RESEARCH ARTICLES

Agrobacterium-mediated transformation of
mature embryos of Triticum aestivum and
Triticum durum
Debasis Patnaik, Dalia Vishnudasan and Paramjit Khurana*
Centre for Plant Molecular Biology, Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110 021, India

One of the key points in these protocols has been the use
Plant regeneration studies in cereals have been under-
taken in immature embryos, scutellum and also in imma- of actively dividing cells/tissues such as immature em-
ture inflorescence tissue. The wheat mature embryos bryos and immature embryo-derived calluses that were
can also be employed for callusing and regeneration, further co-cultivated with Agrobacterium in the presence
as they are available throughout the year and have of potent inducers of virulence genes8. Mooney and cowork-
presently been employed for transformation studies. ers9 were the first to demonstrate the wound-independent
An efficient and reproducible method for Agrobacte- in vitro attachment of Agrobacterium to wheat embryos.
rium-mediated transformation of mature embryos of Subsequently, Chen and Dale10 reported a higher frequency
hexaploid bread wheat (Triticum aestivum) and tetraploid of infection by incubation of exposed apical meristems of
pasta wheat (Triticum durum) is reported. Presence of dry wheat seeds with Agrobacterium. Previous work from
acetosyringone at 200 µM concentration in the bacte- this laboratory also reported the transient expression of gus
rial growth medium, inoculation medium and co-
gene in meristematic leaf bases, calluses, mature seeds
cultivation medium was essential for achieving a 1.5–
2.0 fold increase in transient expression of the introduced and mesocotyl punctured seedlings following co-cultivation
gus gene. Successful generation of T. aestivum and T. with different strains and vectors of Agrobacterium tume-
durum transgenic plants at a transformation frequency faciens11. Stable Agrobacterium-mediated transformation of
ranging from 1.28 to 1.77% has been achieved follow- wheat and transmission of the transgenes to subsequent
ing 2–3 days co-cultivation using mature embryos and generations have now been reported by many workers7,12–15.
also mature embryo-derived calluses with binary Nonetheless, the widescale application of this methodol-
Agrobacterium strain LBA4404 (pBI101 :: Act1) and ogy in diverse genotypes is still restricted.
LBA4404 (p35SGUSINT) respectively. Paromomycin The present study thus focuses on the use of excised mature
and phosphinothricin served as effective selection agents embryos and mature embryo-derived calluses as primary
as they did not adversely affect plantlet regeneration. explants for Agrobacterium-mediated transformation of
Successful integration as well as inheritance of the
bread wheat (Triticum aestivum) and also the macaroni
transgene was confirmed by Southern hybridization
and PCR amplification in T0 as well as T1 generation. wheat (Triticum durum). The optimized protocol was subse-
Optimization of this method facilitated the introduction quently used for the introduction of potato proteinase in-
of bar gene as a selectable marker conferring herbi- hibitor (pin2) gene into T. aestivum and T. durum, the
cide resistance as well as potato proteinase inhibitor successful use of which had conferred insect resistance in
gene (pin2) for insect resistance into wheat. japonica rice16. The use of proteinase inhibitors for genetic
engineering of insect resistance in wheat has also been re-
Keywords: Agrobacterium, embryos, transformation, ported17. Here we report Agrobacterium-mediated transforma-
Triticum aestivum, Triticum durum, wheat. tion of T. aestivum and T. durum using mature embryos as
recepient explants.
GENETIC transformation of crop plants by Agrobacte-
rium-mediated co-cultivation is an efficient and cost- Materials and methods
effective method for gene delivery. Monocotyledonous
plants, including important cereals were earlier thought to be Plant materials and culture conditions
recalcitrant to Agrobacterium-mediated gene transfer1,2, but
the scenario has changed in the last few years. Consistent Seeds of T. aestivum cvs HD2329, CPAN1676, PBW343 and
efforts by researchers on cereal crop plants have resulted T. durum cvs PDW215, PDW233 and WH896 were ob-
in the development of protocols for efficient gene delivery tained from IARI, New Delhi as well as the Directorate of
via Agrobacterium into rice3,4, maize5, barley6 and wheat7. Wheat Research, Karnal, Haryana, India. Seeds were ini-
tially washed with a liquid detergent (Teepol, Reckitt &
*For correspondence. (e-mail: paramjitkhurana@hotmail.com) Coleman of India) for 1 min with running tap water, and

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Table 1. Abbreviations of various media used for wheat regeneration and transformation

Medium Composition

MSE MS medium supplemented with 200 mg/l casein hydrolysate and 100 mg/l inositol
MSE2 MSE supplemented with 2 mg/l 2,4-D
MSER MSE supplemented with 2.22 µM BA and 0.1 µM NAA
MSE2As MSE2 supplemented with 200 µM acetosyringone
MSE2Cef250 MSE2 supplemented with 250 µg/µl cefotaxime
MSE2P5 MSE supplemented with 2 mg/l 2,4-D and 5 mg/l phosphinothricin
MSE2P5Cef250 MSE-2P5 supplemented with 250 mg/l cefotaxime
MSE2Pm100 MSE supplemented with 2 mg/l 2,4-D and 100 mg/l paromomycin
MSE2Pm100Cef250 MSE-2Pm100 supplemented with 250 mg/l cefotaxime
MSERP2.5 MSER supplemented with 2.5 mg/l phosphinothricin
MSERPm50 MSER supplemented with 50 mg/l paromomycin
MS1/2P2.5 MS half supplemented with 2.5 mg/l phosphinothricin

