RESEARCH

High Efficiency Transformation of Banana [Musa acuminataL. cv. Matti (AA)] for Enhanced Tolerance to Salt and DroughtStress Through Overexpression of a Peanut Salinity-InducedPathogenesis-Related Class 10 Protein

Anjana Rustagi • Shalu Jain • Deepak Kumar •

Shashi Shekhar • Mukesh Jain • Vishnu Bhat •

Neera Bhalla Sarin

� Springer Science+Business Media New York 2014

Abstract Bananas and plantains (Musa spp. L.) are

important subsistence crops and premium export com-

modity in several countries, and susceptible to a wide range

of environmental and biotic stress conditions. Here, we

report efficient, rapid, and reproducible Agrobacterium-

mediated transformation and regeneration of an Indian

niche cultivar of banana [M. acuminata cv. Matti (AA)].

Apical meristem-derived highly proliferative multiple

shoot clump (MSC) explants were transformed with the

Agrobacterium strain EHA105 harboring a binary vector

pCAMBIA-1301 carrying hptII and uidA. Sequential agro-

infiltration (10 min, 400 mmHg), infection (additional

35 min, Agrobacterium density A600 = 0.8) and co-culti-

vation (18 h) regimen in 100 lM acetosyringone contain-

ing liquid medium were critical factors yielding high

transformation efficiency (*81 %) corroborated by tran-

sient GUS expression assay. Stable transgenic events were

recovered following two cycles of meristem initiation and

selection on hygromycin containing medium. Histochemi-

cal GUS assay in several tissues of transgenic plants and

molecular analyses confirmed stable integration and

expression of transgene. The protocol described here

allowed recovery of well-established putative transgenic

plantlets in as little as 5 months. The transgenic banana

plants could be readily acclimatized under greenhouse

conditions, and were phenotypically similar to the wild-

type untransformed control plants (WT). Transgenic plants

overexpressing Salinity-Induced Pathogenesis-Related

class 10 protein gene from Arachis hypogaea (AhSIPR10)

in banana cv. Matti (AA) showed better photosynthetic

efficiency and less membrane damage (P \ 0.05) in the

presence of NaCl and mannitol in comparison to WT plants

suggesting the role of AhSIPR10 in better tolerance of salt

stress and drought conditions.

Key words Abiotic stress � Agrobacterium � Arachis

hypogaea � Multiple shoot clumps � Musa spp. �PR proteins � Transgenic

Abbreviations

AhSIPR10 Arachis hypogaea salinity-induced PR class

10 gene

ECS Embryogenic cell suspension

MS Murashige and Skoog medium

MSC Multiple shoot clump

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12033-014-9798-1) contains supplementarymaterial, which is available to authorized users.

A. Rustagi � S. Jain � D. Kumar � S. Shekhar � M. Jain �N. B. Sarin (&)

Plant Developmental Biology and Transformation Lab, School of

Life Sciences, Jawaharlal Nehru University, New Delhi 110067,

India

e-mail: neerasarin@rediffmail.com

Present Address:

A. Rustagi

Department of Botany, Ramjas College, University Enclave,

New Delhi 110007, India

Present Address:

S. Jain

Department of Plant Pathology, North Dakota State University,

Fargo, ND 58102, USA

Present Address:

M. Jain

Department of Plant Pathology, University of Florida,

Gainesville, FL 32610, USA

Present Address:

V. Bhat

Department of Botany, University of Delhi, Delhi 110007, India

123

Mol Biotechnol

DOI 10.1007/s12033-014-9798-1

PR10 Pathogenesis-related (class 10)

WT Wild-type

Introduction

Bananas and plantains (Musa spp. L., Musaceae, Zingibe-

rales) are giant perennial monocotyledonous herbs culti-

vated in nearly 120 countries of the humid and sub-humid

tropical regions. Bananas (and plantains) form the fourth

most important food crop in the developing countries after

rice, maize and wheat, and the second largest fruit crop in

the world. Besides being rich in proteins, vitamins and

essential minerals, bananas also provide a starch staple

across some of the poorest parts of Africa and Asia, and are

a premium export commodity in several countries [1]. India

accounted for a principal 34.5 % share of the global banana

produce of 102 million tons valued at USD 28 billion in

2012 (FAO 2013, http://www.fao.org/corp/statistics/en/

2013). Worldwide, more than a thousand cultivars or

landraces of domesticated bananas have been documented.

