Pak. J. Bot., 48(6): 2467-2475, 2016.

COMPARISON OF AGROBACTERIUM MEDIATED WHEAT AND

BARLEY TRANSFORMATION WITH NUCLEOSIDE
DIPHOSPHATE KINASE 2 (NDPK2) GENE

UMMARA WAHEED1, WENDY HARWOOD2, MARK SMEDLEY2, SANG-SOO KWAK3,

RAZA AHMAD1 AND MOHMMAD MAROOF SHAH1*
1
Biotechnology Program, Department of Environmental Sciences
COMSATS Institute of Information Technology (CIIT), Abbottabad
2
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney, NR4 7UH, UK
3
Plant Systems Engineering Research Center, KRIBB Daejeon 305.806, Korea
*
Corresponding author’s email: [email protected]
Phone: +92-992-383591-7; Fax: +92 (992) 383441

Abstract

An efficient and reliable transformation system is imperative for improvement of important crop species like barley and
wheat. Wheat transformation is complex due to larger genome size and polyploidy while barley has a limitation of genotypic
dependency. The objective of current study was to compare the relative transformation efficiency of wheat and barley using
specific expression vector pBRACT 214-NDPK2 constructed through gateway cloning carrying Nucleoside Diphosphate
Kinase 2 (NDPK2) gene. The vector was used to compare the transformation response in both crops using immature
embryos through Agrobacterium mediated transformation. Both wheat and barley showed different responses towards callus
induction and regeneration. Immature embryos of 1.5 to 2 mm in diameter was found optimum for wheat callus induction
while 1 to 1.5 mm for barley. Both embryogenic and non-embryogenic calli were found in wheat with significantly greater
tendency for embryogenecity in barley. The overall regeneration response was found different for all transformed wheat and
barley cultivars. Wheat cultivars showed good response initially that drastically slowed down in later stages with the
exception of Fielder that reached to the green shoots with good roots. The barley transformed lines showed good
regeneration response as compared to wheat. PCR analysis of putative transformants using genomic DNA showed a
maximum of 27% transformation efficiency in barely. No true transformation response was obtained in all cultivars of wheat
used in this study. The protocol developed for wheat and barley transformation will greatly be helpful in crop improvement
programme through genetic engineering especially in diploid relatives of cereals.

Key words: Callogenesis, Embryogenic, Agrobacterium, NDPK2, Wheat, Barley.

Introduction efficiency due to less complex and smaller genome as

compared to wheat (Harwood, 2011). In this study, the
The family Triticeae includes some of the most transformation efficiency of both wheat and barley was
economically important crop plants including wheat tested with NDPK2 a regulatory protein that retains the
(Triticum aestivum L.) and barley (Hordeum vulgare). ability to induce tolerance against multiple abiotic stresses,
Bread wheat ranked third in world food crop production using Agrobacterium-mediated transformation.
and makes an essential source for our everyday diet An efficient transformation requires the cloning of
throughout the globe. Barley holds the fourth position gene of interest into a suitable plasmid vector (Clough et
among important cereal crops in terms of production. As al., 1998; John et al., 2014; Abbasi et al., 2016).
well as being an important crop in its own right, it is used Traditional methods of cloning including DNA restriction
as a diploid model for the more complex polyploid cereals and ligation is quite lengthy and less efficient (Goderis et
such as hexaploid wheat. Domestication history of
al., 2002; Tzfira et al., 2005). In recent times, Gateway®
Triticeae is thousands years old, therefore, its members
contains a complex genetic record (Purugganan & Fuller, cloning technology (Invitrogen Co.) has proven its role
2009). Divergence of wheat and barley is almost 11.6 for fast and promising cloning that is based on the
million years back and from that time continued into bacteriophage λ site-specific recombination system
species and sub species (Chalupska et al., 2008). Both (Hartley et al., 2000). When compared to conventional
wheat and barley have complex genomes of large size, cloning methods, this method is more convenient and
high repeat contents and complex transposable element efficient because it can clone the gene of interest into
structure (Eilam et al., 2007; Wicker et al., 2011). Wheat, larger binary vectors (5 to 12 kb) (Curtis & Grossniklaus,
a hexaploid, has a larger genome of ~17 Gb, whereas 2003) and does not involve either DNA digestion or
barley is a diploid and contains genome of ~5 Gb ligation (Karimi et al., 2007; Xu & Li, 2008) that can
(Pickering & Johnston, 2005). impede the cloning process. Many Gateway® compatible
Both wheat and barley are being investigated for
binary vectors have been made available (Karimi et al.,
successful and efficient transformation potential. Between
the two, barley transformation efficiency is quite promising 2007; Curtis & Grossniklaus, 2003). In this study plant
as compared to the wheat that is more difficult to transform expression vector carrying NDPK2 gene under the control
due to low efficiency, genotype dependency and its complex of maize ubiquitin promoter with hygromycin resistance
genome (Li et al., 2012). Therefore, barley could be used as gene was constructed using gateway cloning method for
a model for wheat because of its better transformation barley and wheat transformation.
2468 UMMARA WAHEED ET AL.,