surface-sterilized with absolute ethanol for 30 s followed Transformation

by 4% (v/v) sodium hypochlorite for 30 min. The seeds
were thoroughly washed in sterile water prior to mature The bacterial cultures were initiated by inoculating 50 µl of
embryo excision in a laminar flow hood. Mature embryos glycerol stock in 30 ml LB + medium (LB medium supple-
were excised by removing the endosperm part from the mented with 0.5% glucose) with 100 µg/ml rifampicin,
surface-sterilized caryopses with a sterile blade. 100 µg/ml kanamycin sulphate and 200 µM acetosyrin-
MS medium18 supplemented with 200 mg/l casein hy- gone. The cultures were incubated at 28°C/200 rpm. For
drolysate and 100 mg/l inositol designated as MSE19, was plant transformation, bacterial cultures with A600 (absorb-
used in this investigation. The pH of the culture medium ance at 600 nm) value of 0.5 to 1.00 were chosen. The
was adjusted to 5.8 using 1.0 M NaOH/HCl. After ad- excised mature embryos or three-week-old mature em-
justment of pH, Phytagel (0.4%) was added as the gelling bryo-derived calluses were inoculated by immersion in
agent (Sigma, USA) and autoclaved at 121°C and bacterial suspension and incubating for 1 h after which
1.08 kg/cm2 pressure for 15 min. Mature embryos along the explants were transferred to MSE2As medium. Co-
with the attached scutellum were placed on MSE2 me- cultivation was performed by incubating the petri plates
dium with the embryo axis side facing downwards, as at 28°C in dark for 2–3 days.
precocious germination of mature embryos was drastically
reduced when the embryos were placed so. The explants
Selection and regeneration of transformants
were cultured on MSE2 for three weeks in dark at
26 ± 1°C with regular subculturing at weekly intervals.
After 2–3 days of co-cultivation, the explants were washed
The different media used are listed in Table 1.
in liquid MSE2Cef250 to remove Agrobacterium and after
several washes, placed on sterile blotting sheets to remove
Bacterial strain and plasmid vectors excess moisture and transferred to MSE2Cef250 medium
supplemented with 100 mg/l paromomycin (for LBA4404
A. tumefaciens strain20 LBA4404 and the binary vectors (pBI101:Act1) and LBA4404 (p35SGUSINT)) or 5 mg/l
(p35SGUSINT, pBI101 : : Act, and pCAMBIA3301 : : pin2) phosphinothricin for LBA4404 (pCAMBIA3301 : : pin2). The
were employed for Agrobacterium-mediated transformations. mature embryo explants were cultured on MSE2Pm100Cef250
For evaluating the parameters involved in Agrobacte- or MSE2P5Cef250 for two weeks. Explants showing callus-
rium-mediated transformations, the constructs pCAM- ing on the selection medium were transferred to fresh
BIA1301, pCAMBIA2301 and pCAMBIA3301 were also MSE2Pm100Cef250 or MSE2P5Cef250 medium and kept for
used. For construction of the binary vector pBI101 : : Act1, ten days. The mature embryo-derived calluses were also
the rice Act1–5′ region was excised from the plasmid21 cultured on MSE2Pm100Cef250 or MSE2P5Cef250 for three
pDM302 as a 1.5 kb HindIII fragment and cloned in the vec- weeks with one subculture in between.
tor pBI101 (Clontech). For the construction of pCAM- For regeneration, the explants were transferred to MSER
BIA3301 :: pin2, the expression cassette of the potato medium supplemented with 2.5 mg/l phosphinothricin or
proteinase inhibitor (pin2) gene was excised as a 3.0 kb 50 mg/l paromomycin. After 10 days on MSERP2.5 or
PstI fragment from the vector pTWa 16 and cloned in MSERPm50, the explants were transferred to selection-
pCAMBIA3301 (ref. 22). The binary vector23 p35SGUSINT free regeneration medium and after another 10 days, the
was isolated from the Agrobacterium strain GV2260 and mo- regenerated plantlets were transferred to MS1/2 supple-
bilized by triparental mating24,25 into A. tumefaciens strain mented with 2.5 mg/l phosphinothricin or 50 mg/l paromo-
LBA4404. mycin for shoot elongation. The regenerated plantlets were
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transferred to transfer plugs (Sigma) and maintained for Phosphinothricin leaf-paint assay: The progeny of
nearly ten days till the root system established firmly. transgenic plants with bar gene as the selectable marker
Rooted plantlets were transferred to pots containing a was analysed by leaf-painting assay. Leaf painting was
mixture of soilrite (Kel Perlite, Bangalore, India) and garden performed as described by Lonsdale et al.30 A solution of
soil (1 : 1) and grown to maturity in growth chambers phosphinothricin (150 mg/l) and 0.1% Tween-20 was applied
(Conviron, Control Environments Limited, Winnipeg, to leaf sections, three times a week at two-day intervals.
Canada) operating at 21–18°C at 16/8 h light/dark cycle. Absence of necrotic damage as compared to controls was
The plants were supplied with a liquid medium26 recom- taken as evidence for the expression of bar transgene.
mended for growth of wheat plantlets.
DNA isolation and Southern analysis
Enzyme assay
Total genomic DNA was isolated from wheat leaves ac-
GUS assay: The gus reporter gene activity was either local- cording to Dellaporta et al.31. The probe was radio-
ized histochemically in the explants or quantitatively de- labelled using Megaprime DNA Labelling kit (Amersham
termined by spectrofluorometric method according to the International Inc. UK) and (α-32P) ATP (BRIT, Hydera-
protocol described by Jefferson et al.27. Histochemical bad, India) according to manufacturer’s specifications.
localization of GUS was carried out by incubating the tis- Hybridization was carried out for 16–24 h at 37°C with
sue samples overnight at 37°C in histochemical buffer shaking at 40 rpm. The blot was washed in sequence,
(0.1 M sodium phosphate buffer, pH 7.0; 50 mM EDTA; with the following solutions for 10 min each: (i) 50%
0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1% Triton X- formamide, 5X SSC, 0.1% SDS; (ii) 2X SSC, 0.1% SDS;
100, 1 mg/ml X-gluc (Amresco Inc., Ohio, USA)). The ex- (iii) 1X SSC, 0.1% SDS and (iv) O.5 X SSC, 0.1% SDS.
plants were washed thoroughly with 70% ethanol prior to
observations using a stereo-microscope (Nikon, SMZU).
PCR analysis
For quantitative estimation of gus activity, samples were
incubated with 300 µl of GUS assay buffer (1.0 mM MUG
PCR analysis of genomic DNA was carried out using
in GUS extraction buffer) at 37°C for 15 h and 900 µl of
200–300 ng of wheat genomic DNA employing reagents
stop buffer (0.2 M Na2CO3) added. Relative fluorescence
from MBI Fermentas (USA) in a 25 µl reaction volume
of the samples was measured in Silica quartz cuvettes,
according to manufacturer’s instructions. PCR amplifica-
employing a RF540 spectrofluorophotometer (Shimadzu,
tion was performed by initial denaturation at 94°C (5 min
Japan) at an excitation wavelength of 365 nm and emission
hold), followed by 25 cycles at 94°C (30 s), annealing
wavelength of 455 nm. The fluorometric units were converted
(30 s) and 72°C (30 s) and finally holding at 72°C (7 min)
into amount of 4-MU using a calibration curve. Specific
for extension, employing a Perkin-Elmer Gene Amp PCR
activity was expressed in terms of pmol or nmol of 4-MU
system 2400. The forward and reverse primers employed
mg protein/h. Any GUS activity observed in controls was
for the amplification of nptII gene were 5′-TCG GCT ATG
deducted to obtain the final values.
ACT GGG CAC AAC AGA-3′ (nptF) and 5′-AAG AAG
GCG ATA GAA GGC GAT GCG-3′ (nptR) respectively.
NptII dot blot assay: The nptII dot blot assay was per-
Primers used for the detection of bar gene are 5′-ACC
formed according to the protocol of Roy and Sahasrabud-
ATC GTC AAC CAC TAC ATC G-3′ (bar5) and 5′-TCT
dhe29. The blot was wrapped in cling film and exposed to
TGA AGC CCT GTG CCT C-3′ (bar3). Annealing tempe-
an X-ray film (Kodak, India) in Hypercassettes (Amer-
sham, UK) at –20°C for 2–3 days depending on the
counts. The X-ray film was developed to check for nptII
activity in the form of intensity of the dots.
Table 2. Per cent callusing and regeneration response of mature em-
bryos isolated from different varieties of T. aestivum and T. durum on
NptII functional assay: The functional assay of nptII MSE2 and MSER media respectively
gene was performed21 on wheat seedlings at the three-leaf
stage by spraying with a solution of 2% (w/v) paromo- Genotype Callusing (%) Regeneration (%)
mycin and 0.1% Tween-20. Alternatively, 1–2 cm sec- T. aestivum cultivar
tions of leaf tips were painted with paromomycin solution HD2329 92.00 ± 1.73 54.93 ± 3.03
using a cotton bud. The response was observed after seven CPAN1676 78.33 ± 2.60 68.47 ± 2.25
days of paromomycin application. Plants with a functional PBW343 58.00 ± 2.30 58.00 ± 2.30
nptII gene showed little or no damage. However, plants T. durum cultivar
lacking a functional nptII gene were identified by the PDW215 59.67 ± 2.02 58.17 ± 1.96
presence of bleached spots throughout the leaf, which PDW233 60.33 ± 1.76 67.10 ± 2.26
WH896 51.67 ± 1.45 63.83 ± 2.57
subsequently affected their survival.

