Journal of Integrative Agriculture 2018, 17(5): 1128–1136

Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Heterologous expression of Lolium perenne antifreeze protein

confers chilling tolerance in tomato

Srinivasan Balamurugan, Jayan Susan Ann, Inchakalody P Varghese, Shanmugaraj Bala Murugan,
Mani Chandra Harish, Sarma Rajeev Kumar, Ramalingam Sathishkumar

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641046, India

Abstract
Antifreeze proteins (AFP) are produced by certain plants, animals, fungi and bacteria that enable them to survive upon
extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from
cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to
elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical
and physiological analyses. Stable integration and transcription of LpAFP in transgenic tomato plants was confirmed by
Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the
chilling stress induced physiological damage in wild type (WT) plants, while the transgenic plants remained healthy. Total
sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic
plants exhibited remarkable alterations in terms of relative water content (RWC) and electrolyte leakage index (ELI) than
those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants
comparing with WT. Overall, this report proved that LpAFP gene confers chilling tolerance in transgenic tomato plants and
it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.

Keywords: Lolium perenne antifreeze protein, chilling tolerance, genetic transformation, transgenic tomato

Among them, abiotic stresses are very critical, as they are

1. Introduction
Being sessile in nature, plants constantly encounter several
abiotic and biotic stresses that adversely affect plant
growth, development and survival (Smйkalovб et al. 2014).
Received 21 January, 2017 Accepted 16 May, 2017
Correspondence Ramalingam Sathishkumar, Tel: +91-93-
60151669, E-mail: rsathish@buc.edu.in
© 2018 CAAS. Publishing services by Elsevier B.V. All rights
reserved.
doi: 10.1016/S2095-3119(17)61735-0
uncontrollable and unpredictable, which will potentially limit
plant survival and thus lead to reduced yield (Wang et al.
2013; Velada et al. 2014). The temperature stress imposed
on plants has huge effect on agriculture. For instance, it has
been reported that decrease of 1°C in the world average
temperature might result in 40% reduction in rice production
(Hale and Orcutt 1987). Hence, there exists a pressing need
to overcome the crop loss due to negative impact of chilling
stress on plant growth and survival.
Generally, temperate plants can increase their chilling
tolerance by exposing to cold nonfreezing temperature via
an adaptive response called cold acclimation (Thomashow
1999). Whereas, the plants of tropical and subtropical
 Srinivasan Balamurugan et al. Journal of Integrative Agriculture 2018, 17(5): 1128–1136
regions are sensitive to chilling stress and the negative
effects of chilling stress are known to be higher in these
plants, as they lack the cold acclimation mechanism
(Thomashow 1999; Chinnusamy et al. 2007). Moreover,
many important food crops such as rice, maize, soybean,
cotton and tomato are chilling-sensitive, which are unable
to acclimate cold and also they cannot withstand the chilling
stress when exposed to low temperature (Chinnusamy
et al. 2007).
Overwintering plants have natural ability to resist ice
crystal formation in the cell by inhibiting its growth (Pudney
et al. 2003). Proteins isolated from many grass species
have shown to possess antifreeze properties that help
them to cope up with the cold conditions (Antikainen and
Griffith 1997). Antifreeze proteins (AFP) are a class of
proteins found in a range of over-wintering plants that
protect them from the damage imposed by chilling stress
(Kumar et al. 2014). Lolium perenne belongs to the
Poaceae family, which are adapted to grow in the colder
Northern hemisphere (Sandve et al. 2011) and reported to
withstand freezing (Kuiper et al. 2001). Superior antifreeze
protein responsible for this activity was identified from
L. perenne, which possessed two putative opposite-facing
sites with surface complimentary to the prism face of ice
(Kuiper et al. 2001). The LpAFP gene consists of 354 bp
