World J Microbiol Biotechnol (2017) 33:31

DOI 10.1007/s11274-016-2196-z

ORIGINAL PAPER

Elimination and molecular identification of endophytic bacterial

contaminants during in vitro propagation of Bambusa balcooa
Syandan Sinha Ray1 · Md. Nasim Ali2 · Shibasis Mukherjee1 · Gautam Chatterjee1 ·
Maitreyi Banerjee3 

Received: 15 August 2016 / Accepted: 20 December 2016

© Springer Science+Business Media Dordrecht 2017

Abstract Bambusa balcooa is an economically impor-
tant, multipurpose bamboo species, decidedly used in con-
struction industry. Availability of natural bamboo is deplet-
ing very rapidly due to accelerated deforestation and its
unrestrained use. The large number and timely supply of
saplings are the need of the hour for the restoration of bam-
boo stands. Micropropagation, being the potent alternative
for season independent rapid regeneration, is restricted in
bamboo because of endophytic contamination. An in vitro
attempt has been taken to overcome the endophytic contam-
ination by using broad spectrum antibiotics as surface ster-
ilant as well as a media component. Ampicillin sodium salt
(5 mg/ml for 30 min) as a surface sterilant was found as the
best treatment for high bud breaking (80%) coupled with
high branching and low contamination (20%) but it was
found ineffective to control the contamination during mul-
tiplication stage. Then, two endophytes were isolated and
minimum inhibitory concentration was determined through
antibiotic susceptibility test for successful eradication at
multiplication stage. Finally, contamination free cultures
Electronic supplementary material The online version of this
article (doi:10.1007/s11274-016-2196-z) contains supplementary
material, which is available to authorized users.
* Md. Nasim Ali
nasimali2007@gmail.com
1
IRDM Faculty Centre, Ramakrishna Mission
Vivekananda University, Ramakrishna Mission Ashrama,
Narendrapur, Kolkata 700103, India
2
Department of Agricultural Biotechnology, Faculty
of Agriculture, Bidhan Chandra Krishi Viswavidyalaya,
Mohanpur, Nadia, West Bengal, India
3 tion and its unrestrained use (Agnihotri and Nandi 2009).
West Bengal State Council of Science
and Technology, Vigyan Chetana Bhavan, DD-26/B,
Salt Lake, Kolkata 700064, India
were obtained when streptocycline (100  μg/ml) and gen-
tamicin sulphate (75  μg/ml) were added into the medium.
The two isolated endophytes,  BB1 and BB2, were identi-
fied through 16S rDNA techniques and NCBI-BLAST
algorithm with 99% sequence similarity with those of
Janibacter sp. (KX423734) and Serratia marcescens strain
(KX423735). To our knowledge, this is the first report for
B. balcooa where antibiotics were used as surface sterilant
as well as medium component, to control endophytic bacte-
rial contaminants, followed by their identification.
Keywords Micropropagation · Bambusa balcooa ·
Endophytic contamination · MIC · 16S rDNA
Introduction
Bambusa balcooa is an indigenous multipurpose bamboo
species of India (Tewari 1992). This species is widely used
for construction purpose because of having the strongest
and long-lasting stem under the genus Bambusa (Gaintait
et  al. 2016). The tender edible shoots are used in pickle
preparation (Mudoi and Borthakur 2009); the fermented
shoots are used to produce pharmaceutically active ster-
oids (Sarangthem and Singh 2003). This species is in great
demand in making paper, pulp, and handicraft (Sivabalan
et  al. 2014). The multipurpose usage makes B. balcooa
the priority bamboo species by the Food and Agriculture
Organization (FAO) among 18 others globally distributed
bamboo species (Ghosh et  al. 2013). A major bamboo
source in India is natural forests, which are decreasing
day by day due to industrialization vis-à-vis deforesta-
The large number and timely supply of saplings are the
top most priority for the restoration of bamboo stocks.

