Using the NiR gene encoding high enzymatic
activity as a selectable marker, Nishimura et al. succeeded in
transforming a foreign gene into the elite japonica, ‘‘Koshihikari,’’
by the conventional method with a 35–45% transformation fre-
quency. It is not necessary to use selective agents such as kanamycin
or hygromycin in a selection system using the NiR gene because
‘‘Koshihikari’’ carrying its native low NiR activity seldom regener-
ates under conventional culture conditions. Unfortunately, NiR
MATERIALS
REAGENTS
. Mature dry rice seeds m CRITICAL For japonica varieties, the regeneration
frequency of the target variety is key to this transformation method
(see REAGENT SETUP).
. Agrobacterium strain(s), EHA101 (ref. 26), EHA105 (ref. 27) harboring the
gene of interest and an appropriate selectable marker gene, such as
hygromycin resistance (hpt) gene28 or a high-activity NiR gene (GenBank
accession no. NM_001049543) for plant selection in a binary vector
m CRITICAL We usually use the 35S cauliflower mosaic virus (CaMV)
promoter for hpt expression28 and the NiR promoter or rice Actin1
promoter29 (GenBank accession no. S44221) for NiR selection18 (the effect
of NiR and Actin1 promoter for the expression is similar at least in the
‘‘Koshihikari’’ variety)
. Ethanol (KANTO CHEMICAL, http://www.kanto.co.jp, cat. no. 14033-00)
70% (vol/vol) in distilled water
. 1.5% (wt/vol) Sodium hypochlorite (Wako, http://www.wako-chem.co.jp,
cat. no. 197-02206) in sterile distilled water, prepared before use
. Sterilized distilled water
. CHU (N6) Basal salt mixture (Sigma-Aldrich, cat. no. C1416)
. Glycine (Wako, cat. no. 077-00735)
. Nicotinic acid (Wako, cat. no. 142-01232)
. Pyridoxine hydrochloride (Wako, cat. no. 163-05402)
. Thiamine hydrochloride (Wako, cat. no. 201-00852)
. Myo-inositol (Sigma-Aldrich, cat. no. I5125)
. Casamino acid, DAIGO (Wako, cat. no. 392-00655)
. Proline (Wako, cat. no. 165-04605)
. 2,4-Dichlorophenoxyacetic acid (2,4-D; Wako, cat. no. 040-18532)
. Sucrose (Wako, cat. no. 193-09545)
. Gellan gum (Wako, cat. no. 075-03075)
. Dipotassium hydrogenphosphate (KANTO CHEMICAL, cat. no.
32378-00)
PROTOCOL
gene-based improvements of regeneration frequency will not be
applicable to the major indica rice varieties because they already
have high NiR activity (see REAGENT SETUP), and consequently
their low-regeneration frequency will be due to other causes.
(2) Indica varieties: Most indica rice varieties show a low rate of
callus growth or low-regeneration frequencies in conventional
culture using mature seeds on MS16 or N6 medium (see ref. 16
or 17) medium. Some successes have been reported in Agrobacter-
ium-mediated transformations using immature indica embryos as
the starting materials7,19. In comparison to mature seeds, preparing
immature embryos is difficult and much more laborious. There are
also a few reports describing Agrobacterium-mediated transforma-
tion using mature indica seeds, but successes are limited to very
specific varieties6,8. Recently, some improved methods were
reported for Agrobacterium-mediated transformation using mature
elite indica seeds20,21; however, these procedures take a long time
(5–7 months) to obtain transgenic plants because of prolonged
culture selections. Consequently, there is still much opportunity to
make additional improvements to the indica rice transformation
procedure. Further innovations such as in planta transformation of