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ratures for amplification of nptII and bar genes are 57 about 67–68% was observed in T. aestivum cv. CPAN1676
and 50°C respectively. PCR products were run on 1.6% and T. durum cv. PDW233 (Table 2).
agarose gel in 1X TAE along with size markers (Gen-
eRulerTM 1 kb ladder and GeneRulerTM 100 bp ladder,
Factors affecting gene delivery
MBI Fermentas).
The present investigation explores the suitability of ex-
Results cised mature embryos as well as the mature embryo de-
rived calluses towards agrobacterial infection. Excised mature
Regeneration studies in wheat have been mostly confined to embryos were chosen for Agrobacterium-mediated trans-
leaf bases, immature embryos, scutellum and to immature formation, as the regeneration response of this explant
inflorescence tissue32,33. Although mature embryos have been was found to be comparable to that of immature embryos
the explants of choice for regeneration and transforma- in culture. Mature embryos were excised aseptically from
tion experiments34–37, their use is not prevalent. In the surface-sterilized seeds and co-cultivated with Agrobac-
present investigation, mature embryos (ME) have been terium tumefaciens LBA4404 harbouring the binary vec-
used for evaluating the regeneration and transformation tors (pBI101 :: Act1 and pCAMBIA3301 :: pin2). In general,
response in three cultivars each of T. aestivum and T. durum, two significant observations were recorded. First, the co-
employing various media (Table 2). In general, all culti- cultivation duration of 2–3 days was more effective than
vars of T. aestivum and T. durum experimented with dis- others, as evidenced by the quantitative measurement of
played reasonable regeneration response on MSER, and GUS-specific activity (Table 3). Second, the T. aestivum
this has been the medium of choice for regeneration in cultivar CPAN1676 was more receptive to Agrobacte-
the present study. However, some differences in callusing rium-mediated gene transfer in comparison with T. durum
and regeneration of different cultivars were evident, cultivar PDW215, especially in the presence of vir induc-
e.g. T. aestivum cv. HD2329 displayed the highest callus- ers such as acetosyringone (Figure 1). The GUS activity
ing but lowest regeneration. In contrast, in T. durum of an individual explant (mature embryo) of T. aestivum
cv WH896 displayed poor callusing but a relatively was always observed to be more than that of T. durum follow-
higher regeneration percentage. Highest regeneration of ing co-cultivation with Agrobacterium (Figure 2).

Table 3. Gus-specific activity (nmol MU/mg protein/h) of mature embryo-derived calluses of T. aesti-
vum and T. durum varieties using various constructs

Constructs

Cultivar p35SGUSINT pCAMBIA1301 pCAMBIA2301 pCAMBIA3301

T. aestivum
HD2329 26 ± 3.0 50 ± 3.1 58 ± 5.5 44 ± 4.2
CPAN1676 46 ± 4.8 48 ± 3.6 45 ± 3.5 18 ± 3.0
PBW343 38 ± 4.0 49 ± 5.6 30 + 3.0 42 + 5.0
T. durum
PDW215 52 ± 4.3 38 ± 4.3 58 ± 5.2 31 ± 4.8
PDW233 44 ± 6.0 52 ± 7.3 69 ± 6.0 56 ± 5.8
WH896 34 ± 5.2 43 ± 4.7 60 ± 5.7 52 ± 3.8

Figure 1. GUS-specific activity of individual mature embryos after three days of co-cultivation with Agrobacterium tumefaciens LBA4404
(pBI101 :: Act1). a, T. aestivum cv. CPAN1676 and b, T. durum cv. PDW215.

