optimization of factors for efficient recovery of transgenic peanut (arachis hypogaea l.)
TRANSCRIPT
ORIGINAL PAPER
Optimization of factors for efficient recovery of transgenic peanut(Arachis hypogaea L.)
Siddharth Tiwari • Rakesh Tuli
Received: 22 July 2011 / Accepted: 22 October 2011 / Published online: 5 November 2011
� Springer Science+Business Media B.V. 2011
Abstract De-embryonated cotyledon explants of peanut
were co-cultivated under different conditions with Agro-
bacterium tumefaciens harbouring pIG121hm plasmid car-
rying intron-containing b-glucuronidase as a reporter while
hygromycin phosphotransferase and neomycin phospho-
transferase as selectable marker genes. Co-cultivation
duration and temperature, various antioxidants and their
concentrations, bacterial strains and explant characteristics
(incised and non-incised) were examined either alone or in
combinations for optimization of transient expression of the
reporter gene. Up to 81% transformation was recorded when
non-incised explants were co-cultivated with strain EHA101
for 5 days at 21�C on shoot induction medium containing
100 mg/L L-cysteine. Addition of the optimized concentra-
tion of augmentin (200 mg/L) along with cefotaxime
(200 mg/L) to the shoot induction medium not only
effectively eliminated bacterial growth, but also facilitated
high frequency of shoot induction. The 40 mg/L hygromycin
concentration prevented complete shoot regeneration of
non-transgenic explants thus considered for the regeneration
of transgenics. Resistant shoots were successfully trans-
ferred to soil either by grafting or in vitro rooting. Survival
rate of the grafted shoots was nearly 100% in glass-house
conditions. The optimized protocol took around 3 months to
generate healthy plants. Polymerase chain reaction, South-
ern blot hybridization, histochemical tests, segregation and
hygromycin-leaf assays of selected transgenic plants showed
integration of the transgene into peanut genome. No chi-
meras were noticed during the study.
Keywords Agrobacterium tumefaciens �De-embryonated cotyledon � Genetic transformation �Grafting � Peanut
Abbreviations
AS Acetosyringone
BAP 6-Benzylaminopurine
DEC De-embryonated cotyledon
GUS b-Glucuronidase enzyme
hpt Hygromycin phosphotransferase gene
nptII Neomycin phosphotransferase gene
PCR Polymerase chain reaction
SIM Shoot induction medium
uidA b-glucuronidase gene
Introduction
Genetic transformation has been widely exploited to study
plant biology, develop commercial crops with desired
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11240-011-0079-4) contains supplementarymaterial, which is available to authorized users.
S. Tiwari � R. Tuli
Plant Molecular Biology and Genetic Engineering Division,
National Botanical Research Institute, Rana Pratap Marg,
Lucknow 226001, India
Present Address:S. Tiwari � R. Tuli
National Agri-Food Biotechnology Institute (NABI),
Department of Biotechnology, Govt. of India, C-127, Industrial
Area, Phase VIII, SAS Nagar, Mohali 160071, Punjab, India
e-mail: [email protected]; [email protected]
R. Tuli (&)
National Agri-Food Biotechnology Institute (NABI),
Department of Biotechnology Govt. of India, C-127, Industrial
Area, Phase VIII, SAS Nagar, Mohali 160071, Punjab, India
e-mail: [email protected]; [email protected]
123
Plant Cell Tiss Organ Cult (2012) 109:111–121
DOI 10.1007/s11240-011-0079-4
agronomic traits and more recently, in the use of plants as
bio-reactors (Yonekura-Sakakibara and Saito 2006; Tiwari
et al. 2009a, b; http://www.isaaa.org/ISAAA 2010).
Genetic transformation overcomes the constraints encoun-
tered in inter-specific, inter-generic or inter-kingdom gene
transfers. Transgenic peanut plants have been obtained
using Agrobacterium rhizogenes (Medina-Bolivar et al.
2007; Kim et al. 2008), A. tumefaciens (McKently et al.
1995; Li et al. 1997; Sharma and Anjaiah 2000; Qiushen
et al. 2005; Dodo et al. 2008; Tiwari et al. 2008, 2011) and
biolistic (Singsit et al. 1997; Magbanua et al. 2000;
Livingstone et al. 2005; Athmaram et al. 2006; Niu et al.
2009) methods. Biolistic transformation exhibits multiple
copies of transgene integration in peanut genome (Singsit
et al. 1997; Livingstone et al. 2005; Niu et al. 2009). The
A. tumefaciens-mediated peanut transformation is reported
to give low copy transgene integration (Li et al. 1997;
Sharma and Anjaiah 2000; Dodo et al. 2008; Tiwari et al.
2008, 2011). However, low frequency regeneration of
transformants, development of false positive transgenic
‘escapes’ and high incidence of sterility in regenerated
peanut plants are the commonly reported problems. Precise
understanding of the factors influencing the efficiency of
T-DNA transfer, transgene integration and expression in
peanut could considerably facilitate the development of
efficient genetic transformation protocol for this crop.
Several chemical and physical parameters are prereq-
uisites for efficient transformation by A. tumefaciens.
