RESEARCH ARTICLE

Influence of Different Growth Regulators on In VitroMultiplication 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

� 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

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].

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

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-014-0435-3

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

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).

P. Bohra et al.

123

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

shoots

Total

propagation

Shoot

length (mm)

Shoot

diameter (mm)

Number of

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

Micropropagation of Ney Poovan Banana

123

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

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

Table 2 Culture multiplication and shoot growth parameters as affected by different cytokinins using double bud explant

Treatments Number of

shoots

Total

propagation

Shoot length

(mm)

Shoot

diameter (mm)

Number of

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

P. Bohra et al.

123

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

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).

Table 3 In vitro rooting in microshoots as influenced by the kind and concentration of auxins

Treatments Rooting (%) Number of

primary roots

Number of

secondary roots

Length of

longest root (mm)

Basal 71.43 6.60 bc 17.00 e 81.20 d

NAA (0.5 mg/l) 85.71 6.16 c 15.33 e 25.95 f

NAA (1.0 mg/l) 85.71 8.34 a 49.66 b 87.55 c

NAA (2.0 mg/l) 100.00 6.57 bc 40.15 c 78.43 d

IBA (0.5 mg/l) 85.71 8.43 a 34.15 d 102.15 b

IBA (1.0 mg/l) 100.00 4.16 d 66.00 a 110.84 a

IBA (2.0 mg/l) 100.00 7.85 ab 65.00 a 55.43 e

Mean values followed by the same lower case letter in a column did not differ significantly at P \ 0.05

Micropropagation of Ney Poovan Banana

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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