germinating rice seeds22, and the use of genes controlling regenera-
tion frequency in indica rice varieties as described above could be
important factors for improving indica rice transformation.
Here, we describe a transformation protocol routinely used in
our laboratories18,23–25 for japonica varieties (e.g., high-regenera-
tion varieties, ‘‘Nipponbare’’ and ‘‘Taichung 65,’’ and the low-
regeneration variety, ‘‘Koshihikari’’) and limited indica varieties
with high-regeneration ability (e.g., ‘‘Kasalath’’). The method is a
modified version of that of Toki, which can be used with antibiotic
or NiR selection.
. Sodium dihydrogenphosphate dihydrate (Wako, cat. no. 192-02815)
. Ammonium chloride (Wako, cat. no. 017-02995)
. Magnesium sulfate heptahydrate (KANTO CHEMICAL, cat. no. 25034-00)
. Potassium chloride (KANTO CHEMICAL, cat. no. 32326-01)
. Calcium chloride dihydrate (Wako, cat. no. 038-12775)
. Iron (II) sulfate heptahydrate (Wako, cat. no. 094-01082)
. Glucose (Wako, cat. no. 041-00595)
. Agar for bacterial culture medium (Wako, cat. no. 010-08725)
. Kanamycin sulfate (Wako, cat. no. 113-00343)
. Hygromycin B (Wako, cat. no. 085-06153)
. Manganese (II) sulfate pentahydrate (KANTO CHEMICAL,
cat. no. 25079-30)
. Boric acid (KANTO CHEMICAL, cat. no. 04232-00)
. Zinc sulfate heptahydrate (KANTO CHEMICAL, cat. no. 48056-30)
! CAUTION Irritating to eyes and skin. Harmful if inhaled and ingested.
Handle it with suitable protection.
. Disodium molybdate (VI) dihydrate (Wako, cat. no. 196-02472)
. Copper (II) sulfate pentahydrate (KANTO CHEMICAL, cat. no. 07516-30)
! CAUTION Irritating to eyes and skin. Harmful if inhaled and ingested.
Handle it with suitable protection.
. Cobalt (II) chloride hexahydrate (Wako, cat. no. 036-03682)
. Potassium iodide (KANTO CHEMICAL, cat. no. 32351-20)
. Ethylenediamine-N,N,N¢,N¢-tetraacetic acid, iron (III) sodium salt, trihydrate
(Wako, cat. no. 345-01245)
. L-Arginine (Wako, cat. no. 015-04613)
. L-Glutamine (KANTO CHEMICAL, cat. no. 17025-30)
. L-Aspartatic acid (KANTO CHEMICAL, cat. no. 01453-30)
. Acetosyringone (Sigma-Aldrich, cat. no. D134406) m CRITICAL
Acetosyringone is essential for optimizing the Agrobacterium infection
process.
. Dimethyl sulfoxide (DMSO; Wako, cat. no. 043-07216)
NATURE PROTOCOLS | VOL.1 NO.6 | 2006 | 2797
 PROTOCOL
. Carbenicillin sodium salt (Wako, cat. no.
034-18953) m CRITICAL Carbenicillin is used for a b c
eliminating Agrobacterium after the infection
step. Claforan (Cefotaxime sodium salt, Wako,
cat. no. 030-16113) can also be used at a
concentration of 250–500 mg l
1 as an
alternative reagent to eliminate Agrobacterium.
. MS plant salt mixture (NIHON
PHARMACEUTICAL, http://www.nihon-pharm.
co.jp, cat. no. 392-00591) ! CAUTION Hazardous
ingredients, zinc sulfate heptahydrate and copper
(II) sulfate pentahydrate are present in this
mixture. These chemicals are irritating to eyes Figure 1 | Regeneration test of three rice varieties. (a) A japonica variety, ‘‘Nipponbare,’’ that has a high-
and skin, harmful if inhaled or ingested and
regeneration frequency. (b) A japonica variety, ‘‘Koshihikari,’’ that has a low-regeneration frequency.
should be handled with suitable protection.
. 1-Naphthylacetic acid (Wako, cat. no. 148-00092) (c) An indica variety, ‘‘Kasalath,’’ that exhibits a high-regeneration frequency.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