In India, around 12 local and 30 exotic varieties are com-

mercially cultivated in different states. Inter- and intra-

specific hybridization events between two wild diploid

(2n = 2x = 22) species, M. acuminata Colla. (A genome)

and M. balbisiana Colla. (B genome), have yielded seed-

less and parthenocarpic sweet dessert bananas (AAA gen-

ome) and mostly starchy plantains (AAB or ABB genome)

with triploid (2n = 3x = 33) genomic organization. Some

hybrids have also evolved through crossing with M.

schizocarpa Simmonds (S genome, 2n = 2x = 22) and

Australimusa species (T genome, 2n = 2x = 20) [2].

Despite its economic importance, improvement in banana

germplasm has not been adequately supported by a suc-

cessful breeding history. Recalcitrance of banana in tradi-

tional breeding programs has been attributed to polyploidy,

parthenocarpy, male and/or female sterility, limited genetic

variability, long generation time (10–24 months), and large

space requirements (6 m2 per plant). Banana phenotypes

are poor indicator of genetic constitution and this restricts

the development of specific breeding schemes due to

unreliable prediction of combining ability of parental

genotypes. Value added trait integration and transgenic

augmentation of elite germplasm can reinforce sustainable

yield performances through improved fruit quality, adapt-

ability to changes in agricultural climates, disease control

and diminished agrichemical usage in commercial planta-

tions as well as small-holding subsistence farms [3].

Successful genetic transformation of banana is reliant on

several empirical parameters including Agrobacterium

strain and inoculum density, age of embryogenic cell

suspension (ECS) cultures, infection and co-cultivation

duration and co-cultivation medium. Likewise, physical

and chemical treatments such as co-centrifugation of ECS

and Agrobacterium cultures, heat-shock pretreatment of

ECS cultures, microwounding of meristematic explants,

sonication and agro-infiltration, use of surfactants, Vir gene

inducers and polyamines, etc. have been shown to increase

bacterial virulence and accessibility, and enhance trans-

formation-competence of plant cells [4–8]. The current

banana transformation protocols employing ECS cultures

are, however, significantly constrained by low transfor-

mation frequencies, high genotype and cell line depen-

dence, and increased accumulation of somaclonal

variations due to extremely long in vitro culture passage of

ECS lines [9]. Moreover, high incidence of transgene

silencing, yield penalties, and clonal infidelity have been

observed in several plant species following biolistic

transgene delivery [10]. Agrobacterium-mediated trans-

formation and regeneration exploiting direct organogenic

potential of apical or intercalary meristematic explants of

banana offers a relatively rapid and facile method for

potentially achieving low copy number, defined integration

profiles, and stable expression of transgenes [4, 6, 8, 11].

Accumulation of pathogenesis-related (PR) proteins

during microbial infection or under abiotic stress condi-

tions constitutes an integral component of innate immune

responses in plants. The PR proteins have been designated

to 17 families contingent upon their primary structure,

serological relationships and biological activities, the

largest being the PR10 family with more than 100 members

reported across more than 70 plant species [12]. Several PR

proteins display antimicrobial and secondary metabolic

enzyme activities, for example, chitinases (PR3, PR4, PR8,

and PR11 family members) [11, 13], b-1,3-glucanase

(PR2) [11], defensins (PR12) [14], and RNAse (PR10) [15–

17]. PR10 proteins are transcriptionally responsive across

biotic as well as abiotic stress environments including

drought, salinity, low and high temperatures, heavy metals,

wounding, and UV exposure [15, 18–20]. In a previous

study from our laboratory, it was noted that several proteins

with similarities to the PR10 family members were

upregulated in peanut callus cultures subjected to salt stress

[21]. Transgenic overexpression of a salinity-induced PR10

gene (AhSIPR10) from peanut in tobacco exhibited

enhanced tolerance to salt, heavy metal (ZnCl2) and man-

nitol-induced drought stress [19]. India, being one of the

centers of origin of Musa, boasts of a diverse repertoire of

commercial and niche cultivars including a few diploid

varieties under commercial cultivation [22]. Matti (AA

genome) is an indigenous diploid cultivar of choice in

Southern India, which is highly prized for its fragrant fruits

and locally valued for therapeutic purposes such as to treat

upset stomach and lower blood pressure. It is a high

Mol Biotechnol

123

yielding variety and shows tolerance to yellow Sigatoka

leaf spot disease. The objectives of this study were two-

fold: first, standardization of Agrobacterium-mediated

genetic transformation platform for cultivar Matti (AA)

using multiple shoot clumps (MSCs) as the explant source;

and second, to overexpress a peanut PR10 gene (Ah-

SIPR10) [19] in the banana cv. Matti (AA) and validate its

role in conferring tolerance to salt and mannitol-induced

drought stress.

