ORIGINAL ARTICLE

Expression of cold and drought regulatory protein (CcCDR)of pigeonpea imparts enhanced tolerance to major abiotic stressesin transgenic rice plants

Mellacheruvu Sunitha1 • Tamirisa Srinath1 • Vudem Dashavantha Reddy1 •

Khareedu Venkateswara Rao1

Received: 3 January 2017 / Accepted: 3 March 2017 / Published online: 8 March 2017

� Springer-Verlag Berlin Heidelberg 2017

Abstract

Main conclusion Transgenic rice expressing pigeonpea

CcCDR conferred high-level tolerance to different abi-

otic stresses. The multiple stress tolerance observed in

CcCDR-transgenic lines is attributed to the modulation

of ABA-dependent and-independent signalling-pathway

genes.

Stable transgenic plants expressing Cajanus cajan cold and

drought regulatory protein encoding gene (CcCDR), under

the control of CaMV35S and rd29A promoters, have been

generated in indica rice. Different transgenic lines of

CcCDR, when subjected to drought, salt, and cold stresses,

exhibited higher seed germination, seedling survival rates,

shoot length, root length, and enhanced plant biomass

when compared with the untransformed control plants.

Furthermore, transgenic plants disclosed higher leaf

chlorophyll content, proline, reducing sugars, SOD, and

catalase activities, besides lower levels of MDA. Local-

ization studies revealed that the CcCDR-GFP fusion pro-

tein was mainly present in the nucleus of transformed cells

of rice. The CcCDR transgenics were found hypersensitive

to abscisic acid (ABA) and showed reduced seed germi-

nation rates as compared to that of control plants. When the

transgenic plants were exposed to drought and salt stresses

at vegetative and reproductive stages, they revealed larger

panicles and higher number of filled grains compared to the

untransformed control plants. Under similar stress condi-

tions, the expression levels of P5CS, bZIP, DREB,

OsLEA3, and CIPK genes, involved in ABA-dependent

and-independent signal transduction pathways, were found

higher in the transgenic plants than the control plants. The

overall results amply demonstrate that the transgenic rice

expressing CcCDR bestows high-level tolerance to

drought, salt, and cold stress conditions. Accordingly, the

CcCDR might be deployed as a promising candidate gene

for improving the multiple stress tolerance of diverse crop

plants.

Keywords Abiotic stress � Abscisic acid � Cold and

drought regulatory protein � Transgenic rice � Cajanuscajan

Abbreviations

CcCDR Cold and drought regulatory protein

CaMV 35S Cauliflower mosaic virus 35S promoter

MDA Malonaldehyde

Introduction

Terrestrial plants, being sessile, are continually exposed to

numerous changes occurring in various environmental

factors such as drought, salinity, temperature, etc. These

fluctuations in environments often pose serious threats to

the normal growth and development as well as productivity

of diverse crop plants. Different abiotic stresses, in general,

are known to cause more than 50% losses in the yields of

different crop plants worldwide (Boyer 1982; Bray 1997).

M. Sunitha and T. Srinath contributed equally.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-017-2672-1) contains supplementarymaterial, which is available to authorized users.

& Khareedu Venkateswara Rao

rao_kv1@rediffmail.com

1 Centre for Plant Molecular Biology, Osmania University,

Hyderabad 500007, India

123

Planta (2017) 245:1137–1148

DOI 10.1007/s00425-017-2672-1

Abiotic stresses are known to affect various physiological

and biochemical processes of the plants by altering the

regulation of various important genes. Survival of the

plants mainly depends on their innate ability to modulate

various physiological and molecular processes by fine-

tuning the expression of certain key genes (Ingram and

Bartels 1996). Understanding the nature and role of

molecular mechanisms, involved in plants’ ability to

respond and to tolerate abiotic stresses, is important aspects

of plant molecular biology. Efforts have been made in this

direction to analyze the underlying mechanisms of stress

tolerance and to improve the crop productivity under dif-

ferent abiotic stress conditions (Nguyen et al. 2016).

Rice is one of the most important cereal crops and

serves as a major staple food for more than half of the

world’s population. Rice plant is amenable to different

genetic manipulations, and is used as a model system to

study its genome organization and functional genomics

(Bajaj and Mohanty 2005). The genetic improvement of

rice for water-deficit environments was rather slow and

more limited (Evenson and Gollin 2003). However, adop-

tion of genetic engineering approaches offers unique

opportunities to improve the tolerance of rice to different

abiotic stresses (Tyagi and Mohanty 2000). Earlier, pro-

gress was made in developing transgenic rice with signif-

icant tolerance to drought, cold, and salt stresses

(Mellacheruvu et al. 2016). A stress-associated protein

OsiSAP8 of rice could confer salt, drought, and cold stress

tolerance in transgenic tobacco and rice (Kanneganti and

Gupta 2008). Transgenic Arabidopsis and rice plants,

overexpressing rice OsDREB1F gene, revealed higher

tolerance to salt, drought, and low-temperature stresses

(Wang et al. 2008). In transgenic rice, overexpression of

rice ZFP182 protein caused enhanced tolerance to multiple

abiotic stresses (Huang et al. 2012). Expression of Triticum

aestivum salt-induced protein (TaSIP) in Arabidopsis and

rice resulted in better tolerance to salt and drought stresses

(Du et al. 2013).

The cauliflower mosaic virus promoter (CaMV 35S) is

being used most commonly for transgene expression in

various plants. However, the expression of different stress-

responsive genes, under the control of CaMV35S, often

showed undesirable phenotypic effects (Thompson et al.

2000; Priyanka et al. 2010a, b). However, development of

crop plants expressing transgenes exclusively under stress

conditions, sans abnormal phenotypes, is desirable.

Transgenic plants expressing stress related genes, under the

control of rd29A promoter, showed increased tolerance to

different abiotic stresses while minimizing the negative

effects of the transgenes on plant growth and development

(Li et al. 2013; Estrada-Melo et al. 2015).

Reproductive development of plants was found to be

sensitive to various environmental stresses including

drought and high salinity (Jiang et al. 2012). Especially,

stages from meiosis to seed set were found highly vul-

nerable to drought stress conditions (Firon et al. 2012; He

and Serraj 2012). Rice plant was found to be extremely

sensitive to drought stress because of its restricted adap-

tation to water-deficit conditions (Yang et al. 2010),

resulting in the loss of grain yield (Oh et al. 2009). The

molecular mechanisms underlying the reproductive devel-

opment and decrease in grain yield under drought stress

have not yet been fully understood. Different crop plants

conferred with enhanced genetic tolerance to diverse

environmental stresses were found to contribute to

increased yield and productivity under various adverse

abiotic stress conditions (Hou et al. 2009).