Materials and Methods any fungicide. Immature embryos were collected when
they reached to 1.5 to 2 mm in diameter in wheat while 1
A. Construction of plant expression vector to 1.5 mm for barley (Fig. 2a). The immature embryos
were transformed by two protocols of both barley
In order to construct the plant expression vector, full (Adopted from Harwood et al., 2009) and wheat under
length cDNA (accession no. AF017640) of Nucleoside strict aseptic conditions.
Diphostase Kinase 2 (NDPK2) gene from Arabidopsis was
used and cloned into the pBRACT 214 expression vector Media used in transformation: For media preparation,
under the control of ubiquitin promoter in which the phytagel was used as gelling agent, prepared at 2x the
selectable marker encodes hygromycin resistance using required concentration and was autoclaved prior to adding
gateway cloning technology (Invitrogen). Plant expression into the media. All the tissue culture media was sterilized
vector was transformed into the AGL1 Agrobacterium through steritop 0.22 µm filter except the stocks and were
strain through electroporation. Transformation cassette added in double concentration to that of required
formed in this study was used for Agrobacterium based concentration. After warming at 600 C the 2x media and
transformation in barley and wheat. 2x phytagel was mixed well and poured on to the petri
plates. During transfer to regeneration and rooting
B. Plant transformation medium, the plants were transferred to either deep plates
or glass tubes. For barley and wheat transformation,
Plant material and growth conditions: Wheat cultivars different but standard plant tissue culture media were used
Fielder, Siran, Atta Habib, Marvi 2000, MH 97 and barley for each stage including inoculation, callus induction,
cultivar Golden Promise were used in the study. Donor selection and regeneration (Tables 1 and 2).
plants of both wheat and barley were grown in a
controlled environment in growth room at John Innes Growth of Agrobacterium tumefaciens strain AGL1:
Center (JIC), Norwich, UK, for the collection of Agrobacterium culture was prepared from glycerol stock
immature embryos. Growth conditions were maintained at without any antibiotics. After overnight incubation at
24°C day and 18 °C night temperatures (for wheat) and 28°C with shaking (200 rpm) the bacteria were collected
24°C day and 18 °C night (for barley), 80% relative by centrifugation at 2200 rpm for 10 min at 4oC. For
humidity and light levels of 500 μmol/m2 /s1 at the mature barley full strength Agrobacterium culture was used while
plant canopy level. Light was provided by metal halide for wheat, cells were re-suspended into the inoculation
lamps (HQI) supplemented with tungsten bulbs. The medium and left for 3 hours at room temperature with
plants were watered regularly and were not sprayed with slight shaking until the desired OD0.4 was achieved.

Table 1. Media composition for wheat transformation.

