INTRODUCTION
Strawberry (Fragaria ananassa Duch) is propagated

conventionally through runners but it is not feasible for mass
multiplication. Alternative methods of propagation like micro-
propagation have tremendous potential and attempts have
been made for commercially exploitation (Boxus, 1974;
Boxus et al, 1977). In the process of mass multiplication,
the initial step of surface sterilization of explants is one of
the most crucial factors in plant tissue culture. Contamination
in plant cultures generally originates from explants, operators,
laboratory environments or ineffective sterilization
techniques. Microbes existed even among the healthy plants
and their presence is often missed under field conditions,
but the deleterious effects get expressed during in vitro
conditions. Further, enzymatic browning at the initial
establishment of in vitro culture is another primary cause
failure of the cultures, leading to the death explants (Pirttilla
et al, 2008; Zaid, 1984). Thus, establishment of a simple and
effective disinfection protocol for escalating the survival of

Effective decontamination and regeneration protocol for in vitro culture
of strawberry cv. Chandler

S. Diengngan1, B.N.S. Murthy* and M. Mahadevamma1
ICAR-Indian Institute of Horticultural Research

Hesaraghatta Lake Post, Bengaluru-560 089, India
*E-mail: bnsmurthy@iihr.ernet.in

ABSTRACT
Nodal segments of strawberry cv. Chandler were collected from the field and surface sterilization of the explants

was carried out using various levels of decontaminants as different treatments viz. sodium hypochlorite (NaClO)
0.5% for 7, 5 and 3 min and mercuric chloride (Hg Cl2) 0.1% for 5 min, 3 min and 3 min 10 sec. The explants were
excised and cultured for 3 weeks on the initiation medium supplemented with various levels of BAP (0.5, 1, 1.5 and
2mg/l) and its combination with GA (0.5 and 1mg/l). Sub culturing was done and finally rooting was initiated on the
medium supplemented with various levels of IBA (0.5, 1, 1.5 and 2mg/l) for 4 weeks. The results obtained indicated
that, among the decontaminants, treatment of explants with Hg Cl2 0.1% for 3 min 10 sec resulted in the minimum
contamination and browning of explants. Further, maximum shoot proliferation percentage, shoots per explant and
minimum number of days to shoot initiation was observed when explants were cultured on MS medium supplemented
with 1.5 mg/l BAP over other concentrations of either BAP or in combination with GA. However, the maximum length
of shoots was obtained in medium supplemented with 1.5 mg/l BAP + 0.5 mg/l GA. While, medium supplemented with
0.5 mg/l IBA supported highest percentage of rooting, the highest number of roots, maximum root length of micro-
cuttings and minimum number of days to root initiation were observed in medium supplemented with 1mg/l of IBA.

Key words: Decontaminants, growth regulators, in vitro, nodal segments, strawberry

explants is imperative both for research and commercial
production. The growth and development of explants in vitro
is very much influenced by the addition of growth regulators
to the basal medium for culture (Skoog and Miller, 1957).
Morozova (2002) reported that high concentration of
cytokinins (BA) required for strawberry micro-propagation,
while Boxus (1999) suggested lower concentrations
(0.5-1mg/l) of BA. Therefore, the present study was
undertaken to develop an effective disinfection protocol for
nodal segments of strawberry cv. Chandler obtained from
field grown plants and assess the efficacies of various
growth regulators at different levels for efficient in vitro
culture of strawberry cv. Chandler.

MATERIAL AND METHODS
Explant selection and sterilization

Field grown runners (7-10cm size) obtained from
open field (Block-I) of the ICAR-Indian Institute of
Horticultural Research, Hesarghatta, Bengaluru, Karnataka,

1University of Horticultural Sciences, Bagalkot, P.G. Centre, Bengaluru-560 065, India

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127

In vitro culture of strawberry cv. Chandler

India, were used in the present studies. The leaves and root
initials were trimmed off and nodal segments (1-2cm) were
pretreated with a mixture of Bavistin (1.0%), CTAB (Cetyl
Trimethyl Ammonium Bromide- 0.5%) and antibiotic
formulation (streptomycin + tetracycline) 0.1% each for
90min. The explants were then washed off with all the
residues using sterile distilled water three times. Thereafter,
the explants were treated for 1 minute using 75% ethanol
containing 2 drops of tween-20. Subsequently, the explants
were treated with various surface sterilants at different
concentrations and timings as per the treatments. The
surface sterilants used were sodium hypochlorite (Na ClO)
0.5% for 7, 5 and 3 min and mercuric chloride (Hg Cl2)

0.1% for 5 min, 3 min and 3 min 10 sec. After subsequent
washing for six times, exposed ends were trimmed off, and
the excised nodal explants of 1-1.5 cm size were cultured
in vitro (Fig. 1).