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Micropropagation has been established as the most suit-
able alternative for season independent rapid regeneration
in many woody species including bamboo (Negi and Sax-
ena 2011; Patel et al. 2015). The nodal segments are identi-
fied as the best explants for micropropagation (Goyal et al.
2015). But the endophytic contamination (bacteria or fun-
gus) from the nodal explants limits micropropagation in B.
balcooa (Ali et  al. 2009) because it’s elimination through
simple sterilization methods is hardly feasible (Attree and
Sheffield 1986; Reed and Tanprasert 1995; Leifert and Cas-
sells 2001). These endophytic contaminants stay within
the plant cell without showing any immediate manifesta-
tion (Nair and Padmavathy 2014). During advanced stages,
the contaminants grow quickly utilizing culture medium
and plant exudes as nutrient, resulting in tissue mortal-
ity, necrosis and reduced growth of the plant (Leifert et al.
1991). The best possible solution to remove endophytic
bacteria is the use of the broad-spectrum antibiotics with
proper dose and duration (Reed et al. 1998). But, the indis-
criminate use of antibiotics may also lead to microorgan-
ism’s resistance (Pollock et al. 1983) as well as phytotoxic-
ity (Kulkarni et al. 2007). For this reason, characterization
of contaminants (type) using the antibiotic susceptibility
test, is a new promising way to counter the problem lead-
ing to the appropriate selection of antibiotic and minimum
damage of plant. The use of antibiotics as a surface ster-
ilant is reported for tissue culture of B. balcooa (Mudoi and
Borthakur 2009; Sharma and Sarma 2011).
Elimination of microbial contamination using antibiotics
within media during in vitro propagation of different plants
like Hazelnut (Reed et  al. 1998), Jatropha curcus (Misra
et  al. 2010), Banana (Msogoya et  al. 2012), Orange tree
(Citrus sinensis L. Osbeck cv. Madame Vinous) and Sweet
orange trees (C. sinensis cv. Valencia) (Niedz and Bausher
2002) are also reported by several authors. But till now
only one report on bamboo in Guadua angustifolia Kunth
(Nadha et  al. 2012) is available. The antibiotic used as
media component was found more effective than as surface
sterilant in Citrus sp. (Eed et  al. 2010). Gentamicin was
also found effective as media component to control the con-
tamination for B. balcooa (Patel et al. 2015). Though broad
spectrum antibiotics are very commonly used in controlling
endophytic bacteria, rarely contaminants have been identi-
fied. Bacterial identification through 16S rDNA sequence
analysis has been done in Ilex dumosa (Luna et al. 2008),
Aglaonema (Fang and Hsu 2012) and G. angustifolia Kunth
(Nadha et al. 2012).
Keeping this information in mind, the objective of the
present work was to select the best possible dose and dura-
tion of broad-spectrum antibiotics, to control the endo-
phytic bacterial contaminants during micropropagation of
B. balcooa. This is the first report for B. balcooa where the
endophytes were identified through 16S rDNA sequence
13
World J Microbiol Biotechnol (2017) 33:31
analysis and antibiotics were used as a surface sterilant as
well as a media component for their successful elimination.
Materials and Methods
Collection of plant material
Nodal explants of B. balcooa were collected from the green
house of Department of Science and Technology, Salt
Lake, Kolkata during the rainy season (July) of 2014.
Use of antibiotic as surface sterilant
The explants were washed in running tap water to clean
dirt adhering to the plant materials. Before surface steri-
lization, the leaf sheath tissues covering the axillary buds
were removed carefully by sharp scalpel without damag-
ing the buds. In this study, two different concentrations (2.5
and 5  mg/ml) of three different broad spectrum antibiot-
ics i.e. ampicillin sodium salt (Himedia Pvt. Ltd., India),
streptomycin sulphate (Himedia Pvt. Ltd., India) and strep-
tocycline (Hindustan Antibiotics Ltd., India) were used
as surface sterilizing agent. At first, nodal segments were
surface sterilized by 1% bavistin for 10  min followed by
antibiotic treatments as mentioned above for 30 and 45 min
separately and then treated with 0.1% mercuric chloride
(HgCl2) (Himedia Pvt. Ltd., India) for 10 min. Finally, sur-
face sterilized explants were washed four times with ster-
ile double distilled water and the cut ends of the segments
were trimmed and put on MS (Murashige and Skoog 1962)
medium supplemented with 3  mg/l BA (6-benzyladenine,
Merck, India) and 3% (w/v) sucrose (Extra pure, Titan Bio-
tech Ltd., India), gelled with 0.8% (w/v) agar (Plant culture
tested, Himedia Pvt. Ltd., India). The whole inoculation
process was carried out under aseptic condition in a laminar
air flow (Klenzaids Bioclean Device Pvt. Ltd., India). The
pH of the medium was maintained at 5.7 with 1 N NaOH
and 1 N HCl prior to autoclaving at 121 °C for 15 min. All
cultures were maintained at 23 ± 2 °C, under a 16-h photo-
period. After 3  weeks shoots regenerated from node were
transferred to the liquid MS media having 3  mg/l BA for