that encodes a protein of about 118 amino acids (13.5 kDa)
with a semi-conserved seven-amino acid repeat X-X-N-X-V-
X-G on its entire length (Sidebottom et al. 2000; Middleton
et al. 2009). AFP reduces the freezing point of the solute
non-colligative, the process termed as thermal hysteresis
(TH), by inhibiting the recrystallization of the growing ice
crystals (Kuiper et al. 2001). Besides that the AFP was
also described as protector of cells from damage during
non-freezing conditions (Tomczak et al. 2002) mainly due
to their interactions with: i) the integral membrane proteins
(Rubinsky et al. 1991); ii) the membrane lipids (Hays et al.
1996); and iii) the membrane, which modifies the acyl chain’s
order in the bilayer core (Tomczak et al. 2002).
Tomato (Solanum lycopersicum) is one of the most
consumed vegetables worldwide with the production of
1.70 million tons (http://www.fao.org/statistics/en/) in 2014
and is also one of the most preferred garden crops (Fan
et al. 2015). In recent years, the consumption rate of
tomatoes has substantially increased worldwide and during
last two decades, the production and harvesting of tomato
has doubled (Bergougnoux 2014). Across the globe, Asia
dominates the tomato market where India ranked the
second after China in tomato production. Tomato is one
of the preferred vegetables in routine human diet, which
achieved a great familiarity over the last century (Harish
and Sathishkumar 2011; Badimon et al. 2017). Apart from
the dietary source, tomatoes are also considered as an
1129
excellent source of antioxidants, phytochemicals such as
lycopene, β-carotene, ascorbic acid, lutein, tocopherol,
phenolic compounds and it is also cholesterol-free. These
compounds are shown to promote positive health benefits
like antitumorigenic effects in human system, reduced risk
of cardiovascular and different types of cancer (Harish
et al. 2012). Besides its role in human diet, tomato plants
are being extensively used in research, mostly due to its
simple diploid genetics, short life cycle and relatively simple
genome (~900 Mb) (Zhou et al. 2013; Kumar et al. 2014).
Nevertheless, tomato is a thermophilic crop that makes
it more sensitive to chilling conditions (Zhou et al. 2013).
Any modifications in terms of enhancing the chilling-
tolerant ability of tomato plants will represent important
biotechnological breakthrough in high-altitude farming.
Transgenic technologies are presenting promising results in
improving plant traits, such as enhanced chilling tolerance,
by introducing single or multiple genes involved in response
to chilling stress. A number of reports showed that the
transgenic plants expressing AFP from various sources
proved enhanced chilling tolerance (Holmberg et al. 2001;
Zhu et al. 2010; Lin et al. 2011; Denga et al. 2014; Sun et al.
2014). As an attempt to develop chilling-tolerant tomato
plants that could aid in high altitude farming, here we are
reporting for first time the development of transgenic tomato
plants by expressing the AFP isolated from L. perenne and
successfully demonstrated the chilling-tolerant capability of
these transgenic lines.
2. Materials and methods
2.1. Gene isolation, cloning and plant expression
using gateway technology
Young leaves from cold acclimated L. perenne plants were
used for total RNA isolation using Qiagen RNeasy RNA
Isolation Kit (Qiagen, USA). And 1 µg of total RNA was used
for cDNA synthesis using the random hexamer primer and
M-MuLV reverse transcriptase (Fermentas, USA) following
manufacturer’s instructions.
The LpAFP gene (GenBank accession number:
AJ277399) was amplified of each gene-specific primer
(LpAFP-f101 and LpAFP-r102, both flanked by the attB
sequences by Phusion DNA polymerase (New England
Biolabs, USA) using a My-cycler Thermal Cycler (Bio-Rad,
USA). The amplicon generated was purified using PCR
Purification Kit (Qiagen, USA) and cloned into the entry
vector pDONR/Zeo using the BP clonase II (Invitrogen,
USA) following the procedure described by manufacturer’s
protocol. Reaction product was then transformed into
Escherichia coli competent cells. The sequencing-confirmed
plasmid was recombined with the destination vector pEARLY
1130 Srinivasan Balamurugan et al. Journal of Integrative Agriculture 2018, 17(5): 1128–1136