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Figure 2. Agrobacterium-mediated transformation of wheat. Excised mature embryos of T. aestivum cv. CPAN1676 and T. durum cv.
PDW215 were co-cultivated with LBA4404(pBI101 :: Act1), while mature embryo-derived calluses of T. aestivum cv. HD2329 were co-
cultivated with LBA4404 (p35SGUSINT). a, Mature embryo of T. aestivum (control); b, Histochemical localization of GUS expression in
excised mature embryos of T. durum after ten days of co-cultivation; c, Histochemical gus expression in mature embryo-derived callus of
T. aestivum after three weeks on MSE2Pm100; d, e, Control and transformed explants respectively, of T. durum on selection medium
(MSERPm50Cef250). f, Transformants on selection-free half-strength MS medium; g and h. Transformants in seedling tray and pot respec-
tively, i, Fertile T. durum transgenic plant growing in pot, j, Fertile T1 transgenic of T. aestivum CPAN1676; k, GUS histochemical assay
in T. durum T1 transgenics leaf whole mounts (left – control, right – transgenic respectively); l, Result of paromomycin leaf spray on T1
progeny of T. aestivum var. CPAN1676, Control (left).

Acetosyringone was also supplemented in the bacterial observations in case of rice and wheat8,10 transformation.
growth medium, resuspension medium and co-cultivation In the presence of acetosyringone, different plasmid con-
medium as an inducer. This is consistent with the earlier structs show different preferences for different genotypes

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(Table 3). Such genotypic preferences are well docu- particularly prone to damage during the antibiotic washing
mented in the literature. The suitability of mature embryo- step, which is required for elimination of Agrobacterium.
derived calluses as explants for Agrobacterium-mediated While washing resulted in loss of competent cells, insuf-
transformation was evaluated after 2–3 days of co- ficient washing with cefotaxime usually resulted in ne-
cultivation with Agrobacterium. Preliminary experiments crosis and in non-recovery of regenerating calluses from
detected the activity of gus gene up to 45 days after co- co-cultivated explants. Thus in several experiments de-
cultivation of mature embryo-derived calluses of T. aesti- spite the calluses proliferating on the selection medium,
vum cv. HD2329 with LBA4404 (p35SGUSINT). There- no regeneration was obtained from the mature embryo-
fore, this vector–genotype combination was employed for derived calluses. Regeneration of plantlets was obtained
generation of transgenic plants. The genetic transforma- from the mature embryos co-cultivated in several inde-
tion methodologies and conditions were similar to those pendent experiments; however, data presented are from
optimized for mature embryos except that more caution two successful experiments indicating a transformation
was observed to minimize damage at the cefotaxime wash efficiency of 5.57% (16 putative transformants out of 287
step. Thus, three days of co-cultivation was found to be co-cultivated explants) with T. aestivum cv. HD2329,
ideal for successful gene transfer as evidenced by histo- calculated on the basis of paromomycin leaf spraying of
chemical localization of GUS expression and was also re- T0 transformants.
confirmed by GUS fluorometric assay of mature embryo-
derived callus of three cultivars each of T. aestivum and
T. durum using four different constructs (Table 3). PCR analysis

PCR amplification was undertaken to screen for the pres-

Selection and regeneration of transformants ence of bar gene in the transformants transformed with
LBA4404(pCAMBIA3301 :: pin2). Leaf samples were
Mature embryos of T. aestivum cv. CPAN1676 and T. du- collected from putative transformants immediately after
rum cv. PDW215 were aseptically excised from the seeds regeneration, but prior to transferring the plantlets to half-
and co-cultivated with A. tumefaciens LBA4404 (pBI101 :: strength MS medium supplemented with 2.5 mg/l phos-
Act1). After three days of co-cultivation followed by phinothricin. Plants scored as positive (Figure 3 a) on the
thorough washing, the mature embryos of T. aestivum var basis of PCR analysis (presence of an amplified product
CPAN1676 were transferred to MSE2Pm100Cef250 for callus of ~ 296 bp) also survived on MS1/2P2.5, whereas plants
induction for approximately 2–3 weeks. Callus formation scored as negative by PCR analysis were unable to survive
was observed on MSE2Pm100Cef250 in 35 and 16% of co- (25%), thus confirming the practical authenticity of PCR
cultivated explants and untransformed explants respec- and the phosphinothricin selection (Figure 3 a, where
tively. Approximately 80% of mature embryos (control) three-fourths of the plants are PCR-positive). Based on the
formed calluses on MSE2. In T. durum cv. PDW215, cal- result of phosphinothricin leaf paint assay and PCR
lus formation on MSE2Pm100Cef250 was observed in 32 analysis, transformation efficiency by co-cultivation of
and 12% of co-cultivated and untransformed mature em- mature embryos of T. aestivum cv. HD2329 with Agro-
bryos respectively (Figure 2 d and e), and callus induction bacterium strain LBA4404 (pCAMBIA3301 :: pin2) was
was about 78% in control explants on MSE2. Growth of nearly 1.77%. PCR amplification was further undertaken to
untransformed explants was curtailed when the calluses screen for the presence as well as segregation of nptII gene
were subsequently transferred to MSE2Pm100 for further in the T1 progeny of T. aestivum CPAN1676 transformants
multiplication. Untransformed calluses showed no growth transformed with LBA4404(pBI101 :: Act1; Figure 3 b).
on MSE2Pm50; however, calluses derived from co-cultivated The genomic DNA of putative transformants of T. aes-
mature embryos showed growth on MSE2Pm100. After tivum HD2329 (LBA4404(p35SGUSINT)) obtained by
four weeks of co-cultivation, both proliferating and non- co-cultivation of mature embryo-derived calluses was
proliferating calluses were transferred to MSERPm50 for screened by PCR employing primers specific to nptII.
regeneration. After 2–3 weeks, the regenerated plantlets PCR amplification of the nptII gene in genomic DNA
were further transferred to MS1/2 for shoot elongation samples of T0 transformants indicated a successful trans-
(Figure 2 f ) and subsequently relocated to transfer plugs formation event (Figure 3 c), which is evidenced by the
for reinforcement of the root system (Figure 2 g). presence of an amplified product of expected size
Mature embryo-derived calluses are among the preferred (~ 721 bp).
explants with a regeneration response (~ 60%) compara-
ble to that observed from calluses derived from the immature
embryos. However, this explant demands greater care NPTII assay
during handling and physical damage to calluses is reflected
in the reduced regeneration response and transformation The transformed plantlets (8–10 cm long) growing in seedling
efficiency as well. Mature embryo-derived calluses are trays were sprayed with a solution of 2% w/v paromomycin