Media Composition
Liquid inoculation 4.3 g/L MS Basal Salt + 1 ml/L MS vitamins + 10 g Glucose + 0.5 g/L MES + 10 ul Acetosyringone, pH 5.8
4.3 g/L MS Basal Salt + 1 mL/L MS vitamins + 10g Glucose + 0.5 g/L MES + 500 ul Acetosyringone + 2000
Co-cultivation
ul AgN03 + 2000 ul CuSO4 5 H20 + Agarose 8g, pH 5.8
4.3 g/L MS Basal Salt + 1 ml/L MS vitamins + 0.5 g/L Glutamine + 0.1 g/L Casein Hydrolysate + 0.75 g/L
Resting MgCL2-6H20 + 1.95 g/L MES + Maltose 40 g/L + 0.5 mg/L 2,4-D + 2.2 mg/L Picloram + 160 mg/mL
Timentin + 0.84 mg/L AgNO3 + 0.100 mg/L Ascorbic Acid + 5 g Agarose, pH 5.8
4.3 g/L MS Basal Salt + 1 ml/l 100X MS vitamins + 0.5 g/L Glutamine + 0.1 g/L Casein Hydrolysate + 0.75 g
MgCl2-6H20 + 1.95 g/L MES + 0.5 mg/L 2,4-D + 0.5 mg/L Picloram +160 mg/mL Timentin + 0.42 mg/L
Selection
AgNO3 + 0.05 g/L Ascorbic Acid + 40 g/L Maltose + 15 mg/L Hygromycin for Ist selection and 30 mg/L for
2nd selection + 5 g Agarose, pH 5.8
4.3 g/L MS Basal Sal + 10 ml 100X Modified LS vitamins + 20 g/L Sucrose + 0.5 g/L MES+ 0.0025 g/L
Regeneration
CuSO4.5H2O + 400 ul Zeatin + 160 mg/mL + Timentin + 30 mg/L + 5g Agarose, pH 5.8
4.3 g/L MS Basal Salt Mixture (M0221) + 10 ml Modified LS vitamins + 15 g Sucrose + 0.5 g/L MES+ 0.2
Rooting
mg/L IBA+ 160 mg/mL Timentin + 30 mg/l + 5 g Agarose, pH 5.8

Table 2. Media composition for barley transformation.

Media Composition
Callus induction 4.3 g/L MS plant salt base + 30 g/L maltose, 1.0 g/L casein hydrolysate + 350 mg/L myoinositol + 690 mg/L proline +
1.0 m g/L thiamine HCl + 2.5 m g/L dicamba + 1.25 m g/L + CuSO4 .5H2O + 3.5 g/L Phytagel. pH 5.8
2.7 g/L Murashige and Skoog modified plant salt base (without NH4NO3) + 20 g/L maltose + 165 mg/L NH4NO3 + 750
Transition mg/L glutamine + 100 mg/L myoinositol + 0.4 mg/L thiamine HCl, + 2.5 mg/L 2,4-D, 0.1 mg/L 6-benzylaminopurine
(BAP) + 1.25 mg/L CuSO4.5H2O + 3.5 g/L Phytagel. pH 5.8
Regeneration 2.7 g/L Murashige and Skoog modified plant salt base (without NH4NO3) + 20 g/L maltose + 165 mg/L NH4NO 3 + 750
mg/L glutamine + 100 mg/L myoinositol, 0.4 mg/L thiamine HCl, 50 mg/L Hygromycin + 60 mg/L Timentin + 3.5 g/L
Phytagel. pH 5.8
Selection 2.7 g/L Murashige and Skoog modified plant salt base (without NH4NO3) + 20 g/L maltose + 165 mg/L NH4NO3 + 750
mg/L glutamine + 100 mg/L myoinositol + 0.4 mg/L thiamine HCl + 3.5 g/L Phytagel , pH 5.8
TRANSFORMATION EFFICIENCIES OF WHEAT AND BARLEY 2469