Gate 102 (Earley et al. 2006) by LR Clonase II Reaction
(Invitrogen, USA) according to manufacturer’s instructions,
thus enabling the transgene to be driven by the CaMV35S
promoter (Fig. 1-A). Recombinant plasmid pEARLEY-
LpAFP was electroporated into Agrobacterium tumefaciens
strain LBA 4404 (Hoekema et al. 1983) and used for stable
plant transformation.
2.2. In vitro propagation and genetic transformation
of tomato plants
Seeds of S. lycopersicum cv. Pusa Ruby (procured from
National Seed Corporation, New Delhi, India) were used.
These seeds were washed 3–5 times with sterile distilled
water and then surface-disinfected by immersion in 4%
sodium hypochlorite solution for 10 min with continuous
shaking. Subsequently, the seeds were rinsed with sterile
distilled water to remove the traces of excess sodium
hypochlorite and blot dried before inoculation. Surface-
disinfected seeds were then inoculated in Petri dishes
(9.0 cm×1.5 cm) (GenAxy, India) with half-strength
Murashige and Skoog (MS) medium (Duchefa, the
Netherlands) (Murashige and Skoog 1962). Three days
before transformation, cotyledons of previously inoculated
seeds were excised and inoculated in the preculture media
characterized by MS solid media with 2.0 mg L-1 zeatin
and 0.1 mg L-1 indole-3-acetic acid (IAA) (~30 explants per
plate in a total of 20 plates). The precultured cotyledons
were used as explants for Agrobacterium-mediated
genetic transformation. Agrobacterium culture (OD600=0.6)
harboring pEARLY102-LpAFP was harvested at 5 000 r
min-1 for 10 min. The cells were resuspended in liquid MS
solution and the cell density was adjusted to yield a final
OD600 of 0.6 and 100 µmol L-1 acetosyringone was added.
The explants were blot-dried, inoculated in co-cultivation
media and incubated for 3 days. After the co-cultivation
period, the cotyledons were blot-dried on sterile filter paper
for 15 min and inoculated in selection media (MS with 2.0 mg
L-1 zeatin+0.1 mg L-1 IAA+20 mg L-1 glufosinate+300 mg L-1
carbenicillin). After 2–3 rounds of selection in the selection
media, the regenerated putative transformants with 5–6 cm
length were excised and inoculated in rooting medium (1/2
MS media supplemented with 300 mg L-1 carbenicillin).
2.3. T1 seed collection and germination
The transgenic tomato plants (T0) were hardened in the
mixture (coir pith:vermiculite:red soil in 1:1:1 (v/v) ratio).
The surviving transgenic lines were confirmed by genomic
PCR. The PCR confirmed transgenic plants were harvested

