The Effects of Auxins and Cytokinin on Growth and Development of (<i>Musa</i> sp.) Var. “Yangambi” Explants in Tissue Culture

doi:10.4236/ajps.2013.411269

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American Journal of Plant Sciences, 2013, 4, 2174-2180

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411269

Open Access AJPS

The Effects of Auxins and Cytokinin on Growth and

Development of (Musa sp.) Var. “Yangambi” Explants in

Tissue Culture

Munguatosha Ngomuo1, Emerald Mneney2, Patrick Ndakidemi1*

1School of Life Sciences, Nelson Mandela African Institution of Science and Technology, Arusha, Tanzania; 2Mikocheni Agricul-

tural Research Institute, Dar es Salaam, Tanzania.

Email: *[email protected]

Received August 12th, 2013; revised September 12th, 2013; accepted October 21st, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The aim of this study was to investigate the effects of concentration of different growth regulators (auxins and cyto-

kinins) on growth and development of banana shoot tips cultured in vitro. Explants were taken from young suckers of

field grown plants of var. “Yangambi”. The shoot tips were cultured on MS media supplemented with different concen-

trations of BAP (0, 2, 4, 6 and 8 mg/l) with or without IAA at concentration of 0.34 mg/l. At the rooting phase, the me-

dia was supplemented with different concentrations of IBA (0.1, 0.5, 1.0, 1.5 and 2.0 mg/l) with or without BAP at

concentration of 0.2 mg/l. The results indicated that 6.0 mg/l BAP significantly increased the number of shoots formed

and the interaction of 6 mg/l BAP with 0.35 mg/l IAA significantly increased the fresh weight. For rooting, 2.0 mg/l

IBA was more efficient in number and length of roots produced than all other treatments.

Keywords: Bud’s Proliferation; Fresh Weight; In-Vitro Rooting; Root Length; Micro Propagation; Shoot Length;

Var. “Yangambi”

1. Introduction

Growth regulators play a key role for developing a spe-

cific mode of growth in the cultured cells or tissues,

which may be due to accumulation of specific biochemi-

cal contents in them. The single or combination of dif-

ferent hormones in the medium causes maintenance of

specific and balanced inorganic and organic contents in

the growing tissue. This leads the cells or tissues to de-

velop either into shoots/or roots or even death [1].

In tissue culture, plant growth regulators are important

media components in determining the development and

developmental pathway of the plant cells. Growth regu-

lators are used in different proportions to break dor-

mancy and enhance shoot formation since it is well

demonstrated that the apical dormancy is under control

of these growth regulators [2]. The cytokinins and auxins

are of importance in in-vitro culture as the later are con-

cerned with root formation, the former is mainly required

in the media for shoot formation and growth of buds [3].

These growth regulators are required in combination in

the media as it is always the manipulation and variation

of auxins and cytokinins levels that can successfully

change the growth behavior of plant cultures [4].

Cytokinins such as benzyl aminopurine (BAP) and ki-

netin are known to reduce the apical meristem domi-

nance and induce both axiliary and adventitious shoot

formation from meristematic explants in banana [5].

However, the application of higher BAP concentrations

inhibits elongation of adventitious meristems and the

conversion into complete plants [6].

Auxins and other growth regulators such as gibberel-

lins play important roles in the growth and differentiation

of cultured cells and tissues [7,8]. Auxins such as Naph-

talene acetic acid (NAA) have been reported to promote

plant rooting in vitro [9,10].

The use of cytokinin in plant nutrient media for in-vi-

tro culture depends on plant tissue growth stage and ex-

pected end product.

In studies conducted on banana, apical meristems were

*Corresponding author.

The Effects of Auxins and Cytokinin on Growth and Development of

(Musa sp.) Var. “Yangambi” Explants in Tissue Culture

2175

cultured in media of high cytokinin concentration or

lower cytokinin at the first stage then transferred to a

media with higher cytokinin concentration where the

increase in concentration especially BA significantly

enhanced buds proliferation [10].

Apart from the influence of genotypes, shoot prolifera-

tion rate and elongation are influenced by cytokinin types

and their concentration. Adenine-based cytokinins are

used in several Musa spp. for in-vitro propagation [11].