Sterilization of wheat immature embryos: For but was devoid of any growth regulators. On the
sterilization, immature seeds of wheat were treated with 70% formation of roots and when the shoots attained a height
ethanol for two minutes followed by three washes in sterile of about 2-3 cm, the plantlets were shifted to the glass
distilled water and 20% sodium hypochlorite stock solution tubes in callus induction medium containing the timentin
for 7 minutes, followed by a rinse of four changes of sterile and hygromycin at the same level but devoid of dicamba
distilled water. For sterilization of barley immature embryos and growth regulators.
a protocol of Harwood et al. (2014) was adopted. All the
sterilization processes were performed in the laminar flow iii. Plantlets transfer to soil: Plantlets of both wheat and
hood under aseptic conditions. barley with strong root systems in hygromycin were
considered as transformed. Selected plants were removed
Transformation procedure for barley and wheat from the glass tubes gently with the help of long forceps.
The entire medium from the plants was washed away under
i. Inoculation and co-cultivation with Agrobacterium:
a running tap and the plants were transferred to the soil in
Sterilized immature embryos of wheat cultivars were
the respective compost of both wheat and barley. In order
introduced into the inoculum medium containing 200 µM
acetosyringone and centrifuged at 14409 rpm at 4oC. The to make the plants acclimatize, they remained covered with
inoculum medium was replaced by the Agrobacterium propagators for one week after transfer to soil.
solution and incubated in the dark for 10 min. The explants
were taken out and placed on the sterilized filter paper to iv. Analysis of transgenic plants by PCR: Genomic DNA
remove the excessive Agrobacterium solution and dry out. extraction was done from the collected samples of both
Next, the explants were placed on the co-cultivation wheat and barley using Extract-N-Amp kit (Sigma-Aldrich,
medium with the scutellum side down in order to maximize Germany). None transformed wheat and barley DNA was
the chances of scutellum contact with the Agrobacterium. used as a negative control. Putative transformants were
Further, the medium was also supplemented with 200 uM detected by PCR screening. PCR amplification was
acetosyringone. This is very important to induce the performed as: Initial denaturation at 94°C for 3 min,
bacterial vir gene as the cereals are known to be inferior in followed by 33 cycles of denaturation at 94°C for 1 minute,
inducing phenolic compounds. Twenty to twenty five primer annealing at 58°C for 1 minute, and extension at 72°
embryos were placed on each 9 cm plate. Following two
C for 1 min followed by the final extension for 10 minutes.
days co-cultivation, the embryonic axis was removed from
the embryo using the fine forceps under microscope (Fig. PCR products were checked on 1% agarose gel by
2b). Embryos were placed scutellum side down onto resting ethidium bromide staining.
medium for five days, in dark at 25oC.
In barley for inoculation, 200 μL of full strength Results and Discussion
Agrobacterium culture was dropped onto the surface of
about 25 embryos per plate (Fig. 3a). After drying the Wheat and barley transformation: Immature embryos
embryos, the plate was tilted to run excessive Agro of barley and wheat were transformed with
solution; the explants were transferred to the callus Agrobacterium harboring pBRACT 214-NDPK2 vector
induction medium with their scutellum side down. (Fig. 1). Both wheat and barley showed different response
towards different stages of transformation including callus
ii. Regeneration and selection: After five days resting induction, regeneration and selection. Transformation
period, wheat explants were transferred to first selection efficiency in both crops found dependent on many factors
medium supplemented with 15 mg/L hygromycin. In including starting material, media composition and the
addition, the antibiotic timentin was also added at 160 protocols adopted.
mg/L during all selection stages in deep petri plates and
placed in dark for two weeks at 25oC. Viable calli were
selected and transferred to a second selection with the
same medium but adding the double concentration of 30
mg/L hygromycin for further three weeks in the same
conditions as was for the first selection. The calli that
survived from the second selection were transferred to the
regeneration medium supplemented with 0.04 mg/L of
zeatin but with the same level of antibiotics. Onto the
regeneration medium the transformed calli were identified
by producing green shoots. After two weeks, the plantlets
were transferred to the rooting medium supplemented
with the 0.2 mg/L indole butyric acid (IBA).
In barley, selection proceeded in three selection steps
each with 50 mg/L of hygromycin and 160 mg/L of
timentin at two weeks interval. During transfer the entire
embryo was shifted and handled as a single experimental
unit. After giving the selection pressure of six weeks the
explants were transferred to the transition medium for
further two weeks. The selected calli were shifted to the
regeneration medium. Regeneration medium was
supplemented with the same concentration of hygromycin Fig. 1. Schematic representation of pBRACT 214 NDPK2 vector.
2470 UMMARA WAHEED ET AL.,

Fig. 2. Different stages of the wheat transformation process. (a) Sterilized wheat seeds before immature embryo isolation (b) an
immature embryo after embryonic axis removed (c) Initiation of callus formation on callus induction medium (d) embryogenic callus
formation (e) Initiation of green shoots on regeneration medium (f) Plantlet rooting in glass culture before shifting to soil.

