I.J.S.N., VOL.

8 (4) 2017: 892 - 897 ISSN 2229 – 6441

PROTOCOL FOR TISSUE CULTURE PROPAGATION OF BANANA CV.

RAJAPURI BALE (AAB)
Prabhuling, G., Rashmi, H. and Babu, A.G.
Center for Horticulture Biotechnology, Directorate of Research, University of Horticulture Sciences, Bagalkot 587 104, Karnataka State
**Corresponding author email: gprabhuling@gmail.com

ABSTRACT
Banana (Musa spp.) cv. Rajapuri Bale (AAB) is a popular cultivar of banana grown in Northern parts of Karnataka. Tissue
culture propagation in banana is preferred for its faster multiplication rate compared to sucker propagation. The present study
was carried out with the objective to investigate the effect of different ascorbic acid, BAP, IBA and NAA on in vitro
regeneration. Shoot tip explants cultured on MS basal medium supplemented with ascorbic acid 175 mg/l, BAP 2.0 mg/l +
NAA 0.25 mg/l and NAA 1.00 mg/l found effective in controlling browning, shoot multiplication and in vitro rooting,
respectively in banana cv. Rajapuri Bale.

KEY WORDS: Rajapuri Bale, Tissue culture, Shoot tip, Ascorbic acid, Shoot multiplication.

INTRODUCTION propagation is a preferred alternative method. Shoot tip

Banana and Plantains (Musa spp.) are one of the most valued culturing for bananas, provides second advantages that
fruit products. Banana belongs to the family Musaceae and coincide with the farmers demands including, increased
section Eumusa. It signifies variable benefits, both as a multiplication rate, physiological uniformity and the
staple food as well as a major export commodity for many availability of disease-free materials all year round [9].
tropical and subtropical countries. In general, banana However, Rajapuri Bale is highly recalcitrant to tissue
cultivars are considered as good sources of carbohydrates, culture propagation mainly due to exudation of phenolic
proteins, vitamins and minerals. Banana is known for its compounds from tissues and browning of the culture
antiquity and it is interwoven with Indian heritage and medium. This process is initiated by browning of the surface
culture. It is considered as the symbol of ‘prosperity and of plant tissues due to the oxidation of phenolic compounds
fertility’ owing to its greater socio-economic significance resulting in the formation of quinines which are highly
and multifaceted uses and high economic returns it is reactive and toxic to plant tissue. The browning phenomenon
referred to as “Kalpatharu” (a plant of virtues) and occurs in response to oxidation process of released phenolic
Kalpavriksh[16]. Rajapuri Bale (AAB) is a popular cultivar of compounds from injured tissue by phenol oxidase and
banana grown in Northern parts of Karnataka. It is a dwarf formation of quinones [4]. Quinones negatively inhibit cell
variety growing upto 6-8 feet height with a very thick stem growth and can often result in death of cells (necrosis) [11].
and stands up very well to wind. The leaves are wider than Therefore, preconditioning of explants with media
those of most bananas growing upto 3 feet wide. It is the supplements such as ascorbic acid [7] was necessary to limit
best plant to grow in marginal areas or where a grower does the production of these harmful substances. Keeping the
not intend to put much care into cultivation of bananas. The above points in view, the present investigation “Protocol for
bunches weigh about 10-15 kg with 8-10 hands and 90-100 Tissue Culture Propagation in Banana cv. Rajapuri Bale
fingers. Fruits have attractive yellow colour with thick skin (Musa spp., AAB Group)” was carried out with the objective
and good blend of sweet and acidity [13]. Bananas are of minimization of browning, enhancement of shoot
generally propagated vegetative through suckers. proliferation and induction of adventitious roots.
Unfortunately, the traditional methods limited the expansion
of bananas production due to a shortage of healthy plant MATERIALS & METHODS
material availability to farmers. High sterility of most Preparation of explants
cultivated bananas has historically prevented conventional The present study was conducted at the Center for
breeding programs and plant propagation. Moreover, the Horticulture Biotechnology, University of Horticultural
longer time required by bananas to generate makes it even Sciences, Bagalkot, India. Healthy and vigorously growing
more difficult to breed them [15]. The major limitation with sword suckers of cv. Rajapuri (3-4 month age), free from
sucker propagation is the transmission of harmful insects, viruses and other diseases were selected as a source of
nematodes and viral diseases to field grown suckers. To explant. Suckers were washed thoroughly under running tap
overcome these issues and enable rapid multiplication of water for 10-15 min. the suckers were then chopped off
economically important commercial varieties, in vitro about 5-6 cm in length and 3-4 cm in diameter (fig. 1).