Factors like explant type, bacterial strains, co-cultivation
duration, co-cultivation temperature, sonication, vacuum,
selective agent, selection pressure and time of application,
super-binary vectors with enhanced virulence, phenolic
compounds and addition of various antioxidants are
reported to increase transformation efficiency in many
recalcitrant plant species (Egnin et al. 1998; Frame et al.
2002; Olhoft et al. 2003; Qiushen et al. 2005; Opabode
2006; Dutt et al. 2011; Kumar et al. 2011). An ideal system
for the production of transgenic plants should be less-time
consuming, give high frequency of regeneration and enable
the elimination of transgenic chimeras. The direct repeti-
tive organogenesis from de-embryonated cotyledon (DEC)
explants of peanut, reported earlier by us, is ideally suited
for genetic transformation (Tiwari and Tuli 2008). The
reported regeneration protocol is being exploited in the
present study to develop genetically transformed individ-
uals with high frequency shoot formation. The cyclic-
regeneration from the DEC explants also facilitates to
apply continuous antibiotic selection on transformed tis-
sues to eliminate the possibility of transgenic chimeras.
Several reports have been published on genetic trans-
formation of peanut. In contrast to the earlier studies, the
present paper reports up to 81% transformation efficiency,
no chimeras, 100% survival in glass-house and less-time
(*3 months) for the development of healthy transgenic
peanut plants.
Materials and methods
Plant material, explant preparation and culture
conditions
The pods of high yielding peanut cultivar JL-24 exten-
sively cultivated in southern states of India (Maharashtra
and Karnataka), were obtained from the University of
Agricultural Sciences, Dharwad (Karnataka, India). Mature
dry seeds were surface-sterilized in 0.1% aqueous mercuric
chloride for 10 min, rinsed six to seven times with sterile
water and left soaked in sterile distilled water for 2–3 h
before removing the seed coat. Cotyledons were aseptically
separated and the zygotic embryos were removed. Coty-
ledons were sliced vertically to obtain the DEC explants.
Throughout the study, the cultures were incubated at
25 ± 2�C in 80 lmol photon m-2 s-1 light intensity with a
16/8-h light/dark period. The media were solidified with
agar. The pH was adjusted to 5.8 before autoclaving at
121�C for 20 min (Tiwari and Tuli 2008, 2009). All bio-
chemicals and media constituents, unless stated otherwise
were molecular biology/cell culture grade from Sigma
Chemical Company (St. Louis, MO, USA).
Effect of antibiotics on A. tumefaciens and shoot
induction
Disc-diffusion assay was performed to determine the
optimal concentration of augmentin (GlaxoSmithKline,
India) and cefotaxime (Lupin, India) required to eliminate
bacterial over growth. Whatman filter paper discs, 5 mm in
diameter were autoclaved and saturated with 15 lL of the
antibiotic taken alone or in combination. Both the antibi-
otics were tested at 100, 200, 300, 400 mg/L each. Com-
bination of cefotaxime (200 mg/L) along with varying
concentrations (100, 200, 300 and 400 mg/L) of augmentin
were also tested. A. tumefaciens culture (50 lL) OD600 of
1.6 was taken aseptically and spread on the surface of shoot
induction medium (SIM). The SIM contained MS salts
(Murashige and Skoog 1962), B5 vitamins (Gamborg et al.
1968), 100 mg/L myo-inositol, 30 g/L sucrose supple-
mented with 20 mg/L 6-benzylaminopurine (BAP) (for
initial 2 weeks) or 15 mg/L BAP (for the 3rd and
4th weeks). The pH was maintained at 5.8 and the medium
was solidified with 8 g/L agar (Tiwari and Tuli 2008). Four
discs in two replicates were placed on the SIM plates and
kept for 72 h at 28�C. Bacterial inhibition zone was cal-
culated as the distance in mm from the edge of the paper
disc to the edge of the bacterial growth area.
112 Plant Cell Tiss Organ Cult (2012) 109:111–121
123
The effect of cefotaxime and augmentin were also tested
on the shoot regeneration efficiency. Non-transformed
DEC explants were cultured on SIM supplemented
with different concentrations (0, 50, 100, 200, 400 and
800 mg/L) of cefotaxime or augmentin. The effect of
cefotaxime (200 mg/L) along with varying concentrations
(0, 50, 100, 200, 400 and 800 mg/L) of augmentin was also
checked. Shoot induction frequency was scored on explants
incubated on SIM for 4 weeks. These experiments were
performed with four replicates of 20 explants for each
treatment.
Minimal inhibitory concentration of hygromycin
The multiple shoot buds induced from non-transformed
DEC explants were incubated on shoot elongation medium
containing 3 mg/L BAP, 200 mg/L each of cefotaxime and
augmentin with the various concentrations (0, 5, 10, 20, 30,
40 and 50 mg/L) of hygromycin. Regeneration frequency
was scored after 6 weeks of culture on shoot elongation
medium (3 sub-cultures of 2 weeks duration each). The
data are based on four replicates of 20 explants for each
treatment.
Agrobacterium strain, binary vector and preparation
of Agrobacterium suspension
Two A. tumefaciens strains LBA4404 (Hoekema et al.
