Effects of Different Benzyladenine (BA) Concentrations on Growth and

Development of Pisang Berangan AAA (Musa acuminata cv. Berangan) in

vitro Plantlets Culture

YONG Kuok Liang1 and HSIEH Ching-Hsiang2

1
Department of Tropical Agriculture and International Cooperation, National Pingtung
University of Science and Technology, Pingtung, Taiwan R.O.C.

2
Department of Plant Industry, National Pingtung University of Science and Technology,
Pingtung, Taiwan R.O.C.

Abstract

Bananas are grown as staple food, significant cash crops and major export crops in
many tropical and subtropical countries. With the ever increasing demand of banana as an
essential source of nutrients for millions of people, measures to reduce production costs
and to make its production more and more cost-effective while still being able to meet
demands is necessary. The objective of this study is to reduce production cost of
commercial micro-propagation of banana by modifying nutrient media constituents, while
still maintaining optimal or near-optimal growing conditions and development in vitro.
This study assesses shoot multiplication response of ‘Pisang Berangan’ in vitro plantlets
when treated with different concentrations of BA (0, 3, 6 and 9 mg/L), and with or without
100 mg/L myo-inositol. This study incorporates the use of food grade gellan gum, which is
the more affordable alternative to Gelrite. At the end of this study, the treatment of no
myo-inositol + 0 mg/L BA produced the highest mean number of shoots per explant (2.5
per explant), mean shoot length (10.91 cm), and mean number of leaves (8.38 per explant).
However, myo-inositol + 9 mg/L BA produced the highest mean number of roots (21.25
per explant). The treatment of no myo-inositol + 0 mg/L BA and myo-inositol + 9 mg/L
BA were the best treatments in this study.

Keywords: banana, in vitro culture, shoot tips, shoot multiplication

Abbreviation: BA-benzyl adenine

1
 Introduction

Musa spp., banana and plantain, constitute the fourth most important staple food

commodity of the world, after rice, wheat and maize. In Asian and Pacific regions, banana

has great socio-economic significance. The region is the major center of diversity and most

of the edible bananas are believed to have originated in South-east Asia and Western

Pacific regions. Banana is the second most commonly grown fruit crop in Malaysia (Chai

et al., 2004). Overall banana production has decreased due to the increasing threat of

Fusarium wilt disease, high labor costs and marketing issues. Banana shoot-tip cultures

were most suitable for micro-propagation for large-scale plant production. Commercial

companies have adopted this method, and they produce 1.3 million plants annually, with

approximately 0.5% somaclonal variation (Chai et al., 2004). However, the cost of

production of in vitro plants could be reduced by low-cost micro-propagation.

Cytokinins are N6-substituted adenine derivatives and a class of plant hormones

involved in plant growth and development (D’Agostino and Kieber, 1999). Cytokinins

such as benzyl adenine (BA) and kinetin are known to reduce the apical meristem

dominance and induce both auxiliary and adventitious shoot formation from meristematic

explants in banana (Jafari et al., 2011). However, the application of higher BA

concentrations inhibits elongation of adventitious meristems and the conversion into

complete plants (Buising et al., 1994). The most established banana shoot-tip culture

system was achieved by using BA as a supplement to Murashige and Skoog (MS) basal
media (Murashige and Skoog, 1969). The effectiveness of BA over other cytokinins in

inducing multiplication of shoot tip cultures has been reported in different cultivars of

bananas (Rahman et al., 2006; Resmi and Nair, 2007; Farahani et al., 2008; Buah et al.,

2010). BA has a marked effect in stimulating the growth of axillary and adventitious buds

and foliar development of shoot tip cultures (Abeyarante and Lathiff, 2002; Buah et al.,
2010).

2
 Myo-inositol on the other hand, is a pentose sugar of plant origin, and has been

routinely used in a concentration of 100 mg/l in banana tissue culture (Vuylsteke, 1989),

and has accordingly been supplemented to MS medium at the same concentration

(Murashige and Skoog, 1962). Myo-inositol has a diverse biological role and participates

in several cellular processes, including signal transduction, stress response, cell wall

biogenesis, growth regulation, osmo-tolerance, IAA metabolism, membrane trafficking

and in phytic acid synthesis (Irvine and Schell, 2001).

Food grade gellan gum (low acyl) is added to the culture medium as the solidifying

agent. Not only does the use of food grade gellan gum help reduce production cost, it may

also prevent medium blackening. The use of gelrite (gellan gum) in culture initiation

means that the medium is less likely to blacken (from the oxidation of phenolic compounds)

than if agar is used (Lee, 2003). Laboratory grade gellan gum is usually marketed by

suppliers as Gelrite. This study incorporates the use of food grade gellan gum, which is the

more affordable alternative to Gelrite.