. Kinetin (Wako, cat. no. 116-00333)

. Sorbitol (Wako, cat. no. 198-03755)
. Hydrochloric acid (KANTO CHEMICAL, cat. no. 18078-00) ! CAUTION
Toxic by inhalation. Causes severe burns. Harmful if ingested. Reacts with
most metals to form flammable hydrogen gas. Handle it with suitable
protection. Use exhaust ventilation to keep airborne concentrations below
exposure limits. Use only with adequate ventilation.
. Sodium hydroxide (KANTO CHEMICAL, cat. no. 37184-00) ! CAUTION
Causes severe burns. Causes severe respiratory irritation if inhaled. Ingestion
can result in serious delayed effects. Handle with suitable protection. This
reagent is deliquescent and readily absorbs water and carbon dioxide from air.
May form flammable hydrogen gas with aluminum, tin and zinc in moist air.
EQUIPMENT
. Rice husker (FUJIWARA, http://www.fujiwara-sc.co.jp, cat. no. 08101701)
. Laminar flow hood (SANYO, http://www.sanyo-biomedical.jp/bio/jikken/
index.html, cat. no. MCV-B161S)
. Shaker for seeds sterilization (BIO CRAFT, http://www.bio-craft.co.jp/
seihinanni12.html, cat. no. BC-730)
. Autoclave (TOMY, http://bio.tomys.co.jp/product_08-02.html, cat. no. ES-215) 1,0003 AA-1 Dissolve 1g manganese (II) sulfate pentahydrate, 300 mg boric
. Incubator for Agrobacterium preculture and co-culture (YAMATO, http://
www.yamato-net.co.jp/, cat. no. IJ100)
. Growth chamber: continuous light (SANYO, cat. no. MLR-350H)
. Sterile plastic Petri plates: 90  15 mm (BIO-BIK, http://www.bio-bik.co.jp,
cat. no. I-90)
. Sterile plastic Petri plates: 90  20 mm (BIO-BIK, cat. no. I-90-20)
. Forceps m CRITICAL Sterilization is required
. Syringe filter for water (MILLIPORE, cat. no. SLGS033SS)
. Syringe filter for DMSO (PALL, http://www.pall.com/default.asp, cat. no. 4433) 1,0003 AA-4 Dissolve 4g ethylenediamine-N,N,N¢,N¢-tetraacetic acid, iron
. Disposable sterilized syringe (TERUMO, http://www.terumo.co.jp/company/ (III) sodium salt, trihydrate in 90 ml distilled water; adjust the volume to 100 ml
products/index.html, cat. no. SS-30ESZ)
. Micropore surgical tapes (3M, cat. no. 1530-0)
. Sterile plastic tubes: 50 ml (NUNC, http://www.nalgenunc.co.jp, cat. no. 373687) distilled water; adjust the volume to 100 ml with distilled water. Store at 4 1C.
. Disposable sterilized pipette (Eikenkizai, http://www.eikenkizai.co.jp/,
cat. no. CD2000)
. Paper towels (CRECIA, http://www.pro-crecia.jp/frame001.html,
cat. no. 61000) m CRITICAL Sterilization is required.
. Stainless-steel sieve (tea strainer): see Figure 2b m CRITICAL Sterilization is
required.
. Filter paper, 70 mm (ADVANTEC, cat. no. 00021070) m CRITICAL Sterilization 1,0003 Car Dissolve carbenicillin at a concentration of 250 mg ml
1 in
is required.
. Microspatula
. Plant culture box (SUMITOMO BAKELITE, http://www.sumibe.co.jp/sumilon/ 1,0003
tecnopot.html, cat. no. MS-94000) m CRITICAL Sterilization is required.
REAGENT SETUP
Testing regeneration frequency Before attempting transformation, confirm
the regeneration frequency (the number of regenerated shoots per seed) of the
variety of interest using the protocol described below, omitting the transfor-
mation steps (Steps 4–16) and selective reagents. Choose a selection system
based on the regeneration frequency of the strain of interest. We usually use
hygromycin selection for varieties with regeneration frequencies of more than
20% (Fig. 1a,c) and the NiR selection system for japonica varieties with
regeneration frequencies lower than 20% (Fig. 1b).
Measuring NiR activity Should the variety of interest show low regeneration
frequency we recommend measuring the NiR activity to confirm the correlation