multiplication.
Isolation of bacterial isolates
Within 24  h of transfer in liquid media from the solid
media, most of the cultures were found contaminated. Four
times repeated experiments confirmed the source of con-
tamination was endophytic bacteria which were uncontrol-
lable even after antibiotic treatment during surface steri-
lization. This finding compelled to isolate bacteria and to
figure out the minimum inhibitory concentration (MIC)
World J Microbiol Biotechnol (2017) 33:31
for the antibiotic used. The contaminants, having been iso-
lated and streaked on nutrient agar plate, were found as two
morphologically dissimilar (yellow colored and colorless)
endophytes. To standardize the dose of the appropriate
antibiotic against these contaminants, pure cultures were
obtained and were designed as BB1 and BB2 (Fig. 1).
Antibiotic assay and determination of MIC
Antibiotic assay of isolated microorganisms was done
through agar-well diffusion method taking the antibiotic
concentrations following the guideline of PhytoTechnology
Laboratories™, USA (Table A is added as supplementary).
For the antibiotic assay, the selected concentrations i.e. 25,
50, 75 and 100  μg/ml were chosen for ampicillin sodium
salt and gentamicin sulphate whereas in the case of strepto-
cycline, the concentrations were 50, 100, 150 and 200 μg/
ml. MIC determination for these antibiotics was done
according to Andrews (2001). A single colony from both
of BB1 and BB2 were inoculated in nutrient broth sepa-
rately and incubated for 24  h at 37 °C. After 24  h, 100  μl
of both microbial culture broths were spread over the sepa-
rate nutrient agar plate. Using the sterilized cork borer, four
wells (volume 60 μl/well) were cut on each plate. The 60 μl
of each antibiotic was added to the well and were kept in
BOD incubator shaker at 37 °C. Though data on the zone
of inhibition at 24, 48 and 72 h interval were recorded, the
observation after 72  h of incubation has been produced.
Double distilled sterilized water was added to well as
control.
Use of antibiotic in culture medium
The concentrations, at which both microorganisms were
killed, were selected for use in culture medium i.e. MS with
3  mg/l BA. Fresh explants were inoculated in media after
treatments of 1% bavistin and 0.1% mercuric chloride for
10 min and followed by washing with sterilized double dis-
tilled water and data were recorded after 20 days of inocu-
lation. The regenerated cultures were kept under observa-
tion up to 90 days in liquid media. Then, the plants having
Fig. 1 Pure culture of endophytes (BB1 and BB2) isolated from B.
balcooa during in vitro culture
Page 3 of 9 31
2–3 shoots were transferred to rooting media having ½
MS (liquid) + 1 mg/l IBA (indole-3-butyric acid) (data not
given) and were transferred to the field after hardening.
Data analysis
All experiments were conducted with three replications for
each treatment having 10 plants. Statistical analysis of data
was performed with SPSS v16.0 for Windows (SPSS Inc.,
USA) with 5% probability level of statistical significance.
Molecular characterization
Genomic DNA isolation of the selected microorganisms
(BB1 and BB2) was done by lysozyme based protocol
(Moore et  al. 2004) from the overnight grown luria broth
culture. After isolation of genomic DNA, amplification of
16S rDNA was done using the universal primer sets 27F
(5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-TAC
GGTTACCTTGTTACGACTT-3) in a thermal cycler
(Applied Biosystems). The PCR reaction was performed
for reaction mixture of 25 μl in the following manner: After
one cycle of 95 °C for 10 min then 30 cycles of 95 °C for
1 min, 52 °C for 45 s and 72 °C for 2 min, followed by one
final cycle of 72 °C for 7 min (BB1) and for BB2 it was 30
cycles of 95 °C for 30 s, followed by 55 °C for 45 s, 72 °C
for 2 min, followed by one final cycle of 72 °C for 10 min.
Gel extraction was done for both of these microorganisms
using XcelGen DNA Gel/ PCR Purification Mini kit (Cat
No. XG3511-01/3514; Xcelris Genomics, Ahmedabad,
Gujrat) and sequencing was done through Xcelaris Genom-
ics (Gujrat). The nucleotide sequences were compared
with NCBI GenBank entries using the nucleotide BLAST
algorithm (http://www.ncbi.nlm.nih.gov/BLAST) for
identification.
Results
Antibiotics as surface sterilant
The use of antibiotic along with mercuric chloride and
bavistin as a surface sterilant for B. balcooa was found
effective at initiation stage. Initially, treatments of 5 mg/ml
ampicillin sodium salt (for 30 min) and 5 mg/ml streptocy-
cline (for 45  min) were found the best in terms of reduc-
ing the contamination (20%) and bud breaking (80%) for
nodal explants but average number of shoots under strep-
tocycline (5  mg/ml for 45  min) was found comparatively
low (Table 1). Both in vitro contamination (10%) and bud
breaking (20%) were found least at ampicillin sodium salt
(2.5  mg/ml for 30  min). On the contrary, high bud break-
ing (80%) coupled with high contamination rate (60%) was
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31 Page 4 of 9 World J Microbiol Biotechnol (2017) 33:31