In our earlier study, a Cajanus cajan cold and drought

regulatory protein encoding gene (CcCDR) has been iso-

lated from pigeonpea plants (Tamirisa et al. 2014).

Transgenic Arabidopsis and tobacco plants expressing

CcCDR disclosed marked tolerance against multiple abi-

otic stresses (Tamirisa et al. 2014; Srinath et al. 2014). To

characterize the role of CcCDR in major crop plants, it has

been introduced into rice and the transgenic plants have

been evaluated against different abiotic stress conditions.

Stable transgenic rice lines expressing CcCDR exhibited

explicit tolerance against drought, salinity, and cold stres-

ses at different stages of growth and development, and

were able to outperform the untransformed control plants.

Materials and methods

Construction of CcCDR-overexpression vector

for rice transformation

The pigeonpea stress-responsive gene, CcCDR, was cloned

into pSB11 Agrobacterium vector containing the bar gene

as a plant selectable marker (Ramesh et al. 2004) under

CaMV35S/rd 29 promoters for constitutive/stress inducible

expression. Later, the recombinant vectors were mobilized

into Agrobacterium strain LBA4404 by triparental mating

method. The resulting super-binary vectors, pSB111-

CaMV35-bar-CaMV35-CcCDR and pSB111-CaMV35-

bar-rd29-CcCDR, were employed for the development of

transgenic rice plants.

Agrobacterium-mediated transformation

and regeneration of transgenic plants

Seeds of the indica rice cultivar Samba Mahsuri (BPT

5204), obtained from the Indian Institute of Rice Research

(IIRR), Hyderabad, were used for genetic transformation.

Calli derived from the mature embryos were infected with

the Agrobacterium and putatively transformed calli were

1138 Planta (2017) 245:1137–1148

123

selected on the Murashige and Skoog (1962) medium

containing 6.0–8.0 mg/l phosphinothricin (PPT). Plants

regenerated from the selected PPT-tolerant calli were

established in the glasshouse and were grown to maturity.

Putative transgenic rice plants (30–40 days old) along with

untransformed controls were tested for their tolerance to

the herbicide 0.25% Basta (Ramesh et al. 2004).

PCR analysis of transgenic plants

Genomic DNA was isolated from the Basta tolerant and

untransformed control plants as described by McCouch

et al. (1988). PCR analysis was carried out using the pri-

mers corresponding to the expression cassettes (Supple-

mentary Table S1). DNA from the untransformed control

plants was used as a negative control and the plasmid was

used as a positive control.

Southern-blot analysis of transgenic plants

For Southern-blot analysis, about 15 lg of genomic DNA

was digested overnight with HindIII and was resolved on

0.8% agarose gel. After denaturation and neutralization

steps, the DNA was transferred onto a Hybond-N? mem-

brane (Amersham Pharmacia) and then cross-linking was

done by exposing it to UV (1200 lJ for 60 s) as described

by Sambrook and Russell (2001). The membranes were

hybridized with the CcCDR and bar coding regions as

probes and later exposed to X-ray film. DNA probe

preparation, hybridization, and membrane washing were

performed according to the manufacturer’s instructions

(AlkPhos Direct Labeling System Cat. No. RPN3680, GE

Healthcare).

Evaluation of transgenic plants for abiotic stresses

Seeds of T4 homozygous lines of CaMV35-CcCDR (CR1,

CR2, CR3, CR4, and CR5), rd29A-CcCDR (RR1, RR2,

RR3, RR4, and RR5), and untransformed control were used

in the stress tolerance experiments.

Seed germination assay

Seeds were surface-sterilised with 0.1% (w/v) HgCl2 for

7 min followed by three washes with the sterile water. The

sterilised seeds were allowed to germinate on MS basal

medium and medium containing 250 mM mannitol and

250 mM NaCl for drought and salt stress responses,

respectively. For cold stress, seeds were placed on MS

medium at 30 �C for overnight followed by transferring

them to 4 �C for 3 days, and later transferred to normal

(30 �C) conditions. Data on germination frequencies were

recorded after 10 days.

Seedling stage

Rice seedlings grown in Hoagland solution at 30 �C (16 h

light/8 h dark cycle) for 15 days were treated with 250 mM

NaCl and 250 mM mannitol for salt and drought stress,

respectively, for 7 days. For cold stress, seedlings

(15 days) were transferred to an incubator set at 4 �C for

7 days. Later, they were transferred to normal conditions

and allowed to recover. After 7 days of recovery, data on

survival rate, biomass, shoot length, and root length of

seedlings were recorded.

Leaf chlorophyll content

For determination of leaf chlorophyll, leaf discs from

6-week-old transgenic and control plants were floated on

20 ml solution of NaCl (250 mM)/mannitol (250 mM) or

water (experimental control) for 72 h at room (30 �C)temperature. For cold stress, leaf discs were floated on

20 ml of water kept at 4 �C for 72 h. The leaf discs were

ground using liquid nitrogen and dissolved in 80% acetone.

Supernatant was collected after centrifuging the samples at

13,201g for 15 min. The absorbance of filtrate was deter-

mined using the spectrophotometer (Mellacheruvu et al.

2016).

Estimation of proline, reducing sugars,

malondialdehyde, and antioxidant enzyme activities

Two-week-old control and transgenic plants were subjected

to NaCl (250 mM), mannitol (250 mM), and cold (4 �C)independently for 3 days, and were used for estimation of

proline, reducing sugars, catalase, superoxide dismutase,

and malondialdehyde contents.

Measurement of proline content

Proline content was determined as per Bates et al. (1973).

Fifteen-day-old CcCDR-transgenic lines of CR and RR

along with control seedlings were subjected to 250 mM

mannitol, 250 mM NaCl, and 4 �C for 3 days. About 0.5 g

of plant material was homogenized in 5 ml of 3% aqueous

sulfosalicylic acid. The homogenate was placed in a boil-

ing water bath for 10 min and centrifuged at 13,201g; the

supernatant was used for the estimation of proline.