of low regeneration with low NiR activity (see Box 1). For japonica, NiR
selection should be used for varieties with low-regeneration and low NiR
activity. It is not clear whether NiR selection is useful in indica because we have
not seen low-regeneration or low NiR activity in the varieties we have tested
(e.g., ‘‘IR24,’’ ‘‘IR28,’’ ‘‘IR29,’’ ‘‘IR36,’’ ‘‘IR64’’ and ‘‘Habataki’’).
1003 N6-vitamins Dissolve 100 mg glycine, 25 mg nicotinic acid, 25 mg
pyridoxine hydrochloride and 50 mg thiamine hydrochloride in 450 ml distilled
water and adjust the volume to 500 ml with distilled water. Store at 4 1C.
1003 2,4-D Dissolve 100 mg 2,4-D in 1 ml of 1 N sodium hydroxide solution
and adjust the volume to 500 ml with distilled water. Store at 4 1C.
203 AB buffer Dissolve 60 g dipotassium hydrogenphosphate and 20 g
sodium dihydrogenphosphate dihydrate in 800 ml distilled water; Adjust the
pH to 7.2 and adjust the volume to 1 liter with distilled water and autoclave.
Store at room temperature (20–25 1C).
203 AB salts Dissolve 20g ammonium chloride, 6g magnesium sulfate
heptahydrate, 3g potassium chloride, 240 mg calcium chloride dihydrate and
50 mg iron (II) sulfate heptahydrate in 800 ml distilled water; adjust the volume
to 1 liter with distilled water and autoclave. Store at room temperature.
acid, 200 mg zinc sulfate heptahydrate, 25 mg disodium molybdate (VI)
dihydrate, 2.5 mg copper (II) sulfate pentahydrate, 2.5 mg cobalt (II) chloride
hexahydrate and 75 mg potassium iodide in 90 ml distilled water; adjust the
volume to 100 ml with distilled water. Store at 4 1C.
1,0003 AA-2 Dissolve 15g calcium chloride dihydrate in 90 ml distilled water;
adjust the volume to 100 ml with distilled water. Store at 4 1C.
1,0003 AA-3 Dissolve 25g magnesium sulfate heptahydrate in 90 ml distilled
water; adjust the volume to 100 ml with distilled water. Store at 4 1C.
with distilled water. Store in the dark at 4 1C.
1,0003 AA-5 Dissolve 15g sodium dihydrogenphosphate dihydrate in 90 ml
2003 AA-6 Dissolve 20 mg nicotinic acid, 20 mg pyridoxine hydrochloride,
200 mg thiamine hydrochloride and 2g myo-inositol in 90 ml distilled water and
adjust the volume to 100 ml with distilled water. Store at 4 1C.
1003 AA-Sol Dissolve 5.3g L-arginine and 225 mg glycine in 250 ml distilled
water and adjust the volume to 300 ml with distilled water. Store at 4 1C.
1,0003 AS Dissolve acetosyringone at a concentration of 10 mg ml
1 in
DMSO, filter sterilize. Store at
20 1C.
distilled water, filter sterilize. Store at
20 1C.
Hyg Dissolve hygromycin B at a concentration of 25–50 mg ml
1 in
distilled water and filter sterilize. Store at
20 1C.
1,0003 Km Dissolve kanamycin sulfate at a concentration of 25–50 mg ml
1
in distilled water and filter sterilize. Store at
20 1C.
1003 MS-vitamins Dissolve 250 mg nicotinic acid, 500 mg pyridoxine
hydrochloride, 500 mg thiamine hydrochloride and 100 mg glycine in 450 ml
distilled water and adjust the volume to 500 ml with distilled water. Store at 4 1C.
1,0003 NAA Dissolve 40 mg 1-naphthylacetic acid in 3 ml of 0.1 N sodium
hydroxide solution and adjust the volume to 200 ml with distilled water. Store at 4 1C.
503 kinetin Dissolve 50 mg kinetin in 20 ml 0.1 N hydrochloric acid and
adjust the volume to 500 ml with distilled water. Store at 4 1C.
N6D medium (for callus induction) Mix and dissolve 3.98g CHU (N6) basal
salt mixture3, 10 ml of 100 N6-vitamin, 0.1g myo-inositol (not contained in
the Toki’s method3), 0.3g casamino acid, 2.878g proline, 10 ml of 100 2,4-D
and 30g sucrose in 800 ml distilled water. Adjust the pH to 5.8 and add 3g gellan