Fig. 3. Different stages of the barley transformation process. (a) Arrangement of Immature for inoculation (b) formation of
embryogenic callus (c) the initiation of green spots on callus (d) regeneration of transgenic plantlets on regeneration medium (e)
plantlet rooting in glass culture tubes (g) transgenic barley growing to maturity in the glasshouse.
TRANSFORMATION EFFICIENCIES OF WHEAT AND BARLEY 2471

Role of starting material: Immature embryos were Embryogenic callus induction: Callus induction
used as the explant source. For each individual response was different in both barley and wheat in terms
experiment a minimum of 100 embryos were collected of callus quality and its growth period. Among wheat
as the starting material for both barley and wheat. Care cultivars, Fielder initiated callus induction during the
was taken in selecting the starting material because first week (Fig. 2c) while Siran and Atta Habib took two
appropriate size of the explant is of critical importance
weeks to initiate the calli induction. Under the
in inducing the good callus. Embryos smaller than the
appropriate size could not induce high quality callus microscope, callus growth clearly indicated the
while larger embryos were difficult to handle and also embryogenic and non-embryogenic structures in wheat.
produced poor quality callus. The spikes were isolated at Embryogenic calli were found to be pale in color smooth
the stage when the immature embryos were 1.5 to 2 mm and compact structures with globular notches (Fig. 2d
in diameter for wheat while 1 to 1.5 mm for barley. and 3b). On the other hand, non-embryogenic calli were
Previous studies also emphasized that successful found to be of cream in color with watery appearance.
transformation events are based on the availability of the Compactness and presence of globular notches or ridged
explants with appropriate sizes and age. In wheat most of structure are some of the true signs of embryogenic
the transformation experiments the explant choice was callus formation (Satyavathi et al., 2004, Abid et al.,
mainly the freshly isolated immature embryos or the 2014). At the end of second selection, maximum callus
callus derived from such embryos. First successful wheat induction was recorded as 52 % in model cultivar
transformation also witnessed the use of immature Fielder while Atta Habib produced a maximum of 40%
embryos (Cheng et al., 1997). From the time onwards similarly 46, 37 and 24 % was recorded for Siran, Marvi
other researchers also tested immature embryos for wheat 2000 and MH 97 respectively (Table 3).
transformation but could not produce the desirable results Barley cultivar Golden Promise showed healthy,
(Uze et al., 2000; Sarker & Biswas, 2002). compact, nodular and more conspicuous embryogenic
There are different types of explants that are in
notches on callus surface (Fig. 3c). Barley showed a
constant use in barley transformation including microspore
very promising callus induction response with a
(Yao et al., 1997), shoot meristems tissue (Zhang et al.,
maximum of 87 % (Table 4). The callus size of barley
1999) and mature embryos (Park et al., 2006; Um et al.,
2007; Shah et al., 2009) but immature embryos remained as was also larger as compared to wheat. While among
good choice of explant for transformation. The early wheat, cv. Fielder showed the great embryogenecity
studies on the both Agrobacterium-mediated and biolistic followed by the Siran and Atta Habib. The embryogenic
techniques of barley transformation, also reports the notches turned into green leafy structures in later period
successful use of immature embryos as starting material in both barley and wheat cultivar (Fig. 2e, 3c). Among
(Wan & Lemaux, 1994; Tingay et al., 1997). An improved wheat cultivars only Fielder showed a maximum
transformation efficiency in wheat using immature appearance of green notches, other Pakistani cultivars
embryos as explants, is based on the fact that such embryos though did not produce the green spotting. On the other
retains higher rate of cell division that ultimately enhance hand a great trend of embryogenecity was seen in the cv.
the delivery of T DNA (Cheng et al., 1997; Hu et al., 2003; golden promise of barley (Fig. 3c). Such differences in
Wu et al., 2003) and also positively effects the recovery of the observations of callus induction clearly indicate that
the stable transgenic wheat plants. tissue culture response is greatly dependent upon the
In addition to the type of explant used another genotypes. Similar findings were also seen in the
important and determining factor is its age and size. It previous studies conducted by Mahmood et al. (2012).
was investigated that embryos size determines the transfer
of T-DNA, transgene expression and regeneration rate as Regeneration capability: The more resistant,
well. The large sized embryos (larger then 2 mm) though embryogenic and healthy calli with green notches of
shows a marked transient expression but low regeneration both barley and wheat were selected for regeneration.
rate during wheat transformation (Wu et al., 2003). Most The model cultivar Fielder was among the first cultivar
of the studies recommend the use of immature embryos to develop small green shoots on regeneration medium
isolated in early stage of development i.e. 14 to 16 days of in the first week of regeneration period followed by the
anthesis with a size of 0.8 to 1.5 mm (Jones, 2005). Weir cultivar Siran in the late first week (Fig. 2e). While cv.
et al. (2001) produced desirable transformation results by
Atta Habib showed good embryogenic callus but did not
using the nine days old immature embryos. These reports
develop green shoots till the second regeneration week.
are in line with the current studies where the immature
Regeneration response was found greatly dependent on
embryos were collected carefully at specific
genotype. Green shoots producing calli was quite low in
developmental stage and size for both barley and wheat.
In case of barley the optimum size of the immature number as compared to the callus induction frequency.
embryos was found to be as 1 to 1.5 mm. This Fielder showed the maximum number of calli producing
development period was found to be the optimum for green shoots in the regeneration medium in comparison
both calli induction and as well as for the better with other wheat cultivars (Table 3). Previous reports
transformation efficiencies in barley previously (Sharma also observed the genotype dependency of regeneration
et al., 1995; Caswell et al., 2000). in wheat (Aydin et al., 2011; Khalid et al., 2013).
2472 UMMARA WAHEED ET AL.,

