Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.
DOI 10.1007/s40011-014-0435-3
RESEARCH ARTICLE
Influence of Different Growth Regulators on In Vitro
Multiplication of Mixed Diploid Banana (Musa AB)
Pooja Bohra • Ajit Arun Waman • B. N. Sathyanarayana
K. Umesha • Balakrishna Gowda
Received: 30 April 2014 / Revised: 14 June 2014 / Accepted: 15 September 2014
Ó The National Academy of Sciences, India 2014
Abstract The commercial scale banana multiplication
through micropropagation is concentrated mainly on the
Cavendish group of bananas. Other varieties, though possess
a wide array of desirable characters, have been conveniently
neglected owing to their inherent problems such as difficult
aseptic establishment, low multiplication rate, culture
browning, difficult hardening, etc. The mixed diploid bana-
nas including the Ney Poovan are among the choicest of
bananas relished in many parts of the world. To promote its
cultivation, systematic studies were undertaken and the lines
possessing superior attributes have been identified. In order
to multiply the clones in large number, a viable multiplica-
tion protocol is a must. The present study dealt with opti-
mizing the plant growth regulator requirements of the
growing cultures for obtaining superior multiplication and
in vitro rooting. Out of the different cytokinins as benzyl
aminopurine, 2-isopentenyl adenine, meta topolin (mT), and
thidiazuron (TDZ) tested, only TDZ was found to promote
P. Bohra (&)
A. A. Waman
B. N. Sathyanarayana
Plant Tissue Culture Laboratory, Department of Horticulture,
University of Agricultural Sciences, GKVK Campus,
Bengaluru 560065, India
e-mail: poojabohra24@gmail.com
Present Address:
P. Bohra
A. A. Waman
Division of Horticulture and Forestry, Central Island
Agricultural Research Institute, Port Blair 744 101, India
K. Umesha
P.G. Centre, University of Horticultural Sciences (Bagalkot),
GKVK Campus, Bengaluru 560065, India
B. Gowda
Department of Forestry and Environmental Sciences, University
of Agricultural Sciences, GKVK Campus, Bengaluru 560065,
India
•
shoot multiplication in both types of propagules i.e. single
and double bud explants, although shorter plantlets were
noticed in the treatment. In vitro root induction parameters
were found to be superior when indole butyric acid was used
as auxin source. This report could help in improving the
multiplication rate of this least attempted group of bananas.
As per the present knowledge it is the first report on the use of
TDZ and mT in mixed diploid banana.
Keywords Auxin
Cytokinin
In vitro
Meta-topolin

Ney Poovan
Thidiazuron
Introduction
Plant growth regulators (PGRs) have been considered as one
of the most critical inputs for in vitro culture, as the processes
of differentiation, de-differentiation and re-differentiation
are greatly dependent on the quality and quantity of these
components in the culture media [1]. A good number of
micropropagation related reports deal with studying the
effect of PGRs on culture response, as requirement in terms
of kind and concentration vary greatly amongst the crops and
varieties therein [2]. Especially in crops like banana, wherein
a number of genomic groups exist, the PGR requirement may
not necessarily be similar for all members of the group [3, 4].
Thus, screening the efficacy of the PGRs for optimizing their
kind and concentrations will be a continuous process as far as
the new types with potential for commercialization are
identified and added to the global diversity.
The mixed diploid group of banana, Ney Poovan (Musa
AB), though cultivated in a number of countries, is amongst
the rarest of cultivated types [5]. The superior horticultural
and post harvest characters make it a distinct type and is also
known to tolerate drought to a considerable extent [6].
123

