Int. J. Adv. Res. Biol. Sci. (2016).

3(5): 240-253

International Journal of Advanced Research in Biological Sciences

ISSN: 2348-8069
www.ijarbs.com
Volume 3, Issue 5 - 2016
Research Article

SOI: http://s-o-i.org/1.15/ijarbs-2016-3-5-34

Low cost micropropagation package for Banana

(Musa paradisiaca L.)

S. Dhanalakshmi and R. Stephan*

Plant Biotechnology laboratory, PG & Research Department of Botany,
Govt. Arts. College, Ariyalur – 621 713, TN, India.
*Corresponding author: stephan.biotech@gmail.com

Abstract
Banana is one of the important horticultural crops extensively cultivated in India. It has been conventionally propagated through
suckers which is a time consuming method with lower rates of multiplication. To overcome this, the standardized protocols for
shoot-tip culture have been commercially exploited by various tissue culture industries. The LBTM supplemented with different
concentration of growth regulators the high shoot induction frequency was obtained in the combination of BAP 0.2 mg/l + IAA
0.1 mg/l (68.1±4.2) followed by KIN 0.2 mg/l + IAA 0.1 mg (65.2±2.8) respectively. The high multiple shoot induction
frequency was obtained in the combination of BAP 2.0 mg/l + IAA 0.5mg/l combination (18.1±3.6) followed by the combination
of KIN 2.0 mg/l + IBA 0.5 mg/l (13.2±0.8) respectively. In root induction different LBTM combination of IBA + GA3. In this
2.0 mg/l IBA + 0.5 mg/l GA3 produced more number of roots (12.5 ± 2.2) followed by other combination. The overall
percentage of cost reduction was comparatively analysed for banana tissue culture propagation viz. LBTM (73.20%), Instruments
(84.31%) and glassware’s (93.4% ) respectively.

Keywords: MS medium, micro-propagation, variety; banana; Hardening

Introduction
Banana is one of the most important major fruit crops time on a year-round basis anywhere, irrespective of
grown in India. Botanically, banana is a the season and weather (Anonymous, 2004). The most
monocotyledonous herbaceous plant belonging to the widely used MS medium (Murashige and Skoog,
family Musaceae. Almost every part of the banana 1962) is used for commercial production of plantlets
plant is used some way or the other and it is rightly through shoot-apical meristem culture of banana. High
called “poor man’s apple”. It is probably the cheapest cost of plantlet production through micropropagation
fruit available throughout the year. technique is a major concern limiting its wide
application, despite its obvious advantages. Due to the
Commercial production of micropropagated banana is high cost of production, 32 out of 90 commercial
now common in many countries and it is estimated micropropagation units were closed down in India
that 25 million plants are produced worldwide each after the tremendous growth in 1990s (Prakash, 2001
year. This plant tissue culture technique provides a and Savangikar, 2004). In developed countries also
large number of uniform, high quality and disease-free this industry has undergone a pause, as it is difficult to
planting material to meet demand in a short span of