3
 Materials and Methods

Plant Materials

Multiple shoot cultures (fifth generation of subculture) of ‘Pisang Berangan AAA’

(ITC.1287) were obtained from Inno Agrotech (Perak, Malaysia). Shoots were supplied in

polypropylene plastic bags containing full strength MS 30 medium. The medium was

based on the formulation of Murashige and Skoog (MS; 1962), with 30 g/L sucrose, 2

mg/L BA, and solidified with 3 g/L of gellan gum (Caixin Sugar Industry, Henan, PRC) at

pH 5.8. Shoots were incubated in a controlled environment room at 28±2°C with a 16 h

photoperiod (T5 fluorescent lamp 28W). The tested shoot materials were in a range of 2-3

cm in length with 2-3 leaves per shoot.

Medium Composition and Preparation

Basal medium used in the present study was Murashige and Skoog (MS) (1962)

medium. Stock solutions of macronutrients, micronutrients, iron source and organic

supplements were prepared separately for the preparation of MS medium. Sucrose (30 g/L)

was added to the medium as the basic energy source and pH of medium was adjusted to 5.8

with the addition of 1N HCl/NaOH. Gellan gum (3 g/L) was used to solidify the medium.

Each culture flask contains 100 mL of medium. The medium was autoclaved at 121ºC for

45 min at 1 atm pressure.

Explant Culture Conditions

Axenic shoots of ‘Pisang Berangan’ were multiplied by separating shoot clusters into

individual shoots. In vitro plantlets, each approximately 1 cm in length, were excised from

each shoot by cutting the upper pseudostem above the apical meristem. Outer leaf sheaths,

leaves, roots, and any necrotic tissue were removed from the explants. Excised shoot tips
were then cultured on 100 mL of solid medium supplemented with different concentrations

and combinations of BA and myo-inositol; (a) 0, 3, 6 and 9 mg/L BA with 0 mg/L

myo-inositol, and (b) 0, 3, 6 and 9 mg/L BA with 100 mg/L myo-inositol, amounting to

4
eight different treatments. Each treatment was replicated three times with each replicate

having four explants. The dissected plantlets were fully or partially submerged in the

medium with their pseudostems erect. These flasks were placed in a temperature-controlled

room at 25 ± 2ºC with 16 h photoperiod (32.14 uMol m-2 s-1 T5 fluorescence lamp;

Wellpower, Taiwan).

Data Analysis

With four plantlets in each replicate, each was marked (1, 2, 3, and 4) on the outside

of the flask. At 2 and 4 weeks after inoculation (WAI), all three replicates of each treatment

were observed for data analysis. At 2 and 4 WAI, the data collected were number of shoot

per explant, shoot length, number of leaves and number of roots. At 6 WAI, two of the

three replicates of each treatment were randomly selected for data analysis. For 6 WAI,

data collected were shoot per explant, shoot length, number of leaves, number of roots,

root length, fresh weight of shoots and roots, as well as dry weight of shoots and roots. The

data collected and calculated were analyzed using Statistical Analysis Software (SAS), on

Analysis of Variance (ANOVA) for all treatments. The comparison of means were tested

for significance using the least significant difference (LSD) test, at 0.05 level of

probability.

5
 Results and Discussion

At 2 WAI (Table 1 and Figure 2), the mean shoot length response was similar for all

treatments. But at 6 WAI, no myo-inositol + 0 mg/L BA gave the highest mean shoot

length (10.91 cm), followed by myo-inositol + 6 mg/L BA (10.11 cm), and then

myo-inositol + 9 mg/L BA (9.85 cm). According to Figure 2, a clear separation in terms of

shoot length can be seen between the treatments at 4 WAI as well as 6 WAI. Referring to

Figure 3 and 4, there was a clear separation between the treatments in terms of the number

of leaves and roots throughout the study. From the Tables 1, 2, 3a, and 3b, the number of

roots showed significant difference throughout the study, however, number of shoots per

explant and shoot length showed no significant difference.