Considering these attributes, the National Research Centre
for Banana, Trichy, India has recommended its cultivation
especially for meeting the international demands (
www.nrcb.res.in). In the local markets, the fruits are sold at
almost double the rate than the commercial Cavendish types.
The traditional planting material as suckers are not sufficient
to meet the growing demands for establishing new planta-
tions. Also, Fusarium wilt disease has been a major concern
in banana cultivation [7] and the suckers being a source of
transfer of devastating Fusarium wilt disease, micropropa-
gation appears to be the most suitable alternative for
obtaining a large number of disease free plants. As most of
these types suffer from inherent problems such as difficult
aseptic establishment, low multiplication rate, culture
browning, etc. [8], efforts are needed to address these issues
to make the multiplication protocol practicable.
To promote the commercial cultivation of such elite types,
the lines need to be screened for an array of characters in
order to identify near-ideotypic line [9]. Considering this, a
series of studies were initiated at authors’ Institute to identify
elite lines possessing traits of economic importance such as
resistance to rhizome rot [10], Fusarium wilt, superior
agronomic [11] and post-harvest characters [8]. After
screening the natural diversity for various traits of interest,
five lines of variety Elakki Bale exhibiting superiority for a
number of parameters have been identified and forwarded for
further evaluation for possible release. However, lack of
reliable multiplication protocol has been a major impedi-
ment for production of a large number of propagules for
advanced studies and also for distribution of the plants of
newly identified types [12]. An attempt was thus made to
establish the aseptic cultures of Elakki Bale [13]. Pre-
liminary experiments suggested that the multiplying cultures
were highly fragile and were getting separated into small
clumps which upon subculture exhibited slower multiplica-
tion. Though, a report has been dealt with in vitro culture of
AB group of bananas using benzyl aminopurine (BAP) and
kinetin [14], further studies using more potent cytokinins
could help in improving the culture multiplication. Also, the
response from two different varieties of the same group show
considerable variation under in vitro conditions. Thus, the
present report is concerned with the determination of the
PGR requirement for obtaining optimum multiplication
from isolated shoot clumps and their subsequent rooting.
Material and Methods
Standardization of Cytokinin Requirement Under
In Vitro Conditions
Based on the preliminary experiments, Murashige and
Skoog’s (MS) medium [15] with modified level of
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P. Bohra et al.
ammonium nitrate (75 %), 3 % sucrose as carbon source
and 0.7 % agar as gelling agent was employed. The pH of
all the media was adjusted to 5.7 prior to autoclaving at
121 °C for 20 min. Shoot tip cultures of banana variety
Elakki Bale were initiated as detailed in the earlier report
[14]. Propagules containing single and double buds
obtained from fifth subculture used for inoculation onto the
media supplemented with different levels of four cytoki-
nins namely, BAP, 2-isopentenyl adenine (2-iP), meta
topolin (mT; 8.89, 17.78 and 35.56 lM, each) and thidi-
azuron (TDZ; 1.0, 2.0 and 4.0 lM). A constant dose of
0.25 mg/l naphthalene acetic acid (NAA) was supple-
mented in all the treatments and basal MS medium served
as control. Cultures were incubated at 28 ± 2 °C under
14 h photoperiod regime. Twelve replications were main-
tained in each treatment and the experiment was laid in
completely randomized design (CRD). The experiment was
repeated twice.
Standardization of Auxin Source and Its Concentration
for Root Induction in Microshoots
For induction of roots under in vitro condition, microshoots
of ca. 3.0 cm length after seventh subculture were excised
aseptically and inoculated onto the media enriched with
two auxins (indole butyric acid, IBA and NAA) used at
various concentrations (0.5, 1.0 and 2.0 mg/l). Basal MS
medium devoid of auxins was used as control. The
experiment was laid in CRD with seven replications, each
repeated twice.
Collection of Data and Statistical Analyses
For first experiment, observations on various parameters
such as, mean number of shoots produced per culture,
mean number of adventitious buds produced per culture
and mean number of leaves produced per shoot were
recorded at fortnightly intervals. Mean length of shoot
(cm) and mean shoot diameter at the collar region (mm)
were recorded using a measuring tape and digital vernier
caliper, respectively. In case of second experiment, per-
cent rooting, mean number of primary roots produced per
shoot and number of secondary roots per shoot were
recorded after 30 days of culture. Length of longest pri-
mary root (cm) was determined using a measuring tape.
Data obtained from both experiments was subjected to
analysis of variance and the mean separation was done
following least significant difference using Web Agri
Statistical Package (WASP v. 2.0, Indian Council for
Agricultural Research-Research Complex for Goa, Old
Goa, India).
Micropropagation of Ney Poovan Banana