Table 2 Zone of Inhibition after 72 h at different antibiotic concen-

20 ± 2.30d 40 ± 0.58c 60 ± 1.15b 80 ± 0.58a 80 ± 3.64a 80 ± 1.15a

20 ± 2.31c 20 ± 1.73c 40 ± 2.3b 60 ± 1.15a 40 ± 1.15b 20 ± 2.88c
1.25 ± 0.11 1.25 ± 0.14cd 0.8 ± 0.11f 1.6 ± 0.11c 0.6 ± 0.11f 0.8 ± 0.11f 2 ± 0.58b
2.5 mg/ml 5 mg/ml
trations for BB1 and BB2
Concentration Time
72 h

45 min
BB1 BB2

GEN 50 1.43 ± 0.07b 1.97 ± 0.28b

2.5 mg/ml 5 mg/ml
GEN 75 1.56 ± 0.07b 2.43 ± 0.28ab
GEN 100 1.76 ± 0.07a 3 ± 0.28a
Streptocycline

STR 100 1.77 ± 0.17a 1.8 ± 0.11b

STR 150 1.6 ± 0.17a 2.23 ± 0.11a
30 min

STR 200 1.57 ± 0.17a 2.4 ± 0.11a

a
Highest mean and other alphabet representing mean in descending
order. Antibiotic concentrations having similar superscripts are statis-
Table 1 Character means at two concentrations of antibiotics used as surface sterilants at two time intervals in vitro propagation of B. balcooa

5 mg/ml

tically similar
Highest mean and other alphabet representing mean in descending order. Observations having similar superscripts are statistically similar

GEN gentamycin sulphate, STR streptocycline

2.5 mg/ml

observed under treatment of streptocycline (5  mg/ml for

45 min

30 min). Though for 45 min treatment of streptomycin sul-

phate (2.5 and 5 mg/ml), contamination rate was found low
cd
b
40 ± 2.88c

(20%) in comparison to treatment of 30 min (60% and 40%

Streptomycine sulphate

40 ± 1.15
2.5 mg/ml 5 mg/ml

contamination were recorded respectively at 2.5 and 5 mg/

ml), bud breaking was not encouraging (Table  1). From
these findings, it can be concluded that ampicillin sodium
20 ± 1.15d 80 ± 1.73a 60 ± 1.15b 60 ± 2.88b 60 ± 1.73b
a
30 ± 0.58 60 ± 0.89
e

salt (5 mg/ml) for 30 min can be used as the effective steri-

0.4 ± 0.11 0.6 ± 0.14 1 ± 0.14
30 min

lizing agent. As ampicillin sodium salt was found effective

at the initial stage to control contamination (Table 1), vari-
bc

ous concentrations of ampicillin sodium salt were added

f
5 mg/ml

to media. But this attempt was failed to control the in vitro

contamination (data not given). Finally, the pure culture
was isolated from the contaminated vessels and designed
fg
c
2.5 mg/ml

10 ± 0.58 20 ± 1.15 20 ± 0.58

as BB1 and BB2. Then antibiotic susceptibility test was fol-

45 min

lowed to select the proper dose (Fig. 1).