Table 3. Agrobacterium mediated transformation of wheat.

Experiment No. of embryos Callus induction %Calli showing Putative NDPK2
Genotype
No. inoculated frequency (%) regeneration potential transformants positive plants
1 100 40 11 1 0
2 Fielder 200 52 16 0 0
3 200 45 22 2 0
1 100 20 6 0 0
2 Atta Habib 200 40 5 0 0
3 100 32 3 0 0
1 200 32 6 0 0
2 Siran 200 46 12 0 0
3 100 39 11 0 0
1 150 37 0 0 0
Marvi
2 150 30 1 0 0
2000
3 150 29 0 0 0
1 200 20 5 0 0
2 MH 97 100 21 0 0 0
3 100 24 2 0 0

Table 4. Agrobacterium mediated transfromation of barley.

% Calli showing
No. of inoculated Callus induction NDPK2 Total transformation
Exp. No. Genotype regeneration
embryos freq. (%) positive plants efficiency
potential
01 Golden Promise 150 87% 40 40 27%
02 Golden Promise 100 85% 30 27 27%

Wheat cultivars showed rooting after almost six days PCR based confirmation of putative transgenic plant:
of regeneration. Although most of the explant developed PCR analysis showed that out of 150 embryos of barley
roots on regeneration medium, but Pakistani cultivars from one individual experiment, forty independent
developed roots rapidly as compared to the Fielder. Out of putative transformed plants were achieved; this gives a
100 embryos as the starting stuff set in one individual transformation efficiency of 27% (Table 4). Out of forty
experiment, only 25 to 30 showed green spots at the early plants, twenty two were selected for PCR analysis for
stages of the regeneration, where a only 5 or 6 embryos both the transformed genes NDPK2 and hygromycin.
reached to the shoot and roots stage (Fig. 2f). After two Seventeen plant samples were found PCR positive for
weeks, the green shoots were shifted to the rooting NDPK2 gene (Fig. 4a and b) while twenty plants samples
medium, with 0.2 mg/L indole butyric acid with were found positive for the hygromycin (Fig. 5a and b). In
hygromycin concentration of 15 mg/L keeping the contrast wheat showed no true transformants (Tables 3
timentin concentration the same as that of selection and 4).
medium. Development of roots prior to shoot formation is Barley has been explored for biolistic transformation
not a satisfactory indication for the explants to develop methods for transfer of useful genes. Previous studies
into the plantlets. Siran, Atta Habib and Marvi 2000 indicated that overall transformation efficiency was
though showed a good response till the regeneration higher from the Agrobacterium mediated transformation
medium, but these cultivars in the later stages shed off as compared to the other methods of transformation. In
shoots and developed roots. Among all wheat cultivars, addition, transgene silencing was quite frequent in the
only the model cv. Fielder reached to a stage with green progeny raised by particle bombardment as compared to
shoots and roots and was selected for shifting to the soil. the Agrobacterium mediated transformation outcomes
On the other hand, transformed lines of barley grew (Travella et al., 2005).
very rapidly on the regeneration medium. Transformed However, Um et al. (2007) tested barley
plants were recognized by the presence of strong roots, as transformation with plant expression vector provided with
good root development in the presence of antibiotic is NDPK2 gene under the control of stress inducible SWAP2
used to test transgenic plants (Fig. 3e). When the barley promoter of sweet potato and bar gene as plant selection
shoots attained a height of about 2–3 cm in length and marker. From this study, a total of 20 plantlets could be
roots were formed, small plantlets were transferred to the regenerated from a 400 immature embryos that were
culture tubes containing 10 to 12 mL of callus induction cultured on the selection medium. 0.15% transformation
medium containing antibiotic at same level as on selection efficiency was achieved from the two barley cultivars that
medium but without dicamba and growth regulators. were tested for transformation response. In the current
Plants growing well on rooting medium were transferred study, regeneration rate was quite higher, 67 independent
to barley compost respectively (Fig. 3f). Once plants were lines were obtained from a total of 250 and molecular
established in soil, leaf samples were collected for analyses revealed that 27 of 40 plants analyzed, were
molecular analysis. positive transgenic plants.
TRANSFORMATION EFFICIENCIES OF WHEAT AND BARLEY 2473