Materials and Methods

Disease-free plants of banana [Musa acuminata cv. Matti

(AA)] were obtained from National Bureau of Plant

Genetic Resources, New Delhi, India and propagated

in vitro via rooting of shoot tip cultures on Murashige and

Skoog (MS) medium [23] supplemented with 3 % (w/v)

sucrose, 20 mg/l ascorbic acid, 0.4 mg/l indole acetic acid

(IAA), 2.0 g/l gelrite, pH 5.8 (rooting medium). Regener-

ation from shoot tip cultures of cultivar Matti (AA) was

achieved following the procedure of May et al. [4]. Shoot

tip explants (about 1 cm) having the apical dome fully

covered by 3–4 leaf primordia and approximately 3 mm of

corm tissue beneath the apical dome were harvested from

in vitro grown plantlets and used for establishment of

compact meristematic MSCs in MS medium supplemented

with 3 % (w/v) sucrose, 20 mg/l ascorbic acid, 22.5 mg/l

6-benzylaminopurine (BAP), and 0.4 mg/l IAA (shoot

regeneration medium). All the cultures were maintained

under cool white fluorescent light (40 lE/m2/s, 16 h pho-

toperiod) at 26 ± 2 �C.

The disarmed binary vector pCAMBIA-1301 (GenBank

acc. AF234297; Center for the Application of Molecular

Biology of International Agriculture, Black Mountain,

Australia; http://www.cambia.org.au) contains the plant

selection marker hygromycin phosphotransferase (hptII)

and the reporter b-glucuronidase (uidA) gene interrupted

by a modified castor bean catalase intron, both driven by

double enhanced cauliflower mosaic virus (CaMV) 35S

promoter. The Pathogenesis-Related class 10 (PR10) pro-

tein gene (AhSIPR10, GenBank acc. DQ813661) was iso-

lated from a salt tolerant cell line of peanut (Arachis

hypogaea) and subcloned into the pCAMBIA-1302 vector

(GenBank acc. AF234298) to yield pCAM-PR10 [19]. The

plasmid pCAMBIA-1302 contains mgfp5 gene (GenBank

acc. U87973) and the hptII selectable marker gene, both

under the transcriptional control of CaMV35S promoter.

Binary plasmids pCAMBIA-1301 and pCAM-PR10 were

mobilized into the super-virulent Agrobacterium tumefac-

iens strain EHA105 via electroporation. The A. tumefaciens

harboring the binary plasmid was grown in 10 ml selective

YENB medium [0.75 % (w/v) yeast extract, 0.8 % (w/v)

nutrient broth, pH 7.0, 100 mg/l kanamycin] at 28 �C for

24 h, used to inoculate 10 ml of fresh YENB medium

(1 %, v/v) and grown further for 28 �C for 24 h. About

0.5 ml of inoculum from this secondary culture was used to

inoculate 50 ml of fresh YENB medium supplemented

with 100 mg/l kanamycin and 100 lM acetosyringone, and

grown further for 6–8 h at 28 �C till the desired absorbance

(A600) was obtained.

Fifty ml Agrobacterium culture (A600 = 0.8) harboring

the plasmids pCAMBIA-1301 or pCAM-PR10 was har-

vested at 5,000g for 10 min at 28 �C and re-suspended in

50 ml of liquid MS medium containing 3 % (w/v) sucrose,

20 mg/l ascorbic acid, 22.5 mg/l BAP and 0.4 mg/l IAA

(shoot initiation medium), and 100 lM acetosyringone.

MSCs were excised from 3-week-old in vitro grown

plantlets, immersed in Agrobacterium suspension (25–30

explants per 50 ml bacterial suspension, in a 250 ml con-

ical flask) and vacuum infiltrated at 400 mmHg for 10 min.