Estimation of reducing sugars

Leaf tissues of *100 mg were collected from the trans-

genic and control plants, and were frozen with liquid

nitrogen and ground to powder. Reducing sugars were

extracted from the powder twice with 80% ethanol at

95 �C. The supernatant collected was bulked and was

Planta (2017) 245:1137–1148 1139

123

reduced to dryness at 80 �C for 2 h. The residue was dis-

solved in 10 ml of distilled water. The reducing sugar

contents were estimated according to Miller (1959).

Determination of catalase activity

Catalase activity was determined as described by Mel-

lacheruvu et al. (2016). Leaf samples collected from the

stressed plants were homogenized in 50 mM phosphate

buffer (pH 7.0), and the homogenate was centrifuged at

8000g for 20 min at 4 �C. Enzyme extract (20 ll) was

added to 3 ml of hydrogen peroxide-phosphate buffer (pH

7.0), and the time required for decrease in the absorbance at

240 nm from 0.45 to 0.40 was noted.

Determination of the malondialdehyde (MDA)

content

The MDA content of the control and transgenic plants was

measured under salt, drought, and cold stress conditions.

About 100 mg of leaves were homogenized in 10 ml of

10% trichloroacetic acid. The homogenate was centrifuged

at 4000g for 10 min. Then, 2 ml of thiobarbituric acid

(made in 10% trichloroacetic acid) was added to 2 ml of

the supernatant. This mixture was boiled for 10 min and

then cooled on ice quickly. Absorbance values of the

supernatant were read at 532, 600, and 450 nm. The MDA

content was calculated using the equation: 6.45 9 (OD

532 nm - OD 600 nm) - 0.56 9 OD 450 nm and

expressed as lmol g-1 FW.

Superoxide dismutase (SOD)

Superoxide dismutase activity was determined based on the

inhibition of photochemical reduction of nitroblue tetra-

zolium (NBT) at 560 nm. The reaction mixture was pre-

pared by taking 50 ll enzyme extract and adding 1 ml

NBT (50 lM), 500 ll methionine (13 mM), 1 ml ribo-

flavin (1.3 lM), 950 ll (50 mM) phosphate buffer, and

500 ll EDTA (75 mM). The reaction was started by

keeping the solution under 30 W fluorescent lamp and was

stopped 5 min later when the lamp was turned off. The

NBT photoreduction produced blue formazane which was

used to measure increases in absorbance at 560 nm. The

SOD activity was determined and expressed as SOD

IU min-1 mg-1 protein.

Relative water content measurement

Relative water contents (RWC) of the 2-week-old

CaMV35-CcCDR, rd29-CcCDR-transgenic plants, and the

control plants were measured. The fresh weight loss was

calculated relative to the initial plant weight. Plants were

weighed and left at the room temperature until there was no

further loss in weight (desiccated weight). Later, plants

were dried for 24 h at 70 �C and dry weights were recor-

ded. The RWC of the samples were measured using the

formula: RWC (%) = (desiccation weight - dry weight)/

(fresh weight - dry weight) 9 100.

Stress treatments at the vegetative stage of plants

The transgenic and control plants were grown in pots for

60–65 days and were exposed to different stress condi-

tions. For drought stress experiment, water was withheld

for 15 days, and re-watering was initiated when the leaves

of control plants were wrinkled. For salt stress, plants were

exposed to 250 mM NaCl solution for 15 days, and later,

NaCl solution was replaced with fresh water. These plants

were allowed to grow to maturity under normal conditions

(30 �C). Data on panicle length and number of filled grains

per panicle were recorded. In each treatment, ten plants

were used and all the experiments were repeated thrice.

Stress treatments at the reproductive stage of plants

Plants were grown in pots under normal conditions until

the inflorescence meristem started to appear. Drought

(withholding water) and salt (250 mM NaCl) stress treat-

ments were applied at the reproductive stage (90–100 days

old) for 10 days at 30 �C. After treatments, plants were

transferred to normal conditions and allowed to grow to

maturity. Data on panicle length and number of filled

grains per panicle were recorded. In each treatment, ten

plants were used and all the experiments were repeated

thrice.

Construction of GFP::CcCDR fusion construct

and stable transformation of rice

Coding sequence of the CcCDR without the termination

codon was fused with the 50 region of green fluorescent

protein (GFP) driven by CaMV35S promoter. Expression

cassettes, CaMV35S–CcCDR::GFP/CaMV35S–GFP,

were subcloned into pCAMBIA3300 containing bar gene

as a selectable marker, and mobilized into the Agrobac-

terium strain (LBA4404) through triparental mating.

Stable genetic transformation was carried out as described

previously and transformed callus cells were observed

under laser-scanning confocal microscope (Leica

Microsystems).

ABA sensitivity test

Transgenic and control seeds (20 each) were germinated on

the MS medium containing 0, 2, and 3 lM of abscisic acid

1140 Planta (2017) 245:1137–1148

123

(ABA). The germination rates were recorded after 7 days

and all the experiments were performed in triplicate.

Gene expression analysis using quantitative real-

time PCR (qRT-PCR)

RNA isolated from the control and transgenic (CaMV35-

CcCDR) seedlings, subjected to stress for 150 mM man-

nitol treatment for 24 h, along with the unstressed plants

was used in the first-strand cDNA synthesis. Real-time

PCR analysis was carried out using SYBR green master

mix with Applied Biosystems 7500 real-time PCR system

at 94 �C (1 min), 60 �C (1 min), and 72 �C (1 min) for 30

cycles. The products were analyzed by melt curve analysis

to check the specificity of PCR amplification. Experiments

were performed in triplicate and the relative expression

ratio was calculated using 2-DDct method employing actin

gene as a reference (Tamirisa et al. 2014). The primers

used in the real-time PCR experiments are listed in (Sup-

plementary Table S1).

Results

Development of CcCDR-transgenic rice plants

Transgenic rice plants of BPT 5204 were regenerated from

PPT resistant calli obtained after co-cultivation with the

Agrobacterium strain LBA4404 harbouring the super-bi-

nary vector carrying bar and CcCDR (pSB111-CaMV35-

bar-CaMV35-CcCDR/pSB111-CaMV35-bar-rd29A-

CcCDR) genes (Fig. 1a, b). A total number of 26 and 15

stable transformants were obtained from 2342 and 1789

calli infected with the above constructs. Five transgenic

lines for each construct, viz., CR1, CR2, CR3, CR4, and

CR5 (CaMV35S-CcCDR) and RR1, RR2, RR3, RR4 and

RR5 (rd29A-CcCDR), were selected for further analyses

based on their high-level tolerance to herbicide (0.25%)

Basta (Suppl. Fig. S1a).