Results and Discussion
Standardization of Cytokinin Source and Its
Concentration for Maximizing Culture Proliferation
Incorporation of different cytokinins at varied levels
resulted in significant differences in both types of explants
viz., single and double bud explants, tested during the
present investigation (Tables 1, 2). In general, cultures of
Elakki Bale responded poorly to the exogenous supple-
mentation of cytokinins in the culture media. Cytokinins
represent a group of PGRs, which are known to have
profound influence on shoot proliferation and elongation of
banana [12]. Conventionally employed cytokinins for
in vitro culture of banana include BAP [16, 17], kinetin
[18], 2-iP [14] and zeatin [19]. TDZ and recently, mTs have
been used to a lesser extent [1, 20, 21]. However, each type
of cytokinin has differential ability to induce shoot prolif-
eration at different concentrations [22], which could be
attributed to factors such as stability, mobility and oxida-
tion of cytokinins in the culture medium [4]. Thus, the
resultant in vitro response is a complex interaction between
all these factors.
Shoot Multiplication Related Parameters
In many cases, culture medium did not support any pro-
liferation, irrespective of type of explants used for inocu-
lation. In case of single bud explants, the number of
inoculated shoots either remained the same as observed in
case of those cultured on basal medium, all levels of 2-iP
and moderate levels of BAP and TDZ; or even decreased
due to degeneration as noticed in explants cultured on
media supplemented with low level of BAP and moderate
to high levels of mT (Table 1). Incorporation of TDZ at
low concentration (1 lM) alone could induce multiplica-
tion in the cultured explant.
Double bud explants derived cultures were observed to
be more responsive than those obtained from single bud
explants (Table 2). Cultures multiplied on media contain-
ing low to moderate levels of BAP, higher level of TDZ
and low level of mT failed to induce any multiplication.
Other treatments such as basal medium, low level of TDZ
and moderate level of 2-iP exhibited significant reduction
over the initial inoculum. As high as 4.33 shoots were
produced when explants were cultured on higher concen-
tration of BAP, which was superior to any other treatment
studied.
Total propagation i.e., summation of number of shoots
and buds produced, was significantly influenced by the
treatments employed. Significantly superior multiplication
was noticed in TDZ supplemented media in both the
explants tried (Tables 1, 2). Lower level of TDZ could
improve multiplication in single bud explants (6.33),
whereas moderate level was required for achieving higher
multiplication of 6.00 per inoculated clump containing
double bud explant (Fig. 1). Though observed to be sig-
nificantly lower than the best treatment as described above,
higher level of TDZ and BAP were also noticed to be
significantly superior over rest of the treatments in
improving the total propagation for both kinds of explants
studied. None of the other cytokinins studied were effective
in inducing multiple shoot and bud formation in both kinds
of explants.
Response of different genotypes to the kind and con-
centration of exogenously applied cytokinins exhibit great

Table 1 Culture multiplication and shoot growth parameters as affected by different cytokinins using single bud explant
Treatments Number of Total Shoot Shoot Number of
shoots propagation length (mm) diameter (mm) leaves/shoot

Control 2.00 d 2.00 cd 73.67 b 3.58 cde 5.33 ab

BAP (8.89 lM) 1.67 e 1.67 d 57.17 d 3.88 bcd 4.83 bcd
BAP (17.78 lM) 2.00 d 2.00 cd 51.00 e 2.83 g 3.50 e
BAP (35.56 lM) 2.67 b 2.67 c 40.58 g 3.48 def 4.33 cde
2-iP (8.89 lM) 2.00 d 2.00 cd 82.83 a 4.78 a 4.67 bcd
2-iP (17.78 lM) 2.00 d 2.00 cd 84.67 a 5.12 a 5.83 a
2-iP (35.56 lM) 2.00 d 2.00 cd 70.00 bc 3.98 bc 5.00 abc
TDZ (1.00 lM) 3.00 a 6.33 a 35.83 h 3.08 fg 4.60 bcd
TDZ (2.00 lM) 2.00 d 2.00 cd 46.00 f 3.15 efg 5.00 abc
TDZ (4.00 lM) 2.33 c 5.00 b 30.97 i 3.03 g 5.06 abc
mT (8.89 lM) 2.33 c 2.33 cd 67.42 c 4.06 b 4.44 cd
mT (17.78 lM) 1.67 e 1.67 d 60.17 d 3.53 de 4.00 de
mT (35.56 lM) 1.67 e 1.67 d 73.25 b 3.73 bcd 5.33 ab
Mean values followed by the same lower case letter in a column did not differ significantly at P \ 0.05

123
 P. Bohra et al.

Table 2 Culture multiplication and shoot growth parameters as affected by different cytokinins using double bud explant
Treatments Number of Total Shoot length Shoot Number of
shoots propagation (mm) diameter (mm) leaves/shoot