Ampicillin sodium salt

Antibiotic susceptibility test and MIC determination

a
2.5 mg/ml 5 mg/ml

0.3 ± 0.09 3 ± 0.58

Among the three antibiotics considered for the study, strep-

tocycline and gentamicin sulphate were found effective to
d

control both the isolates successfully at different time inter-

30 min

val. Data regarding the antibiotic assay after 72  h of the

experiment is given in Table  2. Ampicillin sodium salt at
60 ± 1.73b
80 ± 1.89a
1.25 ± 0.14e
HgCl2 + 1%

higher concentration (100 μg/ml) as well as gentamicin sul-

bavistin

phate (25 μg/ml) and streptocycline (50 μg/ml) were failed

0.1%

to inhibit the growth of isolates after 24 h. Gentamicin sul-

phate 100  μg/ml against both isolates and streptocycline
100  μg/ml against BB1 and 200  μg/ml against BB2 were
No. of avg. branches/

found most effective after 72 h of the experiment (Table 2).

contamination %
Bud breaking %
Time duration

Antibiotics as a medium component

plant

There was significant variation at 5% level among

Dose

the selected antibiotics concentrations for in  vitro

a

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World J Microbiol Biotechnol (2017) 33:31 Page 5 of 9 31

contamination, buds breaking of nodal explants and
branches/plant were observed (Table  3). Streptocycline
150 μg/ml was found (4.16%) better followed by gentamicin
sulphate 50  μg/ml (16.67%) and streptocycline 100  μg/ml
(16.67%) to control the endophytic contamination. Maxi-
mum bud breaking (83.33%) was found in streptocycline
150 μg/ml followed by streptocycline 100 μg/ml and ampi-
cillin sodium salt 100 μg/ml (79.17%). The branching was
found maximum (1.92) in ampicillin sodium salt 100 μg/ml
after 3 weeks of incubation (Table 4). It was clear that the
ampicillin sodium salt (100  μg/ml) was attributed to bet-
ter branching but with higher contamination in compari-
son to streptocycline or gentamicin sulphate (Table 4). It is
mention worthy that reduction of contamination was also
found at ampicillin sodium salt 100 μg/ml in comparison to
75 μg/ml. On the other hand, though the low contamination
was found at a high concentration of gentamicin sulphate
(100 μg/ml) and streptocycline (200 μg/ml), the growth of
plant was reduced (Fig. 2). The problem of reduced growth
at a high concentration of gentamicin sulphate and strp-
tocycline could be overcome using a higher dose of BA
(since the whole experiment done on MS + 3 mg/l BA).
The most striking findings of this study on antibi-
otic as medium component were—(1) negative correla-
tion between contamination and antibiotic concentration
i.e. as an increase of antibiotic concentration leads to a

Table 3 Analysis of variance Source of variation df Bud breaking % Average number Contamination % No. of Plant died
for effect of treatments on of shoots/plant
response variables
Treatments 5 390.62** 0.353** 16.99** 0.49NS
Error 12 78.125 0.054 0.44 0.22

NS non significant
**Significant at 5% level

Table 4 Character means Treatments Bud breaking % Avg. no. of shoots/plant Contamination % No. of
in B. balcooa at different Plant
concentrations of antibiotic died
treatments
GEN 50 75.17 ± 8.33a 1.12 ± 0.07c 16.67 ± 4.17c NS
GEN 75 58.33 ± 4.17b 1.62 ± 0.22ab 12.50 ± 7.22c NS
AMP 75 58.33 ± 4.17b 0.96 ± 0.04c 83.33 ± 4.17a NS
AMP 100 79.17 ± 4.17a 1.92 ± 0.04a 45.83 ± 4.17b NS
STR 100 79.17 ± 4.17a 1.37 + 0.12bc 16.67 ± 4.17c NS
STR 150 83.33 ± 4.17a 1.37+ 0.19bc 4.16 ± 4.17c NS
STR 200 58.33 ± 4.17a – 4.16 ± 4.17c NS
a
Highest mean and other alphabet representing mean in descending order. Antibiotic concentrations having
similar superscripts are statistically similar