Molecular confirmation of transgenic rice lines

PCR analysis of transgenic rice plants generated 666 and

282 bp products when bar-nos and CcCDR gene specific

primers were employed, representing bar-nos and CcCDR

coding sequences, while the control plants failed to show

such amplification (Suppl. Fig. S1b). Southern-blot analy-

sis of transgenic plants, using CcCDR and bar coding

regions as probes, revealed the presence of hybridizable

bands of about 1.0/1.3 kb and different sizes ranging from

[4.0 to 10.0 kb, respectively, whereas the untransformed

control plants failed to show any hybridizable bands

(Fig. 1c–f).

Nuclear localization of CcCDR::GFP fusion protein

Subcellular localization of the CcCDR::GFP fusion protein

and GFP alone was assessed in the stably transformed

callus-derived cells of rice. Examination of the green flu-

orescence by laser-scanning confocal microscope showed

ample fluorescence in both nucleus and cytoplasm of the

cells expressing GFP alone, while cells expressing the

fusion protein exhibited green fluorescence predominantly

in the nucleus (Fig. 1g–i).

Functional validation of CcCDR-transgenic lines

for multiple abiotic stress tolerance

Five transgenic lines each from CaMV35-CcCDR (CR1,

CR2, CR3, CR4, and CR5) and rd29A-CcCDR (RR1, RR2,

RR3, RR4, and RR5) were selected for analyzing their

stress tolerance against drought, salt, and cold stresses.

Seed germination rates of transgenics under stress

conditions

Under different stress conditions, seeds of different trans-

genic lines exhibited higher seed germination rates as

compared to the control seeds. Seeds of transgenic lines

when germinated on mannitol (250 mM) showed higher

(85–89%) germination rates compared to the control seeds

(Fig. 2a). Similarly, seeds of different transgenic lines

grown on the medium containing NaCl (250 mM) showed

*74 to *81% germination rates as compared to the

control seeds (Fig. 2a). Likewise, seeds of transgenic lines

subjected to cold (4 �C) stress exhibited greater (85–90%)

germination rates as compared to the control (Fig. 2a).

Stress tolerance of CcCDR transgenics at seedling

stage

Transgenic lines grown under different stress conditions

exhibited higher plant survival rate, increased shoot and

root length, and enhanced plant biomass when compared to

the control plants (Suppl. Fig. S2). The transgenic lines

subjected to mannitol (250 mM) stress showed higher

(*73 to *83%) survival rates in comparison with the

control plants (Fig. 2b). Transgenic lines also exhibited

higher biomass with substantial increases in shoot length

and root length as compared to the control plants (Fig. 2c–

e). Similarly, transgenic plants grown under salt stress

revealed higher (*66 to 75%) survival rates when com-

pared to the control plants (Fig. 2b). Furthermore, trans-

genics disclosed enhanced plant biomass with significant

increases in shoot and root lengths (Fig. 2c–e). Higher

plant survival rates (*83 to 90%) were also observed in

transgenic lines subjected to cold (4 �C) stress compared to

Planta (2017) 245:1137–1148 1141

123

the control plants (Fig. 2b). Moreover, marked increases in

plant biomass, shoot length, and root length were observed

in different transgenics (Fig. 2c–e).

Stress tolerance of CcCDR transgenics at vegetative

stage

Two-month-old transgenic and control plants were sub-

jected to drought (water withholding) and salt (250 mM

NaCl) stresses for 15 days. After stress treatments, the

transgenic plants were able to reach the reproductive stage

and set normal seeds, while control plants failed to reach

reproductive stage.

Stress tolerance of CcCDR transgenics

at reproductive stage

To evaluate stress tolerance at the reproductive stage,

drought (water withholding) and salt (250 mM NaCl)

stresses were imposed for 15 days on transgenic and con-

trol plants. Different transgenic (CR and RR) lines showed

longer panicles both under drought and salt stresses when

compared to the control plants (Fig. 3a). Similarly, trans-

genic plants also exhibited higher number of filled grains

per panicle under drought and salt stresses compared to that

of control plants (Fig. 3b).

Chlorophyll content in transgenic plants

under stress conditions

Under normal conditions, no significant differences were

observed in chlorophyll contents between control and

transgenic plants. However, under stress conditions, the

chlorophyll content of control plants was significantly

lesser than that of transgenic plants. The mean chlorophyll

contents of transgenic plants were found to be higher under

drought, salt, and cold stress conditions compared to the

control plants (Fig. 4a).

Fig. 1 Southern-blot analysis of CcCDR-transgenic rice lines and

subcellular localization of CcCDR:GFP fusion protein. a, b Restric-

tion maps of T-DNA region containing CaMV35S-CcCDR and

rd29A-CcCDR expression units along with bar selectable marker

gene. c, e Southern-blot analyses of CaMV35S-CcCDR-transgenic

lines (lanes 1, 2, 3, 4, and 5) and d, f of rd29A-CcCDR-transgeniclines (lanes 1, 2, 3, 4, and 5). P represents 1.0 and 1.3 kb regions of

CaMV35S-CcCDR-nos and rd29-CcCDR-nos expression units. C

represents untransformed control plants. Each lane was loaded with

15 lg of genomic DNA digested with HindIII enzyme and probed

with initially with CcCDR sequence and later with bar coding

sequence. g, h Restriction maps of T-DNA region containing

CaMV35S–CcCDR::GFP and CaMV35S–GFP. i Image represents

individual rice cells expressing GFP and CcCDR:GFP fusion protein.

1, 4 bright field; 2, 5 combined; and 3, 6 dark field. Bar 100 lm

1142 Planta (2017) 245:1137–1148

123

bFig. 2 Evaluation of CcCDR -transgenic lines against different

abiotic stress. a Seed germination rates of transgenics and control

subjected to drought, salt, and cold treatments. Seed germination

ability was tested on MS medium supplemented with mannitol

(250 mM) and NaCl (250 mM). For cold stress, seeds were placed on

MS medium at 30 �C for overnight and later transferred to 4 �C for

3 days. After 1 week of stress treatments, data were recorded.