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Figure 3. a, PCR analysis of genomic DNA samples of T0 transformants of T. aestivum cv. HD2329 using primers specific to bar gene. The T0
transformants were obtained by co-cultivation of mature embryos with A. tumefaciens strain LBA4404(pCAMBIA3301 :: pin2). The plasmid
pCAMBIA3301 :: pin2 and genomic DNA from an untransformed plant were used as positive and negative control respectively. b, T1 progeny:
PCR analysis of T. aestivum cv. CPAN1676 transformed with LBA4404(pBI101 :: Act1) using primers specific to nptII gene. The plasmid
pBI101 :: Act1 and genomic DNA isolated from an untransformed plant were used as positive and negative controls respectively. c, PCR analysis of
genomic DNA samples of T0 transformants of T. aestivum cv. HD2329 using primers specific to nptII gene. The T0 transformants were obtained by
co-cultivation of mature embryo-derived calluses with A. tumefaciens strain LBA4404 (p35SGUSINT). The plasmid p35SGUSINT and genomic
DNA from an untransformed plant were used as positive and negative control respectively.

sulphate and 0.1% Tween-20. After seven days, trans-

formed plants displayed little to no damage following
paromomycin spraying. Untransformed control plants de-
veloped bleached spots and subsequently displayed sig-
nificant growth inhibition (Figure 2 l). The activity of nptII
gene was further confirmed by dot-blot assay in the trans-
formants of T. aestivum var CPAN1676 and T. durum cv.
PDW215 tested (Figure 4 b), which were selected on the
basis of paromomycin leaf-spraying results. Transformation
frequencies observed in case of T. aestivum and T. durum
were 1.6 and 1.28% respectively, using the construct
LBA4404 (pBI101 :: Act1). When the construct LBA4404
(pCAMBIA :: pin2) was used, transformation efficiency
of 1.77 and 1.54% was observed in T. aestivum and T.
durum respectively. However, the growth response of T0
transformants was poor compared to the untransformed
control plants. Seed setting was observed in only 40 and
33.33% of transformants of T. aestivum and T. durum res-
pectively, and even the quality of seeds was not satisfac-
tory. This could probably be due to the non-availability
of optimal growth conditions.

Southern analysis of T0 transformants

Genomic DNA of T0 transformants of T. durum obtained
after co-cultivation of mature embryos with LBA4404 Figure 4. a, Southern analysis of T0 transformants of T. durum cv.
PDW215 co-cultivated with A. tumefaciens LBA4404(pBI101 :: Act1).
(pBI101 :: Act1) was digested with EcoRI and blotted Lanes 1 and 2, Untransformed control (undigested and digested respec-
onto nylon membrane and hybridized with a gus-specific tively); Lanes 3–14, Genomic DNA of putative transformants digested
probe. Digestion of pBI101 :: Act1 releases a ~ 3.3 kb with EcoRI. Digestion of pBI101 :: Act1 with EcoRI releases a ~ 3.3 kb
fragment containing gus-coding region, nos terminator and part of
fragment which has the gus coding region, a portion of Act1-5′ region. Numerals on the left indicate size of the DNA frag-
rice Act1-5′ region and nos terminator. A specific fragment ments of the length standard in kb (λ DNA, HindIII digest). Hybridiza-
hybridizing to GUS was observed in the transgenic lines tion was performed by BamHI–SacI fragment of pAct1-F, which spans
the gus coding region. b, Autoradiograph showing NPTII activity in dot
tested. This observation confirms the presence of the gus blots of T0 transformants obtained by co-cultivation with A. tumefa-
gene in the screened transformants (Figure 4 a). ciens LBA4404(pBI101 :: Act1).

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DNA blot of primary transformants of T. aestivum cv. one of the T1 plants developed extensive yellow patches
HD2329 transformed with LBA4404 (pCAMBIA3301 :: on the leaves, while the other two plants suffered little or
pin2) was probed with a 1.5 kb BamHI fragment of no damage (Figure 2 l). Presence of nptII gene in two of
pCAMBIA3301 :: pin2 which contains the pin2 coding the T1 plants was also confirmed by PCR amplification of
region and also a pin2-3′ terminator. Southern analysis of genomic DNA samples using nptII-specific primers (Figure
BamHI-digested samples revealed the presence of hybri- 3 b). T1 plants also displayed better growth than T0 plants
dization signals in the 1.5 kb range in the T0 transfor- (Figure 2 j), on pots containing a mix of garden soil and
mants tested. EcoRI-digested genomic DNA samples show soilrite.
the presence of hybridizing bands (Figure 5), demonstrat-
ing the presence of pin2 gene in the T0 regenerants.
Discussion

Progeny analysis Cereals, especially wheat, were once considered recalcitrant

to Agrobacterium-mediated transformation. However, since
As mentioned earlier, the T. aestivum T0 transformants the first successful report of fertile transgenic wheat
produced few seeds and most of the seeds were poorly plants7, there has been considerable progress. Various factors
developed. From one representative T0 plant, three out of influencing successful Agrobacterium-mediated genetic
ten seeds were successfully germinated on half-strength transformation of wheat have been explored by several
MS. After paromomycin spraying of seven-day-old seedlings, workers in a quest to achieve higher transformation effi-

Figure 5. Southern analysis of T0 transformants of Triticum aestivum cv. HD2329 co-cultivated with A. tumefaciens LBA4404 (pCAM-
BIA3301 :: pin2). UD, Undigested; E, EcoRI; B, BamHI. Numerals on the left indicate size of the DNA fragments of the length standard in kb (λ
DNA, HindIII digest). Hybridization was performed by 1.5 kb BamHI fragment of pCAMBIA3301 :: pin2, which spans the pin2 coding-pin2 termi-
nator region.