N6-benzylaminopurine (BAP) is the most commonly

preferred cytokinin [9]. The others are isopentyladenine

(2-ip), zeatin and kinetin [12]. The concentration of ex-

ogenous cytokinin appears to be the main factor affecting

multiplication.

Many other studies have reported the use of auxins and

cytokinin in tissue culture. Gubbuk and Pekmzci (2004)

[11] reported that moderate concentrations of cytokinins

increased the shoot proliferation rate, but very high con-

centrations decreased multiplication and especially de-

pressed shoot elongation. Also they reported higher shoot

proliferation and elongation with Thidiazuron (TDZ)

than with BAP. However, BAP above 20 μM and TDZ

over 2 μM decreased shoot elongation. The use of TDZ

is known to inhibit shoot elongation. In another study, it

was found that TDZ at 0.91 μM induced the largest

number of shoots, but at higher concentration of TDZ

(9.1 μM), elongation of shoots was inhibited and clumps

of small globular buds appeared at the base of shoots

[13].

In a study on effects of auxin/cytokinin combination

on shoot proliferation on banana cultivars, Arinaitwe et

al. reported that incorporation of a strong auxin in the

media suppressed the shoot proliferation rates of the ba-

nana cultivars [15]. On media modified with low cyto-

kinin/auxin ratios, for example 16.8/1.0 and 16.8/1.2

ZN/NAA combinations, the East African Highland ba-

nana (AAA-EA) cultivars showed single shoot develop-

ment and callus induction due to apical dominance re-

sulted from increased level of auxin concentration [15].

In another study, Buah et al. [15] demonstrated that dif-

ferences exist in the relative strengths of different cyto-

kinin types in inducing shoots. This differential ability of

different hormones in inducing shoots in vitro may be

attributed to factors such as stability, mobility and the

rate of conjugation and oxidation of hormones.

The concentration and combination of auxins and cy-

tokinins in the nutrient mediums is an important factor

which determines successful plant regeneration [16].

Thus for efficient in-vitro propagation of banana the

study of optimum combination of cytokinins and auxins

and their interaction in a tissue culture medium for a spe-

cific cultivar is necessary.

2. Materials and Methods

2.1. Plant Materials and Sterilization

Young suckers of Musa var. “Yangambi” were collected

from a healthy true to type mother plants. After removing

the leaves and the roots, the suckers were thoroughly

washed with tape water and liquid soap to remove ad-

hering soil. The suckers were trimmed to size by remov-

ing layers of the developing leaves. Then the suckers

were rinsed with clean tape water and soaked in 1 g/l

ascorbic acid for one hour before transfer to laminar flow.

The shoot apices explants were sequentially treated with

70% alcohol for 30 seconds, then treated with 100% (v/v)

hypochlorite (the active ingredient was 3.85% sodium

hypochlorite) mixed with few drops of Tween 20 for one

hour in order to sterilize the surface. This was then fol-

lowed with treatment of 50% (v/v) hypochlorite of the

same active ingredient for 30 minutes. The explants were

further trimmed to remove the remaining hypochlorite

and rinsed with sterile distilled water before initiation.

2.2. Culture Conditions and Media for Buds

Proliferation

The explants were placed in culture vessels containing 20

mls of culture media containing MS basal salts

supplemented with 20 g/l sucrose, vitamins; glycine 2 g/l,

pyridoxine 0.5 g/l, Nicotinic acid 0.5 g/l, Thiamine 0.1

g/l and Myo inositol at 0.1 g/l. The media was also sup-

plemented with different concentrations of BAP and IAA

as shown in (Table 1) and solidified with 4.5 g/l of agar.

The pH was adjusted to 5.8 prior to autoclaving at 121˚C

for 15 min.

2.3. Roots Initiation Media

Good established shoots were transferred to root initia-

tion media. This media consisted of MS basal salts with

Table 1. Different concentrations of BAP and IAA used for

buds proliferation.

