Indian Journal of Biotechnology

Vol 7, October 2008, pp 465-471

Overexpression of tobacco osmotin gene leads to salt stress tolerance in

strawberry (Fragaria × ananassa Duch.) plants

Amjad Masood Husaini and Malik Zainul Abdin*

Centre for Transgenic Plant Development, Department of Biotechnology, Jamia Hamdard, New Delhi 110 062, India

Received 26 June 2007; revised 26 February 2008; accepted 29 April 2008

Transgenic plants of strawberry tolerant to salt stress were produced using Agrobacterium mediated gene transfer

technology. Leaf discs of in vitro grown plantlets of strawberry cv. Chandler were used as explants. Agrobacterium

tumefaciens strain GV2260 harbouring osmotin gene under the control of CaMV 35S promoter in a binary vector system

pBinAR was used in transformation experiments. Transgene integration and copy number were assessed by PCR and

Southern hybridization confirming single copy as well as multiple copies of transgene integration in 10 different lines of

transgenic strawberry. Expression of osmotin gene was confirmed in transgenic lines TL3, TL5, TL9 using Northern

hybridization, while biochemical analysis revealed enhanced levels of proline, total soluble protein and chlorophyll content

as compared to the wild plants. Leaf disc assays performed using both wild-type and transgenic plants had shown that these

transgenic lines were tolerant to salt stress.

Keywords: Agrobacterium, proline, stress, strawberry, transgenic plants.

Introduction It is known for a long time that the concentration of

proline increases in a large variety of plants under

stress, up to 100 times the normal level1, which makes

up to 80% of the total amino acid pool. This dramatic

increase occurs over several hours2 or days

3. Stresses,

such as, cold4, heat

5, salt

6, drought

7, UV

8 and heavy

metal9 cause significant increase in the proline

concentration in a variety of plants. The increase in

proline content under salt stress has been correlated

with the increased activity of ∆-pyrroline-5-

carboxylate reductases10

and γ-glutamyl kinase11

, and

with the low activity of the degrading enzymes, viz.,

proline oxidase and proline dehydrogenase11

. Proline

is synthesized from glutamate by the catalytic action

of enzyme ∆1-pyrroline-5-carboxylate synthase

(P5CS) and the over-expression of this enzyme in

transgenic tobacco lead to increased levels of proline

and consequently improved growth, enhanced

biomass production, and flower development under

drought and salinity stress conditions12

. Accumulation

of proline has also been reported in transgenic

tobacco over-expressing osmotin gene; hence,

osmotin has been implicated in conferring tolerance to

osmotic stress13,14

. Transgenic plants that are not able

to produce proline, have a significantly lower stress

tolerance15,16

.

Osmotin is a stress responsive multifunctional 24

kDa basic protein17

belonging to PR-5 protein family

providing osmotolerance18–20

to plants. Osmotin could

be involved in osmotic adjustment by the cells either

by facilitating the accumulation or compartmentation

of solutes13

, or by being involved in metabolic or

structural alterations during osmotic adjustment21

.

Besides, osmotin shows antifungal activity also22

,

which is correlated with plasma membrane

permeabilization and dissipation of the membrane

potential of sensitive fungi23

. In this paper, authors

communicate the development of transgenic

strawberry overexpressing osmotin gene

constitutively under the control of CaMV 35S

promoter and its effect on proline accumulation and

salt tolerance.

Materials and Methods

Kanamycin Sensitivity of Leaf Discs

To identify the lethal concentration of kanamycin

for effective selection of putative transgenic plants,

regeneration medium with MS salts + B5 vitamins+

2% glucose + TDZ (4 mg/L) with different

concentrations of kanamycin (0-90 mg/L) were tested.

Transformation of Strawberry Plants

Leaf discs of strawberry (Fragaria × ananassa

——————

*Author for correspondence:

Tel: 91-11-26059688 ext. 5583/5594; Fax: 91-11-26059663

E-mail: mzabdin@yahoo.com

Abbreviations: WT, wild-type; TL, transgenic line; ROS, reactive

oxygen species

INDIAN J BIOTECHNOL, OCTOBER 2008

466

Duch. cv. Chandler) were transformed with

Agrobacterium tumefaciens strain GV2260 containing

732 bp osmotin gene24

under the control of CaMV

35S promoter, in the binary vector pBinAR. npt II

gene was used as a plant selectable marker.