Agrobacterium suspension was decanted off after total

45 min and the MSC explants were co-cultivated for 18 h

in liquid shoot initiation medium containing acetosyrin-

gone (100 lM) with gentle shaking at 70 rpm in dark. At

the end of co-cultivation period, the explants were washed

twice for 60 min each in liquid shoot initiation medium

supplemented with 200 mg/l cefotaxime, and cultured on

same medium in the presence of 10 mg/l hygromycin.

Viable explants were dissected into smaller pieces (con-

taining 3–4 shoot buds) and subcultured on selection

medium for additional 4 weeks. Elongation of transgenic

shoot buds was achieved on selection medium containing

2.25 mg/l BAP and 0.4 mg/l IAA. After 4 weeks on

elongation medium, putative transgenic shoots were indi-

vidually excised and transferred to semi-solid MS medium

supplemented with 0.4 mg/l IAA and 10 mg/ml hygro-

mycin for rooting. Transgenic banana plants were clonally

propagated to yield at least three sister plantlets prior to

greenhouse acclimatization. Plantlets with well-established

roots were transferred to plastic pots containing agropeat

and kept for 2 months in a growth room maintained at

26 ± 2 �C under white fluorescent light. The hardened

plantlets were finally transferred to clay pots containing

soil and agropeat (1:1, v/v) and grown in a greenhouse

maintained at 26 ± 2 �C with 70 % relative humidity.

Young banana leaves were used for the extraction of

high molecular weight genomic DNA and total RNA using

the DNAeasy Plant Mini Kit (Qiagen Inc., CA, USA) and

TriPure reagent (Roche, IN, USA), respectively. Five lg

total RNA was reverse-transcribed using the AccuScript

High Fidelity reverse transcriptase (Stratagene, CA, USA).

PCR reactions were carried out using either genomic DNA

or the first strand cDNA template and gene specific prim-

ers: uidA, 50-GCCATTTGAAGCCGATGTCACGCC-30

(forward) and 50-GTATCGGTGTGAGCGTCGCAGAAC-30

Mol Biotechnol

123

(reverse); AhSIPR10, 50-ATGTCCATGGATGGGCGTC

TTCACTTTCGAG-30 (forward) and 50-GATGACTAGT

TAATATTGAGTAGGGTTGG-30 (reverse); actin, 50-AG

TAAGGTGACCTTGCAATTACTTTAGACTTCACCG-30

(forward) and 50-AAAGGCTAGCGTTGAAGATGCCTC

TGCCGAC-30 (reverse). The thermal cycling (MJ

Research, Waltham, MA, USA) protocol entailed activa-

tion of Platinum Taq DNA polymerase (Invitrogen,

Carlsbad, CA) at 94 �C for 5 min, followed by 30 cycles of

denaturation at 94 �C, primer annealing at 56 �C for 15 s,

and extension at 72 �C for 30 s each. The amplification

reactions were finally extended for 10 min at 72 �C and

held at 4 �C. The amplified products were electrophoresed

on 1 % agarose gel and visualized under UV light by

ethidium bromide staining. Southern blot analysis was

performed with 10 lg DNA, following the procedure by

Sambrook et al. [24] using radiolabeled probe.

The histochemical assay for transient GUS gene

expression in MSC explants was performed 1 week after

co-cultivation according to the modified procedure of Jef-

ferson [25]. Meristematic shoot clumps, shoots, and leaves

from the control and putative transgenic plants were briefly

rinsed in sterile 50 mM potassium phosphate buffer (pH

7.0), immersed in GUS staining solution [1 M NaH2PO4

(pH 4.0), 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6]�3H2O, 1 mM EDTA, 0.1 % Triton X-100, 1 mg/ml

5-bromo-4-chloro-3-indolyl glucuronide (X-gluc), 0.02 %

sodium azide] and vacuum infiltrated for 5 min

(200 mbar). The tissues were incubated overnight at 37 �C

and later rinsed extensively with 70 % (v/v) ethanol to

remove chlorophyll. For leaf senescence assay, leaf sec-

tions of equal diameter were excised from the fifth leaf

from top of transgenic and wild-type untransformed control

(WT) banana plants and floated on liquid MS basal med-

ium containing NaCl (200–800 mM) or mannitol

(200–600 mM). Chlorophyll content in the leaf sections

was estimated after 5 days according to the procedure of

Arnon [26]. The method described by Sairam and Sri-

vastava [27] was used to determine electrolyte leakage.

