Advances in Bioscience and Biotechnology, 2010, 1, 409-416 ABB
doi:10.4236/abb.2010.15054 Published Online December 2010 (http://www.SciRP.org/journal/abb/).
Published Online December 2010 in SciRes. http://www.scirp.org/journal/ABB
Rapid regeneration of stable transformants in cultures of
potato by improving factors influencing
Agrobacterium-mediated transformation
Bipasha Chakravarty, Gefu Wang-Pruski
Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, Canada.
Received 4 August 2010; revised 25 August 2010; accepted 8 September 2010.
ABSTRACT
An efficient and rapid Agrobacterium tumefaciens-
mediated transformation protocol was developed to
generate activation-tagged mutant lines with the aim
of large-scale functional analysis of the potato ge-
nome. The explants were inoculated with an Agro-
bacterium strain harboring the binary plasmid
pSKI074 containing four CaMV 35S enhancers in the
T-DNA region which activates the downstream genes
in the host plant after its integration. Various pa-
rameters investigated to increase transformation ef-
ficiency were the type and age of explant, cultivar,
hormone combinations, preculture of explants, pe-
riod of co-cultivation with bacteria and concentration
of bacterial cultures used for transformation. Stem
explants from 5 week old plantlets of cv. Bintje which
had undergone phytohormone pretreatment for 4
days, inoculation with diluted bacterial co ncentration
of OD600 = 0.2 containing acetosyringone followed by
2 days of co-cultivation and selection in media with
IAA and trans-zeatin all helped in greatly improving
the transformation efficiency. The total time required
from infection to rooted shoots was 6-7 weeks. Initial
evidence for stable integration and expression of the
transgenes by PCR analysis showed that over 93% of
the regenerated lines were transgenic and this was
confirmed by Southern hybridization.
Keywords: Bacterial Concentration; Cultivar; Explant;
Potato T ransformation; Preculture
1. INTRODUCTION
Although there are numerous regeneration and transfor-
mation protocols reported in potato, many of these have
long regeneration periods and low frequecies [1-3]. In
many reports an intermediate callus phase occurs [4]
which increases regeneration time and also promotes
dedifferentiation leading to somaclonal variants since
potato is extremely sensitive to somaclonal variation in
culture [5]. Moreover, transformation protocols in potato
are genotype-dependent, limiting their usage and making
practical applications not easily adaptable to all geno-
types [6-8]. Previous reports indicated that the compe-
tence of plant cells for transformation via Agrobacte-
rium-mediated gene transfer could be modified by ad-
justing physical conditions of the explant or media
composition just prior to or during T-DNA delivery [9].
This paper describes the efforts on assessing and im-
proving previous protocols and also selection of the best
cultivar and explant to develop a rapid and efficient
transformation and regeneration system in potato. The
aim of the present investigation was thus to develop a
protocol that could produce large numbers of potato
transformants with stable integration of the transgenes
within a short time. This system was used to generate a
large number of activation-tagged mutant lines with the
goal of large-scale functional analysis of the potato ge-
nome.
Using several cultivars and explants, the effectiveness
of various factors that increase competence for trans-
formation in recalcitrant explants or genotypes such as
phytohormone pretreatment, hormone combinations in
the regeneration medium, increasing period of co-culti-
vation, concentration of bacterial cultures, and age of
explants was explored to select the best conditions for
rapid transformation and regeneration in potato. The
stable integration of transgenes in the transformed lines
was analyzed by PCR and further confirmed by Southern
hybridization.
2. MATERIAL AND METHODS
2.1. Stock Plants and Explant
Five different cultivars of potato, Solanum tuberosum L:

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 410
Superior, Bintje, Atlantic, Shepody and Russet Burbank,
were evaluated for their regeneration ability. Since
transformation rate greatly depends on the cultivars,
these five cultivars which are widely grown in Atlantic
Canada were assessed for their ability to give the best
results in our study. Sterile stock plantlets of potato were
initially provided by New Brunswick Department of
Agriculture and Fisheries, and the explants (4-6 mm)
were cut from internodal stem and leaf sections. The
stock plants were maintained in plantlet growth medium
(PGM) containing MSMO salts (MS minimal organics,
Sigma, Oakville, ON) with 3% sucrose, pH of 5.7 and
solidified with 0.22% gelrite (Sigma, Oakville, ON).