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remain cost-effective (Govil and Gupta, 1997; control (Table- 1). Among the different low-cost
Dhanalakshmi and Stephan, 2014). alternative viz. macronutrients , micronutrients, iron
source, vitamin source, carbon source, growth
The shoot multiplication rate obtained in the low cost regulators and solidifying agent, The selected low-
options was not less when compared to the other in cost nutrients and their composition are standardized
vitro multiplication trails. (Ganapathi et al., 1995) using sword sucker explants of Musa paradisiaca L.
used tap water, commercial grade sugar and reduced The standardized low cost nutrients are listed in the
the salt components in medium for banana plantlet Table -1. This medium composition i.e LBTM (low
production and achieved a maximum cost reduction of cost banana tissue culture medium) was used
31.2 %. Use of the media, culture vessel and low cost throughout the banana micropropagation. (Table- 1).
substitutes for mass propagation was successful in
several other species (Sujatha and Chandran, 1997; Cost analysis (2015-2016)
Varshney et al., 2000; Kodym and Arias, 2001
;Kadota and Niim, 2004; Piatezak et al.,2005; Hung et The market price quotation were obtained for 2015-16
al., 2006). from the reputed companies for the conventional and
alternative (low cost) sources of medium components,
In order to increase the tissue culture technology in instruments and glasswares. Based on the quotation of
banana farming, innovative approaches are needed to the manufacture quantities used per liter of the
lower the cost of micropropagule production. Banana medium, per unit cost differences were evaluated by
plant production via low cost technology in which cost the following formula. (Table- 2), For instruments
reduction is achieved by improving process efficiency and glassware’s based on the Quotation comparative
and better utilization of resources is reported by statement was used. (Table- 3,4).
Savangikar (2002). Low cost options should lower the
cost of production without compromising the quality (Amount used in the culture medium (g/l) × Price of
of the micropropagules and plants (Prakash et al., amount bought (Rs)
2004). It is necessary to develop low cost technologies Amount bought (g)
by improving the process efficiency and better
utilization of resources. Keeping the above facts Sterilization of explants
present study was aimed to reduce the cost of banana
tissue culture by using alternative nutrient sources. Sword suckers of Musa paradisiaca L. was collected
from the college garden and used as a explants source.
Materials and Methods The sword suckers with medium size were carefully
removed from field grown banana plant. The older
Plant materials parts were excised with stainless steel knife. The shoot
tips about 3-4 cm length were excised. They were
To develop a low cost tissue culture techniques in washed thoroughly with a solution of Tween - 20. (2-3
Musa paradisiaca L. var.Monthan (Fig-1).The sword drops in 500 ml water). All traces were removed by
sucker of this plant were collected from National repeated washings under running tap water to remove
Research Centre for Banana (NRCB), Thogamalai dust particles for 30 min, and then treated with liquid
road, Tiruchirappalli, Tamil Nadu, India. And these detergent for 30 min, and finally rinsed three times
varieties are maintained in the college garden, PG and with RO water. Then the explant was treated with an
Research Department of Botany, Government Arts antimicrobial agent (Bavistin) for 1 min and again
College, Ariyalur and were used as a source of mother rinsed three times with distilled water. Further,
plant in this, the sword suckers were used as a source sterilization treatments were done under a laminar-air
material for in vitro studies. flow chamber. The explants were then disinfected with
0.1% (w/v) Mercuric chloride (HgCl2) for 3 minutes
Low cost Banana Tissue culture Medium (LBTM) under aseptic conditions. After these explants were
preparation thoroughly washed 3-4 times with sterilized distilled
water to remove the traces of Mercuric chloride, then
In the present study two tissue culture medium were the explant was ready for inoculation.
used for the in vitro studies of Musa paradisiaca L.
The first one was MS medium (Murashige and Skoog,
1962) and this medium composition was used as the
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Inoculation: the mixture of red soil, sand and saw dust in 1:1:1
ratio in the plastic cups for 10 days and subsequently
Inoculation was carried out in a sterile laminar airflow transferred to pots. The plants need 90-100% humidity
hood chamber. Surface sterilization was achieved and they were covered with plastic bags with
through spraying 70% (v/v) ethanol. The sword sucker perforation or holes (Table -8). After 20 days the
cuttings were dissected from four weeks-old in vitro plantlets in the plastic cups were transferred into
plantlets using sterile blade and forceps. The single potting mix.
shoots cuttings were inoculated into a test tube
containing 10 ml LBTM. All cultures were incubated Statistical analysis
at 25 ±100C with a 16-hr photoperiod (2000 lux). The
materials were sub-cultured at 30 day interval in same The well-developed cultures were examined
medium to produce multiple shoots. periodically and morphological changes were recorded
on the basis of visual observations. Whenever possible
Effect of different growth regulators on the effects of different treatments were quantified on
micropropagation. the basis of percentage of cultures showing the
responses per culture. The experimental design was
In Musa paradisiaca L. sword suckers explants var. completely randomized design (CRD) and factorial
Monthan are supplemented with different with Auxin and cytokinin as independent variables.
combinations of Auxin and Cytokinin. The Sword All the experiments were repeated thrice. The data
suckers explants were cultured on LBTM pertaining to frequencies of shoot induction, root
supplemented with various concentration of BAP and induction, number of shoots was subjected to
IBA (0.1, 0.2, 0.3, 0.4 and 0.5 mg/1) with 0.1 mg/l statistical analysis with Standard Deviations.
IAA for shoot induction (Table-5). The effect of
growth regulators on the culture development response Results
was studied and the effort was made to determine the
optimal shoot growth combination. In the present study the MS nutrient composition was
replaced by low cost alternatives (Table -1) i.e called
Effect of different growth regulators on multiple the LBTM (Low cost Banana Tissue culture Medium).
shoot induction The LBTM quantity per litre compostion was slightly
increased, in compare with the convetinal MS medium
The well-developed shoots were sub cultured on composition (Table – 1).
LBTM (Low cost Banana Tissue culture Medium),
supplemented with various concentration of BAP Comparative cost analysis of low cost medium and
(1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) with 0.5 mg/l KIN for conventional MS medium
shoot multiplication. The effect of growth regulators
on the shoot multiplication response was studied and In the present study the MS nutrient composition plus
the effort was made to determine the optimal shoot low cost nutrient composition (LBTM) was
multiplication combination. (Table- 6), comparatively analyzed cost wise as per the price list
of 2015-16 (Table -2). In comparison with the
Root induction individual requirement per litre of conventional MS
medium and LBTM composition was cost wise
Well-developed shoots were sub cultured into LBTM compared per litre conventional medium Rs. 152.48
(Low cost Banana Tissue culture Medium), was required for banana tissue culture propagation. In
supplemented with different concentrations of IBA contrast using LBTM (Low cost Banana Tissue culture
and GA3 (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l) for root Medium) Rs. 40.86 is required per litre. The overall
induction (Table -7). Rooting was observed at 10 to 15 percentage of cost reduction for banana tissue culture
days. propagation was 73.20% respectively (Table –2 ).