Basal 1.00 f 1.00 f 57.33 c 3.40 bc 4.33 ab

BAP (8.89 lM) 2.00 de 2.00 de 44.75 e 3.58 bc 3.25 de
BAP (17.78 lM) 2.00 de 2.00 de 49.75 d 3.60 bc 3.50 cde
BAP (35.56 lM) 4.33 a 5.00 b 34.52 f 3.07 cd 4.49 a
2-iP (8.89 lM) 2.33 cd 2.67 cd 69.01 a 3.72 ab 4.59 a
2-iP (17.78 lM) 1.67 e 1.67 ef 42.90 e 2.45 ef 3.00 e
2-iP (35.56 lM) 2.67 c 3.00 c 64.50 b 4.30 a 4.22 ab
TDZ (1.00 lM) 1.67 e 2.00 de 34.15 f 3.53 bc 4.33 ab
TDZ (2.00 lM) 3.33 b 6.00 a 31.31 f 2.68 def 4.06 abc
TDZ (4.00 lM) 2.00 de 4.67 b 26.83 g 2.40 f 4.00 abc
mT (8.89 lM) 2.00 de 2.00 de 53.23 cd 3.47 bc 4.33 ab
mT (17.78 lM) 2.33 cd 2.33 cde 50.33 d 3.81 ab 4.25 ab
mT (35.56 lM) 2.33 cd 2.67 cd 51.00 d 3.02 cde 3.72 bcd
Mean values followed by the same lower case letter in a column did not differ significantly at P \ 0.05

Fig. 1 Culture multiplication in

single bud explants (a) and
double bud explants (b) on
media supplemented with 1.0
and 2.0 lM TDZ, respectively

variations [12, 16]. In the present study, TDZ was found to
promote shoot proliferation as well as total multiplication
in both single bud explant and double bud explant at much
lower concentration than other cytokinins used. This could
be due to the ability of TDZ to enhance the endogenous
biosynthesis of adenine based cytokinins [23], thus
improving shoot proliferation in recalcitrant banana variety
Elakki Bale. Similar was the case in other banana cultivars,
wherein TDZ was observed to be the better cytokinin
source over BAP and 2-iP in terms of shoot multiplication
[3]. The roles of TDZ in promoting shoot proliferation in
banana varieties (Topala, Fougamou, Gros-Michel)
exhibiting weak multiplication has been reported by Yo-
umbi et al. [1]. Such increased multiplication was, how-
ever, accompanied by reduced shoot length [21] and more
number of leaves per plant, as observed in the present
investigation.
Shoot Growth Related Parameters
In general, shoot length was significantly influenced by
both quantity and type of cytokinin used in the multipli-
cation medium. In cultures obtained from single bud
explants, maximum shoot elongation was recorded in