Fifteen-day-old seedlings of transgenics and control were transferred

to the Hoagland solution containing mannitol (250 mM) or NaCl

(250 mM) and were allowed to grow for 7 days. For cold treatment,

plants of the same age were transferred to the incubator set at 4 �C for

7 days, and moved to normal (30 �C) temperature. After 7 days of

recovery, data on survival rate (b), biomass (c), shoot length (d), androot length (e) were recorded. In each treatment, ten seedlings of

control and transgenic lines were used. Bar represents mean and I

represents SE from three independent experiments. Asterisk indicates

significant differences in comparison with C (control plants) at

P\ 0.05. CR1, CR2, CR3, CR4, and CR5 35S transgenic lines, RR1,

RR2, RR3, RR4, and RR5 rd29A transgenic lines, C control plants,

FW fresh weight

Fig. 3 Analysis of CcCDR-transgenic lines for drought and salt stress

tolerance at the reproductive stage. Transgenic and control

(90–100 days old) plants were subjected to drought (withholding

water) and salt (250 mM NaCl) stress treatments for 10 days at

30 �C, and the data on panicle length (a) and grain number/panicle

(b) were collected. In each treatment, ten plants were used and all the

experiments were repeated thrice. Bar represents mean and I

represents SE from three independent experiments. Asterisk indicates

significant differences in comparison with C (control plants) at

P\ 0.05. 1, 2, 3, 4, and 5 35S transgenic lines, 6, 7, 8, 9, and 10

rd29A transgenic lines, C control plants

Planta (2017) 245:1137–1148 1143

123

Estimation of proline and reducing sugars

in transgenic plants

Under normal conditions, no significant differences were

observed between proline and reducing sugars of trans-

genics and control plants. When 15-day-old transgenic

plants were subjected to mannitol (250 mM) stress, higher

accumulation of proline and reducing sugars were observed

(Fig. 4b, c). Whereas, under similar stress conditions,

control plants showed lower levels of proline and reducing

sugars (Fig. 4b, c). Under NaCl 250 mM (salt) stress,

transgenic lines accumulated higher levels of proline and

reducing sugars, while control plants showed lower levels

of proline and reducing sugars. Likewise, under cold (4 �C)stress, transgenics revealed higher levels of proline and

reducing sugars, while control plants showed lower levels

of proline and reducing sugars (Fig. 4b, c).

Estimation of relative water content and MDA

in transgenic plants

Higher relative water contents were observed in transgenic

lines as compared to the control plants (Fig. 5a). Further-

more, MDA contents in transgenic plants subjected to

drought, salt, and cold stresses were significantly lower as

compared to the control plants. However, under normal

conditions, no significant differences were observed in the

MDA levels between transgenics and control plants

(Fig. 5b).

SOD and catalase activities in transgenic plants

SOD and catalase activities were significantly increased in

the transgenic plants under salt, drought, and cold stresses

compared to activities recorded in the control plants under

similar stresses (Fig. 5c, d).

ABA sensitivity of CcCDR-transgenic plants

Seed germination rates of transgenic plants were compa-

rable to that of control plants in the absence of ABA.

However, seeds of transgenic plants showed decreased

germination rates in the presence of ABA as compared to

the control plants (Fig. 5e).

Expression analysis of stress-responsive genes

in CcCDR-transgenic plants

RT PCR analysis was carried out to analyze the mRNA

expression levels of selected stress-responsive genes, viz.,

P5CS (pyrroline-5-carboxylate synthetase), bZIP (bZIP

transcription factor), OsDREB, OsLEA3 (late embryogen-

esis abundant protein), and CIPK (calcineurin B-like pro-

tein-interacting protein kinases), under stress and

unstressed conditions. Under similar stress conditions, the

expression levels of P5CS, bZIP, DREB, OsLEA3, and

CIPK genes were found higher in the transgenic plants as

compared to the control plants (Fig. 6).

Fig. 4 Estimation of chlorophyll, proline, and reducing sugars in

CcCDR-transgenic plants. For determination of leaf chlorophyll

content, leaf discs transgenic and control plants were floated in

solution containing NaCl (250 mM)/mannitol (250 mM)/4 �C or

water for 72 h at room temperature (30 �C). Proline and reducing

sugars estimation was done on 2-week-old control and transgenic

plants subjected to NaCl (250 mM) and mannitol (250 mM) and

4 �C, independently for 3 days. Bar represents mean, and I represents

SE from three independent experiments. For each treatment, ten

seedlings were used. Asterisk indicates significant differences in

comparison with C (control plants) at P\ 0.05. C control plants,

CR1, CR2, CR3, CR4, and CR5 35S transgenic lines, RR1, RR2, RR3,

RR4, and RR5 rd29A transgenic lines

1144 Planta (2017) 245:1137–1148

123

Discussion

Transgenic rice plants expressing pigeonpea CcCDR gene,

under the control of CaMV35S and rd29A promoters, have

been generated in popular indica rice (BPT5204) variety.

bFig. 5 Estimation of relative water content, MDA, SOD, and catalase

activities in CcCDR-transgenic plants. a Relative water contents

(RWCs) of the 2-week-old CcCDR-CaMV35 and CcCDR-rd29

transgenics and control plants grown under similar conditions were

measured. Two-week-old control and transgenic seedlings were

transferred to the Hoagland solution with mannitol (250 mM) or

NaCl (250 mM) for 3 days or cold stress (4 �C for 3 days) for

estimation of MDA (b), SOD (c), and catalase (d). e Seed germination

rates of transgenics on ABA medium. Bar represents mean, and I

represents SE from three independent experiments. For each

treatment, ten seedlings were used. Asterisk indicates significant

differences in comparison with C (control plants) at P\ 0.05.

C control plants, CR1, CR2, CR3, CR4, and CR5 35S transgenic lines,

RR1, RR2, RR3, RR4, and RR5 rd29A transgenic lines

Fig. 6 Real-time PCR analysis of P5CS, bZIP, OsDREB, OsLEA3,

and CIPK genes in transgenic and control plants under normal and

drought stress conditions. Relative transcript levels of genes in control

(C) and transgenic (T) seedlings subjected to 150 mM mannitol stress

and under unstressed conditions were analyzed using Real-time PCR.