Cultures were incubated under a 16/8 h light/dark pho-
toperiod at a temperature of 20 ± 2℃.
2.2. Agrobacterium Strain and Culture
The Agrobacterium tumefaciens strain GV3101 harbor-
ing the plasmid vector pMPRK90 was obtained from the
Salk Institute, Cologne. The plasmid pSKI074 from E.coli
JM109 strain was introduced into the Agrobacterium
cells by electroporation. The T-DNA construct contained
four 35S enhancers as well as the nptII gene for kana-
mycin resistance. It was grown on YEB medium con-
taining 5 mg.l-1 beef extract, 1 mg.l-1 yeast extract, 1
mg. l -1 peptone, 5 mg.l-1 sucrose and 10 mg.l-1 MgSO4.7
H2O, pH of 7.3. Antibiotics used for selection of the
plasmid in bacteria were 100 mg.l-1 ampicillin and 50
mg. l -1 gentamycin. Bacterial suspensions of transformed
Agrobacterium colonies initiated from –80℃ glycerol
stocks were derived from overnight cultivation of single
colonies in liquid YEB media at 28℃ on a rotary shaker
at 200 rpm. Cultures were centrifuged at 3000 rpm for
10 min to pellet the cells, resuspended and diluted in
three volumes to adjust cell density to OD600 = 0.2, 0.4,
0.6 for inoculation of the explants.
2.3. Preculture and Co-Cultivation
Explants were excised from 4-8 week old sterile plant-
lets and precultured on callus growth medium (CGM)
containing MSMO salts, 2% glucose, 0.02% L-glutamine,
0.04% adenine sulphate, 0.05% of 2-N-morpholinoethane
sulphonic acid (MES) and 0.05% of polyvinyl pyrroli-
done (PVP) with a pH of 5.7 and solidified with 0.22%
gelrite. The phytohormones: trans-zeatin, IAA, NAA,
2,4-D, BAP and zeatin riboside were filter sterilized and
added after autoclaving the medium in five different
combinations and concentrations as described in Table 4.
Preculturing was carried out for 0-6 days in CGM con-
taining the auxins and cytokinins for callusing and re-
generation. After preculture, explants were immersed in
infection medium (IM) containing MSMO salts, 3%
sucrose, 0.05% MES, 2% mannitol with pH of 5.5. To
this medium 0.148M acetosyringone was added and dif-
ferent volumes of bacterial suspension were adjusted for
OD600 to 0.2-0.6. The explants were immersed in IM for
2-5 min, blotted dry on sterile filter paper and placed on
CGM for co-cultivation. Bacterial co-cultivation was
performed at 20 ± 2℃ in the dar k fo r 2- 3 days.
2.3. Selection and Regeneration of
Transformants
The explants incubated with bacteria were rinsed thor-
oughly with sterile distilled water containing 300 mg.l-1
claforan (Aventis Pharma Inc. Canada) to prevent further
bacterial growth, blot-dried and transferred to callus se-
lection medium (CSM) which was similar to CGM but
with addition of the antibiotics kanamycin (100 mg.l-1)
and claforan (300 mg.l-1) for selection of transformed
regenerants. Regenerating explants were transferred
from CSM after 3-4 weeks to shoot growth medium
(SGM) containing the same composition as CSM but
with absence of auxin for further growth and elongation
of shoots. Shoots with 2-3 leaflets were cut out and
placed for rooting on the medium without hormones
(PGM) and containing 300 mg.l-1 claforan. All cultures
were maintained under 16 h photoperiod at a light inten-
sity of 100 μE m-2s-1 and a temperature of 20 ± 2℃.
Each experiment was performed with 100 explants with
20 explants contained in each Petriplate. The total num-
ber of explants showing callusing, shoot-bud regenera-
tion and total regenerated plantlets from each experiment
using 100 explants was recorded.