1983) and EHA101 (Hood et al. 1986), harbouring the
binary vector pIG121hm were used to optimize transfor-
mation. The T-DNA of pIG121hm contains nos promoter
driven neomycin phosphotransferase (nptII) gene,
CaMV35S promoter driven b-glucuronidase (uidA) gene
interrupted by a modified castor bean catalase intron and
CaMV35S promoter driven hygromycin phosphotransfer-
ase (hpt) gene (Ohta et al. 1990, Fig. 1).
A single Agrobacterium colony was inoculated in
50 mL YEP medium. The bacterial cultures were grown to
an OD600 of 1.4–1.6 at 28�C in a shaker at 200 rpm and
centrifuged at 5,000 rpm. The pellet was resuspended in
100 mL of liquid induction medium containing MS salts,
B5 vitamins, 100 mg/L myo-inositol, 20 g/L glucose and
100 lM acetosyringone (AS). The induction medium was
kept at 28�C for 30 min and used for explants infection.
Co-cultivation of explants with A. tumefaciens
The DEC explants were rinsed 3–4 times with half-strength
MS liquid medium (pH 5.6) containing 100 lM AS.
Agrobacterium infection was carried out in the induction
medium for 20 min at room temperature under continuous
shaking. The explants were blot dried on sterilized blotting
discs for 10–15 min and transferred to co-cultivation
medium consisting of MS salts, B5 vitamins, 100 mg/L
myo-inositol, 30 g/L sucrose, 20 mg/L BAP, pH 5.8 and
solidified with 6 g/L agar. After co-cultivation, the
explants were rinsed 5–6 times with autoclaved double
distilled water and blot dried to remove excess bacterial
suspension. Co-cultivation was done in dark and various
factors were evaluated on the basis of transient expression
of the reporter gene.
Optimization of factors enhancing transient
transformation efficiency
Factors were optimized for the efficiency of transformation
on the basis of transient expression of the transgene uidA
using DEC explants. Different factors were analysed in the
following order, co-cultivation duration (1–5 days) at 21�C
with and without 100 mg/L of antioxidants (L-cysteine,
sodium thiosulfate, dithiothreitol, L-glutathione and L-
ascorbic acid), co-cultivation for 5 days at 21�C with and
without different concentrations (100, 200, 300 and
400 mg/L) of the antioxidants, Agrobacterium strains
(EHA101 and LBA4404), co-cultivation temperature (18,
21, 25�C), duration (4–7 days) and explant characteristics
(incised and non-incised). Antioxidants were added in
autoclaved co-cultivation medium. The transient transfor-
mation frequency was calculated on the basis of histo-
chemical b-glucuronidase enzyme (GUS) assay and
recorded as percentage of the total number of co-cultivated
explants. In each experiment (repeated three times), 10
DEC explants were co-cultivated.
RBnptII Tnos Tnos TnosP35S P35S
HindIIISalI Sac I
SalIBamHIEcoRI
LBPnos uidA hpt
Xba I
Intron
0.19kb 1.2kb 0.26kb 0.8kb 0.2kb 2.0kb 0.26kb 0.8kb 1.1kb 0.26kb
Fig. 1 Binary vector pIG121hm. RB right border, LB left border,
Pnos and P35S are nos and CaMV35S promoters, Tnos nos terminator,
nptII neomycin phosphotransferase gene, uidA b-glucuronidase gene
and hpt hygromycin phosphotransferase gene. The gap between XbaI
and SalI indicates the position of intron in uidA. Restriction sites
HindIII, XbaI, SalI, SacI, EcoRI and BamHI are shown
Plant Cell Tiss Organ Cult (2012) 109:111–121 113
123
Selection of transformants and multiplication
The optimized conditions were applied to 120 DEC
explants for the development of stable transgenic plants.
Multiple shoot buds induced on SIM were excised and
cultured on the elongation medium containing cefotaxime
(200 mg/L), augmentin (200 mg/L) and hygromycin
(40 mg/L). At 2 weeks interval, multiple shoot buds and
grown-up shoots were sub-cultured onto fresh medium.
The proliferated shoot buds were maintained for several
months on MS medium supplemented with BAP (3 mg/L),
cefotaxime (200 mg/L) and hygromycin (20 mg/L).
Transgenic plant development and growth conditions
After 3 cycles (2 weeks of each), 3–5 cm grown-up shoots
were excised and transferred onto MS medium supple-
mented with 1 mg/L a-naphthalene acetic acid and
200 mg/L cefotaxime to induce roots. To check the sur-
vival and healthy growth of transgenic plants, parallel
experiments were performed to graft the transgenic shoots
onto non-transformed germinated seedlings. Ten to fifteen
days old non-transformed seedlings, germinated on sterile
filter paper bridges in test tubes containing half-strength
MS liquid medium, were decapitated and a vertical incision
made from the top end of rootstocks. The bottom end of
transgenic shoot (height of 3–5 cm) was cut into a wedge
(‘V’) shape, and placed ex-vitro into the incision of non-
transformed rootstock. The plantlets were acclimatized in
irrigated plastic pots containing Soilrite mix (Keltech
Energies Ltd., Bangalore, India) at 25 ± 2�C with a 16/8-h
light/dark period. For the next 15 days, acclimatization
hoods were gradually raised to decrease the humidity. After
3 weeks, the plants were planted in sandy loam soil and
kept in glass-house till maturity.