A Kpnl Msel

LB BAR 35S LpAFP HA OCS RB

B
1 2 3 4 M

LpAFP
C

1 2 3
26S rRNA

LpAFP

Fig. 1 Molecular evaluation of transgenic lines. A, schematic vector map of pEarly102-LpAFP showing restriction enzymes used
for Southern blot. B, Southern blot analysis. Lane 1, negative control (wild type); lanes 2 and 3, transgenic tomato lines showing
the integration of AFP in transgenic lines Tm31 and Tm35, respectively; lane 4, positive control (pEarly102-LpAFP vector digested
with KpnI and MseI); lane M, marker. C, RT-PCR analysis showing mRNA expression pattern of AFP in transgenic tomato lines.
Lanes 1 and 2, transgenic tomato lines; lane 3, wild type. 26S rRNA was used as an internal reference gene. LB, left border; BAR,
basta (herbicide) resistance gene for selection of transgenics; 35S, the Cauliflower mosaic virus 35S promoter; HA, haemagglutinin;
OCS, 3´ sequences of the octopine synthase gene, including polyadenylation and termination sequences; RB, right border.
 Srinivasan Balamurugan et al. Journal of Integrative Agriculture 2018, 17(5): 1128–1136
individually. The transgenic lines were grown to maturity;
flowers were self-pollinated and covered with a polythene
bag to avoid cross contamination. Seeds were collected
and harvested in order to further generate the in vitro T1
transgenic lines. Seeds of T1 tomato lines (Tm5, Tm12,
Tm17, Tm24, Tm27, Tm31 and Tm35) were randomly
selected and cultivated after surface-disinfection in vitro in
1/2 MS media+20 mg L-1 glufosinate. Then 4–5 weeks-old
plantlets were selected by genomic PCR as mentioned
earlier and subjected to chilling treatment and further
molecular analysis.
2.4. Molecular characterization of transgenic plants
Gene expression analysis of AFP by RT-PCR The
cDNA was synthesized from the total RNA as described
earlier in Section 2.1. The synthesized cDNA was used as
template for the RT-PCR using AFP gene specific primers
(LpAFP-f201 and LpAFP-r202). The native 26S rRNA
gene was used as reference gene with a forward (Ref-f)
and reverse primer (Ref-r). All the oligonucleotide primers
employed in this study are provided in Appendix A.
Southern blot analysis of transgenic tomato lines Genomic
DNA from the selected transgenic T1 tomato lines were
isolated using DNeasy Maxi Kit (Qiagen, USA). The
genomic DNA (about 30 µg) from transgenic lines, wild
type (WT) lines (negative control) and pEARLY-LpAFP
plasmid (1 µg positive control) were digested with restriction
enzymes (KpnI and MseI) to release the LpAFP and the
digested product was electrophoresed at 25 V for 18 h on
a 0.8% agarose gel and then transferred to nitrocellulose
membrane (BioTrace NT, Pall, USA) by capillary blotting
(Sambrook et al. 1989). Hybridization, biotin labeling of
probe, membrane washing and detection were carried out
as per manufacturer’s instructions (Thermo, USA).
2.5. Physiological characterization of transgenic
plants
Physiological indexes such as electrolyte leakage index
(ELI), relative water content (RWC) and estimation of total
sugar content was performed for the T1 transgenic lines
under chilling and normal conditions to study the chilling
tolerance capability.
Chilling treatment The T1 transgenic lines (5 weeks old)
were subjected to chilling treatment at 4°C in the growth
chamber and the leaves were harvested every 24 h up to
5 days. The control samples were taken from the plants
grown in the green house at normal conditions. The
collected samples were used for further analysis.
Total soluble sugars Total soluble sugar content was
measured according to the protocol described by Parida
1131
et al. (2007) with slight modifications. Briefly, after chilling
stress, the leaf samples (~100 mg) were ground in liquid
nitrogen and added with 10 mL of 75% ethanol and
incubated overnight in room temperature (RT) at 150 r min-1.
Thereafter, the samples were centrifuged at 12 000 r min-1
for 5 min and 1 mL of freshly prepared anthrone reagent
(0.2%) was added to the supernatant, incubated in boiling
water bath for 15 min and cooled to room temperature. The
absorbance was measured at 625 nm.
RWC RWC (%) was measured in the T1 transgenic and
WT leaves as per the protocol described by Balestrasse
et al. (2010).
RWC (%)=(Fresh weight-Dry weight)/Dry weight×100
ELI Electrolytic leakage or ionic leakage index was
calculated from both the WT and transgenic lines as
described by Wu et al. (2012). Leaf discs of all the samples
(transgenic and WT plants) were collected and equilibrated
in sterile deionized water for 4 h and initial conductivity
was measured. The samples were then boiled and the
final conductivity was measured using the formula. ELI
(%)=Initial conductivity/Final conductivity×100
2.6. Data analysis
Data were represented as means±standard deviation of
mean (SDM). All the experiments were repeated three
times. Data were statistically analyzed. The data obtained
were subjected to one-way analysis of variance (ANOVA)
followed by Duncan’s multiple range test (DMRT) using
SPSS ver. 13.0 (SPSS Inc., USA).
3. Results and discussion
3.1. Genetic transformation of tomato using LpAFP
A. tumefaciens LBA 4404 strain harboring the vector
pEARLY102-LpAFP under the control of CaMV35S promoter
was used for genetic transformation. After co-cultivation
period of 3 days, the cotyledons were placed on the selection
media (MS+zeatin (2 mg L–1)+ IAA (0.1 mg L–1)+glufosinate
(20 mg L–1)+crbenicillin (300 mg L–1)) to regenerate the
transformants. Multiple shoots were induced in selection
media within 3 weeks. Individual shoots were separated
and inoculated in rooting media. Rooting was observed
after 1 week and the plants with well-developed roots were
hardened after 3 weeks. The fruits were collected and seeds
were harvested to generate T1 transgenic lines (Appendix
B). Several independent transgenic lines were analyzed in
terms of molecular and biochemical analyses (Appendices
C, D and E). For clarity of presentation, results from two
representative transgenic tomato lines are given.
1132 Srinivasan Balamurugan et al. Journal of Integrative Agriculture 2018, 17(5): 1128–1136