PCR and Southern Blot Analysis

Genomic DNA was isolated from in vitro

developed plantlets of control and transgenic plants25

.

Integration of the transgene into the plant genome was

screened by PCR using osmotin gene specific primer

pair. The PCR products were then separated on 0.8%

agarose gel.

For Southern blot analysis, genomic DNA (10 µg)

was digested with BamH1, resolved on a 0.7%

agarose gel, and blotted on a nylon membrane (Roche

Diagnostics, Indianapolis). The blot was probed with

digoxigenin labeled osmotin at 46ºC. The probe was

labeled with digoxigenin using the DIG High Prime

DNA labeling and detection starter kit (Roche

Diagnostics, Indianapolis). The filter was washed and

the probe detected following the manufacturer’s

instructions.

Analyses of Transgenic Plantlets for Tolerance Against Salt

Stress

Leaf Disc Senescence Assay

The leaves of transgenic and wild-type plantlets

were removed and leaf segments of 1 cm2 were

floated in Petriplates having different concentrations

(0, 50, 100, 150, 200 mM) of NaCl in deionized

milliQ water. Chlorophyll estimation was done from

the samples after 7 d incubation at 25ºC under 16-h

photoperiod. Total chlorophyll content of the leaves

was estimated according to the method of Hiscox and

Israelstam26

.

Estimation of Chlorophyll, Total Soluble Protein, Proline and

Leaf Relative Water Contents in plantlets

Transgenic and wild-type plantlets were exposed to

different concentrations (0, 50, 100, 150, 200 mM) of

NaCl under in vitro conditions. The leaves of these

plantlets were then collected after 1 and 2 wk.

Chlorophyll, total soluble protein, proline and relative

water contents were determined using the methods of

Hiscox and Israelstam26

, Bradford27

, Bates et al28

and

Barr and Weatherley29

, respectively. All

measurements were carried out in triplicate.

Measurement of Shoot and Root Length

At every sampling, plantlets were taken from each

treatment and separated into roots and shoots. The

length of roots and shoots was measured. All

measurements were carried out in triplicates.

Results

Kanamycin Sensitivity of Leaf Discs

Sensitivity of leaf discs to kanamycin was

established prior to actual transformation experiments

in order to determine the effective concentration for

selection. In the absence of kanamycin, the leaf discs

regenerated normally and produced multiple shoots

on the periphery. The shoot regeneration capacity of

leaf discs was inhibited even at 30 µg/mL of

kanamycin, but they expanded to some extent before

bleaching. However, the growth of leaf discs was

considerably restricted and bleaching was complete at

50 µg/mL kanamycin within 4 wk. Hence, this

concentration was used for selection of transformed

cells in leaf discs. Higher kanamycin concentrations

were too toxic to leaf discs and caused immediate

browning.

Transformation of Strawberry with Tobacco Osmotin Gene

Transformation of strawberry was carried out with

the osmotin gene using the construct as shown in

Fig. 1. This resulted in the regeneration of several

putative transgenic shoots, of which only 17 survived

and the rest perished in the different stages of their

growth and development. The young shoots from

these 17 putative transgenic lines were analyzed for

osmotin gene presence using PCR. Ten transgenic

lines tested positive, whereas wild-type control shoots

were negative. Transgenic plants showed 0.73 kb

amplification products when amplified with osmotin

gene specific primers (Fig. 2). Southern hybridization

confirmed single and multiple copy integration of the

transgene in different transgenic lines (Fig. 3).

Analysis of Transgenic Strawberry Plantlets for Tolerance to

Salt Stress

The plants from transgenic line, TL3 were analysed

for tolerance to NaCl stress by the leaf disc

senescence assay. Leaf discs were floated in NaCl

solution (0, 50, 100, 150, 200 mM) and after one wk

of stress clear difference was visible as the leaf discs

from transgenic plants remained green, whereas the

leaf discs from wild-type plantlets showed complete

senescence (Fig. 4A,B). The visual phenotype was

confirmed by estimating total chlorophyll content, and

it was found that transgenic seedlings had

significantly higher chlorophyll than wild-type

seedlings when exposed to NaCl stress (Fig. 5).