Histochemical GUS expression analysis
Histochemical GUS assay was performed to determine the
uidA transgene expression (Jefferson et al. 1987). The tis-
sues incubated with X-Gluc solution at 37�C for 24 h in
dark were washed with sterile water. Subsequently, chlo-
rophyll was removed by soaking the tissue in 70% ethanol
and finally preserved in absolute ethanol and photographed.
The uidA gene in pIG121hm is interrupted by a modified
castor bean catalase intron, thus the GUS activity was
derived only from plant protein synthesis machinery.
Polymerase chain reaction (PCR)
Total genomic DNA was isolated from fully expanded
2nd and 3rd node leaves from glass-house-grown plants
following the DNeasy Plant Maxi kit (Qiagen) as per
manufacturer’s instructions. Genomic DNA was quantified
by Fluorometer DyNA QuantTM 200 (Hoefer, Pharmecia
Biotech). Putative (T0) transgenic plants were screened for
the presence of the uidA and hpt genes by PCR. The bac-
terial virG gene specific primers were used to exclude any
false positives due to persistent Agrobacterium cells in T0
plants. The primer pair used for the amplification of the
450 bp virG amplicon was (Forward) 50-CTG GCG GCA
AAG TCT GAT-30 and (Reverse) 50-TGT CGT AAA CCT
CCT CGT-30. The uidA primers (Forward) 50-ATG CGG
ACT TAC GTG GCA AAG GA-30 and (Reverse) 50-GCC
AAC GCG CAA TAT GCC TTG-30 amplified 800 bp from
the uidA gene, while the hpt primers (Forward) 50-CTA
TTT CTT TGC CCT CGG ACG-30 and (Reverse) 50-ATG
AAA AAG CCT GAA CTC ACC G-30 amplified the
1,027 bp hpt gene. Each PCR reaction was performed in
25 lL volume, consisting of 19 reaction buffer, 2.0 mM
MgCl2, 400 lM dNTPs, 10 pmol of each primer, 1.5 units
of Taq DNA polymerase, and 100 ng of plant genomic
DNA. The amplification reactions were carried out in a MJ
Research PTC 200 Peltier thermal cycler under the fol-
lowing conditions: 94�C for 5 min followed by 30 cycles at
94�C for 1 min, 58�C (hpt/uidA) for 1 min, 72�C for 1 min
30 s with a final extension at 72�C for 5 min. The PCR
product was fractionated by electrophoresis on a 0.8%
agarose gel, detected by ethidium bromide staining and
photographed under ultraviolet light.
Southern blot hybridization analysis
PCR positive five transgenic and one non-transformed
control plants were subjected to Southern blot hybridiza-
tion analysis. About 15–20 lg of genomic DNA was
digested with HindIII, separated by electrophoresis on a
0.8% agarose gel and alkali blotted onto positively charged
nylon membrane (Hybond N?, Amersham Life Sciences,
USA) by vacuum transfer. The blots were probed with
800 bp of the uidA coding PCR amplified sequence. The
PCR product was radiolabelled with a a P32-dCTP and
added in hybridization solution. As there was no HindIII
cleavage site within the selected uidA probe, the number of
hybridising fragments indicated the number of insertion
events. Hybridisation was carried out at 65�C for 18 h. The
blot was washed for 10 min in solution containing 29
saline sodium citrate (SSC) buffer ? 0.1% sodium dodecyl
sulfate (SDS) at room temperature, followed by 20 min in
19 SSC ? 0.1% SDS at 65�C and 20 min in 0.1%
SSC ? 0.1% SDS at 65�C. After washing, the blot was
exposed to Fuji screen for 24 h and scanned on phospho-
imager (Molecular Imager FX; Bio-Rad, Hercules, CA).
114 Plant Cell Tiss Organ Cult (2012) 109:111–121
123
Transgene segregation analysis
Transgenic progenies from the selected five T0 lines were
subjected to hygromycin-based segregation analysis.
Transgenic (T1) seeds were sterilised and soaked overnight
in sterile water. The seeds were germinated on sterile filter
paper bridges in test tubes containing half-strength MS
liquid medium with 10 mg/L hygromycin for 15 days at
25 ± 2�C under 16/8-h light/dark period. The seedlings
germinating in the presence of hygromycin were scored to
analyse the segregation of hpt transgene in the progeny.
Hygromycin-based leaf assay
The transgenic nature was further confirmed through leaf
inoculation from seedlings of T1 transgenic line on MS
basal medium containing 10 mg/L hygromycin. After
15–20 days of incubation on this medium, hygromycin
positive leaves were visually selected and scored. Hygro-
mycin resistant seedlings were transferred to glass-house
for further growth.
Results
Agrobacteria elimination
The concentration and combinations of the antibiotics
tested by disc-diffusion assay revealed that augmentin
([100 mg/L) was more effective and showed more than
20 mm bacterial inhibition area (Fig. 2). The most effec-
tive combination however was cefotaxime (200 mg/L) and
augmentin ([100 mg/L), where more than 25 mm inhibi-
tion zone was observed (Fig. 2). Cefotaxime alone was
not very effective and showed smaller inhibition zone
(\25 mm) even at 400 mg/L concentration.