2.4. PCR Analysis and Southern Hybridization
Transformed bacterial colonies used for the infection of
explants were subjected to PCR analysis after 3 days
growth in YEB medium just prior to infection. Colony
PCR of bacterial colonies was carried out following a
standard method [10]. The presence of the plasmid was
detected by PCR amplification of a 450bp fragment us-
ing forward FRED primer (Sequence 5’- GCG TGG
CTT TAT CTG TCT TTG TAT TG - 3’ ) and revers e R E D
primer (Sequence 5’- GGC CTA CTT TAA TTG CTT
CCA GTG TTA -3’) to confirm the presence of the
plasmid pSKI074. PCR analysis was performed in a
BioRad iCycler and the PCR product was visualized on
an ethidium bromide stained agarose gel. Since the en-
hancers are very unstable, fresh plates were streaked
from –80℃ stock for each experiment. The putative
transformants were also subjected to PCR analysis by
extracting the total genomic DNA of individual plants
following the CTAB extraction method [11] and con-
firming the presence of the plasmid. To ascertain that
amplification is caused by the plasmid and not endoge-

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 411
nous Agrobacterium colonies, the transformed plantlets
were also subjected to PCR using virG primer which
amplifies only the virulence region of the bacteria. For
Southern blot analysis, 1-2 μg of total genomic DNA
isolated from the plantlets (see method above) was di-
gested by the restriction enzyme EcoRI, for 16 h at 37℃
before separation by electrophoresis on a 0.7% (w/v)
agarose gel at 50 V for 6 h. The enzyme EcoRI has two
cutting sites in the T-DNA at 8750 and 9740bp releasing
a 990 bp fragment. About 25 pg of pSKI074 cut by
EcoRI was used as positive control. The DNA was de-
natured and blotted onto a positively charged Hy-
bond-XL nylon membrane (Amersham Biosciences, UK)
and fixed to the membrane by baking at 80℃ for 1 h.
The 990 bp fragment from pSKI074 harvested and puri-
fied was labeled with [32P]-dCTP and used as probe.
Hybridization was carried out at 45℃ for 16-18 h and
the blots washed twice with 2xSSC, 0.1% SDS for 5 min
each time, followed by washing with 1xSSC, 0.1% SDS
for 5 min and a final wash at 0.1xSSC, 0.1% SDS for 15
min all at 47℃. The blots were exposed to X-ray film
(Kodak XAR) for 14 h at –80℃.
2.5. Statistical Analysis
Each experiment was initiated with 100 explants and
was replicated three times. The percentage of callusing
and regeneration was scored on the basis of number of
explants showing the response in each Petriplate con-
taining 20 explants. The final number of plantlets regen-
erating from a total of 100 explants was noted at the end
of each experiment. Statistical analysis was carried out
for callus induction and shoot regeneration data for in-
teractions between type of explants (leaf or stem) and
cultivars using two-way ANOVA. All significant differ-
ences between means were found at P < 0.05. The Sta-
tistical Analysis System (SAS for Windows, Ver. 8.2,
2001) was used for ANOVA (Proc GLM) and compari-
son of means.
3. RESULTS AND DISCUSSION
3.1. Influence of Explants and Hormones
Callusing and regeneration response was more rapid in
the stem explants than leaf explants in all the cultivars
studied and in the medium reported previously [4].
Moreover, leaf explants are delicate and more easily
injured during manipulation especially in potato plantlets
which result in low transformation rate and higher
somaclonal variations [12]. Stem explants not only
showed better in vitro response but are reportedly more
convenient for conducting transformation experiments
[13]. Of the different cultivars studied, Bintje showed
the best regeneration response although Shepody showed
best callusing (Ta bl e 1 ). The quality of callus formed in
Shepody was white, soft and watery whereas Bintje
formed green, nodular and friable callus (Figure 1(a))
that showed better regeneration ability. Callus formation
was very prolific in Shepody and Superior, but these
calli did not show good regeneration potential. The cal-
lus produced in Bintje, although showing less growth,
gave better regeneration results and initiated more shoots
on transfer to shooting medium within 4 weeks (Table
1).
Thus, due to the high quality of callus and regenera-
tion ability, the cv. Bintje and stem explants were se-
lected for all further experiments. These results con-
firmed the genotypic variations in responses observed
previous ly in potato [14] and also shows that the type of
callus formed influences the regeneration poten tial of th e
plant [15]. Statistical analysis as shown by two-way
ANOVA, revealed highly significant effects of cultivars
and explants and their interaction s on both callusing and
regeneration responses (Tables 2 and 3).