Shoot proliferation and root initiation

Surface sterilized nodal segments were cultured on
MS medium containing 0.5, 1.0, 1.5 or 2.0 mg/l of BAP.
Another set of treatments consisting of 0.5 or 1 mg/l of GA
in combination of the four concentrations of BAP mentioned
above with were also tested. The cultures were examined
regularly and the data on shoot proliferation was recorded
after 3 weeks of culture. These shoots were used as micro-
cuttings and sub-cultured on fresh medium for further shoot
proliferation and root initiation. Growth characteristics such
as percentage of explants showing shoot proliferation, days
to shoot initiation, number of shoots per explant and average
length of shoot were recorded. Further, the micro-cuttings
obtained from the shoot induction treatments were
transferred to MS media containing 0.5, 1.0, 1.5 and 2.0
mg/l of IBA and the percentage of rooting, number of days
to root initiation, number of roots per micro-cutting and
average root length were recorded in each treatment after
4 weeks of sub-culturing and the data were subjected to
appropriate statistically analysis.

Hardening

Hardening of the rooted plantlets was carried out on
the medium consisting of a mixture of sterilized sand, soil
and cocopeat (1:1:2). One third of the plastic cups punched
with drainage holes were filled with hardening media. After

Fig. 1. Decontamination protocol of strawberry nodal explants in
vitro culture.

Fig. 2. Efficacy of decontaminants in reducing the contamination
and browning of explants

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the medium was saturated with water to field capacity, the
plantlets were transplanted in to plastic cups after thorough
washing and finally the cups were covered with an inverted
punched transparent cups to maintain humidity and placed
under cool fluorescent light in the culture room. After one
week, the covered plastic cups covers were removed to
permit further hardening, and after one month, hardened
plantlets were relocated to glasshouse conditions.  Such
hardened plantlets of two months old were ready for transfer
to the field.

Statistical analysis

Analysis of variance was carried out using Completely
Randomized Design (CRD) using SPSS statistics version
17.0. For each of the treatment combinations, ten
replications were maintained and the test for significance
(p d”0.05) was conducted among different surface sterilants
and plant growth regulators treatments.

RESULTS AND DISCUSSION
The efficacy of various decontaminants in reducing

the contamination of explants of strawberry cv. Chandler
for in vitro culture was evaluated. The data on percentage
of contamination showed significant differences among the
surface sterilants, and it was observed that explants treated
with Na ClO 0.5% for 7 min resulted in zero contamination,
but the browning percentage was highest. Treatment of
explants with HgCl2 0.1% 3 min 10 sec gave the minimum
percentage of contamination and browning compared to
other treatment combinations. The best decontamination
protocol suggested by Ying Ko et al (2009) using sodium
hypochlorite (0.5%) for 7 min, Biswas et al (2007) with
0.1% HgCl2 for 5 min and Sharma et al (2009) with 0.1%
HgCl2 for 3 min were also tried in the present study but of
little help in overcoming contamination. Since, the
decontamination treatment identified in the present study
worked efficiently with the explants collected from the open
field, it saves the cost for installation of protected structure
for raising plants as source of explants, thereby increasing
the economic return.

Significant differences in shoot proliferation of
strawberry cv. Chandler to in vitro culture were observed
under the influence of growth regulator treatments (Table
1). It was observed that medium supplemented with 1.5mg/
l BAP resulted in the highest percentage of shoot
proliferation and minimum number of days to shoot initiation.
Maximum number of shoots per explant was obtained on

medium supplemented with 1.5mg/l BAP and was
significantly higher than the other concentrations of BAP
alone or in combinations with GA. The maximum number
of days taken for shoot initiation and minimum number of