Fig. 4. PCR amplified bands of NDPK2 gene in barley plants Fig. 5. PCR amplified bands of hygromycin gene in barley
regenerated following Agrobacterium inoculation. A negative plants regenerated following Agrobacterium inoculation. A
control (non-transgenic wild type plant DNA of cv Golden negative (-) control (non-transgenic plant DNA of cv Golden
Promise (GP), molecular weight markers (M) (Invitrogen Promise), molecular weight markers (M) (Invitrogen PCR
Lambda DNA markers, 1 kb plus ladder), and the expected markers 1-kb ladder), and the expected PCR hph product (353
NDPK2 PCR product (544 kb) are indicated. DNA samples used bp) are indicated. DNA samples used for PCR reaction were
for the PCR reaction were taken from plants regenerated from taken from plants regenerated from immature embryos (a)
immature embryos (a) (Lanes 1-13) and (b) (Lanes 14-22). (Lanes 1-13) and (b) (Lanes 14-22). Hygromycin positive plants
NDPK2 positive plants exhibiting bands of expected size. exhibiting bands of expected size.

The improved transformation efficiency is due to 2009) and also proved good for selection of barley in the
many factors, including transformation method, the current study as well. On contrary, for wheat, the
protocol and the promoter in the construct. Hygromycin hygromycin selection though been in constant use
was used in the present study as plant selection marker. (Hauptmann et al., 1987) but it is less efficient and plants
The hygromycin selection process was found to be very can still occasionally grow on hygromycin leading to the
effective during many cereals transformation, as this regeneration of ‘escape’ plants.
selection system allows rare chances of escapes during
plant selection (Bartlett et al., 2008). In addition, the Conclusion
promoter used in the current study was of monocot origin
that generally gives better transformation efficiency as Gateway cloning method proved to be reliable and
compared to the driving promoter of dicot origin. Above efficient strategy for vector construction. Transformation rate
all, the Agrobacterium-mediated transformation was in barley was promising. The negative wheat transformation
successfully used in the current study to transform gene may be due to the transcription factor NDPK2. The
that was well known for its economic importance in constitutive expression of NDPK2 might have caused lethal
comparison to biolistic transformation in addition to the effect on the regeneration. The protocol developed and
efficient transformation efficiency. optimized for wheat and barley transformation will greatly
In case of wheat, five putative transformants were help crop improvement programmes through genetic
checked through PCR but no true transformants could be engineering especially in diploid relatives of cereals.
recovered. Negative transformation response in wheat can
Acknowledgment
be attributed to many factors. One of the most important
factors is that over expression of the targeted gene The authors greatly appreciate and acknowledge the
NDPK2 may have interfered with the growth of wheat contributions of Higher Education Commission (HEC) of
plants in some way that they could not be recovered. Pakistan for generously funding the project under
Therefore the only plants recovered on regeneration International Research Support Initiative Programme (IRSIP)
medium were escapes. This is because some of the stress to accomplish this research at John Innes Center, UK.
responsive transcription factors pose negative or
unwanted side effects in the host plants, like yield loss References
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2474 UMMARA WAHEED ET AL.,

Abid, N., A. Maqbool and K.A. Malik. 2014. Screening Karimi, M., A. Bleys, R. Vanderhaeghen and P. Hilson. 2007.
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(Received for publication 12 October 2015)