Effect of cefotaxime and augmentin concentrations
on shoot induction
Shoot induction frequency of DEC explant varied,
depending upon the concentration of cefotaxime and aug-
mentin in the media. Cefotaxime and augmentin alone or
combination (up to 200 mg/L) showed [90% shoot
induction which was similar to control (Fig. 3). Consider-
able inhibitory effect on regeneration frequency started
from 400 mg/L of augmentin and 800 mg/L cefotaxime,
where the frequency of shoot induction decreased gradu-
ally. A combination of cefotaxime (200 mg/L) with aug-
mentin (200 mg/L) showed[90% shoot induction (Fig. 3).
This combination of the antibiotics was also effective
in eliminating overgrowth of Agrobacterium. Therefore,
200 mg/L cefotaxime along with 200 mg/L augmentin was
used for Agrobacterium elimination without effecting high
regeneration frequency of the explants.
Effect of hygromycin on plant regeneration
The regeneration frequency decreased gradually at higher
hygromycin (10–50 mg/L). Addition of [30 mg/L hygro-
mycin to the elongation medium, shoot regeneration turned
to stop (Fig. 4). Hygromycin had a strong toxic effect on
cultures at 50 mg/L. After 10–15 days of proliferation, the
regenerated shoots turned brown (necrotic) and died.
Despite good regeneration frequency at 30–40 mg/L, the
induced shoots stopped further growth and ultimately died.
The shoots regenerated on the elongation medium supple-
mented with 20–30 mg/L hygromycin were weak, had
white leaves and died after a short period of time. There-
fore, 40 mg/L hygromycin was suitable as a selective
antibiotic for 6–7 weeks in the transformation experiments,
0
5
10
15
20
25
30
35
40
45
100 200 300 400
Concentration (mg/L)
Zon
e of
inhi
bitio
n (m
m)
CefotaximeAugmentin
Augmentin*
Fig. 2 Disc-diffusion assay. Agrobacterium strain EHA101 chal-
lenged with antibiotics. Bacterial growth is represented as zone of
inhibition (mm). Data are means of 2 replicates ± SD. {Augmentin*
represents the combination of cefotoxime (200 mg/L) with augmentin
(100–400 mg/L)}
Fig. 3 Effect of the three antibiotics on shoot induction frequency
of non-transformed DEC explants on SIM. Data presented in
percent frequency of four replicates ± SD. {Augmentin* represents
the combination of cefotoxime (200 mg/L) with augmentin
(0–800 mg/L)}
Plant Cell Tiss Organ Cult (2012) 109:111–121 115
123
while reduced concentration up to 20 mg/L was suitable
for long term maintenance of the transgenic culture.
Effect of duration of co-cultivation and antioxidants
The transient transformation frequency of DEC explants
co-cultivated with and without antioxidants (100 mg/L) for
1–5 days is shown in Fig. 5a. The highest ([75%) trans-
formation efficiency was recorded after 5 days co-cultiva-
tion of explants in the presence of 100 mg/L L-cysteine.
The optimized co-cultivation duration (5 days) was used to
check the effect of different concentrations (100–400 mg/L)
of antioxidants (Fig. 5b). The 100 and 200 mg/L
L-cysteine gave better (C55%) results as compared to the
other treatments. However, 100 mg/L L-cysteine showed
higher efficiency at 70% transformation frequency
(Fig. 5b).
Effect of co-cultivation temperature and period
The transient transformation frequency of explants
co-cultivated on SIM containing 100 mg/L L-cysteine at
different temperatures (18, 21 and 25�C) for 4–7 days is
shown in Fig. 5c. Highest frequency (81%) was recorded at
21�C with 5 days co-cultivation. Co-cultivation for more
than 5 days resulted in Agrobacterium growth around the
explants. The transformation frequency decreased drasti-
cally after 7 days co-cultivation. Explants also became
necrotic and turned brown.
Effect of bacterial strains, explants characteristics
and temperature
Agrobacterium strains EHA101 and LBA4404 co-culti-
vated for 5 days with DEC explants (incised and non-
incised) at 21 and 25�C showed GUS expression (Fig. 5d).
Incised explants co-cultivated for 5 days showed hyper-
sensitivity, resulting in tissue death. Nearly 1.5 times
higher GUS expression was recorded with non-incised
explants co-cultivated at 21�C with EHA101 as compared
to LBA4404 (Fig. 5d).
0
20
40
60
80
100
Hygromycin (mg/L)
Shoo
t reg
ener
atio
n (%
)
0 5 10 20 30 40 50
Fig. 4 Influence of hygromycin level on regeneration frequency of
non-transformed DEC explants grown on shoot elongation medium.