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FIGURE 1. Mother plant and sword suckers of cv. Rajapuri Bale

The plant material obtained from the field was thoroughly 0.05% streptocycline for eight hours. After thoroughly
washed in running tap water followed by washing with a washing with double distilled water, they were trimmed
detergent solution to remove adhering soil particles. Later, again, so that trimmed suckers were of 2-3 cm in length and
rhizomes were kept immersed in a fungicide solution of 1 % 2-2.5 cm in diameter. These shoot tips were soaked in 0.05
bavistin for half an hour, to further clean the planting % cetrimide for 30 minutes. After removing one more layer,
material. The outer leaves, leaf base and corm tissue were the shoot tips were surface sterilized with 0.1% mercuric
trimmed using a sterilized stainless steel knife until the chloride in a closed container for 10 minutes. Further
length of explant was 4-6 cm and the diameter, 3-4cm. operations such as washing several times with sterile
These trimmed suckers enclosing the shoot tip were washed distilled water to remove all traces of chlorine, trimming of
with double distilled water. After trimming one more outer explants and inoculation were carried out under laminar air
layer, they were soaked in a solution of 0.50 % bavistin + flow chamber (Fig. 2 A-C).

FIGURE 2. Establishment of aseptic culture: A) Trimming of suckers; B) Trimmed shoot tip; C) Trimming of shoot tip under
laminar airflow; D) Inoculated shoot tips on liquid MS media; E) Established shoot tip.

Initiation of aseptic culture as important criteria for assessing the success in

Shoot tip explants were inoculated in MS liquid medium establishment (Fig. 2E). Shoot tips that had turned dark
containing 2 mg/l BAP, 35 mg/L adenine sulphate and brown/black and which did not swell were considered as
Different concentration of ascorbic acid concentration (25, non-established. Healthy and contamination free explants
50, 75, 100, 125, 150,175, 200 mg/l) (Fig. 2D) for two were excised by removing discolored tissue and transferred
weeks maintaining standard culture conditions of 25 ±20C to the semisolid medium supplemented with BAP (2 mg/l)
temperature, 70 % RH and photoperiodic cycle of 16 hours and adenine sulphate (35 mg/l) and incubated for four weeks
light and 8 hours dark period. After two weeks of maintaining standard culture conditions. The browning
incubation, all the explants were evaluated for their percent degree in initiation stage was scored visually as: +++ = High
response, browning and percent contamination in liquid browning; ++ = Moderate browning and - = No browning.
medium. Greening and swelling of the explants were utilized

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Effect of cytokinin andauxin on shoot proliferation in attached to the roots. Observations were recorded onper cent
Rajapuri Bale (AAB) rooting, number of roots, length of primary roots and length
Established aseptic cultures were transferred onto of secondary roots. Immediately after washing they were
multiplication media for shoot multiplication and transferred to protrays containing a sterilized cocopeat and
development. The medium used for banana multiplication kept under poly tunnel for weeks. Later plantlets transferred
was Murashige & Skoog Medium (MS)[6]. Different topoly bags containing sand, red soil and compost in 1:1:1
concentration of 6-Benzyl amino urine (BAP), Naphthalene ratio (v/v). They were kept under shade house and sprayed
acetic acid (NAA) hormones were used for shoots induction with water regularly to maintain high humidity around the
and shoots multiplication. Individual shoots were inoculated plantlets.
onto half strength MS medium containing different Statistical analysis
concentration of Indole-3-butyric acid (IBA), Naphthalene The data were taken at four week intervals after incubation.
acetic acid (NAA) and activated charcoal 2 mg/l. All the There were five explants for each treatment and three
media were autoclaved at 15 psi and 121 ˚C for 20 minutes. replications. Data recorded for different parameters were
The culture jars containing explants were incubated in subjected to completely randomized design (CRD).
growth chamber at 26 ±2°C with 16 hour photoperiod Statistical analysis was done by (ANOVA) using software
(approximately 2000 lux) provided by cool white fluorescent Wasp developed by ICAR Research Complex, Goa.
tubes. The materials were 6 times sub cultured at a regular
interval of four weeks onto same medium to produce RESULTS & DISCUSSION
multiple shoots. Observations were recorded of percent The effect of ascorbic acid on browning of medium
response, number of shoots per explants, length of shoot Browning phenomena is one of the most common problems
(cm) and number of leaves per shoot. The regenerated associated with in vitro establishments of shoot tip explant.
plantlets after developing sufficient root system were In the present investigation, the effect of antioxidant
deemed ready to transfer in soil. The plantlets were carefully ascorbic acid on browning and growth of banana cv.
removed from the culture vessels. The roots of the plantlets Rajapuri Bale were studied.
were gently washed under running tap water to remove agar