Leaves from WT and transgenic plants were collected and

any surface-adhered electrolyte was washed off with

deionized water. Leaves were immersed in 10 ml of

deionized water in a test tube and the electrical conduc-

tivity (EC1) was measured. Electrical conductivity after

incubation at 55 �C for 30 min (EC2) was determined

again. The total conductivity after heating samples in boing

water for 1 h (EC3) was measured in the solution. Relative

ion leakage was calculated as a percentage of the total

conductivity using the following formula: Relative Elec-

trolyte Leakage = [(EC2 - EC1)/EC3] 9 100. Malondi-

aldehyde (MDA) content was measured in leaves by

following the procedure of Heath and Packer [28]. The

leaves from WT and transgenic plants were ground to a fine

powder in liquid nitrogen. Three ml of 10 % trichloroacetic

acid was added to 200 mg leaf powder and incubated at

4 �C for overnight. Supernatant was collected after cen-

trifugation at 1,000g for 20 min. Two ml of 0.6 % thio-

barbituric acid (TBA) mixed thoroughly to 2 ml of the leaf

supernatant and heated in boiling water for 15 min.

Supernatant was cooled and centrifuged. Absorbance val-

ues of the supernatant were recorded at 532 and 450 nm

wavelengths using water as blank. The following formula

for the calculation of MDA content was used: MDA con-

tent (lmol/l) = 6.45 9 OD532 - 0.56 9 OD450. Results

were analyzed by the Student’s t test at significant level of

P value (\0.05).

Results and Discussion

Meristems and shoot tips are, unarguably, the most widely

used explant material for mass clonal propagation, germ-

plasm conservation, and recovery of axenic plant material

in banana. Shoot tip explants (Fig. 1a) were excised from

in vitro grown plantlets under aseptic conditions and cul-

tured for 2 months. Approximately 92 % explants devel-

oped dense, proliferative MSCs (with an average number

of 28 shoot buds per MSC) on shoot regeneration medium

(Fig. 1b). Adventitious shoot bud clusters were longitudi-

nally excised and further elongated in the presence of

reduced BAP concentration (2.25 mg/l) (Fig. 1c) and

eventually rooted in the presence of 0.4 mg/l IAA

(Fig. 1d). Preliminary experiments were carried out to

optimize the conditions for Agrobacterium-mediated

transformation of MSC explants of cultivar Matti (AA)

(Fig. 2). As expected, transformation efficiency (based on

transient GUS expression assayed 3 days after co-cultiva-

tion period) was significantly affected by the growth stage

of Agrobacterium culture used for transformation. At

bacterial density (A600) of 0.8 and following a co-cultiva-

tion regimen in liquid shoot initiation medium for 45 min,

56–58 % MSC explants were routinely scored as GUS

positive. However, it was extremely important to let the

Agrobacterium cultures grow to the required density, rather

than concentrating or diluting the bacteria following har-

vest and resuspension in the shoot regeneration medium.

Transformation efficiency was further enhanced to

*81 %, if the MSC explants were agro-infiltrated for

10 min (400 mmHg vacuum) and incubated further in the

Agrobacterium suspension for additional 35 min, followed

by co-cultivation for 18 h in liquid shoot initiation medium

supplemented with 100 lM acetosyringone. Ten mg/l hy-

gromycin (LD50 value) was considered optimum for the

selection and recovery of putative transgenic events (Sup-

plementary Fig. 1). Although, any differences in the initial

shoot bud initiation response of MSCs were indiscernible

Mol Biotechnol

123

during the first week of culture in the presence of 5 or

10 mg/l hygromycin, significantly compromised initiation

and growth of shoot buds and necrosis of *50 % explants

was observed after 10 days of culture on 10 mg/l hygro-

mycin. The explants cultured on medium containing higher

concentrations of hygromycin (15–40 mg/l) showed

extremely retarded growth of multiple shoot primordia and

necrosis within the first week of culture.