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ciency9,13,12,35. The advantages of using the Agrobacte- bacterium infection. Since there were two limiting steps,
rium-mediated approach involve the ability to transfer agroinfection and regeneration from the transformed cal-
large segments of DNA with minimal rearrangement, with luses, approaches such as transformation of immature/
fewer copies at a higher efficiency incurred with minimal mature embryos instead of immature/mature embryo-
cost. Thereby, such an approach results in simple segrega- derived calluses limit the time duration without hamper-
tion pattern, better transformation frequency and also the ing the regeneration ability of the explants. In the present
absence of vector backbone sequences. Furthermore, in investigation, callusing and regeneration were optimized
Agrobacterium-mediated transformation DNA generally to attain reproducible, standardized transformations, as
inserts into transcriptionally active regions38 via illegitimate loss of regeneration potential over time has been a major
recombination, thereby ensuring the active transcription/ limitation in most wheat-transformation experiments.
expression of the transgenes. This loss of regeneration potential, especially during pro-
Transformation efficiency in wheat may be influenced longed culture, has been overcome by supplementing the
by many factors such as genotype, type of explant, opti- regeneration media with polyamine spermidine to improve
mized conditions of inoculation and co-cultivation, opti- recovery of regenerants from transformed calluses14.
mized callusing and plant regeneration, selection media, However, in the present approach direct transformation of
vector and Agrobacterium strain, etc.13. The present investiga- the mature embryos was undertaken to minimize the loss
tion was initiated to develop an efficient gene delivery of regeneration potential due to prolonged culture.
protocol for bread wheat and durum wheat. There have In the present investigation, LBA4404 was employed
been limited studies on genetic transformation of wheat as the Agrobacterium strain of choice. Successful produc-
via Agrobacterium-mediated co-cultivation and few re- tion of transgenic plants was achieved by co-cultivation
ports demonstrate the successful production of transgenic of mature embryos and mature embryo-derived calluses.
wheat plants. Most Agrobacterium-mediated research has Our investigations support the observations of other re-
employed immature embryos, precultured immature embryos, searchers, who recommend the use of acetosyringone and
and their embryogenic calluses for co-cultivation7,16,35,36. glucose for inclusion in the inoculation and co-culture
For efficient introduction of T-DNA genes into recalcitrant medium7. Acetosyringone was employed in both the in-
explants, several groups have employed different ap- oculation as well as the co-cultivation medium, resulting in
proaches such as enhancing the vir gene expression (by 1.5-2-fold increase in transient expression of gus activity,
wounding) or promoting the adherence of Agrobacterium thereby validating the observation of other workers42,43.
to the plant tissues. Various approaches include mechanical Durum wheat transformation has been accomplished success-
abrasion of wheat seeds11, use of surfactants7, agrolistics39, fully using the biolistics approach37,44–48, but Agrobacte-
use of silicon carbide fibres for wounding of immature rium-mediated transformation is yet to be accomplished.
wheat embryos40, and subjecting the plant tissues to brief The vectors successfully employed in this study are
periods of ultrasound in the presence of Agrobacterium conventional binary vectors, while the supervirulent vec-
(sonication-assisted Agrobacterium-mediated transforma- tors (having extra copies of virB, virC and virG of A281)
tion)41. The procedure reported here involves excision of that have been extensively used for transformation of rice
mature embryos: it is simple and fulfils the basic wound- and maize4,5 do not appear to be essential for wheat trans-
ing requirement and does not require special media. In formation, as this observation is also shared by other co-
the present investigation, our transformation efficiencies workers7. In the binary vector pBI101 : Act1, the gus gene
(1.28–1.77%) are comparable with those reported ear- is under the control of a monocot promoter (rice Act1) for
lier7,12,13,42,43, though we have not been able to characterize specific expression in plant tissues and the gus gene con-
in detail the segregation patterns due to paucity of trans- tains an intron in the vectors pCAMBIA3301 :: pin2 and
genic events. Agrobacterium-mediated transformation has p35SGUSINT, which efficiently blocks its expression in
been reported to achieve a maximal of 4.4% transformation bacteria, thus excluding false positives. The antibiotic/
efficiency using the construct aroA :: CP4 and immature herbicide-resistant nature of transgenic tissues proves that
embryos as explants12. the selectable marker genes are active in wheat. The results
The key factor in the successful generation of transgenic of antibiotic and herbicide resistance assays were observed
plants is the optimization of conditions for regeneration to be consistent with those of molecular analysis. The
of plantlets after several rounds of selection with herbi- transformants that scored positive on the basis of assays
cide/antibiotic, which is also necessary for elimination of for antibiotic and herbicide resistance were also detected
escapes. Our work also emphasizes on the prospects of positive by Southern and PCR analysis with the respective
paromomycin and phosphinothricin as effective selection genes. A direct correlation was observed (data not pre-
agents for wheat transformation, and this is consistent sented) between expression of pin2 in T. durum and resis-
with the observations of earlier investigators2,17. Strate- tance against the cereal cyst nematode, Heterodera
gies towards enhancing the transformation frequency involve avenae49.
optimization of parameters such as the use of suitable ex- In our laboratory, the suitability of mature embryo as a
plants along with optimizing factors that promote Agro- starting explant for wheat transformation has been demon-
CURRENT SCIENCE, VOL. 91, NO. 3, 10 AUGUST 2006 315
RESEARCH ARTICLES