3.2. Molecular analysis revealed the successful in-
tegration, expression and inheritance of transgene
in the host genome
Southern blot hybridization In order to reinforce the
integration of the transgene in the host genome, Southern
blot analysis was carried out in the transgenic lines that are
preliminary confirmed with genomic PCR (data not shown).
The results showed the stable integration of LpAFP gene
in all the tomato lines. The hybridization signal observed
in the transgenic lines corresponds to the size of LpAFP
gene, whereas no signal was observed in WT (Fig. 1-B).
The above data demonstrated that the transgene LpAFP
was stably integrated in the transgenic tomato.
RT-PCR The stable expression of LpAFP in the T1 lines
was analyzed by RT-PCR. The expected band size of
354 bp confirmed the transcription of LpAFP gene in the
transgenic plants, whereas no such amplicon was detected
in WT. The result showed that the levels of transcription of
LpAFP gene in the transgenic lines were same. This might
be due to the presence of constitutive promoter CaMV35S;
hence the expression pattern was found to be same in all
the lines. The 26S rRNA gene (500 bp) was used as an
internal standard (Fig. 1-C).
3.3. Analyses of physiological indices revealed that
LpAFP conferred chilling-tolerance in transgenics
Chilling-tolerant ability of transgenic lines was evaluated by
phenotypic analysis under 4°C. The results of the chilling
treatments confirmed that transgenic lines carrying LpAFP
exhibited greater chilling tolerance. The phenotype analysis
showed that the WT plants could not survive at 4°C. At the
end of the 5th day, the transgenic lines did not show any
phenotypic variations and found to be healthy (Fig. 2).
Total soluble sugar Chilling stress in plants induces
osmotic and oxidative stress that eventually leads to the
accumulation of compatible solutes and elevates the anti-
oxidative mechanisms in order to protect the plants from
the stress-induced damage. The RWC, ELI and total sugar
content were recognized as the important indicators of
membrane integrity and chilling-tolerant ability of the plants
(Wu et al. 2012). In this study, transgenic tomato lines were
subjected to chilling treatment at 4°C and total sugar content
was measured in both transgenic and WT plants. The total
soluble sugar was increased during the chilling treatment in
both WT and transgenic lines. Sugar content was increased
up to 3.3-fold after 1 day of chilling treatment. After the 2nd
day, sugar content increased gradually till the 5th day of
treatment (Fig. 3). Significant modifications in the total sugar
content were reported in many of the plants exposed to low
temperatures, which was correlated to the increased soluble
sugar level (Wang et al. 2013). Congruently, generation of
transgenic tomato by overexpressing AtGRXS17 resulted in
increased total soluble sugar content during chilling stress
(Xin and Browse 2000).
Sugars are known to act as cryoprotectants and
osmolytes that protect the cell by stabilizing the membrane,
thus prevent the cellular dehydration during chilling
conditions (Sasaki et al. 1996). Among the various
physiological responses during freezing tolerance, sugar
content in plants is one of the major factors that determine
the extent of freezing tolerance. Earlier studies showed that
the exposure of plants to low temperatures led to fructan
synthesis (Antikainen and Pihakaski 1994), then led to
the accumulation of sucrose, fructose or glucose inside