Actin gene was used as a reference. Bar represents mean, and I

represents SE from three independent experiments, Asterisk indicates

significant differences in comparison with C (control plants) at

P\ 0.05. C control plants

Planta (2017) 245:1137–1148 1145

123

Transgenic lines were evaluated against three abiotic

(drought, salt, and cold) stresses at various growth stages,

and were found to exhibit marked tolerance against these

stresses. Molecular analyses of different transgenics by

PCR and Southern-blots confirmed the unequivocal pres-

ence and integration of bar and CcCDR genes in the rice

genome. For evaluation of abiotic stress tolerance of rice

plants, single copy containing CcCDR-transgenic lines was

selected. Southern analysis of these plants indicated the

presence of single copy nature of introduced T-DNA when

the genomic DNA of the plants was probed with the

CcCDR and bar gene sequences (Fig. 1). Single copy

carrying transgenics are preferable to avoid the problem of

gene silencing, since multiple copies of transgene are often

responsible for gene silencing (Yarasi et al. 2008).

The CcCDR-transgenic lines revealed higher seed ger-

mination and plant survival rates when compared to the

untransformed control plants under drought, salt, and cold

stress conditions (Fig. 2). Transgenics also exhibited

higher shoot length, root length, and enhanced plant bio-

mass. Seed germination stage is considered as one of the

most sensitive to various environmental stresses compared

to other growth stages of the plant. Under stress conditions,

the transgenics showed higher percentage of seed germi-

nation and increased stress tolerance of seedlings as com-

pared to the control, suggesting that the CcCDR plays a

unique role in the abiotic stress tolerance. In rice, overex-

pression of the stress-associated OsiSAP8 protein was

found to promote increased seed germination rate and

improved seedling growth under salt, drought, and cold

stresses (Kanneganti and Gupta 2008).

Higher chlorophyll contents were observed in different

CcCDR transgenics when subjected to abiotic stress con-

ditions as compared to the control plants, thus demon-

strating the increased ability of transgenic plants to protect

the chlorophyll pigments required for photosynthesis under

environmental stress conditions. Transgenic rice and Ara-

bidopsis expressing pigeonpea CcHyPRP gene could

maintain higher chlorophyll contents when grown under

different abiotic stress conditions (Priyanka et al. 2010a, b;

Mellacheruvu et al. 2016).

Proline and soluble sugar levels were found to be higher

in CcCDR-transgenic lines, indicating that CcCDR regu-

lates accumulation of both free proline and reducing sugars

in the growing rice plants under stress conditions. The

presence of higher amounts of proline and soluble sugars

contributes to osmotic adjustment as well as maintenance

of the membrane integrity of the cells under stress condi-

tions (Huang et al. 2012). Transgenic rice plants expressing

wheat TaSTRG gene exhibited higher proline and soluble

sugar contents resulting in enhanced tolerance to multiple

abiotic stresses (Zhou et al. 2009). The transgenic rice

overexpressing zinc finger protein (ZFP245) showed

increased proline levels and elevated expression level of

pyrroline-5 carboxylate synthetase gene under stress con-

ditions which resulted in enhanced tolerance to cold,

drought, and oxidative stress conditions (Huang et al.

2009).

The CcCDR-transgenic lines, grown under abiotic stress

conditions, revealed higher levels of catalase and SOD

activities when compared to untransformed control plants

(Fig. 5). Catalase is an important antioxidant enzyme

which mediates the decomposition of H2O2 and thus plays

a major role in controlling the homeostasis of ROS,

whereas SOD is the first enzyme that acts in the detoxifi-

cation as it converts O2- radicals into H2O2 (Du et al.

2008). Furthermore, in our study, lower levels of MDA and

higher relative water contents were observed in the trans-

genic rice lines when they were grown under different

abiotic stress conditions (Fig. 5). MDA is widely used as a

reliable marker for oxidative lipid injury in plants’

response to both biotic and abiotic stresses (Davey et al.

2005). In the present study, transgenic plants proved to be

more effective in detoxifying the ROS and as such they

could maintain better membrane stability and water content

when grown under stress conditions. Under water-deficit

conditions, mature leaves from OsiSAP1 transgenic rice

plants exhibited decreased rate of water loss at the vege-

tative stage compared to that of WT plants (Dansana et al.

2014).

The CcCDR-transgenic rice plants, when subjected to

drought and salt stresses, at vegetative and reproductive

stages disclosed increased panicle size besides higher

number of filled grains as compared to the untransformed

control plants (Fig. 3). Mild abiotic stresses irreversibly

affected grain yield, without altering the plant survival and

the vegetative plant parts, since the rice plants are highly

vulnerable to the water stress at the reproductive stage

(Matsushima 1966). The marked ability of CcCDR-trans-

genic plants to survive and set seed even under severe

stress conditions affirms that CcCDR plays a major role in

protecting the rice plants during different stages of growth

and development. Overexpression of OsLEA3 in rice could

significantly improve the drought tolerance of plants under

field conditions (Xiao et al. 2007). It was reported that

overexpression of OsbZIP23 in rice conferred increased

drought and salinity tolerance to plants especially at the

reproductive stage (Xiang et al. 2008).

The seeds of CcCDR-transgenic lines were found

hypersensitive to ABA at the germination stage and

exhibited lesser germination rates as compared to the

control seeds. It is known that ABA could regulate various

processes of plant growth and development (Tang et al.

2012). ABA was found to be an important phytohormone

that helps in the adaptation of plants to environmental

stresses by regulating the expression of diverse genes (Sah

1146 Planta (2017) 245:1137–1148

123

et al. 2016). As such, in rice, the CcCDR might be acting as

a positive regulator in the ABA-mediated abiotic stress

tolerance. Transgenic rice plants overexpressing OsbZIP72

gene revealed hypersensitivity to ABA and showed higher

expression levels of different ABA responsive genes (Lu

et al. 2009).

In the present study, the fusion protein CcCDR-GFP was

predominantly visualized in the nucleus, suggesting that

the CcCDR enters into the nucleus, and interacts with

various other proteins, thereby regulating gene(s) expres-

sion. To know the mechanism of CcCDR in transgenic rice

plants, real-time PCR analysis was carried out with the

selected gene expression profiles (Fig. 6). Under stress

conditions, the expression levels of P5CS were upregulated

in transgenic rice plants which accorded with their higher

proline contents (Fig. 4).

The bZIP transcription factor gene OsABI5 and DREB

transcription factor gene OsDREB showed higher expres-

sion levels in CcCDR-transgenic rice plants as compared to

the control plants (Fig. 6). The bZIP transcription factors

were often found involved in different developmental and

physiological processes in response to various stresses, and

are thus known to play diverse roles in confronting adverse

environmental conditions (Jakoby et al. 2002). In rice,

OsABI5 gene was found to be involved in the adaptive

stress response as well as plant fertility (Zou et al. 2008).