Treatments Concentration (mg/l)

MS + BAP 0

MS + BAP 2

MS + BAP 4

MS + BAP 6

MS + BAP 8

MS + (BAP + IAA) 0 + 0.35

MS + (BAP + IAA) 2 + 0.35

MS + (BAP + IAA) 4 + 0.35

MS + (BAP + IAA) 6 + 0.35

MS + (BAP + IAA) 8 + 0.35

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The Effects of Auxins and Cytokinin on Growth and Development of

(Musa sp.) Var. “Yangambi” Explants in Tissue Culture

2176

20 g/l sucrose, vitamins glycine 2 g/l, pyridoxine 0.5 g/l,

Nicotinic acid 0.5 g/l, Thiamine 0.1 g/l and Myo inositol

at 0.1 g/l. The media also contained 0.8 g/l of activated

charcoal (AC) to mimic the soil environment. It was sup-

plemented with different concentrations of auxin as

treatments for rooting; IBA with or without BAP as

shown in (Table 2) and solidified with 4.5 g/l of agar.

Each treatment was replicated five times and one explant

was cultured in each culture bottle. The pH of the media

was adjusted to 5.8 before addition of agar. The media

were autoclaved at 121˚C and 1.05kg/cm3 for 15 minutes.

The cultures were incubated at 25˚C ± 1˚C and 16 and 8

hrs light and darkness respectively.

2.4. Data Collection and Analysis

After four weeks the following parameters were meas-

ured; fresh weight (g), number of shoots, height (cm),

roots length (cm) and number of roots. The data collected

were analyzed for statistical significance using analysis

of variance (ANOVA). These computations were done

by using a statistical software program STATISTICA

version 2013 (StatSoft Inc., Tulsa, OK, USA). Fisher

least significance was used to compare means at p = 0.05

level of significance.

3. Results and Discussion

3.1. The Effect of BAP Concentration on Bud

Proliferation Rate

The findings of this study demonstrated the effects of

cytokoinins on shoots formation and multiplication. In

this experiment, the use of BAP alone or in combination

with IAA had no significant (p ≤ 0.05) effect on buds

proliferation (Table 3). However, different concentra-

tions regimes of BAP with and without IAA had signifi-

cant effect on the number of buds produced as well as

Table 2. Different concentrations of IBA and BAP used for

rooting.

Treatments Concentration (mg/l)

MS + IBA 0

MS + IBA 0.5

MS + IBA 1.0

MS + IBA 1.5

MS + IBA 2.0

MS + (IBA + BAP) 0 + 0.2

MS + (IBA + BAP) 0.5 + 0.2

MS + (IBA + BAP) 1.0 + 0.2

MS + (IBA + BAP) 1.5 + 0.2

MS + (IBA + BAP) 2.0 + 0.2

fresh weight increase in buds produced. It was observed

that the number of buds produced increased with increase

in concentration. A significantly (p ≤ 0.001) highest

number of buds were observed when 6mg/l was used

(Table 3). Slightly increase in number of buds was also

observed in other treatments with low concentrations

compared with the control. Bhosale et al. [17], in a study

on in vitro shoot multiplication of different species of

banana similarly reported increased average number of

shoots produced at nearly same level of BAP (7 mg/l). In

another study, Sajid et al. [18], found that presence of

cytokinin in the media did not only determined regenera-

tion response of banana meristem cultures but also af-

fected the mode of regeneration. The initial response of

explants to shoot formation due to addition of cytokinin

is mediated by an increase in the cytosolic calcium con-

centration which is promoted by its high uptake from the

media. This affects cytoskeleton and regulates exocytosis

[19].

Other studies by [12,20,21] observed that 5mg/l BAP

was the most efficient concentration for in vitro bud

proliferation of many banana cultivars. In a review on

banana cell and tissue culture, Strosse et al. [22] in-

dicated that for multiplication of propagules, a medium

containing a range of concentration 0.1 - 20 mg/ of BA is

added to the media.

In this study, higher concentration beyond 6 mg/l did

not enhance fresh weight or number of buds produced.

At concentration of 8 mg/l the number of buds produced

was less compared with concentrations of 4 mg/l and 6

mg/l. Higher concentrations of cytokinin tend to have an

adverse effect on the multiplication rate and morphology

of the culture [5,22].

Generally, this study indicates that increasing concen-

tration of BAP for this particular variety enhanced the

fresh weight and buds formations. Addition of 6 mg/l to

the growth media showed best results compared with all

other treatments (Figure 1). This seems to be the optimal

concentration for this variety. In vitro buds proliferation

of banana is reported to be cultivar dependent [23]. Also

Strosse et al. [24] indicated that the rate of shoots

multiplication depends both on the cytokinin concen-

tration and the genotype of banana.