Following the optimum procedure for infection and co-

cultivation of MSC explants with A. tumefaciens strain

EHA105 harboring the plasmid pCAMBIA-1301 as

described above, the MSC explants were finally cultured on

semi-solid shoot initiation medium for 8 weeks in the

presence of 10 mg/l hygromycin and 200 mg/l cefotaxime

to prevent bacterial overgrowth (Fig. 1e). The hygromycin

resistant MSC explants with proliferative shoot bud initials

were further excised into smaller pieces (containing 3–4

shoot bud initials) and transferred to fresh selection med-

ium for additional 4 weeks. The rapidly growing shoot

buds were elongated in the presence of 2.25 mg/l BAP and

0.4 mg/l IAA for 4 weeks (Fig. 1f) and transferred to the

rooting medium containing 0.4 mg/l IAA (Fig. 1g), in the

presence of selective hygromycin concentration. Putative

transgenic shoots rapidly regenerated vigorous root sys-

tems within 3–4 weeks on selection medium, and were

transferred to the greenhouse (Fig. 1h). Histochemical

characterization of GUS activity in MSC explants, shoot

buds, leaves, and leaf sheath confirmed stable integration

and expression of the uidA reporter gene in the plantlets

regenerated on hygromycin selection (Fig. 1i–n). Molecu-

lar integration of transgene was further confirmed by PCR

amplification of *1 kb fragment in 10 randomly selected

putative transgenic lines of banana overexpressing uidA

gene, and by Southern blot hybridization in nine out of 10

PCR-positive transgenic events (Supplementary Fig. 2).

Likewise, MSC explants were transformed with Agrobac-

terium harboring the binary vector pCAM-PR10. Based

upon Southern blot hybridization and RT-PCR data,

I J K

L M N

H

E

F G

A B C D

Fig. 1 Regeneration and selection of transgenic banana [Musa

acuminata cv. Matti (AA)] plants transformed with A. tumefaciens

strain EHA105 harboring binary plasmid pCAMBIA-1301. a Shoot tip

explant (bar = 0.5 cm); b 1-month-old shoot meristem-derived

proliferative multiple shoot clump (MSC) on shoot initiation medium

containing 22.5 mg/l BAP and 0.4 mg/l IAA (bar = 1.0 cm); c elon-

gation of adventitious shoots on 2.25 mg/l BAP and 0.4 mg/l IAA

(bar = 1.0 cm); d rooting in the presence of 0.4 mg/l IAA

(bar = 1.0 cm); e MSC explants 10 days-post-infection on shoot

initiation medium supplemented with 10 mg/l hygromycin (explants

on the left represent wild-type untransformed control (WT) plants)

(bar = 1.0 cm); f elongation of 2-month-old hygromycin resistant

shoot buds (bar = 1.5 cm); g rooting of putative transgenic shoots in

selection medium (bar = 1.5 cm); h greenhouse-acclimatized trans-

genic banana plants (bar = 14 cm); i–n histochemical GUS assay to

confirm stable integration and expression of uidA gene in (i) MSC

explant 1 week-post-infection (bar = 0.5 cm); j developing shoots

on the selection medium (bar = 1.0 cm); k developing shoot from

WT explant (bar = 1.0 cm); l leaves and (bar = 1.5 cm); m leaf

sheath from the putative transgenic plantlets (bar = 1.0 cm); and n a

leaf from WT plant showing absence of blue stain (bar = 1.0 cm)

Mol Biotechnol

123

indicating stable integration and expression of AhSIPR10

(Fig. 3), five transgenic lines of banana were further

selected for validating the role for AhSIPR10 gene in

alleviation of abiotic (salt and drought) stress-induced

damage in leaf bioassays. All the transgenic lines over-

expressing either AhSIPR10 were transferred to green-

house, and were similar in morphology and growth

characteristics to the wild-type untransformed plants. The

ameliorative effects of AhSIPR10 overexpression on

senescence and loss of chlorophyll due to salt and manni-

tol-induced drought stress were observed in transgenic

plants (Fig. 4a–d). Leaf disc senescence assays showed

bleaching and significant loss of chlorophyll in WT leaf

discs as compared to T4 and T9 transgenic leaves under

high salt (200–800 mM NaCl) and mannitol

(200–600 mM) treatments (P \ 0.05). While WT leaf

discs showed 86 and 69 % decline in total chlorophyll

content due to 800 mM NaCl and 600 mM mannitol

treatments, 57 and 62 % decline due to salt treatment and

34 and 41 % decline due to mannitol treatment was

observed in the chlorophyll content in the leaf discs excised

from T4 and T9 leaves, respectively. The electrolyte

leakage from the leaf tissues of WT and transgenic lines

(T4 and T9) were assessed. No significant difference was

observed between WT and transgenic plants under non-

stress conditions. However, electrolyte leakage increased

to 77–79 % in WT plants as against an increase to 57 %

(T4) and 59 % (T9) in transgenic lines exposed to NaCl

and 61 % (T4) and 60 % (T9) under mannitol treatments

(Fig. 5a). The MDA level, an indicator of membrane

damage due to lipid peroxidation was measured in WT and

transgenic plants under salt and drought stress conditions.