At 4 WAI (Table 2), no myo-inositol + 0 mg/L BA produced the highest mean number

of shoots per explant (2.0 per explant), mean shoot length (6.11 cm), and mean number of

leaves (4.92 per explant). However, myo-inositol + 9 mg/L BA produced the highest mean

number of roots (14.25 per explant), followed by no myo-inositol + 0 mg/L BA (13.67 per

explant), and then by no myo-inositol + 0 mg/L BA (19.00 per explant) according to Table

3a. At the end of this study (6 WAI), no myo-inositol + 0 mg/L BA produced the highest

mean number of shoots per explant (2.5 per explant), mean shoot length (10.91 cm), and

mean number of leaves (8.38 per explant). However, myo-inositol + 9 mg/L BA produced

the highest mean number of roots (21.25 per explant), followed by myo-inositol + 6 mg/L

BA (20.00 per explant), and then by no myo-inositol + 0 mg/L BA (19.00 per explant)

according to Table 3a. This results showed that the positive effects of myo-inositol on root

formation was only observed later, at 4 and 6 WAI (Figure 4).

Referring to Table 3a at 6 WAI, increasing BA concentration from 0 to 9 mg/L +

myo-inositol had a progressive positive effect on root formation. In other words, the higher

the BA concentration, the higher the number of roots formed. In the four treatments with

the addition of myo-inositol, the concentration of 6 mg/L BA proved to be the most

optimum for the formation of leaves (7.88 per explant) compared with no myo-inositol + g
mg/L BA (6.38 per explant) at 6 WAI.

6
 From Table 3b, no myo-inositol + 0 mg/L BA produced the highest fresh weight of

both shoot (2.49 g) and root (1.46 g) as well as highest root dry weight (80.96 mg)

compared to other treatments. However, myo-inositol + 6 mg/L BA produced the highest

shoot dry weight (163.70 mg). The fresh and dry weight values of all treatments had no

significant difference as indicated at Table 3b.

In agreement with (Vuylsteke, 1989; Hussein, 2012), it was evident that the role of

myo-inositol was not essential and should not necessarily be included in Musa acuminata

cv. Berangan tissue culture. Therefore, some carbon sources are plant-specific and their

role on in vitro culture is likely to be related to tissue type and physiological age.

Table 1. In vitro plant growth responses under different medium treatments (2 WAI).
Treatment No. of Shoot Length No. of Leaves No. of Root
Shoot/Explant (cm)
No M* + 0 mg/L BA 1.75 a 1.69 a 1.83 bc 8.67 a
No M* + 3 mg/L BA 1.50 a 1.73 a 2.58 a 6.83 abc
No M* + 6 mg/L BA 1.33 a 1.45 a 2.67 a 6.08 bcd
No M* + 9 mg/L BA 2.00 a 1.80 a 2.25 ab 7.25 ab
M* + 0 mg/L BA 1.58 a 1.37 a 1.50 c 5.08 cd
M* + 3 mg/L BA 1.75 a 1.33 a 1.50 c 4.58 d
M* + 6 mg/L BA 1.67 a 1.45 a 1.67 bc 6.17 bcd
M* + 9 mg/L BA 1.78 a 1.50 a 1.58 c 5.50 bcd
M- myoinositol
Values within the same column with the same letter are not significantly different at P <
0.05 according to the LSD test. Each treatment was replicated three times with each
replicate having four explants.

7
Table 2. In vitro plant growth responses under different medium treatments (4 WAI).

Treatment No. of Shoot Length No. of Leaves No. of Root

Shoot/Explant (cm)
No M* + 0 mg/L BA 2.00 a 6.11 a 4.92 a 13.67 ab
No M* + 3 mg/L BA 1.83 a 5.25 a 4.83 a 10.33 c
No M* + 6 mg/L BA 1.33 a 4.74 a 4.33 a 10.42 c
No M* + 9 mg/L BA 2.08 a 5.68 a 4.92 a 11.00 bc
M* + 0 mg/L BA 1.75 a 4.52 a 4.42 a 9.47 c
M* + 3 mg/L BA 1.92 a 3.71 a 3.33 a 9.92 c
M* + 6 mg/L BA 2.00 a 4.88 a 4.50 a 12.67 abc
M* + 9 mg/L BA 2.33 a 5.23 a 4.50 a 14.25 a
M- myoinositol
Values within the same column with the same letter are not significantly different at P <
0.05 according to the LSD test. Each treatment was replicated three times with each
replicate having four explants.

Table 3a. In vitro plant growth responses under different medium treatments (6 WAI).