Data present percent frequency in four replicates ±SD
0
20
40
60
80
100
Non-incised Incised Non-incised Incised
EHA101 LBA4404
GU
S ex
pres
sion
21°C
25°C
d
0
20
40
60
80
100
GU
S ex
pres
sion
Antioxidants
0
100mg/L
200mg/L
300mg/L
400mg/L
b
0
20
40
60
80
100
1 2 3 4 5
Co-cultivation period (Days)
Negative controlNo treatmentL-cysteineSodium thiosulfateDithiothreitolL-glutathioneL-ascorbic acid
a
0
20
40
60
80
100
4 5 6 7
GU
S ex
pres
sion
Co-cultivation period (Days)
18°C
21°C
25°C
c
% E
xpla
nts
with
% E
xpla
nts
with
% E
xpla
nts
with
GU
S ex
pres
sion
% E
xpla
nts
with
Fig. 5 Effect of different factors on the transient transformation of
the DEC explant. The expression frequency examined by histochem-
ical GUS assay was scored visually. Bars represent % frequency of
transformation ± SD. a Effect of co-cultivation duration (1–5 days)
with (100 mg/L each) and without antioxidants. b Effect of
antioxidants at different concentrations (0–400 mg/L), following
5 days of co-cultivation. c Effect of co-cultivation duration
(4–7 days) and temperature (18, 21 and 25�C). d Effect of bacterial
strains (EHA101 and LBA4404), explant characteristics (Non-incised
and incised) and co-cultivation temperature (21 and 25�C)
116 Plant Cell Tiss Organ Cult (2012) 109:111–121
123
Development of transgenic plants
The optimized conditions for transformation (strain
EHA101 of A. tumefaciens, 5 days co-cultivation, 21�C,
SIM containing 100 mg/L L-cysteine and non-incised
explant) were used to infect the DEC explants. Explants
cultured on SIM for 4 weeks showed 91% frequency of
shoot induction. Induced multiple shoot buds cultured on the
elongation medium containing hygromycin showed 80%
survival of transgenic shoots. Transgenic shoot buds were
continuously multiplied and maintained for several months
on the selection medium (Fig. 6a). Elongated shoots took
nearly 2 months to develop healthy lateral roots on the
rooting medium. Proliferation of compact callus at the
crown (root–shoot junction) was noticed in some plantlets
(Fig. 6b). Acclimatized in vitro rooted plants showed 62%
survival after plantation in the glass-house. The grafted
transgenic plantlets kept for 3 weeks in acclimatization
hoods looked healthy (Fig. 6c) and showed 100% survival
in glass-house conditions. The acclimatized plants grew
normally (Fig. 6d) and produced flowers and pods within
3–4 months. The number of pods developed from T0
transgenic plants was low as compared to that from the non-
transformed plants. However, in subsequent generations, the
transgenic plants performed as well as the non-transformed
plants (data not shown). The histochemical GUS expression
was uniformly observed as dark blue colour in the trans-
formed leaf and shoot while, non-transformed (control)
tissues did not show the expression of GUS (Fig. 6e, f).
Molecular analyses of transformants
The PCR analysis of genomic DNA of eight uidA T0
transgenic plants obtained as independent transformation
events, showed amplification of the predicted 800 bp uidA
as well as 1,027 bp hpt fragments of the transgenes
(Fig. 7a). The positive control (PCR with plasmid
pIG121hm) also gave similar size amplicons (lane no. 4).
The bacterial virG gene specific primers did not show
amplification of the 450 bp amplicon. This was performed
to eliminate the detection of false positives due to persis-
tent contaminating Agrobacterium cells in T0 plants. The
results of Southern blot hybridization, establishing geno-
mic integration of uidA gene in five T0 transgenic plants,
are shown in Fig. 7b. Different sizes of the inserts estab-
lished that the transgene insertion was at different loci in
different transgenic plants. Among the selected transgenic
plants, G1/1, G2/1, G3/1 and G4/1 (lane nos. 1, 2, 3 and 4)
revealed single-copy insertion, while G5/1 (lane no. 5) had
two copies of uidA (Fig. 7b).
Gus +ve Control
e
db ca
Gus +ve
f
Control
Fig. 6 Transgenic peanut plant
regeneration and histchemical
GUS analysis. a Shoot
elongation and multiplication on
hygromycin selection medium.
b Transformed plantlets with
lateral roots on the rooting
medium. c Grafted transgenic
shoots onto non-transformed
healthy rootstock. d Tissue
culture raised acclimatized
healthy transgenic peanut plant.
e The blue color showing the
expression of uidA reporter gene
in leaf and f shoot of T0
transgenic plants analyzed by
histochemical GUS assay
Plant Cell Tiss Organ Cult (2012) 109:111–121 117
123
Transgene inheritance analyses
The phenotypic segregation of hygromycin resistance in
progeny was tested by Chi-square test (Supplementary
Table 1). The T1 progeny of 4 transgenic lines (G1/1, G2/1,
G3/1 and G4/1) exhibited a segregation ratio close to 3:1,
suggesting a single integration event. The fifth transgenic
line (G5/1) suggested multiple integrations.
Hygromycin-based leaf assay further confirmed trans-
genic nature of the plants. Primary leaves from positive
seedlings were inoculated on MS basal medium containing
hygromycin at 10 mg/L for 2 weeks, remained green while
non-transformed control leaves turned brown and finally
died.
The flow chart summarized the optimized protocol for
the developments of transgenic peanut plants are shown in
Fig. 8.