123
Micropropagation of Ney Poovan Banana
treatments involving low and moderate levels of 2-iP
(Table 1). Higher concentration of TDZ drastically reduced
the shoot length (30.97 mm) as compared to the basal
medium (73.67 mm). Similar trend was also noticed in
double bud explants wherein lower concentration of 2-iP
could support shoot elongation and higher concentration of
TDZ hindered it (Table 2).
Mean diameter of shoot also followed the similar pattern
as in case of shoot length (Table 1). In case of cultures
derived from single bud explants, thickest shoots were
noticed in medium supplemented with low (4.78 mm) and
moderate levels of 2-iP (5.72 mm). Moderate level of BAP
and all concentrations of TDZ hindered shoot thickening
and the differences remained non-significant amongst
them. In case of double bud explants, cultures multiplied
on low (3.72 mm) and high (4.30 mm) levels of 2-iP and
moderate level of mT (3.81 mm) showed thicker shoots
than other treatments (Table 2). Shoots were comparatively
thinner in treatments involving use of moderate and high
levels of TDZ which remained at par with those obtained
from moderate levels of 2-iP.
Though the leaf production was significantly affected by
the treatments studied, none of the treatments was rated
superior to the cultures multiplied on basal medium
(Tables 1, 2), irrespective of the explant type used.
In both explants, shoots obtained from media supple-
mented with 2-iP exhibited maximum shoot length, how-
ever, without appreciable shoot multiplication. The results
corroborated with those obtained by Buah et al. [4]. The
use of aromatic cytokinin, mT, has been documented to
promote shoot proliferation in a number of plant species
including banana [20, 24]. However, in the present study, it
was of no practical utility as explants cultured in most of
the mT containing media did not show any multiplication.
It was surprising that the cytokinin improving multiplica-
tion in banana cultivars belonging to different genomic
groups viz., CEMSA 3/4 (AAB), Williams and Grande
Naine (AAA) was not suitable for the AB genome of
Elakki Bale, resulting into poor multiplication observed in
Table 3 In vitro rooting in microshoots as influenced by the kind and concentration of auxins
Treatments Rooting (%)
Basal 71.43
NAA (0.5 mg/l) 85.71
NAA (1.0 mg/l) 85.71
NAA (2.0 mg/l) 100.00
IBA (0.5 mg/l) 85.71
IBA (1.0 mg/l) 100.00
IBA (2.0 mg/l) 100.00
Mean values followed by the same lower case letter in a column did not differ significantly at P \ 0.05
the present study. Further detailed investigations may be
needed to understand the cause for this deviation.
Standardization of Auxin Source and Its Concentration
for Root Induction in Microshoots
The extent of rooting varied significantly among the
treatments studied during the experimentation (Table 3).
Lowest percentage of rooting was observed in microshoots
cultured on basal medium and incorporation of auxin had
marked influence on rooting. In terms of root induction
efficiency, IBA was superior to NAA. Among various
levels of NAA tried in the experiment, culture of micro-
shoots on medium containing high level of NAA induced
rooting with 100 % success. The other auxin, IBA, was
also effective at 1.0 and 2.0 mg/l concentrations and all the
shoots subjected to these treatments could induce roots.
Number of primary roots produced at different levels of
auxins did not follow any specific trend (Table 3). Mod-
erate level of NAA (1.0 mg/l) and low level of IBA
(0.5 mg/l) were found to support production of higher
number of roots per microshoot. However, further increase
in the concentration of auxin was not desirable as the root
production was hindered at higher concentration. Number
of secondary roots was significantly superior in most of the
treatments involving supplementation of auxins in the
culture media to that produced in basal medium (Table 3).
Incorporation of IBA was more effective when compared
to NAA as highest number of secondary roots were formed
in the shoots cultured on media containing moderate
(66.00) or high (65.00) levels of IBA.
Longest roots were reported at 1.0 mg/l concentration,
irrespective of the kind of auxin used and further addition
of auxin hindered root elongation (Table 3). In general,
IBA was superior in producing longer roots than NAA.
Supplementation of culture medium with 1.0 mg/l IBA
supported maximum root elongation (110.84 mm) while
shortest roots were observed in medium containing low
level of NAA (25.95 mm).
Number of Number of Length of
primary roots secondary roots longest root (mm)
6.60 bc 17.00 e 81.20 d
6.16 c 15.33 e 25.95 f
8.34 a 49.66 b 87.55 c
6.57 bc 40.15 c 78.43 d
8.43 a 34.15 d 102.15 b
4.16 d 66.00 a 110.84 a
7.85 ab 65.00 a 55.43 e
123

Once sufficient number of subcultures are over (here
seven subcultures), the regenerated microshoots need to be
transferred to the auxin supplemented rooting medium.
Auxins have been reported to influence the root initiation
process in many crops including banana [25]. Reports are
available on use of various auxins viz., IAA [16], IBA [16,
25] and NAA [12, 18, 26] for banana varieties belonging to
similar or different genomic groups. In the present study,
IBA was found to be the better auxin source than NAA for
induction of rooting, number of secondary roots as well as
length of root. Superiority of IBA over NAA has also been
reported in other banana cultivars [26]. In general, 1.0 mg/l
concentration was found to be better than other two levels,
irrespective of the auxin source. The results were in com-
pliance with those obtained by Akbar and Roy [18].
Conclusion
From the present investigation, it could be concluded that
the mixed diploid Ney Poovan banana could be multiplied
more efficiently, if TDZ is used as cytokinin in the mul-
tiplication medium. The cytokinin was potent enough to
induce shoot multiplication in both types of explants tried.
For root induction, IBA was found to be better auxin source
than NAA. Thus, the present report would help in aug-
menting the possibilities of multiplying this variety through
micropropagation in a large scale.
Acknowledgments PB is thankful to the Department of Science and
Technology, Government of India for providing financial assistance
in the form of an INSPIRE Fellowship (IF-10077).
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