strated also for cellular permeabilization experiments in- 3. Chan, M., Cheng, H., Ho, S., Tong, W. and Xu, S., Agrobacte-
volving mechanically isolated embryos with membrane rium-mediated production of transgenic rice plants expressing a
chimeric α-amylase promoter/β-glucuronidase gene. Plant Mol.
permeabilizing agents35 and for particle bombardment of Biol., 1993, 22, 491–506.
basal embryo-derived calluses48. Therefore, it appears that 4. Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T., Efficient trans-
mature embryos are an excellent system for Agrobacte- formation of rice (Oryza sativa L.) mediated by Agrobacterium
rium-mediated transformation, as they are not only con- and sequence analysis of the boundaries of the T-DNA. Plant J.,
venient due to their ease of availability and but also for 1994, 6, 271–282.
5. Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T. and Kuma-
handling large numbers within a reasonable time, thus aid- shiro, T., High efficiency transformation of maize (Zea mays L.)
ing recovery of transgenics. In the present study, most of mediated by Agrobacterium tumefaciens. Nature Biotechnol.,
the regenerated plants reached maturity and set seeds, and 1996, 14, 745–750.
the further analysis of the transformants indicated absence 6. Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thornton,
of vector backbone sequences and confirmed T-DNA integra- S. and Brettell, R., Agrobacterium tumefaciens-mediated barley
transformation. Plant J., 1997, 11, 1369–1376.
tion in low copies, thereby resulting in simple segregation 7. Cheng, M. et al., Genetic transformation of wheat mediated by
patterns. Genetic transformation techniques have often Agrobacterium tumefaciens. Plant Physiol., 1997, 115, 971–980.
been associated with aberrations in morphology and fertility 8. Hiei, Y., Komari, T. and Kubo, T., Transformation of rice medi-
of plants. The T0 transformants obtained in the present study ated by Agrobacterium tumefaciens. Plant Mol. Biol., 1997, 35,
displayed poor growth compared to plantlets regenerated 205–218.
9. Mooney, P. A., Goodwin P. B., Dennis, E. S. and Llewelleyn, D.
from untransformed calluses on selection-free medium. J., Agrobacterium tumefaciens-gene transfer into wheat tissues.
However, the T1 progeny appeared to be normal pheno- Plant Cell Tissue Organ Cult., 1991, 25, 209–218.
typically and similar in morphology to seed-derived con- 10. Chen, D. F. and Dale, P. J., A comparison of methods for deliver-
trol plants. ing DNA to wheat: the application of wheat dwarf virus DNA to
seeds with exposed apical meristems. Transgenic Res., 1992, 1,
93–100.
Conclusion 11. Mahalakshmi, A. and Khurana, P., Agrobacterium-mediated gene
delivery in various tissues and genotypes of wheat (Triticum aesti-
vum L.). J. Plant Biochem. Biotechnol., 1995, 4, 55–59.
A reproducible method for efficient production of trans- 12. Hu, T. et al., Agrobacterium-mediated large-scale transformation
genic wheat plants using A. tumefaciens with a frequency of wheat (Triticum aestivum L.) using glyphosate selection. Plant
of nearly 1.28–1.77% has been achieved in both bread Cell Rep., 2003, 21, 1010–1019.
wheat as well as durum wheat. Although no significant 13. Wu, H., Sparks, C., Amoah, B. and Jones, H. D., Factors influenc-
difference was observed in the transformation efficiencies ing successful Agrobacterium-mediated genetic transformation of
wheat. Plant Cell Rep., 2003, 21, 659–668.
under similar conditions, the level of transient gus gene 14. Khanna, H. K. and Daggard, G. E., Agrobacterium tumefaciens-
expression was always observed to be higher in T. aesti- mediated transformation of wheat using a superbinary vector and a
vum varieties compared to T. durum varieties. The reason polyamine-supplemented regeneration medium. Plant Cell Rep.,
for this is difficult to ascertain and probably resides in the 2003, 21, 429–436.
better optimized regeneration capabilities of T. aestivum 15. Chugh, A. and Khurana, P., Herbicide-resistant transgenics of
bread wheat (T. aestivum) and emmer wheat (T. dicoccum) by par-
compared to T. durum. Agrobacterium-mediated trans- ticle bombardment and Agrobacterium-mediated approaches.
formation efficiency can be enhanced by factors that activate Curr. Sci., 2003, 84, 78–83.
the vir genes and enhance the susceptibility of plant cells 16. Duan, X., Li, X., Xue, Q., Abo-El-Saad, M., Xu, D. and Wu, R.,
against Agrobacterium such as acetosyringone, monosac- Transgenic rice plants harboring an introduced potato proteinase
charides (including phytohormonal, CaCl2 and osmotic inhibitor II gene are insect resistant. Nature Biotechnol., 1996, 14,
494–498.
treatments). The present study focuses on the hitherto un- 17. Altpeter, F., Diaz, I., McAuslane, H., Gaddour, K., Carbonero, P.
explored usage of mature embryos as suitable explants for and Vasil, I. K., Increased insect resistance in transgenic wheat
Agrobacterium-mediated transformation. The factors that stably expressing trypsin inhibitor CMe. Mol. Breed., 1999, 5, 53–63.
impact T-DNA delivery and regeneration were similar to 18. Murashige, T. and Skoog, F., A revised medium for rapid growth
the ones observed when immature embryos are employed, and bioassays with tobacco tissue cultures. Physiol. Plant., 1962,
15, 473–497.
such as duration of pre-culture and also inoculation and 19. Iglesias, V. A., Gisel, A., Potrykus, I. and Sautter, C., In vitro
co-cultivation parameters13. Nonetheless, we have devel- germination of wheat proembryos to fertile plants. Plant Cell Rep.,
oped a method for production of transgenic wheat by A. 1994, 13, 377–380.
tumefaciens applicable for both bread wheat and durum 20. Hoekama, A., Hirsch, P. R., Hooykaas, P. J. J. and Schilperoort,
wheat, thus demonstrating considerable genotypic inde- R. A., A binary plant vector strategy based on the separation of vir
and T-region of the Agrobacterium tumefaciens Ti plasmid. Na-
pendence. ture, 1983, 303, 179–180.
21. Cao, J., Duan, X., McElroy, D. and Wu, R., Regeneration of her-
1. Smith, R. H. and Hood E. E., Agrobacterium tumefaciens trans- bicide resistant transgenic rice plants following microprojectile-
formation of monocotyledons. Crop Sci., 1995, 35, 301–309. mediated transformation of suspension culture cells. Plant Cell
2. Mahalakshmi, A. and Khurana, P., Agrobacterium-mediated cereal Rep., 1992, 11, 586–591.
transformation: A critical appraisal. Indian J. Exp. Biol., 1997, 35, 22. Center for the Application of Molecular Biology to International
416–426. Agriculture, www.cambia.org