The MDA content in the WT plants increased by 1.8-fold

under salt condition and by 2-fold under drought stress

compared to that in the WT plants grown under normal

non-stress condition. However, the increase in MDA con-

tent was only 1.4- and 1.45-fold under salt and 1.56- and

1.63-fold under drought stress in the transgenic lines T4

and T9, respectively (Fig. 5b). Similar data were obtained

for the transgenic lines T1, T2 and T6 (data not shown),

thus implicating a role for constitutive overexpression of

AhSIPR10 in mitigation of stress-induced damage in the

transgenic plants.

Successful transformation in banana has been achieved

for several triploid cultivars belonging to the genomic

groups AAA [4, 6, 7, 9, 13, 29] and AAB [5, 8, 11, 30].

0.0 20.0 40.0 60.0 80.0 100.0

A

Agr

obac

teri

umde

nsity

(A

600)

(Inf

ectio

n tim

e =

45

min

)

1.0

0.9

0.8

0.7

0.6

0.5

0.4

B60

45

30

15

Infe

ctio

n tim

e (m

in)

(A60

0=

0.8

)

D

Infection (45 min)

Vacuum infiltration (10 min)+ infection (35 min)

Explants (%) showing transient GUS expression

CSemi-solid co-cultivation medium+ 100 µM acetosyringone

Liquid co-cultivation medium

Liquid co-cultivation medium+ 100 µM acetosyringone

Semi-solid co-cultivation medium

Fig. 2 Conditions optimized on

multiple shoot clump (MSC)

explants of banana [Musa

acuminata cv. Matti (AA)] for

transformation with

Agrobacterium strain EHA105

harboring the binary plasmid

pCAMBIA-1301. The data

represented are average (±SD)

for three independent

experiments, each with 100

explants and results were

analyzed at significance level of

P \ 0.05. a Effect of

Agrobacterium inoculum

density (infection

time = 45 min). b Effect of

Agrobacterium infection time

(bacterial A600 = 0.8). c Effect

of co-cultivation medium (MS

medium supplemented with 3 %

sucrose, 20 mg/lascorbic acid,

22.5 mg/l BAP, and 0.4 mg/l

IAA) (bacterial A600 = 0.8,

infection time = 45 min).

d Effect of vacuum

(400 mmHg) infiltration for

10 min during Agrobacterium

infection (bacterial A600 = 0.8,

total infection time = 45 min,

liquid co-cultivation medium,

100 lM acetosyringone)