2798 | VOL.1 NO.6 | 2006 | NATURE PROTOCOLS

 PROTOCOL

BOX 1 | MEASURING NIR ACTIVITY.

NiR activity is assayed by the reduction of NO2
in the assay mixture30. If the low-regeneration variety of interest shows low NiR activity similar
to Koshihikari (we usually observe an NiR activity in Koshihikari lower than 0.5 mmol NO2
reduced per mg protein per hour18), you should try to
use the NiR selection system for the transformation.
1. Prepare supernatant from the callus homogenate by standard methods.
2. Determine protein content of the supernatant by using the Bradford method32.
3. Mix 50 ml of the supernatant and 850 ml of assay mixture (50 mM Tris–HCl pH 7.5, 0.5 mM NaNO2, 1 mM methyl viologen).
4. Add 100 ml 0.12 M Na2S2O4 dissolved in 0.2 M NaHCO3 and incubate at 30 1C for 1 h.
5. Vortex until the light blue color of methyl viologen disappears completely.
6. Add 100 ml Zn(CH3COO)2 and centrifuge at 10,000g for 10 min at room temperature.
7. Determine the NO2
content by a colorimetric31 or fluorescence method33.
8. The NiR activity is expressed as reduced NO2
per mg protein per hour.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

9. Compare the activity with that of Koshishikari.

gum (2g in the Toki’s method3). Adjust the volume to 1 liter with distilled water
and autoclave. Cool the medium to about 50 1C and pour into 25 sterile plastic
Petri plates (90  20 mm) in a laminar flow hood, solidify and dry3.
AB medium (for Agrobacterium culture) Dissolve 5g glucose and 15g agar
in 800 ml distilled water and adjust the volume to 900 ml with distilled water4.
After autoclaving, add 50 ml of 20 AB buffer, 50 ml of 20 AB salts and the
appropriate antibiotics (add 1,000 Km at a concentration of 25–50 mg l
1, and
if necessary, add 1,000 Hyg at a concentration of 25–50 mg l
1). Pour the
medium into sterile plastic Petri plates (90  15 mm) (ref. 3). m CRITICAL
Confirm the appropriate antibiotics for your construct and Agrobacterium strain.
AAM medium (for infection) Mix and dissolve the following reagents in
800 ml distilled water: 1 ml each of 1,000 AA-1 to AA-5, 5 ml of 200 AA-6,
10 ml of 100 AA-Sol, 0.5g casamino acids, 68.5g sucrose, 36g glucose, 0.9g
L-glutamine, 0.3g L-aspartic acid and 3g potassium chloride4. Adjust the pH to
5.2 and the volume to 1 liter with distilled water and autoclave1.
2N6-AS medium (for co-cultivation) Mix and dissolve 3.98g CHU (N6) basal
salt mixture, 10 ml of 100 N6-vitamins, 0.1g myo-inositol, 0.3g casamino acid,
10 ml of 100 2,4-D, 30g sucrose and 10g glucose in 800 ml distilled water4.
Adjust the pH to 5.2 and add 3g gellan gum (2g in the method of Hiei et al.1);
adjust the volume to 1 liter with distilled water and autoclave. Cool the medium
to about 50 1C and add 1 ml of 1,000 AS. Pour the medium into 25 sterile
plastic Petri plates (90  20 mm) in a laminar flow hood, solidify and dry1.
N6D-S medium (for selection) Prepare 1 liter N6D medium and add
2 ml of 1,000 Car before pouring into plates. For NiR selection, it is not
necessary to add any selective reagents, but for hygromycin selection, add
1,000 Hyg at a concentration of 25–50 mg l
1 before pouring into plates3.
m CRITICAL Confirm the appropriate selection medium for your construct
and rice varieties. Using the hygromycin resistance gene (hpt) driven by the
35S CaMV promoter, we observed similar results with ‘‘Nipponbare’’ and