Discussion
The present study describes a rapid and efficient protocol
for the development of escape-free transgenic peanut
plants. A number of potentially useful genes have been
introduced into peanut genome either by A. tumefaciens
(Li et al. 1997; Sharma and Anjaiah 2000; Dodo et al.
2008; Tiwari et al. 2008, 2011) or biolistic (Singsit et al.
1997; Magbanua et al. 2000; Livingstone et al. 2005;
Athmaram et al. 2006; Niu et al. 2009) mediated genetic
transformation. The development of less-time consuming,
fertile and more efficient protocol which could prevent
regeneration of possible non-transformed plants and chi-
meras remain important gaps to be overcome in peanut
transformation. Kanamycin-based selection systems do
not eliminate the non-transformed cells completely
(McKently et al. 1995; Cheng et al. 1996; Sharma and
800bp
1027bp
a G1/1 G2/1 G3/1 G4/1 G5/1 G6/1 G7/1 G8/1
1 2 3 4 5 6 7 8 9 10 11 12
21.2kb5.1kb/3.5kb2.0kb/1.9kb1.5kb/1.3kb0.9kb/0.8kb0.56kb
(uidA)
(hpt)21.2kb5.1kb/3.5kb2.0kb/1.9kb1.5kb/1.3kb0.9kb/0.8kb0.56kb
b
21.2kb
5.1kb
4.2kb
3.5kb
G1/1 G2/1 G3/1 G4/1 G5/1 1 2 3 4 5 6
Fig. 7 Detection of transgene
integration in genomic DNA
isolated from transgenic peanut
plants. a PCR analysis of
transgenic plants for the b-
glucuronidase (uidA) and
hygromycin phosphotransferase
(hpt) genes. Agarose gel
electrophoresis shows Lane 1lambda DNA HindIII & EcoRI
digested markers, Lane 2without template (DNA), Lane 3non-transformed plant, Lane 4positive control (pIG121hm
vector), Lane 5–12: transgenic
plants. The arrows show the
positions of the expected 800
and 1,027 bp amplicons for the
uidA and hpt genes,
respectively. b Southern blot
analysis of HindIII digested
genomic DNA from T0
transgenic plants. The blot was
hybridized with a P32-dCTP
labeled 800 bp PCR amplified
fragment of uidA gene. Lane1–5 transgenic plants, Lane 6non-transformed plant
118 Plant Cell Tiss Organ Cult (2012) 109:111–121
123
Anjaiah 2000; Dodo et al. 2008) and could produce
escapes or chimeric plants. The hygromycin-based step-
wise antibiotic selection regime optimized here resulted in
the recovery of a large number of putative transformants
in a short-period. This could also reduce the probability of
inducing somaclonal variation (Olhoft et al. 2003). The
short transformants-regeneration cycle has also additional
advantage to improve the recovery of fertile transgenic
peanut as reported by Dodo et al. (2008). Low concen-
tration of hygromycin selection applied in this study gave
transformed multiple shoot buds that could be maintained
as continuous source of shoot primordia without devel-
oping of transgenic chimeras.
Effective elimination or suppression of Agrobacterium
overgrowth without inhibiting the regeneration potential of
the transformed cells is essential to successful recovery
of healthy transgenic plants. Antibiotics like, cefotaxime,
carbenicillin, timentin and augmentin are most commonly
used to counter select Agrobacterium (Antunez de Mayolo
et al. 2003; Ieamkhang and Chatchawankanphanich 2005;
da Silva Mendes et al. 2009). These antibiotics have a
broad spectrum of activity against bacteria and a low tox-
icity to eukaryotes. The present study demonstrates that the
optimized concentration of augmentin along with cefo-
taxime not only effectively eliminated bacterial growth, but
also maintained the high frequency of multiple shoot
induction.
Several parameters optimized here established up to
81% efficiency of genetic transformation in peanut. The
mature dry seeds used for preparing the DEC explant in the
present study confer an advantage of storage, easy handling
and round the year availability. The cut surface of DEC
explants provides competent cells for both Agrobacterium
infection and shoot induction. It also helped to develop
multiple independently transformed shoots per explants.
Co-cultivation temperature and duration considerably
influence T-DNA delivery (Uranbey et al. 2005; Opabode
2006). The vir genes play important role in T-DNA
delivery in plant tissue and their expression depends on the
optimal temperature (Uranbey et al. 2005). The present
study showed that co-cultivation of DEC explants at 21�C
for 5 days significantly improved transient transformation
frequency. The results also demonstrated that A. tumefac-
iens the strain EHA101 was superior to LBA4404 for the
infection of DEC explants. The high level of virG
expression in hyper-virulence strain EHA101 may be the
basis for efficient transformation. This observation is in
line with earlier reports on the superiority of EHA101 in
peanut transformation (Mckently et al. 1995; Egnin et al.
1998).
The cut surface of DEC explants and Agrobacterium
infection may induce the production of free radicals
which result in oxidative stress. This limits the growth
potential of Agrobacterium infected plant cells in
co-cultivation medium. Antioxidants protect cells from
oxidative stress by scavenging free radicals (Dutt et al.