A B C D

E F G H I

Fig. 2 Phenotypic analysis of 5-week-old tomato lines after 5 days under chilling-treatment. A-G, transgenic lines; i.e., Tm5,
Tm12, Tm17, Tm24, Tm27, Tm31, and Tm35, respectively. H and I, wild type (WT) lines.
 Srinivasan Balamurugan et al. Journal of Integrative Agriculture 2018, 17(5): 1128–1136
Tm31 Tm35 WT
600
Total sugar content (mg g–1)
500
*
400
*
300 *
200
100
0
0 1 2 3 4 5
Days after treatment (d)
Fig. 3 Measurement of total sugar content in transgenic
plants under chilling treatment. Each bar value represents
mean±SD, n=3. Significant difference between wild type
(WT) and transgenic lines (Tm31 and Tm35) are indicated by
*
, P<0.05 level.
the cell; however, the type of sugar accumulation varies
among different plant species (Rizhsky et al. 2004). The
accumulation of sugar in turn protects the plasma membrane
and other macromolecules from the effects of freezing or
dehydration although the mechanism is still unclear. During
deacclimation, sugar content of cold-acclimated plants was
reduced, which proved that sugars were possibly one of the
major factors in chilling tolerance (Antikainen and Pihakaski
1994). These results indicated that the excess content of
sugar in both the transgenics and WT plants under chilling
treatment might play a vital role in development of cold
hardiness in plants and also suggested that total sugar
content alone was not responsible for the cold tolerant ability
of the transgenic plants.
RWC In the present study, RWC was found to decrease in
both the WT and transgenic lines. There was no significant
difference for RWC among the plants just before chilling
treatment. Interestingly the transgenic lines were found
to have higher RWC level when compared to that of
WT lines throughout the chilling treatment. Just before
chilling treatment, i.e., the water contents of WT lines
and transgenic lines were found to be 84.0 and 88.0% at
26°C, respectively. After 5 days of chilling treatment, water
content of the WT lines was observed as 24.0%, while
the transgenic lines were found to maintain the higher
water content when compared with the WT lines (Fig. 4).
Thus the observed reduction could be correlated to the
increase in the solutes such as RNA, sugars, proteins
and accumulation of osmoprotectants, which helps the
plant defense mechanism (Campos et al. 2003). Similar
reduction in the water content during chilling stress was
also reported in transgenic tobacco plants (Wu et al. 2012).
Thus, the RWC in plants was inversely proportional to
1133
Tm31 Tm35 WT
**
100 **
***
** ***
** ***
80 **
***
***
60
RWC (%)
40
20
0
0 1 2 3 4 5
Days after treatment (d)
Fig. 4 Measurement of relative water content (RWC) in
transgenic plants under chilling treatment. Each bar value
represents mean±SD, n=3. Significant difference between wild
type (WT) and transgenic lines (Tm31 and Tm35) are indicated
by **, P<0.01 and ***, P<0.001 levels, respectively.
the freezing injury induced by low temperature, i.e., the
presence of large amount of water inside the cell is prone
to be more susceptible for chilling injury and vice versa.
ELI Plasma membrane plays a vital role in the structure
and function of all the cells. During low temperature, it
undergoes phase transitions from liquid crystalline to rigid
gel phase and streaming of intracellular water and electrolyte
through plasma membrane resulting in increased electrolyte
and conductivity (Hu et al. 2015). ELI was found to be a
versatile and sensitive assay to study cold acclimation of
plants (Yang et al. 2011). There was a significant increase
in electrolyte leakage in the WT lines during the chilling
treatment, whereas the transgenic lines maintained the
lower level of electrolyte leakage during the chilling treatment
up to the 5th day of treatment, which was 50% lower than