Mol Biotechnol

123

Broad spectrum abiotic stress tolerance in a fertile A

genome diploid cultivar such as Matti which is amenable to

conventional breeding, is noteworthy. Besides biolistic

transformation of M. acuminata cv. Mas (AA) [31],

Agrobacterium-mediated transformation in a diploid A

genome cultivar Matti is being reported here for the first

time. Regeneration of Agrobacterium-infected meriste-

matic explants offers a simple, efficient and relatively rapid

method for the recovery of transgenic events in banana,

irrespective of the ploidy and genotypic variants [6, 8]. The

transformation and regeneration protocol described in the

present report, allowed for recovery of putative transgenic

shoots within few weeks and yielded robust rooted trans-

genic plantlets in as little as 5 months that were readily

acclimatized under greenhouse conditions. Enhanced bac-

terial virulence and accessibility to the transformation-

competent meristematic cells were dependent upon two

critical experimental parameters, namely, agro-infiltration

and co-cultivation of Agrobacterium and MSC explants in

liquid medium containing the chemotropic bacterial Vir

gene inducer acetosyringone contributing favorably to

successful transformation and recovery of putative trans-

genic events in a transient GUS assay. Previously, Huang

et al. [31] had shown liquid co-cultivation routine to be the

most important factor determining successful Agrobacte-

rium-mediated transformation of ECS cultures of banana

cv. Mas (AA). Subramanyam et al. [8] achieved stable

Agrobacterium-mediated transgene delivery following a

sequential sonication and vacuum infiltration treatment and

liquid co-cultivation regimen for sucker explants in the

cultivar Rasthali (AAB). High transformation efficiency

coupled with a two-step procedure for the multiplication

and selection of hygromycin resistant shoot buds ensured

Fig. 3 a Southern blot with 10 lg DNA digested overnight with

restriction enzymes NcoI and SpeI and using radiolabeled 474 bp

PCR-amplified AhSIPR10 probe. b RT-PCR analyses confirming

stable integration and expression of AhSIPR10 in young fully

expanded leaves of transgenic banana [Musa acuminata cv. Matti

(AA)] plants. P, pCAM-PR10 plasmid; WT, wild-type untransformed

control; T1, T2, T3, T4, T5, T6, T8 and T9, independently

transformed primary transgenic lines of banana. Actin was used as

the reference gene for normalizing the transcript profiles

Fig. 4 Leaf disc senescence

assay showing arbitration of

bleaching and loss of

chlorophyll due to (a,

b) 200–800 mM salt (NaCl),

and (c, d) 200–600 mM

mannitol-induced drought stress

in the leaves of transgenic

banana [Musa acuminata cv.

Matti (AA)] overexpressing

AhSIPR10. The data were

scored after 5 days of treatment,

and represent the average

(±SD) for three experiments for

two (T4 and T9) independently

transformed primary transgenic

lines of banana (WT, wild-type

untransformed control) at

significance level of P value

(P \ 0.05)

Mol Biotechnol

123

that chances for recovery of chimeric transgenic plants

were minimal.

A role of AhSIPR10 in alleviation of abiotic stress-

induced damage to photosynthetic apparatus was func-

tionally validated through its transgenic overexpression in

banana cv. Matti (AA), corroborating our previously pub-

lished work in tobacco [19]. Despite being ubiquitously

present across several plant species, and transcriptionally

responsive to a melange of abiotic stress conditions and

stress/defense signaling molecules, the precise biological

functions of PR10 proteins remain sparsely understood.

Several stress and pathogen inducible PR10 homologs have

been shown to possess antimicrobial ribonuclease activity.

Constitutive expression of a ribonuclease-active pea (Pi-

sum sativum) PR10 protein (PR10.1) gene in Brassica

napus seedlings enhanced endogenous cytokinin pool

while promoting seedling germination and growth rates

under saline conditions [32]. Krishnaswamy et al. [33]

speculated that PR10 proteins may modulate cytokinin

levels through an, hitherto, uncharacterized mechanism

including the possible degradation of tRNAs containing

cytokinin moieties. In addition to their signaling function

in regulation of plant growth and development, cytokinins

have also been accepted as integral components of plant

defense repertoire and abiotic stress responses [34]. Rivero

et al. [35] observed that transcriptional activation of chlo-

rophyll biosynthesis and light reaction-related genes, and

increased photorespiration contributes to protection of

photosynthetic processes during cytokinin-induced drought

tolerance in transgenic tobacco plants. It is therefore safe to

presume that AhSIPR10 expression may mitigate salt and

mannitol stress-induced oxidative burden in the transgenic

banana plants and afford protection in the leaf senescence

bioassays, by virtue of its high binding affinity and provi-

sion of cytokinins. Currently, AhSIPR10 overexpressing

transgenic banana lines are under extended trials for cor-

roborating stability of transgene expression through ratoon

progenies. Finally, a male/female fertile and seeded diploid

cultivar such as Matti (AA) can be suitably integrated into

banana breeding programs aimed at developing superior

diploid or triploid germplasm to address the specific needs

for sustainable organic and marginal banana cultivation.

Acknowledgments This research work was supported by Depart-

ment of Biotechnology, India (Grant no BT/PR10231/AGR/02/555/

2007) to NBS. A.R. acknowledges the financial support from Uni-

versity Grant Commission (UGC). The authors also wish to thank Dr.

Anuradha Agarwal, and Dr. Kailash Chander Bansal, Director,

National Bureau of Plant Genetic Resources, New Delhi, India for

disease-free banana plants. Research in the laboratory of NBS is

supported by U.G.C.-C.A.S., U.G.C.-R.N.W., Department of Science

and Technology (D.S.T.)-F.I.S.T., and D.S.T.-PURSE.

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