2011). In the present study, several antioxidants were
analysed for the co-cultivation of explants. The inclusion
of 100 mg/L L-cysteine for 5 days at 21�C significantly
enhanced (up to 81%) transient transformation, following
agroinoculation. The transient expression in optimized
conditions showed around 4.5-fold increase in transfor-
mation efficiency. L-cysteine has been used successfully
to minimize oxidative damage to the host cells during
co-cultivation. It has been reported to enhance transfor-
mation efficiency in several important crop plants (Frame
et al. 2002; Olhoft et al. 2003; Kumar et al. 2011). For
instance, Frame et al. (2002) reported co-cultivation of
immature zygotic embryos of maize on medium supple-
mented with 400 mg/L L-cysteine, increased T-DNA
delivery to embryogenic-scutellum cells. They suggested
that L-cysteine could minimize cell death caused by the
hypersensitive response. This helped in post-infection
survival of embryogenic competent cells, thus increased
stable transformation efficiency. Olhoft et al. (2003)
suggested co-cultivation of cotyledonary node explants of
soybean in the presence of mixtures of the thiol com-
pounds (L-cysteine, sodium thiosulfate, and dithiothreitol).
These combined with hygromycin based selection strategy
significantly increased the regeneration of transformed
soybean plants, with no chimeras. Kumar et al. (2011)
reported a 2.9-fold increase in sorghum transformation
Mature dry seeds
Seeds sterilization (0.1% HgCl2) for 10 min and pre-soak in sterile water for 2-3 h
De-embryonated cotyledon (DEC) explants preparation
Rinse with half-strength MS liquid medium containing 100 µM acetosyringone (AS)
Incubate in Agrobacterium induction medium containing 100 µM AS for 20 min
Blot dry and place on co-cultivation medium containing 100 mg/L L-cysteine
Incubate in dark at 21oC for 5 days
Wash with sterile water for 5-6 times
Blot dry and place explants on proximal end down to shoot induction medium (SIM) containing augmentin and cefotaxime (200 mg/L each) for 4 weeks
Induced shoot buds transfer to selective shoot elongation medium containing augmentin and cefotaxime (200 mg/L each) and hygromycin (40 mg/L)
(3 cycles, each 2 weeks)
Multiple shoot buds maintained on shoot elongation medium
containing cefotaxime (200 mg/L) and hygromycin (20 mg/L)
Grafting of transgenic shoots onto non-transformed
rootstocks
In-vitro root development
Fig. 8 Flow chart of the transformation protocol for the development
of transgenic peanut plants
Plant Cell Tiss Organ Cult (2012) 109:111–121 119
123
efficiency in the presence of L-cysteine during co-culti-
vation of immature embryo explants.
Poor in vitro rooting, low fertility, low recovery and low
survival following acclimatization of transformed plants
are the major constraints on genetic transformation of
peanut (Li et al. 1997; Magbanua et al. 2000; Livingstone
et al. 2005; Anuradha et al. 2006; Dodo et al. 2008). The
proliferation of compact callus was noticed by us, at the
crown (root–shoot junction) position of some of the
plantlets when inoculated in the rooting medium (Fig. 6b).
The roots growing from this area were not attached directly
to the shoot. Such rooted plantlet failed to survive on
transfer to soil. The compact callus could interrupt the flow
of nutrients, resulting in low survival of in vitro rooted
transgenic plants. The problem remained same even when
the transgenic shoots were rooted in different hormone
regimes. To eliminate the possibility of compact callus,
shoots were sub-cultured 2–3 times (each for 3 weeks) on
the rooting medium. The compact callus at the crown
position was removed during each sub-culture. This
resulted in healthy roots without compact callus and
improved the survival of rooted plants (data not shown).
Grafting of in vitro grown shoots onto healthy rootstock
gave an excellent alternative to enhance the survival of
regenerating and transgenic plantlets in the soil. The suc-
cess of in vitro methods in many crops including wild
peanut species (Still et al. 1987), cotton (Luo and Gould
1999; Jin et al. 2006), chickpea (Krishnamurthy et al. 2000;
Chakraborti et al. 2006), citrus (Ballester et al. 2008) and
safflower (Belide et al. 2011) has been reported to increase
by grafting. Following grafting, grown plants in the present
study took around 3–4 weeks to acclimatize in soil-pots
and showed 100% survival in the glass-house. The most
critical factor for successful grafting was the height of
scion (3–5 cm) and age of rootstock (10–15 days).
The histochemical, PCR, Southern blot, gene segrega-
tion and hygromycin-leaf assays established stable inte-
gration of the transgene into peanut genome. Despite being
very simple, the leaf assay was also very efficient in
identifying the transgenic peanut plants.
In conclusion, the optimized genetic transformation
protocol describes here is more efficient, reliable and time-
saving as compared to the previous reported protocols. The
genotype-independent cyclic-regeneration of shoots from
the DEC explants applied in this study to an elite cultivar
(JL-24) could be applicable for genetic transformation of a
number of agronomically important cultivars of peanut.
Acknowledgments The authors express their gratitude to National
Botanical Research Institute, Council of Scientific and Industrial
Research, Lucknow, India for providing research facilities and to the
Department of Science and Technology, Government of India, for a
JC Bose Fellowship to Rakesh Tuli.
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