TABLE 1: Effect of ascorbic acid on browning intensity in banana cv. Rajapuri Bale (AAB)
Treatments Browning intensity
T1: MS B + BAP 2 mg/l (Control) +++
T2: MS B + BAP 2 mg/l + Ascorbic acid 25 mg/l +++
T3: MS B + BAP 2 mg/l + Ascorbic acid 50 mg/l +++
T4: MS B + BAP 2 mg/l + Ascorbic acid 75 mg/l +++
T5: MS B + BAP 2 mg/l BAP+ Ascorbic acid 100 mg/l +++
T6: MS B + BAP 2 mg/l BAP+ Ascorbic acid 125 mg/l ++
T7: MS B + BAP 2 mg/l BAP+ Ascorbic acid 150 mg/l ++
T8: MS B + BAP 2 mg/l BAP+ Ascorbic acid 175 mg/l -
T9: MS B + BAP 2mg/l BAP + Ascorbic acid 200 mg/l -
MS B: Murashige & Skoog Basal Medium
+++ = High browning; ++ = Moderate browning; - = No browning

High browning war observed with Ms medium without the ascorbic acid. There was no browning when the MS medium
ascorbic acid (Control) (Table 1 & Fig. 3 A). The browning was supplemented with ascorbic acid 175 mg/l and 200 mg/l
intensity reduced with increase in the concentrations of (Table 1& Fig. 3 B-C).

FIGURE 3. Intensity of browning in Rajapuri Bale on liquid MS medium: A) Control; B) Ascorbic acid 175 mg/l; C) Ascorbic
acid 200 mg/l.

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Highest browning intensity in control was attributed to culture systems lessen the efficiency of explants initiation,
oxidation phenols by oxygen radicals resulting in oxidative growth and development [5]. One of the major problems for
injury. Inhibition of browning at higher concentration of several tissue culture system, is the lethal browning which
ascorbic acid was mainly due reducing activity of ascorbic result in death of the cultured explants that depend on the
acid, therefore, cells are protected from oxidative injury. rate of oxidation of phenolic compounds, as well as the
Phenolic secretions and other exudates in plants tissue quality of the total phenols [10].

Effect of cytokinin and auxin on shoot proliferation

TABLE 2: Effect of cytokinin and auxin on shoot growth in banana explants cv. RajapuriBale (AAB)
Treatments Percent response Number of Length of Number of
shoots shoots (cm) leaves
T1: MS B + BAP 2.0 mg/l (Control) 100 (89.53) * 3.61 4.33 3.61
T2: MS B + BAP 2.0 mg/l + 2 weeks dark 100 (89.53)
5.41 8.05 5.41
incubation + 1 Week light incubation
T3: MS B + BAP 2.0 mg/l + NAA 0.25 mg/l 100 (89.53) 6.27 8.17 6.27
T4: MS B + BAP 2.0 mg/l + NAA 0.50 mg/l 100 (89.53) 4.07 5.09 4.57
T5: MS B + BAP 2.0 mg/l + NAA 0.75 mg/l 100 (89.53) 3.00 5.16 4.21
T6: MS B + BAP 2.0 mg/l + NAA 1 mg/l 100 (89.53) 3.50 3.77 3.50
T7: MS B + BAP 3.0 mg/l + NAA 0.25 mg/l 100 (89.53) 3.22 3.99 3.22
T8: MS B + BAP 3.0 mg/l + NAA 0.50 mg/l 100 (89.53) 3.78 3.55 3.78
T9: MS B + BAP 3.0 mg/l + NAA 0.75 mg/l 100 (89.53) 2.87 4.66 2.87
T10: MS B + BAP 3.0 mg/l + NAA 1 mg/l 100 (89.53) 3.37 3.17 3.37
T11: MS B + BAP 4.0 mg/l + NAA 0.25 mg/l 100 (89.53) 3.60 4.40 3.60
T12: MS B + BAP 4.0 mg/l + NAA 0.50 mg/l 100 (89.53) 4.44 8.10 4.44
T13:MS B + BAP 4.0 mg/l + NAA 0.75 mg/l 100 (89.53) 3.16 3.41 3.16
T14: MS B + BAP 4.0 mg/l + NAA 1 mg/l 100 (89.53) 3.60 3.21 3.60
T15: MS B + BAP 5.0 mg/l + NAA 0.25 mg/l 100 (89.53) 3.53 3.84 3.54
T16: MS B + BAP 5.0 mg/l + NAA 0.50 mg/l 100 (89.53) 3.47 3.71 3.47
T17: MS B + BAP 5.0 mg/l + NAA 0.75 mg/l 100 (89.53) 3.44 3.44 3.44
T18: MS B + BAP 5.0 mg/l + NAA 1 mg/l 100 (89.53) 3.59 3.21 3.58
SE m ± - 0.23 0.32 0.37
CD at 1% NA 0.92 1.31 1.52
*Figures in parenthesis are arc sin transformation