HUSAINI & ABDIN: SALT TOLERANCE IN STRAWBERRY

467

To further confirm the ability of plants from

transgenic lines, TL3, TL5 and TL9 over-expressing

osmotin gene to tolerate salt stress, the shoots of

transgenic and wild-type seedlings were cultured on

MS salts + B5 vitamin + glucose (2%) + Kn (1 mg/L)

supplemented with NaCl (0, 50, 100, 150, 200 mM)

(Fig. 6). Variation in chlorophyll and total soluble

protein in the leaves of such plantlets was determined

at 1 and 2 wk intervals (Figs 7 & 8). As osmotin has

been implicated to increase proline content, proline

content was measured in the transgenic and wild-type

plants. Interestingly, 4 to 6-fold increase in proline

content over the wild-type plants was observed in

leaves of transgenic plants without being subjected to

stress (Fig. 9).

Fig. 1—Osmotin gene construct. The gene is cloned under the

control of CaMV 35S promoter in binary vector Bin AR

containing nptII gene as selection marker.

Fig. 2—PCR analysis in 17 putatively kanamycin resistant plants

of strawberry for presence of osmotin gene. A fragment of 730 bp

corresponding to the coding region of osmotin gene was amplified

in 10 transgenic lines. Lane 1 : DNA molecular weight marker

(100 bp), Lane 2: positive control (osmotin plasmid DNA), Lane

3: negative control (wild-type plant DNA), and Lane 4–20

putative transgenic plant DNA.

Fig. 3—Southern analysis of 10 individual transgenic strawberry

plants. Genomic DNA was digested with Bam H1 and probed

with osmotin specific gene probe. Lane 1: Positive control

(Plasmid containing osmotin gene), Lane 2: Negative control

(DNA from non-transgenic strawberry plant), and Lane 3-12:

DNA sample from transgenic strawberry plants.

Fig. 4—Difference in chlorophyll pigment leaching from the leaf

discs of wild-type and transgenic plants (TL3) in response to NaCl

stress at (A) 100 mM and (B) 150 mM NaCl.

Fig. 5—Variation in chlorophyll content (mg g-1 fresh wt) of leaf

discs in “Leaf discs senescence assay” after 1 wk of exposure to

NaCl. The bars represent mean ± standard error

INDIAN J BIOTECHNOL, OCTOBER 2008

468

Authors observed significant differences in the

growth and development between wild-type and

transgenic plants. Shoot length and root length of

transgenic plants was greater than that of wild-type

plants even after 2 wk of exposure to salt stress (Figs

10 & 11).

Discussion Of 2445 leaf discs that were co-cultivated with

genetically engineered Agrobacterium, only 17

putative transgenic shoots could be regenerated. The

young transformed shoots were analyzed for nptII

gene presence using PCR. Ten transformants tested

positive, whereas untransformed control shoots were

negative. These findings are supported by the results

obtained by James et al30

who had screened 15

kanamycin resistant shoots of strawberry cultivar

Rapella, of which only 10 tested positive by Southern

hybridization. Of these ten transgenic lines only three

lines, which showed single (TL3) and double copy

(TL5, TL9) integration, were able to show expression at

metabolite levels.

Osmotin gene is induced by salt, water and low

temperature stress24,31,32

. With regard to osmotin,

numerous studies have been attempted to determine

the physiological role in stress tolerance33

, but the

mechanism of its action still remains unknown. As

osmotin is a small protein of 24 kDa mol wt, it may

have a role in signaling especially in the upregulation

of proline biosynthetic pathway. The susceptibility in

Saccharomyces cerevisiae to osmotin is controlled by

Fig. 6—In vitro developed strawberry transgenic plantlets after 2

wk on different concentrations of NaCl (0-200 mm): Wild-Type

(A) Transgenic line TL3 (B).

Fig. 7—Chlorophyll content (mg g-1 fresh wt) of the leaves of

strawberry plantlets (wild and transgenic) exposed to varied

concentrations of NaCl under in vitro conditions. The bars

represent mean ± standard error

Fig. 8—Total soluble protein content (mg g-1 fresh wt) of the

leaves of strawberry plantlets (wild and transgenic) exposed to

varied concentrations of NaCl for 1, 2 wk under in vitro

conditions. The bars represent mean ± standard error

Fig 9—Proline content (µg g-1 fresh wt) of the leaves of

strawberry plantlets (wild and transgenic) exposed to varied

concentrations of NaCl for 1, 2 wk under in vitro conditions. The

bars represent mean ± standard error

HUSAINI & ABDIN: SALT TOLERANCE IN STRAWBERRY

469

activation of a heteromeric G-protein and MAP kinase

pathway34

, and since a number of abiotic stress

factors, such as, wounding, low temperature, high

osmolarity, high salinity and reactive oxygen species,

act as a signal in activating MAP kinase cascade35

,

osmotin could be associated with MAP kinase

signaling, regulating the cellular redox state.