‘‘Taichung 65’’ when using a range of hygromycin concentrations from
25 to 50 mg l
1. If other rice varieties or other promoters are used for hpt
expression, the appropriate concentration of hygromycin in the selection
medium should be established.
MS-NK medium (for regeneration) Mix and dissolve 4.6g MS plant salt
mixture, 10 ml of 100 MS-Vitamins, 0.1g myo-inositol, 2g casamino acid, 1 ml
of 1,000 NAA (0.1 ml in the Toki’s method3), 20 ml of 50 kinetin, 30g
sucrose and 30g sorbitol in 800 ml distilled water. Adjust the pH to 5.8 and add
3g gellan gum (4g in the Toki’s method3). Bring the volume to 1 liter with
distilled water and autoclave. Cool the medium to about 50 1C and add 1 ml of
1,000 Car. Pour the medium into 25 sterile plastic Petri plates (90  20 mm) in
a laminar flow hood, solidify and dry. For regeneration from hygromycin-
selected calli, add 1,000 Hyg at a concentration of 25–50 mg l
1 before pouring
into plates, but for NiR selection, it is not necessary to add any additional
reagents. m CRITICAL Confirm the appropriate regeneration medium for your
construct and rice varieties. For regenerating hygromycin-selected plants using
the hygromycin resistance gene (hpt) driven by the 35S CaMV promoter, we
observed similar results using 25–50 mg l
1 hygromycin with cultivars ‘‘Nip-
ponbare’’ and ‘‘Taichung 65.’’ If other rice varieties or other promoters are used
for hpt expression, the appropriate concentration of hygromycin required in the
selection medium should be established.
MS-HF medium (for plant growth) Mix and dissolve 4.6g MS plant salt
mixture, 10 ml of 100 MS-vitamins, 0.1g myo-inositol (not contained in the
Toki’s method3) and 30g sucrose in 800 ml distilled water. Adjust the pH to 5.8
and add 3g gellan gum (2g in the Toki’s method3); adjust the volume to 1 liter
with distilled water and autoclave. Cool the medium to about 50 1C and pour
into plant culture boxes in a laminar flow hood, solidify and dry. For growing
hygromycin-selected plants, add 1,000 Hyg at a concentration of 25–50 mg l
1
before pouring into boxes, but for NiR selection, it is not necessary to add any
additional reagents. m CRITICAL Confirm the appropriate plant growth medium
for your construct and rice varieties.

PROCEDURE

Induction of calli TIMING 3–4 weeks
1| Dehusk mature healthy seeds with a rice husker and surface-sterilize in 70% ethanol for 30 s, then in 1.5% sodium
hypochlorite for 30 min with vigorous shaking, followed by five rinses in sterile water.
2| Place ten sterilized seeds per plate on the surface of N6D medium and seal the plate with surgical tape.
3| Incubate the seeds on the medium at 29.5 1C at about 3.5 klux for 3–4 weeks. Calli are formed from the scutella after
3–4 weeks in culture (Fig. 2a).
m CRITICAL STEP Seeds are occasionally contaminated at this step. Check the culture plates often and transfer uncontaminated
seeds onto new plates immediately in such cases.
? TROUBLESHOOTING
Preparation of calli and Agrobacterium TIMING 3 days 
4| Collect the actively growing calli (yellowish white, relatively dry and about 1–3 mm in diameter; see Fig. 2a) from Step 3
and subculture on fresh N6D medium at 29.5 1C at about 3.5 klux for 3 days.
m CRITICAL STEP We routinely remove and discard seeds that have developed seedlings or brown calli from culture plates before
collecting the calli for subculture. This callus selection is a key point for efficient transformation.