3.2. The Effect of BAP and IAA Concentration

on Fresh Weight and Shoot Length

Generally, the results indicated that like in buds prolif-

eration, fresh weight increase was significantly (p ≤ 0.01)

better at higher concentration (6 mg/l) (Table 3). Treat-

ments supplied with 6 mg/l showed best results in terms

of fresh weight followed by treatments with 2 mg/l and 8

mg/l. Other studies reported that combinations of BAP

with IAA or IBA were more efficient for in vitro multi-

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The Effects of Auxins and Cytokinin on Growth and Development of

(Musa sp.) Var. “Yangambi” Explants in Tissue Culture

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Table 3. Effect of BAP concentration with and without IAA on the number of buds formed, fresh weight and shoot length.

Growth Parameters

Treatment Number of Buds Fresh Weight Shoot Length

Growth Regulators

BAP 5.42 ± 0.47a 3.26 ± 0.23a 3.66 ± 0.30a

BAP + IAA 6.06 ± 0.56a 3.54 ± 0.28a 3.79 ± 0.28a

Concentration

0 mg/l 1.45 ± 0.21e 2.40 ± 0.27b 3.09 ± 0.39a

2 mg/l 4.45 ± 0.61d 3.70 ± 0.38ab 3.23 ± 0.35a

4 mg/l 7.30 ± 0.54bcd 3.14 ± 0.24b 4.11 ± 0.33a

6 mg/l 9.55 ± 0.66ab 4.19 ± 0.76ab 4.45 ± 0.65a

8 mg/l 5.95 ± 0.61cd 3.56 ± 0.39ab 3.82 ± 0.47a

2-Way ANOVA (F – Statistic)

Growth Regulators 1.66ns 0.70ns 0.11ns

Concentration 29.99*** 3.17** 1.63ns

Growth Regul * Conc 0.56ns 2.66** 0.41ns

**p ≤ 0.01; ***0.001. Values (Mean ± SE) Followed by dissimilar letter(s) in a column are significantly different by Least Significant Difference test at P = 0.05.

ns = non-significant.

Figure 2. Interactive effect of BAP with IAA on fresh

weight of buds produced.

Figure 1. Some of banana shoots developed in vitro at con-

centration of 6 mg/l BAP and 0.35 mg/IAA.

cultured banana is also reported by Hussein [10], where-

by supplementing NAA to increasing concentration of

BA (from 0.2 to 0.4 mg/l) resulted in higher fresh weight

and plant height.

plication of bananas and plantains [24]. As reported ear-

lier, increase in fresh weight may also be attributed to

increased cytosolic calcium concentration resulting from

enhanced uptake from the media due to the use of higher

amount of BAP. In terms of shoot length increase, there was no

significant difference across all the treatments, though

slight increases were observed in the treatments with

relatively high concentration. These findings did not

agree with the results of Al-Amin et al. [25], where the

MS media supplemented with BAP and NAA in their

study showed different results for increasing shoot length

Interactive effect of BAP and IAA was also observed

in fresh weight (Figure 2). The media with 6 mg/l of

BAP in combination with 0.35 mg/l IAA, significantly (p

≤ 0.01) resulted in high fresh weight as compared with

other treatments (Table 3). The interactive effect of

cytokinin and auxin in enhancing growth of tissue

The Effects of Auxins and Cytokinin on Growth and Development of

(Musa sp.) Var. “Yangambi” Explants in Tissue Culture

2178

which was significantly influenced by different con-

centration of these hormones.

3.3. The Effect of IBA and BAP Concentration

on the Number of Roots per Explant

Produced in Vitro

In this study, there was no significant difference between

MS media supplemented with IBA alone and media sup-

plemented with IBA and BAP in terms of number of

roots produced per explant (Table 4). However, signifi-

cant (p ≤ 0.001) increase in number of roots produced

was observed with increased concentration. The concen-

tration of 2.0 mg/l exhibited superiority over all other

treatments in terms of number of roots produced (6.1

roots per explant) (Figure 3(a)). This was followed by

concentration of 1.5 mg/l while the concentrations of 1.0

mg/l and 0.5 mg/l statistically produced same number of

roots per explant. In vitro rooting of banana can be in-

duced by transferring the explants to the basal media

alone [26,27]. Gubbuk and Pekmezci [11], reported that

activated charcoal was added to the media in replacement

of auxins such as IAA or IBA. However, auxins are

known to induce quick and further roots initiation [9].