Identification of osmotin-like protein from

intercellular space of a halophyte,

Mesambryanthemum crystallnum and association of

osmotin protein with tonoplast of tobacco21,36

support

the contention that osmotin is involved in intracellular

compartmentation of Na+

ions both to the vacuole as

well as to the intercellular space and, thereby,

minimizing the buildup of Na+

ions in the cytoplasm

or reducing the uptake of Na+

ions in the cell36

.

The leaching of chlorophyll during the ‘leaf disc

senescence assay’ of strawberry might be due to

increased membrane permeability under high NaCl

concentration37

, and the difference in the extent of

leaching between wild and transgenic strawberry may

be due to osmotin’s role in sequestering Na+ ions both

in the vacuoles as well as intercellular spaces and

enhancing the stability of membranes.

In general, there was some reduction in the amount

of chlorophyll of the strawberry plantlets treated with

NaCl. This reduction in chlorophyll could be due to

the interference of Na+ and Cl

- ions with the enzymes

associated with chlorophyll biosynthetic pathway or

to a disturbance in the integration of chlorophyll

molecules into stable complexes. Transgenic

strawberry lines overexpressing osmotin were able to

maintain higher chlorophyll content under salt stress

probably due to its ability in enhancing their proline

content, which in turn significantly lowers the level of

reactive oxygen species and alleviates salt stress

induced enhancement in ribulose oxygenase activity38

.

Maintenance of high chlorophyll content in the leaves

of transgenic strawberry plantlets even at high NaCl

concentrations is of special significance, as low

concentration of chlorophyll has been reported to

directly limit the photosynthetic potential and primary

productivity in plants39,40

.

Protein synthesis is rapidly inhibited by salinity

stress41

and overall protein content is reported to

decrease under severe stress, as proteins being

macromolecules do not contribute much towards

colligative properties like osmolarity. In wild-type

strawberry the decrease in protein content can be

attributed to the fact that abiotic stresses enhance the

protein degradation process as a result of reactive

oxygen species generation, increased protease activity

and inhibited transcription, translation and

polyribosome activity42,43

. However, since transgenic

strawberry plantlets overexpressing osmotin show

reduced level of stress even at high salinity, their

ability to maintain higher protein content may be due

to osmotin-induced proline accumulation, and hence

preventing the buildup of proteases by ROS

scavenging and membrane stabilization. Proline has

been suggested to directly influence protein solvation

by preventing dehydration-induced thermodynamic

perturbations in proteins44

, which is due to its ability

to induce the formation of strong H-bonded water

around the proteins45

under water stressed conditions.

The higher protein content of transgenic plants under

unstressed condition may be due to both reduced

degradation of protein and accumulation of osmotin

Fig. 10—Shoot length (cm plant-1) of strawberry plantlets (wild

and transgenic) exposed to varied concentrations of NaCl under in

vitro conditions. The bars represent mean ± standard error

Fig. 11—Root length (cm plant-1) of strawberry plantlets (wild

and transgenic) exposed to varied concentrations of NaCl under in

vitro conditions. The bars represent mean ± standard error

INDIAN J BIOTECHNOL, OCTOBER 2008

470

protein in response to its overexpression under CaMV

35S promoter.

The results in transgenic tobacco13

, tomato46

and

strawberry (present study) showing improved

tolerance to salt stress, in combination with the results

showing antifungal action of osmotin23,47

indicate the

usefulness of osmotin in developing transgenic plants

for tolerance against both abiotic and biotic stresses.

In particular, transgenic strawberry plants produced in

the present study with enhanced ability to grow under

long period of NaCl exposure provide a way of

achieving a significant yield gain in salinity affected

areas.

Acknowledgement

The authors are thankful to Dr K C Bansal,

Scientist, National Research Centre for Plant

Biotechnology, Indian Agricultural Research Institute,

New Delhi for providing the osmotin gene construct.

The first author is thankful to Council of Scientific

and Industrial Research, New Delhi for the award of

junior and senior research fellowships.

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