Fig. 2 Effect of different

antibiotics; streptocycline (S),
gentamycin sulphate (G) and
ampicillin sodium salt (A)
on the culture of B. balcooa
after 2 weeks of inoculation.
Explants were contaminated
when it was treated with 1%
bavistin and 0.1% HgCl2

13
31 Page 6 of 9 World J Microbiol Biotechnol (2017) 33:31

reduction of in  vitro contaminations. (2) Shoot length

also inversely proportional to the antibiotic concentra-
tion i.e. as high concentration of antibiotic reduced the
shoot length of the plant in spite of high bud breaking
in nodal explants (Fig.  2). To check the contamina-
tion rate, the explants which had sufficient shoots were
transferred to liquid media containing MS + 3  mg/l BA
for long durations (Fig.  3). Successive changes in fresh
liquid media (subculturing) were done in the 3-week
interval to avoid browning of plants. After 90  days of
culture, there was no contamination in plants generated
from streptocycline 100  μg/ml and ampicillin sodium
salt 100  μg/ml containing medium. While plants from
gentamicin sulphate containing medium died during the
time of experiment limiting the use of gentamicin as a
media component. Finally rooted plantlets were obtained
through culturing in rooting medium (Fig.  4). From the
study, streptocycline 150  μg/ml, gentamicin sulphate at
75 μg/ml and also ampicillin sodium salt 100 μg/ml was
found suitable for culture of B. balcooa due to low phy-
totoxicity to plant. Fig. 4 Rooting in in vitro plants of B. balcooa

Molecular characterization Discussion

The isolated bacteria designed as BB1 and BB2 were
identified using 16S rDNA sequencing and NCBI-
BLAST algorithm. The sequence of BB1 and BB2 were
identified at 99% similarity with 16S rDNA sequence
of Janibacter sp. (KX423734) and Serratia marcescens
strain (KX423735). Table B is added as supplementary.
Use of antibiotic as surface sterilant was reported by several
authors (Mudoi and Borthakur 2009; Ali et  al. 2009) for
bamboo in  vitro propagation. The combination of mercu-
ric chloride, bavistin, and streptomycin sulphate was found
effective as successful surface sterilants for B. nutans after
pretreatment with detergent (Tween 20) and ethanol (Mehta
et  al. 2011). The addition of sodium hypochloride instead
of ethanol for D. membranaceus was found effective to

Fig. 3 Effect of streptocycline 100 μg/ml (STR 100) and ampicillin sodium salt 100 μg/ml (AMP 100) on the culture of B. balcooa during mul-
tiplication up to 90 days at liquid MS medium