Vuylsteke and De Langhe [12] found that optimal con-

centration for IBA was 1 µM (≈0.2 mg/l) for banana cul-

tivars they were testing. Due to cultivar dependence of

response to growth regulators of banana, each cultivar

responds differently to similar concentration of growth

regulators. In terms of rooting, our experiment indicated

Table 4. Effect of IBA concentration with and without BAP

on the number of roots formed and average root length.

Growth Parameter

Treatment Number of roots Average root length

Growth Regulators

IBA 3.4 ± 0.34a 5.0 ± 0.34a

IBA + BAP 3.9 ± 0.45a 5.4 ± 0.55a

Concentrations

0 mg/l 2.3 ± 0.47c 3.0 ± 0.75d

0.5 mg/l 2.9 ± 0.40bc 4.0 ± 0.71cd

1.0 mg/l 2.9 ± 0.52bc 5.0 ± 0.67bc

1.5 mg/l 4.1 ± 0.48b 6.0 ± 0.58b

2.0 mg/l 6.1 ± 0.52a 8.0 ± 0.61a

2-Way ANOVA

(F-Statistic)

Growth regulators 1.09ns 0.46ns

Concentrations 10.35*** 8.59***

Growth regulators*Conc 1.68ns 0.42ns

***0.001. Values (Mean ± SE) Followed by dissimilar letter(s) in a column

are significantly different by Least Significant Difference test at p = 0.05. ns

= non-significant.

(a)

(b)

Figure 3. (a) Some of the explants with the roots produced

in vitro at (2.0 mg/l IBA and 0.2 mg/l BAP (b) Measurement

of root length.

that better response to roots initiation of this variety was

2 mg/l.

3.4. The Effect of IBA and BAP Concentrations

on the Length of Roots Produced in Vitro

Root length varied with different concentrations of IBA

and BAP (Table 4 ). The results indicated that there were

increasing trend of root length with increasing concentra-

tion. The highest root length was observed in the treat-

ment with concentration of 2 mg/l, where the number of

roots produced per explant was 8.0. This was followed

by treatment with concentration of 1.5 mg/l which pro-

duced 6.0 roots per explant.

Generally, this trend showed that auxin was essential

for quick induction of banana roots in vitro as compared

with the control treatment. The necessity of using auxins

for roots induction in banana tissue culture is also

reported by Raut and Lokhande [28].

Rahman et al. (2013) in their study indicated that

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The Effects of Auxins and Cytokinin on Growth and Development of

(Musa sp.) Var. “Yangambi” Explants in Tissue Culture

2179

highest root length of 3.69 cm was achieved under 1.0

mg/l IBA for excised shoots of banana [29]. This may be

due to genotype of their cultivar and the relatively low

concentration of IBA used, given that the experiment was

conducted for six weeks.

In our study, it was evident that 2.0 mg/l can increase

the average root length within short time and reduce the

costs associated with tissue culture. This may have a

positive influence on the survival of the explants.

4. Conclusions

The optimum concentration of BAP for buds prolifera-

tion of this variety (Yangambi) was 6 mg/l as reflected

by increased number of buds and fresh weight. The use

of BAP in combination with IAA (BAP * IAA) was

found to enhance fresh weight at concentration of 6 mg/l:

0.35 mg/l respectively. Therefore, due to the genotype

specificity on response of many banana cultivars to tissue

culture media, it is recommended that this concentration

be used for in vitro propagation of this variety.

As for rooting, supplementing a media for tissue

culture of banana variety “Yangambi” with 2.0 mg/l of

IBA increased the number and length of roots formed

and reduce the time required to wait for roots to be

formed in basal media or media with activated charcoal.

5. Acknowledgements

This study was funded by the Nelson Mandela African

Institute of Science and Technology through research

funds from Commission for Science and Technology

(COSTECH) in Tanzania.

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