The DREB proteins specifically interact with various

dehydration responsive cis-acting elements and thereby

control the expression of several stress inducible genes

(Dubouzet et al. 2003). Overexpression of OsDREB gene in

transgenic rice could significantly improve the tolerance of

plants to water-deficit stress conditions (Chen et al. 2008).

The levels of OsLEA3 and CIPK transcripts were found

upregulated in the CcCDR-transgenic plants (Fig. 6). In

plants, late embryogenesis abundant genes coding for

diverse proteins played an important role in the embryo

maturation process (Bartels et al. 1988). In general, LEA

and CIPK genes were induced by ABA treatment and

various abiotic stresses in both reproductive and vegetative

tissues, and were found confer tolerance to drought, cold,

and high salinity. The transgenic rice plants overexpressing

OsLEA3 showed vigorous plant growth under salinity

stress conditions as compared to the control plants (Godoy

et al. 1990; Duan and Cai 2012). Rice plants expressing

OsCIPK03 and OsCIPK12 proteins accumulated signifi-

cantly higher amounts of proline and soluble sugars, and

showed increased tolerance to cold, drought, and salt

stresses (Xiang et al. 2007).

The overall results demonstrate that the expression of

pigeonpea CcCDR gene has profound effects on the abiotic

stress tolerance of transgenic rice plants. Although the

exact mechanism of action of CcCDR protein is not known

in the stress tolerance of plants, it is presumed that it is due

to the modulation of various genes involved in both ABA-

dependent and-independent signal transduction pathways.

However, further investigations on the mode action of

CcCDR might provide additional insights into its precise

role in multiple stress tolerance. As such, the CcCDR

seems promising as a potential candidate for enhancing the

tolerance of different crop plants against multiple abiotic

stresses.

Author contribution statement KVR, VDR, MS, and TS:

conceived and designed the experiments; MS and TS:

performed the experiments; MS, TS, and KVR: analyzed

the data and wrote the paper.

Acknowledgements This project is supported by grants from the

Osmania University, Hyderabad, India. MS and TS are thankful to the

University Grants Commission, New Delhi, for the award of a

Research Fellowship. The authors are grateful to Prof. T. Papi Reddy

of the Department of Genetics, Osmania University, for his

suggestions.

Compliance with ethical standards

Conflict of interest Authors do not have any conflict of interest.

References

Bajaj S, Mohanty A (2005) Recent advances in rice biotechnology—

towards genetically superior transgenic rice. Plant Biotechnol J

3:275–307

Bartels D, Singh M, Salamini F (1988) Onset of desiccation tolerance

during development of the barley embryo. Planta 175:485–492

Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free

proline for water stress studies. Plant Soil 39:205–207

Boyer JS (1982) Plant productivity and environment. Science

218:443–448

Bray EA (1997) Plant responses to water deficit. Trends Plant Sci

2:48–54

Chen XP, Meng Y, Zhang M, Xia X, Wang P (2008) Over-expression

of OsDREB genes lead to enhanced drought tolerance in rice.

Biotechnol Lett 30:2191–2198

Dansana PK, Kothari KS, Vij S, Tyagi AK (2014) OsiSAP1

overexpression improves water-deficit stress tolerance in trans-

genic rice by affecting expression of endogenous stress-related

genes. Plant Cell Rep 33:1425–1440

Davey MW, Stals E, Panis B, Keulemans J, Swennen RL (2005)

High-throughput determination of malondialdehyde in plant

tissues. Anal Biochem 347:201–207

Du YY, Wang PC, Chen J, Song CP (2008) Comprehensive

functional analysis of the catalase gene family in Arabidopsis

thaliana. J Int Plant Biol 50:1318–1326

Du HY, Shen YZ, Huang ZJ (2013) Function of the wheat TaSIP gene

in enhancing drought and salt tolerance in transgenic Arabidop-

sis and rice. Plant Mol Biol 81:417–429

Duan J, Cai W (2012) OsLEA3-2, an abiotic stress induced gene of

rice plays a key role in salt and drought tolerance. PLoS One

7:e45117

Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S,

Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB

genes in rice, Oryza sativa L., encode transcription activators

Planta (2017) 245:1137–1148 1147

123

that function in drought, high-salt- and cold-responsive gene

expression. Plant J 33:751–763

Estrada- Melo AC, Chao Reid MS, Jiang CZ (2015) Overexpression

of an ABA biosynthesis gene using a stress-inducible promoter

enhances drought resistance in petunia. Hortic Res 2:15013.

doi:10.1038/hortres.2015.13

Evenson RE, Gollin D (2003) Assessing the impact of the green

revolution, 1960–2000. Science 300:758–762

Firon N, Nepi M, Pacini E (2012) Water status and associated

processes mark critical stages in pollen development and

functioning. Ann Bot 109:1201–1214

Godoy JA, Pardo JM, Pintor-Toro JA (1990) A tomato cDNA

inducible by salt stress and abscisic acid: nucleotide sequence

and expression pattern. Plant Mol Biol 15:695–705

He H, Serraj R (2012) Involvement of peduncle elongation, anther

dehiscence and spikelet sterility in upland rice response to

reproductive-stage drought stress. Environ Exp Bot 75:120–127

Hou X, Xie K, Yao J, Qi Z, Xiong L (2009) A homolog of human ski-

interacting protein in rice positively regulates cell viability and

stress tolerance. Proc Natl Acad Sci USA 106:6410–6415

Huang J, Sun SJ, Xu DQ, Yang X, Bao YM, Wang ZF, Tang HJ,

Zhang H (2009) Increased tolerance of rice to cold, drought and

oxidative stresses mediated by the overexpression of a gene that

encodes the zinc finger protein ZFP245. Biochem Biophys Res

Commun 389:556–561

Huang J, Sun S, Xu D, Lan H, Sun H, Wang Z, Bao Y, Wang J, Tang

H, Zhang H (2012) A TFIIIA-type zinc finger protein confers

multiple abiotic stress tolerances in transgenic rice (Oryza sativa

L.). Plant Mol Biol 80:337–350

Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance

in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403

Jakoby M, Weisshaar B, Droge-Laser W, Vicente-Carbajosa J,

Tiedemann J, Kroj T, Parcy F (2002) bZIP transcription factors

in Arabidopsis. Trends Plant Sci 7:106–111

Jiang SY, Bhalla R, Ramamoorthy R, Luan HF, Venkatesh PN, Cai

M, Ramachandran S (2012) Over-expression of OSRIP18

increases drought and salt tolerance in transgenic rice plants.