Variable number of shoots were produced per explant in MS treatment BAP 2.0 mg/l + 2 weeks dark incubation +1 Week
media supplemented with different concentrations of BAP light incubation (5.41 shoots per explant and 8.05 cm shoot
and NAA. Among the different concentrations, MS B+ BAP length). Next best results were achieved with BAP 4.0 mg/l
2.0 mg/l + NAA 0.25 mg/l showed significantly maximum + NAA 0.50 mg/l which was superior as compared to control
number of shoot per explants (6.27) and the length of the treatment (Table 2 & Fig. 4 A-D).
shoots (8.17 cm) which was statistically on par with the

FIGURE 4. Shoot growth on media containing different concentration ofcytokinin and auxin: A) BAP 2.0 mg/l (Control); B)
BAP 2.0 mg/l + NAA 0.25 mg/l C) BAP 2.0 mg/l + 2 weeks dark incubation + 1 Week lightincubation; D) BAP 4.0 mg/l +
NAA 0.50 mg/l.

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Enhanced shoot growth induced by BAP 2.00 mg/l + NAA auxin interaction in which cytokinin is required to activate a
0.25 mg/l suggest that there was strong synergistic effect of protein expressed in response to auxin [2].
BAP-NAA interaction. Similar findings were also reported
by Srangsam and Kanchanapoom [17,8] in banana. The Effect of auxins on rooting of microshoots cv. Rajapuri
concentration and combination of auxin and cytokinin in the bale (AAB)
nutrient medium is key factor which determines successful The data pertaining to the response of different auxins (IBA
plant regeneration [14]. Synergistic mechanism of cytokinin- and NAA) on in vitro rooting of banana buds are presented
in Table 3.

TABLE 3. Effect of auxins on in vitro rooting of banana plantlets cv. Rajapuri Bale (AAB)
Treatment (mg/l) Percent Number of Length of Number secondary
response primary primary roots/plantlet
roots/plantlet roots (cm)
T1: MS B + IBA 0.50 100 (89.41)* 2.07 5.63 9.10
T2: MS B + IBA 1.00 100 (89.41) 2.10 5.41 8.36
T3: MS B + IBA 1.50 100 (89.41) 2.67 7.73 8.55
T4: MS B + IBA 2.00 100 (89.41) 2.56 7.62 8.19
T5: MS B + NAA 0.50 100 (89.41) 2.14 5.45 6.44
T6: MS B + NAA 1.00 100 (89.41) 3.32 7.63 8.59
T7: MS B + NAA 1.50 100 (89.41) 1.82 4.33 6.83
T8: MS B + NAA 2.00 100 (89.41) 1.67 4.01 4.37
T9: MS Basal media 100 (89.41) 1.32 3.41 6.62
(Control)
S.Em ± - 0.12 0.19 1.23
CD@ 1% NS 0.41 0.52 4.23
*Figures in parenthesis are arc sin transformation

Media containing different concentration of auxins showed control showed poor rooting (Table 3 & Fig. 5). Superiority
variable effects on rooting. MS medium supplemented with of NAA may to be effective absorption, translocation and
NAA 1 mg/l showed maximum number of primary roots per utilization as compared to other types of auxins [3]. These
shoots (3.32) and good length of primary roots (7.63 cm) results are conformity with the findings of [1, 3 &12] in banana.
and number of secondary roots (8.59), while, untreated

FIGURE 5. Effect of auxins on rooting: A) Control; B) MSB+ IBA 1.50 mg/l; C) MSB+ NAA 1.00 mg/l.

CONCLUSION ACKNOWLAGEMENT
The findings of present study indicates that control of lethal Authors are thankful to Karnataka Biotechnology and
phenolic browning, increased shoot proliferation and in vitro Information Technology Services (KBITS), Department of
rooting could be achieved with MS medium +175mg/l Information Technology, Biotechnology and Science &
ascorbic acid, MS medium + BAP 2.0 mg/l+ NAA 0.25 mg/l Technology, Government of Karnataka for financial
and half strength MS medium +NAA 1.00 mg/l. This assistance (KBITS/126/BFC/5/2013-14).
protocol can be employed for large scale production of
disease free planting material of banana cv. Rajapuri Bale.

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