316 CURRENT SCIENCE, VOL. 91, NO. 3, 10 AUGUST 2006

 RESEARCH ARTICLES
23. Vancannyet, G., Schmidt, R., O’Connor-Sanchez, A., Wilmitzer, 38. Ingelbrecht, I., Breyne, P., Vancomperonolle, A., van Montagu, J.
L. and Rocha-Sosa, M., Construction of an intron containing M. and Depicker, A., Transcriptional interferences in transgenic
marker gene: Splicing of the intron in transgenic plants and its use plants. Gene, 1991, 109, 239–242.
in monitoring early events in Agrobacterium-mediated plant trans- 39. Hansen, G. and Chilton, M. D., ‘Agrolistic’ transformation of plant
formation. Mol. Gen. Genet., 1990, 220, 245–250. cells: Integration of T-strands generated in planta. Proc. Natl.
24. Van Haute, K. H., Maes, S., Warren, G., Van Montagu, M. and Acad. Sci. USA, 1996, 93, 14978–14983.
Schell, J., Intergenic transfer and exchange recombination of re- 40. Singh, N. and Chawla, H. S., Use of silicon-carbide fibres for
striction fragments cloned in pBR322: A novel strategy for the re- Agrobacterium-mediated transformation in wheat. Curr. Sci.,
versed genetics of Ti plasmids of Agrobacterium tumefaciens. 1999, 76, 1483–1485.
EMBO J., 1983, 2, 411–417. 41. Trick, H. N. and Finer, J. J., SAAT: Sonication-assisted Agrobac-
25. Walkerpeach, C. R. and Velten, J., Agrobacterium-mediated gene terium-mediated transformation. Transgenic Res., 1997, 6, 329–336.
transfer to plant cells: cointegrate and binary vector systems. Plant 42. Guang-Min, X. I. A., Zhong-Yi, L., Chen-Xia, H., Hui-Min, C.
Mol. Biol. Man., 1994, 1, 1–19. and Brettel, R., Transgenic plant regeneration from wheat (Triti-
26. Lee, B., Murdoch, K., Kreis, M. and Jones, M. G. K., A method cum aestivum L.) mediated by Agrobacterium tumefaciens. Acta
for large-scale progeny screening of putative transformed cereal. Phytophysicol. Sin., 1999, 25, 22–28.
Plant Mol. Biol. Rep., 1989, 7, 129–134. 43. Mi, Q. L., Velazhahan, R., Muthukrishnan, S. and Liang, G. H.,
27. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., GUS fu- Wheat transformation mediated by Agrobacterium tumefaciens.
sions: β-glucuronidase as a sensitive and versatile gene fusion Ann. Wheat Newsl., 2000, 45, http://wheat.pw.usda.gov/ggpages/
marker in higher plants. EMBO J., 1987, 6, 3901–3907. awn/45/Textfiles/USKS.html#anchor367934.
28. Bradford, M. M., A rapid and sensitive method for the quantifica- 44. Bommineni, V. R., Jauhar, P. P. and Peterson, T. S., Transgenic
tion of microgram quantities of protein utilizing the principle of durum wheat by microprojectile bombardment of isolated scutella.
protein-dye binding. Anal. Biochem., 1976, 72, 248–254. J. Hered., 1997, 88, 475–481.
29. Roy, P. and Sahasrabuddhe, N., A sensitive and simple paper 45. He, G. Y. et al., Transformation of pasta wheat (Triticum tur-
chromatographic procedure for detecting neomycin phosphotrans- gidum L. var. durum) with high-molecular weight glutenin subunit
ferase II (nptII) gene expression. Plant Mol. Biol., 1990, 14, 873– genes and modification of dough functionality. Mol. Breed., 1999,
876. 5, 377–386.
30. Lonsdale, D. M., Lindup, S., Moisan, L. J. and Harvey, A. J., Using 46. Tosi, P., Napier, J. A., D’Ovidio, R., Jones, H. D. and Shewry, P.
firefly luciferase to identify the transition from transient to stable R., Modification of the LMW glutenin subunit composition of du-
expression in bombarded wheat scutellar tissue. Physiol. Plant., rum wheat by microprojectile-mediated transformation. R. Soc.
1998, 102, 447–453. Chem., 2000, 261, 93–96.
31. Dellaporta, S. L., Wood, J. and Hicks, J. B., A plant DNA mini- 47. Pellegrineschi, A., Brito, R., Velazquez, L., Noguera, L., Pfeiffer,
preparation: version II. Plant Mol. Biol. Rep., 1983, 4, 19–21. W., McLean, S. and Hoisington, D., The effect of pretreatment
32. Mahalakshmi, A., Khurana, J. P. and Khurana, P., Rapid induction with mild heat and drought stresses on the explant and biolistic
of somatic embryogenesis by 2,4-D in leaf base cultures of wheat transformation frequency of three durum wheat cultivars. Plant
(Triticum aestivum L.). Plant Biotechnol., 2003, 20, 267–273. Cell Rep., 2002, 20, 955–960.
33. Patnaik, D. and Khurana, P., Wheat biotechnology: A minireview. 48. Chugh, A. and Khurana, P., Regeneration via somatic embryo-
Electron. J. Biotechnol., 2001, 4, 1–29. http://www.ejb.org/content/ genesis from leaf basal segments and genetic transformation of
vol4/issue2/full/4/. bread and emmer wheat by particle bombardment. Plant Cell Tis-
34. Ozgen, M., Turet, M., Altinok, S. and Sanzak, C., Efficient callus sue Organ Cult., 2003, 74, 151–161.
induction and plant regeneration from mature embryo culture of 49. Vishnudasan, D., Tripathi, M. N., Rao, U. and Khurana, P., Assess-
winter wheat (Triticum aestivum L.) genotypes. Plant Cell Rep., ment of nematode resistance in wheat transgenic plants expressing
1996, 18, 331–335. potato proteinase inhibitor (PIN2) gene. Transgenic Res., 2005,
35. Mahalakshmi, A., Chugh, A. and Khurana, P., Exogenous DNA 14, 665–675.
uptake via cellular permeabilization and expression of foreign gene in
wheat zygotic embryos. Plant Biotechnol., 2000, 17, 235–240.
36. Khurana, J., Chugh, A. and Khurana, P., Regeneration from ma- ACKNOWLEDGEMENTS. This work was financially supported by
ture and immature embryos and transient gene expression via the Department of Biotechnology, New Delhi. We thank Drs Jun Cao,
Agrobacterium-mediated transformation in emmer wheat (Triticum Xiao lan Duan, David McElroy and Ray Wu for the plasmid construct
dicoccum Schuble). Indian J. Exp. Biol., 2002, 40, 1295–1303. pDM302 and Ray Wu for the plasmid construct pTWa. D.P. and D.V.
37. Patnaik, D. and Khurana, P., Genetic transformation of Indian thank the Council of Scientific and Industrial Research, New Delhi for
bread (T. aestivum) and pasta (T. durum) wheat by particle bom- award of fellowships.
bardment of mature embryo-derived calluses. BMC Plant Biol.,
2003, 3, 5. Received 9 May 2005; revised accepted 6 April 2006

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