Treatment No. of Shoot No. of No. of Root Length

Shoot/Explant Length (cm) Leaves Root (cm)
No M* + 0 mg/L BA 2.50 a 10.91 a 8.38 a 19.00 ab 12.39 b
No M* + 3 mg/L BA 2.25 a 8.40 a 7.75 a 14.63 bc 10.49 bc
No M* + 6 mg/L BA 1.25 a 7.84 a 6.38 a 13.75 c 11.50 bc
No M* + 9 mg/L BA 2.00 a 7.95 a 7.25 a 13.88 c 10.53 bc
M* + 0 mg/L BA 2.25 a 8.05 a 7.63 a 14.50 bc 9.81 c
M* + 3 mg/L BA 2.13 a 7.19 a 6.63 a 16.88 abc 9.59 c
M* + 6 mg/L BA 2.25 a 10.11 a 7.88 a 20.00 a 12.56 c
M* + 9 mg/L BA 2.13 a 9.85 a 6.75 a 21.25 a 15.19 a
M- myoinositol
Values within the same column with the same letter are not significantly different at P <
0.05 according to the LSD test. Only the data from two of the three replicates of each
treatment were randomly selected for this table.

8
Table 3b. In vitro plant fresh and dry weight under different medium treatments (6 WAI).
Treatment Shoot Fresh Shoot Dry Root Fresh Root Dry
Weight (g) Weight (mg) Weight (g) Weight (mg)
No M* + 0 mg/L BA 2.49 a 154.84 a 1.46 a 80.96 a
No M* + 3 mg/L BA 1.67 a 110.21 a 1.03 a 51.06 a
No M* + 6 mg/L BA 1.80 a 110.19 a 0.89 a 53.51 a
No M* + 9 mg/L BA 1.95 a 119.06 a 0.85 a 50.63 a
M* + 0 mg/L BA 1.59 a 103.50 a 0.59 a 38.91 a
M* + 3 mg/L BA 1.38 a 130.88 a 0.68 a 42.21 a
M* + 6 mg/L BA 2.22 a 163.70 a 1.00 a 62.98 a
M* + 9 mg/L BA 2.25 a 149.05 a 1.29 a 74.59 a
M- myoinositol
Values within the same column with the same letter are not significantly different at P <
0.05 according to the LSD test. Only the data from two of the three replicates of each
treatment were randomly selected for this table

3
Number of shoot per explant

No M + 0 mg/L BA
No M + 3 mg/L BA
No M + 6 mg/L BA
2 No M + 9 mg/L BA
M + 0 mg/L BA
M + 3 mg/L BA
M + 6 mg/L BA
M + 9 mg/L BA

1
0 2 4 6
WAI
Figure 1. Mean number of shoots induced per explant at 2, 4, and 6 WAI.

9
 11

9
No M + 0 mg/L BA
No M + 3 mg/L BA
Shoot Length (cm)

No M + 6 mg/L BA
7
No M + 9 mg/L BA
M + 0 mg/L BA
5 M + 3 mg/L BA
M + 6 mg/L BA
M + 9 mg/L BA
3

1
0 2 WAI 4 6

Figure 2. Mean of shoots length of each treatment at 2, 4, and 6 WAI.

7
No M + 0 mg/L BA
6
No M + 3 mg/L BA
Number of leaves

5 No M + 6 mg/L BA
No M + 9 mg/L BA
4
M + 0 mg/L BA

3 M + 3 mg/L BA
M + 6 mg/L BA
2
M + 9 mg/L BA

0
0 2 4 6
WAI
Figure 3. Mean number of leaves formed from each treatment at 2, 4, and 6 WAI.

10
 22

20

18

16 No M + 0 mg/L BA

14 No M + 3 mg/L BA
Number of root

No M + 6 mg/L BA
12
No M + 9 mg/L BA
10 M + 0 mg/L BA
8 M + 3 mg/L BA
M + 6 mg/L BA
6
M + 9 mg/L BA
4

0 0 2 4 6
WAI
Figure 4. Mean number of roots formed from each treatment at 2, 4, and 6 WAI.

11
 Conclusion and Suggestions

Culture medium without the addition of myo-inositol and BA was one of the most

optimum treatment, as expressed by highest mean number of shoots per explant (2.5 per

explant), mean shoot length (10.91 cm), mean number of leaves (8.38 per explant), highest

fresh weight of both shoot (2.49 g) and root (1.46 g) as well as highest root dry weight

(80.96 mg). Myo-inositol + 9 mg/L BA is also considered with similar beneficial effect in

this study. For lowering the cost of production, it is therefore recommended that

myo-inositol and BA be excluded from Musa acuminata cv. Berangan culture medium as a

measure to reduce economic cost as well as likely, negative chemical effects on culture

medium.

12
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