Table 1. Influence of plant growth regulators on the number of
days to shoot initiation, number of shoots per explant and shoot
length of nodal segments of strawberry cv. Chandler
Plant growth Days to Number of Shoot
regulators (mg/l) shoot shoots per length

initiation explant  (cm)
Control 10.50±0.45 2.30±0.21 1.06±0.09
BAP 0.5 9.20±0.47 3.60±0.16 1.63±0.10
BAP1 8.90±0.43 5.00±0.26 2.42±0.14
BAP 1.5 8.00±0.26 6.30±0.15 3.47±0.10
BAP 2 8.90±0.41 5.00±0.21 2.72±0.06
BAP 0.5 + GA 0.5 9.50±0.22 3.50±0.17 3.10±0.07
BAP 0.5 + GA 1 9.40±0.43 3.70±0.15 3.64±0.07
BAP1 + GA 0.5 9.10±0.41 4.10±0.23 4.00±0.06
BAP 1 + GA 1 8.50±0.45 4.50±0.17 4.43±0.07
BAP 1.5 + GA 0.5 8.00±0.26 5.50±0.17 5.20±0.05
BAP 1.5 + GA 1 8.40±0.48 4.80±0.20 4.77±0.07
BAP 2 + GA 0.5 9.20±0.36 4.20±0.20 4.10±0.04
BAP 2 + GA 1 9.90±0.35 3.60±0.16 3.66±0.07
CD (P=0.05) 1.10 0.54 0.22

Table 2. Rooting responses of micro-cuttings of strawberry cv.
Chandler under the influence of various concentrations of IBA
IBA Days to Number of Root length
concentrations root roots per (cm)
(mg/L) formation micro-cutting
IBA 0 15.40±0.34 1.80±0.13 1.07±0.09
IBA 0.5 12.00±0.47 3.60±0.27 2.20±0.08
IBA 1 9.30±0.21 5.50±0.22 3.47±0.11
IBA 1.5 9.40±0.22 4.40±0.22 2.97±0.08
IBA 2 10.70±0.33 3.60±0.27 2.18±0.07
CD (P=0.05) 0.94 0.65 0.25

Fig. 3. Effect of plant growth regulators on shoot proliferation
percentage of explants of strawberry cv. Chandler

Diengngan et al

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shoots per explant were recorded in basal MS medium.
Biswas et al (2008) however reported that 0.5 mg/l BAP in
MS medium gave the best response for multiple shoot
induction. The higher requirement of concentration for shoot
proliferation in the present study could be attributed to
genotypic differences as genotypes responds differently in
vitro (Passey et al, 2003). Supplementation of GA to the
BAP containing medium did not improve the rate of shoot
proliferation and is in accordance with the reports by Sakila
et al (2007). However, the medium supplemented with
1.5mg/l BAP + 0.5mg/l GA exhibited the highest shoot length
in explants in the present investigation and the minimum
length of proliferated shoots were observed in basal MS
medium thus GA contributed to elongation of proliferated
shoots and again similar observation was reported by Sakila
et al (2007).

On perusal of data, it was found that 0.5mg/l IBA
when supplied to the medium resulted in the highest response
to in vitro rooting reflected in highest percentage of rooting.
The lowest percentage of rooting in micro-cuttings was
observed on basal MS medium (Fig. 4). However, the
minimum number of days taken to root formation was
observed in medium supplemented with 1 mg/l IBA and this
treatment also resulted in highest number of roots per micro-
cutting.  Maximum number of days taken for rooting and
least number of roots per explant were observed in basal
MS medium and interestingly, the average root length of
micro-cuttings also recorded maximum in 1mg/l IBA, which
was significantly superior to other IBA concentrations.
Overall, results indicated that supplementation of 1mg/l to
the media resulted in the highest rooting responses in micro-

cuttings of strawberry cv. Chandler. Similar results have
been obtained by Hemant et al (2001), Ritu et al (2001),
Sakila et al (2007) on other cultivars of strawberry and
Mante et al, 1989 in Prunus sp.

ACKNOWLEDGEMENT
The authors gratefully acknowledge the Vice

Chancellor, The Dean (PGs), University of Horticultural
Sciences, Bagalkot, Karnataka and The Director, ICAR-
Indian Institute of Horticultural Research, Bengaluru, India
for providing necessary facilities requisite for completion of
the experiment and encouragement.

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In vitro culture of strawberry cv. Chandler

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(MS Received 07 December 2014, Revised 12 December 2014, Accepted 13 December 2014)

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