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 PROTOCOL

a b c d

e
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Figure 2 | Procedure for producing transgenic rice plants. (a) Embryogenic calli generated from the scutella of
mature seeds. Arrowheads show actively growing calli appropriate for Agrobacterium inoculation. (b) Inoculation of
calli with A. tumefaciens. (c) Proliferation of hygromycin-resistant calli on N6D-S medium 3 weeks after transfer.
Left calli are resistant to hygromycin and right calli are sensitive. (d) Shoot regeneration from calli resistant to
hygromycin on MS-NK medium 3 weeks after transfer. (e) Rooting and growth of transgenic rice plants.

5| Streak a single colony of Agrobacterium harboring the gene of interest in a binary vector on AB medium, supplemented
with the appropriate antibiotics for binary vector selection. Incubate at 28 1C for 3 days.

Infection of callus with Agrobacterium TIMING 3 days

6| Collect Agrobacterium cells on a microspatula and suspend in 30 ml AAM medium containing 30 ml of 1,000 AS
(OD600¼0.05–0.1). Gently but completely resuspend the cells with a disposable sterilized pipette.
m CRITICAL STEP Make sure no cells aggregate in the suspension medium. Thorough resuspension prevents local excess growth of
Agrobacterium during co-cultivation.
7| Collect subcultured calli (from Step 4) in sterilized stainless-steel sieves held over plastic Petri plates (90  20 mm).

8| Pour the Agrobacterium cell suspension prepared in Step 6 into the plate from Step 7. Immerse calli in the Agrobacterium
solution for 90 s, briefly shaking the sieve (Fig. 2b).
9| Blot the sieve containing calli dry on sterilized paper towels to remove excess Agrobacterium.
m CRITICAL STEP This process is important to prevent excess Agrobacterium growth that can result in damage to calli.

10| Drip 1 ml AAM containing 30 ml 1,000 AS onto a sheet of filter paper on 2N6-AS medium and transfer calli onto the filter
paper.
11| Wrap the plate with aluminum foil and incubate at 28 1C in the dark for 48–60 h.
m CRITICAL STEP Agrobacterium growth is rarely observed after co-cultivation. Excess growth of Agrobacterium causes necrosis of
calli and reduces the transformation efficiency.
? TROUBLESHOOTING

Washing of infected calli, selection and regeneration of transgenic plants TIMING 6–8 weeks 
12| Collect the infected calli in a 50 ml sterile tube and wash with sterile water by shaking the tube. Repeat washes until the
rinse water is clear.

13| Rinse the calli with sterile water, adding 1,000 Car at a concentration of 500 mg l
1 to kill Agrobacterium cells. Pour the
callus suspension through sterilized stainless-steel sieves held over plastic Petri plates (90  20 mm).
14| Place stainless-steel sieves containing calli onto paper towels and dry well.
m CRITICAL STEP It is important to remove excess water from calli to ensure active culture growth.