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World J Microbiol Biotechnol (2017) 33:31
control contamination rate up to 77.8% (Brar et al. 2013).
These findings suggested that only antibiotic with HgCl2
and bavistin is not sufficient to control the in vitro contami-
nation, a pretreatment prior to sterilization is much more
useful for getting lesser contamination. The pretreatment of
Savlon and Tween 20 (1%) for 10 min coupled with 5 mg/
ml ampicillin sodium salt for 30 min was found ineffective
at advanced stages of micropropagation. So, long duration
pretreatment followed by antibiotic and mercuric chloride
may be more effective for highly susceptible in  vitro con-
tamination in bamboo (Mishra et  al. 2008; Anand et  al.
2013). Long duration pretreatment with antibiotics reported
in B. balcooa (Mudoi and Borthakur 2009; Sharma and
Sarma 2011), B. nutans (Sharma and Sarma 2014). But
long duration treatments of chemicals have a toxic effect on
explants and may reduce the growth of the plant (Thakur
and Sood 2006). In that case, the addition of antibiotic
within media instead of surface sterilants for a long dura-
tion may be another alternative approach (Eed et al. 2010)
who reported that antibiotic within media was much more
effective to control the in vitro contamination than as sur-
face sterilants. Rapid use of antibiotics without selective
dose often leads to major changes in the plant as well as
promotes microbial resistant (Niedz 1998). That is why to
select a proper dose, antibiotic susceptibility test was fol-
lowed before going to second part of the experiment.
MIC is considered as the lowest concentration of an
antimicrobial compound at which the visible growth of
a microorganism after overnight incubation is inhibited
(Andrews 2001). Ampicillin sodium salt was found worst
performer during antibiotic susceptibility test on two micro-
bial strains isolated from B. balcooa in the experiment.
Similar results were shared by Nadha et al. (2012) for the
two different isolates during in  vitro culture of G. angus-
tifolia Kunth. Twelve antibiotics including gentamicin and
ampicillin were reported by Kulkarni et al. (2007) to deter-
mine the MIC value against the endophytic contaminants
isolated from the contaminated Withania somnifera, Piper
nigram, Piper colubrium and taxus Baccata sub sp. Wilson
and Power (1989) used streptomycin to determine the MIC
value against the contaminants isolated from rubber (Hevea
brasiliensis Muell.Arg.). A similar approach was also taken
by Misra et al. (2010) for the contaminants isolated from J.
curcus during in vitro propagation.
Using gentamicin sulphate  as a medium component in
B. balcooa, better bud breaking, shoot numbers/plant and
contamination rate was found (Patel et  al. 2015). At high
concentration of antibiotic streptocycline and gentamicin
sulphate, lesser contamination was attributed to the slow
plant growth and decreased shoot numbers as compared
to the low concentration of antibiotic (Fig.  2). This find-
ing again was in agreement with Nadha et  al. (2012) and
confirms the phytotoxicity of chemicals at a higher dose.
Page 7 of 9 31
Streptocycline at all the selected concentrations were
effective for contamination free culture. Though ampicil-
lin sodium salt was unable to control the both of BB1 and
BB2 during antibiotic susceptibility test, it was found effec-
tive as medium component resulting into contamination
free culture (~50%). A higher dose of BA may be used to
compensate the reduced plant growth (Fig. 2) at high con-
centration of gentamicin sulphate (100 μg/ml) and strepto-
cycline (200  μg/ml). Keeping phytotoxicity in mind, it is
suggested to use streptocycline (100 μg/ml) or gentamicin
sulphate (75 μg/ml) or ampicillin sodium salt (100 μg/ml)
in medium to control the contamination of endophytic bac-
teria during micropropagation of B.balcooa.
The 16S ribosomal DNA (16S rDNA) based identifica-
tion are used in identifying bacterium because of its univer-
sal distribution among bacteria (Weisburg et al. 1991) and
the presence of species-specific variable regions (Hugen-
holtz 2002). By estimating the sequence homology with
16S rDNA sequences deposited in biological databases, the
identification was done to the closest affiliation of a new
sequence (Woese et al. 1990). Till now for bamboo, a sin-
gle report by Nadha et al. (2012) in G. angustifolia Kunth
at identified the endophytes through 16S rDNA sequencing.
Janibacter sp. which was found as one of the endophytes in
the study also was reported as endophytic contaminants in
Aglaonema (Fang and Hsu 2012). The presence of Serra-
tia sp. during in vitro propagation of plant is considered as
a failure of proper surface sterilization reported by Leifert
and Cassells (2001). However, the present study confirmed
Janibacter sp. and Serratia marcesens as endophytic inhab-
itants of B. balcooa, which can be controlled during micro-
propagation by incorporating antibiotics in culture media.
To the best of our knowledge, this is the first report on Jani-
bacter sp. and S. marcesens as endophytes of B. balcooa.
Conclusion
Surface sterilization using common chemicals or antibiot-
ics are not sufficient to eliminate the endophytic contami-
nants. Use of antibiotics as a medium component was found
effective to control those contaminants. The present study
revealed that streptocycline and ampicillin sodium salt were
more useful as a media component. Keeping phytotoxicity
in mind, it is suggested to use streptocycline (100  μg/ml)
or gentamicin sulphate (75  μg/ml) or ampicillin sodium
salt (100  μg/ml) in medium to control the contamination
of endophytic bacteria during micropropagation of B. bal-
cooa. The isolated bacteria designed as BB1 and BB2 were
identified using 16S rDNA sequencing and NCBI-BLAST
algorithm. The sequence of BB1 and BB2 were identified
at 99% similarity with 16S rDNA sequence of Janibacter
sp. (KX423734) and S. marcescens (KX423735).
13
31 Page 8 of 9
Acknowledgements The authors are highly indebted to DST, Govt.
of West Bengal, India for providing a fund to this project.
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