Transgenic Res 21:785–795

Kanneganti V, Gupta AK (2008) Overexpression of OsiSAP8, a

member of stress associated protein (SAP) gene family of rice

confers tolerance to salt, drought and cold stress in transgenic

tobacco and rice. Plant Mol Biol 66:445–462

Li F, Han Y, Feng Y, Xing S, Zhao M, Chen Y, Wang W (2013)

Expression of wheat expansin driven by the RD29 promoter in

tobacco confers water-stress tolerance without impacting growth

and development. J Biotechnol 163:281–291

Lu G, Gao C, Zheng X, Han B (2009) Identification of OsbZIP72 as a

positive regulator of ABA response and drought tolerance in

rice. Planta 229:605–615

Matsushima S (1966) Crop science in rice: theory of yield determi-

nation and its application. Fuji Publishing, Tokyo, p 365

McCouch SR, KochertG YuZH, Wang ZY, Kush GS, Coffman WR,

Tanksley SD (1988) Molecular mapping of rice chromosomes.

Theor Appl Genet 76:815–829

Mellacheruvu S, Tamirisa S, Vudem DR, Khareedu VR (2016)

Pigeonpea hybrid-proline-rich protein (CcHyPRP) confers biotic

and abiotic stress tolerance in transgenic rice. Front Plant Sci

6:1167. doi:10.3389/fpls.2015.01167

Miller GL (1959) Use of dinitrosalicylic acid reagent for determina-

tion of reducing sugar. Anal Chem 31(3):426–428

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

Nguyen D, Rieu I, Mariani C, van Dam NM (2016) How plants

handle multiple stresses: hormonal interactions underlying

responses to abiotic stress and insect herbivory. Plant Mol Biol

91:727–740. doi:10.1007/s11103-016-0481-8

Oh S-J, Kim YS, Kwon C-W, Park HK, Jeong JS, Kim J-K (2009)

Overexpression of the transcription factor AP37 in rice improves

grain yield under drought conditions. Plant Physiol 150(3):

1368–1379

Priyanka B, Sekhar K, Reddy VD, Rao KV (2010a) Expression of

pigeonpea hybrid-proline-rich protein encoding gene

(CcHyPRP) in yeast and Arabidopsis affords multiple abiotic

stress tolerance. Plant Biotechnol J 8:76–87

Priyanka B, Sekhar K, Sunita T, Reddy VD, Rao KV (2010b)

Characterization of expressed sequence tags (ESTs) of pigeon-

pea (Cajanus cajan L.) and functional validation of selected

genes for abiotic stress tolerance in Arabidopsis thaliana. Mol

Genet Genomics 283:273–287

Ramesh S, Nagadhara D, Reddy VD, Rao KV (2004) Production of

transgenic indica rice resistant to yellow stem borer and sap-

sucking insects, using super-binary vectors of Agrobacterium

tumefaciens. Plant Sci 166:1077–1085

Sah SK, Reddy KR, Li J (2016) Abscisic acid and abiotic stress

tolerance in crop plants. Front Plant Sci 7:571. doi:10.3389/fpls.2016.00571

Sambrook J, Russell DW (2001) Molecular cloning: a laboratory

manual, 3rd edn. Cold Spring Harbor Laboratory Press, NewYork

Srinath T, Reddy VD, Rao KV (2014) Ectopic expression of

pigeonpea cold and drought regulatory protein (CcCDR) in

yeast and tobacco affords multiple abiotic stress tolerance. Plant

Cell Tissue Organ Cult 119:489–499

Tamirisa S, Vudem DR, Khareedu VR (2014) Overexpression of

pigeonpea stress-induced cold and drought regulatory gene

(CcCDR) confers drought, salt, and cold tolerance in Arabidop-

sis. J Exp Bot 65:4769–4781

Tang N, Zhang H, Li X, Xiao J, Xiong L (2012) Constitutive

activation of transcription factor OsbZIP46 improves drought

tolerance in rice. Plant Physiol 158:1755–1768

Thompson AJ, Jackson AC, Symonds RC, Mulholland BJ, Dadswell

AR, Blake PS, Burbidge A, Taylor IB (2000) Ectopic expression

of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes

over-production of abscisic acid. Plant J 23(3):363–374

Tyagi AK, Mohanty A (2000) Rice transformation for crop

improvement and functional genomics. Plant Sci 158:1–18

Wang Q, Guan Y, Wu Y, Chen H, Chen F, Chu C (2008)

Overexpression of a rice OsDREB1F gene increases salt,

drought, and low temperature tolerance in both Arabidopsis

and rice. Plant Mol Biol 67:589–602

Xiang Y, Huang Y, Xiong L (2007) Characterization of stress-

responsive CIPK genes in rice for stress tolerance improvement.

Plant Physiol 144:1416–1428

Xiang Y, Tang N, Du H, Ye H, Xiong L (2008) Characterization of

OsbZIP23 as a key player of the basic leucine zipper transcription

factor family for conferring abscisic acid sensitivity and salinity

and drought tolerance in rice. Plant Physiol 148:1938–1952

Xiao B, Huang Y, Tang N, Xiong L (2007) Over-expression of a LEA

gene in rice improves drought resistance under the field

conditions. Theor Appl Genet 115:35–46

Yang S, Vanderbeld B, Wan J, Huang Y (2010) Narrowing down the

targets: towards successful genetic engineering of drought-

tolerant crops. Mol Plant 3:469–490

Yarasi B, Sadumpati V, Immanni CP, Reddy VD, Rao KV (2008)

Transgenic rice expressing Allium sativum leaf agglutinin

(ASAL) exhibits high-level resistance against major sap-sucking

pests. BMC Plant Biol 8:102

Zhou W, Li Y, Zhao BC, Ge RC, Shen YZ, Wang G, Huang ZJ

(2009) Overexpression of TaSTRG gene improves salt and

drought tolerance in rice. J Plant Physiol 166:1660–1671

Zou M, Guan Y, Ren H, Zhang F, Chen F (2008) A bZIP transcription

factor, OsABI5, is involved in rice fertility and stress tolerance.

Plant Mol Biol 66:675–683

1148 Planta (2017) 245:1137–1148

123