15| Transfer calli onto N6D-S medium. Up to 16 calli can be placed on one plate.
m CRITICAL STEP The composition of N6D-S medium used at this step differs depending on whether hygromycin selection or NiR
selection is used. Ensure that the appropriate selection medium is used for each construct and rice variety.
16| Seal plates with surgical tape. Incubate at 29.5 1C at about 3.5 klux for 3–4 weeks (Fig. 2c).
m CRITICAL STEP Agrobacterium cells can occasionally re-grow at this step. Check the culture plates often and re-wash
contaminated calli or transfer uncontaminated calli onto new plates immediately in such cases.
? TROUBLESHOOTING

2800 | VOL.1 NO.6 | 2006 | NATURE PROTOCOLS

 PROTOCOL

17| Transfer the growing calli to MS-NK medium for shoot regeneration.
m CRITICAL STEP The composition of MS-NK medium used at this step differs depending on whether hygromycin selection or NiR
selection is used. Ensure that the appropriate selection medium is used for each construct and rice variety. Furthermore, if calli are
placed on regeneration medium plates at high density, the regeneration efficiency of some varieties, especially those with rapidly
growing calli, will decline. Callus growth rates of the variety of interest should be tested or routinely plated at a low density. We
routinely place up to seven calli populations originating from a single co-cultured callus on one plate for ‘‘Nipponbare,’’ ‘‘Taichung
65’’ and ‘‘Kasalath’’ that have calli with high growth rates. In contrast, if calli are transferred at high density on regeneration medium
for NiR selection, there can be a high incidence of non-transformed calli producing shoots because nearby transformed calli with
higher NiR activity can prevent the accumulation of toxic nitrite. Thus, we recommend one callus population per regeneration plate
in this case. Six-well culture plates are especially convenient for this purpose.
18| Seal plates with surgical tape. Incubate at 29.5 1C at about 4.5 klux for 3–4 weeks (Fig. 2d).
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

? TROUBLESHOOTING
19| Transfer shoots of 3–4 cm in length to MS-HF medium in plant boxes (Fig. 2e).
m CRITICAL STEP The composition of MS-HF medium used at this step differs depending on whether hygromycin selection or NiR
selection is used. Ensure that the appropriate selection medium is used for each construct and rice variety.
? TROUBLESHOOTING
20| Transplant rooted plantlets to soil pots, with one plantlet per pot.

 TIMING
The above procedure will take approximately 10–13 weeks to complete.
Induction of calli: 3–4 weeks
Preculture of calli and Agrobacterium: 3 days
Inoculation of Agrobacterium and co-cultivation: 3 days
Selection of transformed cells: 3–4 weeks
Regeneration of transgenic plants: 3–4 weeks

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
TABLE 1 | Troubleshooting table.
Step Problem Possible reasons Solutions
3 Contamination of many seeds with Inadequate sterilization of seeds Use higher concentrations of sodium
saprophytes hypochlorite and/or extend the sterilization
time, alternatively, use a biocide, PPM
(plant preservative mixture; NACALAI TESQUE,
http://www.nacalai.co.jp, cat. no. 26062-84)
Seed contamination owing to Dry seeds well as soon as harvested, store dry
inappropriate storage at 4 1C
11 Excess growth of Agrobacterium High concentration of Agrobacterium in Decrease concentration of Agrobacterium in
infection AAM medium infection AAM medium
16 Re-growth of Agrobacterium on Inadequate washing of calli Wash until the rinse solution is
the surface of many calli completely clear
Inappropriate carbenicillin concentration Check the carbenicillin concentration
No callus proliferation Incorrect construct Confirm the construct by sequencing
Failure of Agrobacterium infection Confirm that Agrobacterium cells contain
the plasmid
Inappropriate selection reagents Check selection reagents and concentrations
18 No or little shoot regeneration Incorrect medium composition or contamina- Check the composition of regeneration medium
tion with inhibitory substance(s) and all reagents used in previous steps.
Regeneration process of rice is very sensitive
to culture conditions
19 All plants dead Regeneration of non-transformed plants Confirm the introduction of desired DNA into
rice genome before Step 17 or 19

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ANTICIPATED RESULTS
This transformation method should routinely yield 30–50 transformants for scutella-derived calli from 30 to 50 mature seeds
with a transformation efficiency of 30–50%. However, the efficiency will vary depending on the regeneration ability of
individual rice varieties, as described above.
ACKNOWLEDGMENTS We thank Sakimi Watanabe and Sachiko Nomura for their
technical assistance in establishing this protocol.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
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