Different concentrations and combinations of auxins
and cytokinins were investigated according to previously
reported pro tocols. The hormones NAA at 0.1 mg.l-1 and
Table 1. Percentage of callus and regeneration response in different explants and cultivars after 4 weeks of culture.
% Callusing * % Regeneration **
Cultivar Stem Leaf Stem Leaf
Superior 60.2 ± 6.15bA 0dB 23.8 ± 4.38bA 0cB
Bintje 27.4 ± 3.36cA 21.2 ± 2.58bA 45.1 ± 4.30aA 14.8 ± 2.38aB
Atlantic 31.2 ± 2.86cA 12.8 ± 1.92cB 0cA 10 ± 3.8bB
Shepody 86.7 ± 9.51aA 32.8 ± 4.96aB 20 ± 7.17bA 0cB
Russet Burbank 33.4 ± 7.3cA 12.4 ± 3.64cB 25.2 ± 3.03bA 8.6 ± 4.82bB
*Number of explants with callus/total number of explants per Petriplate. **Number of explants with shoot-buds/total number of explants per Petriplate. Small
letters denote significance between cultivars whereas capital letters denote significance between explants. Values followed by the same letter are not signifi-
cantly different from each other.

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 412
(a) (b)
(c) (d)
(e) (f)
Figure 1. Stages of regeneration after transformation events. (a) Callus initiated from cultivar Bintje stem explant. (b) Shoots re-
generating from stem explants with minimal callus in CGM media with IAA and t-zeatin without antibiotics. (c) Explants showing
shoots with callus in medium with NAA and t-zeatin. (d) Shoots growing on SGM medium with t-zeatin. (e) Shoots regenerating
from the two ends of stem explants in CSM media with IAA and t-zeatin containing kanamycin. (f) A complete transformed plant-
let showing rooting in medium without hormones.
trans-zeatin at 1 mg.l-1 showed better response than the
same hormones used in reverse concentrations. But an
intermediate callus phase was formed that took more
time to regenerate into shoots (Figure 1(c)) on transfer
to the shooting medium without NAA. Addition of the
hormones BAP and 2,4-D to the medium showed good

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 413
Table 2. Two-way analysis of variance of the cultivars and
explants on the percentage of callusing based on data presented
in Table 1.
Variable df SS MS F P
Cultivar 4 9961.3 2490.3 99.65 < 0.0001
Explant 1 12640.5 12640.5 505.82 < 0.0001
Cultivar x
explant 4 5540.2 1385.1 55.42 < 0.0001
Error 40 999.6 25.0
Table 3. Two-way analysis of variance of the cultivars and
explants on the percentage of regeneration based on data pre-
sented in Table 1.
Variable df SS MS F P
Cultivar 4 3635.88 908.97 63.79 < 0.0001
Explant 1 3152.18 3152.18 221.21 < 0.0001
Cultivar x
explant 4 2343.72 585.93 41.12 < 0.0001
Error 40 570.00 14.25
callusing in the transformed tissues, but on transfer to
shooting medium devoid of 2,4-D, the regeneration fre-
quency was low (Tab le 4). Using the hormone combina-
tions of 2,4-D and zeatin riboside showed a higher re-
generation rate compared to other protocols but an in-
termediate callus phase increased the time required for
regeneration and also increases the likelihood of obtain-
ing somaclonal variants. Direct shoot regeneration from
the explants without introducing a callus phase gives a
much higher regeneration efficiency [16,17]. The addi-
tion of t-zeatin or zeatin riboside to the medium was
found to be more effective for regeneration of shoots and
also produced less calli (Figure 1(b)) as compared to
those without zeatin [18 ]. Another hormone co mbination
with GA and BAP [19] did not show promising results.
The combination of IAA and t-zeatin in different con-
centrations were tested and the best result was obtained
at 0.1 mg.l-1 of each (Ta b l e 4) showing direct shoot re-
generation from explants with minimal callus (Figure
1(e)). This hormone combination was finally selected
and applied in CGM and CSM and followed for all fur-
ther experiments. Further growth and elongation of
shoots could be promoted after 3 weeks on transfer of
explants to SGM with ab sence of IAA.