the WT lines (Fig. 5). This results proved the protective
nature of AFP in transgenic lines. Wu et al. (2012) reported
that WT lines showed significant increase in electrolyte
leakage as compared to transgenic tobacco lines expressing
CbCOR15b gene. Similarly, overexpression of transcription
factor TERF2/LeERF2 increased electrolyte leakage in
transgenic plants and conferred freezing tolerance through
ethylene signaling pathway in both transgenic tomato and
tobacco (Zhang and Huang 2010). In our previous report
(Kumar et al. 2014), we have shown that the T0 transgenic
tomato plants expressing carrot AFP can grow in the chilling
conditions up to 48 h as compared to WT. However, in the
present study, T1 transgenic lines expressing LpAFP has
shown to tolerate chilling treatment even after 5 days and
did not show any phenotypic abnormalities as compared to
that of WT. This result clearly proved the better protective
1134 Srinivasan Balamurugan et al. Journal of Integrative Agriculture 2018, 17(5): 1128–1136
Tm31 Tm35
100
Electrolyte leakage index (%)
80
***
***
60 ***
***
40
20
0 the funds from the University Grants Commission-Special
0 1 2 3
Days after treatment (d)
Fig. 5 Measurement of electrolyte leakage index (ELI) in
transgenic plants under chilling treatment. Each bar value
represents mean±SD, n=3. Significant difference between wild
type (WT) and transgenic lines (Tm31 and Tm35) are indicated
by ***, P<0.001 level.
ability of LpAFP under chilling condition, which could be
due to the presence of two ice-binding domains as against
the single domain present in carrot AFP (Kuiper et al. 2001;
Pudney et al. 2003; Kumar et al. 2014).
Once the temperature is reduced, the transgenic lines
retained the stamina as against WT lines and no phenotypic
abnormalities were observed in the transgenic lines.
Even though function of AFPs showed that, this protein
would inhibit the ice recrystallization at subzero conditions
(Chinnusamy et al. 2007), function of this proteins may
differ with respect to the chilling tolerance ability of the
plant system (Zhang and Huang 2010). Besides the
freezing condition, AFP was reported to protect the cells
from chilling injury (Tomczak et al. 2002). This protective
function of AFPs at chilling temperatures is mainly due to
the interaction of AFP with the integral membrane proteins
(Rubinsky et al. 1991), membrane lipids (Hays et al. 1996)
and the membrane itself, which modifies the acyl chain’s
order in bilayer core (Tomczak et al. 2002). Taken together,
our study proved that LpAFP confer the chilling tolerance
ability to transgenic tomato lines.
5. Conclusion
In the study, we successfully generated transgenic tomato
plants expressing LpAFP with enhanced tolerance to chilling
stress. Our data proved the protective role of LpAFP in
transgenic tomato plants under chilling conditions and
also proved that LpAFP is a potential candidate to confer
chilling tolerance, probably by upholding the membrane
integrity of the transgenic plants. This is the first report of
WT LpAFP expression in transgenic tomato lines to increase
the chilling tolerance. The outcome will significantly help
*** the high altitude farming of economically important food
***
***
crops in long term.
***
Acknowledgements
The research was supported by the Senior Research
Fellowship from the Council of Scientific and Industrial
Research-Human Resource Development Group (CSIR-
HRDG), New Delhi, India (09/472(0164)/2012-EMR-I), and
4 5 Assistance Programme (UGC-SAP) and the Department
of Science and Technology-Fund for Improvement of S&T
Infrastructure (DST-FIST), Bharathiar University, Tamil
Nadu, India.
Appendices associated with this paper can be available on
http://www.ChinaAgriSci.com/V2/En/appendix.htm
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