3.2. Pretreatment Studies
To inve stigate the effects of preculture in phytohormones,
the stem explants were precultured on CGM containing
0.1 mg.l-1 each of IAA and t-zeatin for 0-6 days. The
explants with preculture of 2, 4 or 6 days showed
10-30% increase in regeneration response in comparison
to non-precultured (0 day) explants (Figure 2(a)) that
has also been observed in other plants [20,21]. The
highest shoot regeneration ability was observed after 4
days of preculture and a further period of preculture did
not show any enhancement unlike previous report that
indicates decline in differentiation rates after 2 days of
preculture [22]. The precultured explants not only gave
better shoot regeneration, but also showed less bleaching
in kanamycin-containing medium and less susceptibility
to overgrowth of Agrobacterium cultures. The preculture
of explants in phytohormones before co-cultivation is
important for transformation and may have an effect at
various steps including during T-DNA integration. Phy-
tohormone pretreatment activates cell division and the
phase of cell-cycle influences stable transformation,
since formation of new and thin cell walls probably in-
fluences specific attachment capacity to Agrobacterium
[23]. The presence of an induction agent, such as aceto-
syringone in the inoculation media was also crucial for
efficient T-DNA delivery and improving transformation
rate as reported earlier [24]. Acetosyringone reportedly
activates the transcription of Agrobacterium virulence
genes that can greatly increase the transformaton rate.
The cell-density for inoculation and period of co-cul-
tivation with bacterial culture also influences the trans-
formation efficiency. Although prolonging the inocula-
tion and co-cultivation time period usually yields more
efficient T-DNA delivery, but higher cell-damage and
necrosis occurs and leads to death of tissues. Thus, 2
days of co-cultivation showed better resu lts as compared
to 3 days (Figure 2(b)) and diluting the bacterial sus-
pension to OD600 = 0.2 (Figure 2(c)) significantly in-
creased the transformation rate which confirms previous
findings [24-26]. Diluting the bacterial concentration
also reduced the number of explants with overgrowth of
bacteria thus increasing the viability period of the ex-
plants in regeneration media. Another factor influencing
T-DNA delivery was the age of the explants. Explants
taken from 5 week old plantlets (Figure 2(d)) showed
optimum results whereas explants from older plantlets
had more hard tissues that decreased regeneration re-
sponse and greatly brought down the transformation
efficiency. The results showed that physiological state of
the starting material is also important to ensure success-
ful transformation.
Shoots regenerated from the stem explants in selection
medium containing kanamycin (CSM) with minimal
calli within 4 weeks (Figure 1(e)) and subculture of the
regenerating explants to shooting media without auxin
(SGM) could promote further elongation and growth of
shoots. Although numerous shoots were produced from

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 414
Table 4. Regeneration response of stem explants in medium with different hormone combinations and concentrations.
Hormones combinations and concentrations
CGM (mg.l-1) SGM (mg.l-1) Callus (%)*Shoots (%)** #Plants*** Rgn time in
weeks
NAA (0.1)+t-zeatin (1) t-zeatin (1) 82 ± 3.6 22.8 ± 4.1 19 10
BAP (0.5) +2,4-D (2) BAP (0.5) 67 ± 3.2 20 ± 2.7 15 18
2,4-D (2) + ZR (0.8) ZR (0.8) 88 ± 4.9 42 ± 5.5 32 10
No hormones BAP(2.5) + GA(1) 76 ± 5.4 15 ± 3.6 8 14
IAA (0.1)+t-zeatin (0.1) t-zeatin (0.1) 50.4 ± 4.3 38.2 ± 3.8 25 7
*Number of explants with callus/total number of explants. **Number of explants with shoot-buds/total number of explants. ***Total number of plants regener-
ating in each experiment. CGM indicates medium for callus a n d SGM medium for shoot initiation.
(a) (b)
(c) (d)
Figure 2. Bar charts showing variation in the percentages of regeneration of shootbuds and total plants regenerated from stem ex-
plants due to the effect of different factors. (a) Effect of preculture. (b) Effect of co-culture. (c) Effect of bacterial concentration and
(d) Effect of age of explants.
each explant, only one shoot was taken from each end to
generate independent transgenic events. This signicantly
avoided the collection of duplicated clones. Frequent
subcultures to fresh media every 2-3 weeks were neces-
sary to prevent growth of claforan-resistant bacteria on
the explants. Frequent transfers to fresh selection me-
dium also enhanced the transformation rate probably
because untransformed tissues die and release toxic
compounds. About 85-90% of shoots showed rooting
after transferring to media devoid of hormones and de-
veloped into complete plantlets within 7 weeks of cul-
ture (Figure 1(f)). Cultures over 8 weeks old started
showing bleaching in the explants due to presence of
kanamycin in the medium and had to be discarded. Some
of them showed abnormal albino shoots and did not root,
which were probably non-transgenic escapes. Vigorous

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 415
rooting and fu rther growth in selection media wer e good
indicators of successful transformation.
3.3. PCR and Southern Hybridization Results
PCR analysis confirmed the presence of 450 bp product
indicating the presence of the plasmid in the regenerants
(Figure 3(a)). A total of 80 regenerated plants were sub-
jected to PCR analysis and of these, 75 were PCR-posi-
tive indicating that over 93% of the regenerated plants
were transgenic with successful integration of the actva-
tion-tagged plasmid into the genome of the plants. Of
these plants, 60 were regenerated from precultured ex-
plants, and 58 were PCR positive, whereas of 20 plants
regenerated from non-precultured explants, 17 were
positive. The use of virG primer which amplifies only
the virulence region of the plasmid showed negative
results for all the transformed plants (Figure 3(b)) indi-
cating that the amplification is not due to the presence of
endogenous bacterial colonies in the transformants. Re-
sults of Southern hybridization experiments have shown
the presence of the 990bp fragment in the transformed
plantlets (Figure 3(c)) thus confirming the presence of
the plasmid and reinstating successful and stable integra-
tion of the transgene.
4. CONCLUSIONS
In conclusion, rapid transformation and regeneration in
potato with minimal callus phase can be achieved by
manipulating media and culture conditions before and
during T-DNA delivery. This protocol not only reduces
the regeneration time and callus phase but also gives
greater number of shoots and stable transformed potato
plants. Due to its high cooking and processing quality,
Bintje is commercially a preferred cultivar in many
European countries, but previous efforts have reported
difficulty in regenerating large number of transgenic
plants in this cultivar. The development of an efficient
transformation protocol for this potato cultivar to gener-
ate a large number of independent transgenic lines as
described here is crucial to carry out further work on
functional genomics. The stable integration of trans-
genes in the transformed potato plants by this method as
confirmed by PCR and Southern hybridization is due to
the absence of dedifferentiation steps that are common
(a) (b)
(c)
Figure 3. Verification of gene insertion by PCR (a) PCR analysis of transformants using 074 FRED and RED
primers. Lane 1- 5: transformed plants #1,2,3,4 and 5, C: control non-transformed plant, M: 100 bp ladder DNA
molecular weight marker. (b) PCR analysis of transformed plants using virG primer. Lane 1-6: transformed
plants #1, 2,3,4,5 and 6. C: control non-transformed plant. P: plasmid pSKI074. M: High molecular weight 1 kbp
DNA marker. (c) Southern hybridization analysis of EcoRI cut genomic DNA of plantlets transformed with
pSKI074. Samples S1-S6 indicate transformed plants #1,2,3,4,5 and 6. C- indicates the negative control untrans-
formed plant and C2-C25 indicates the positive control EcoRI cut plasmid pSKI074 releasing a 990bp fragment
with 2, 5, 10 and 25pg of DNA respectively.

B. Chakravarty et al. / Advances in Bioscience and Biotechnology 1 (2010) 409-416 416
during initiation of callus.
5. ACKNOWLEDGEMENTS
Financial support for the Canadian Potato Genome Project from Ge-
nome Atlantic is gratefully acknowledged. We thank Shirlyn Coleman
from New Brunswick Department of Agriculture, Fisheries and Aqua-
culture, for providing the potato stock plants.
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