Hardening Comparative cost analysis of low cost instruments

Plantlets with well-developed roots were removed In banana tissue culture laboratory setting purpose
from culture tubes and transferred to greenhouse after several high costs and low-cost instruments were
washing their roots in running tap water, and grown in analyzed. As per the comparative price list 2015-16,

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Table: 1. Low cost medium and conventional medium composition

Conventional MS medium Amou Low cost Banana tissue Amount

(Murashige&Skoog nt culture medium (mg/l)
composition, 1962) (mg/l) composition (LBTM)
Macro nutrients Macro nutrients
Ammonium Nitrate 1650 Ammonium nitrate fertilizer 2200
(NH4NO3) Calcium Chloride fertilizer 460
Calcium chloride (CaCl2) 440 Potassium Nitrate fertilizer 2100
Potassium Nitrate (KNO3) 1900 Magnesium Sulphate 420
Magnesium Sulphate fertilizer
(MgSO4) 370 Single super Phosphate 210
Potassium dihydrogen
Phosphate (KH2PO4) 170 Micro nutrients
Micro nutrients
Potassium iodide (KI) 0.83 Potassium Iodide(LR) 0.1
Boric oxide (H3BO3) 6.2 Power B-boran, Boric 15.0
Manganese Sulphate powder
(MnSO4.4H2O) 22.3 Manganese Sulphate 27.0
Zinc Sulphate (ZnSO4.7H2O) 8.6 fertilizer
Sodium Molybdate Zinc Sulphate fertilizer 10.0
(Na2 MOO4.2H2O) 0.25 Adbor powder 0.50
Copper Sulphate (CuSO4. Chelated fertilizer 0.025
5H2O) 0.025 Grandular/ powder 0.025
Cobalt chloride (COCl2) 0.025
Iron Nutrient Iron Nutrient
Ethylene diamine tetra acetic 1.9 Ethylene diamine tetra acetic 2.1
acid (EDTA) acid (EDTA)
Ferrous Sulphate 1.39 Ferrous Sulphate fertilizer 1.6
(FeSO4.7H2O)
Vitamins Vitamins
Myo Inositol 100 BecosulesB-complex Tablets 15.0
Glycine 2 containing
ThiamineHcl 0.1 Thiamine
Nicotinic acid 0.5 Riboflavin
Pyridoxine Hcl 0.5 Pyridoxine HCl
Ascorbic acid
Growth regulators * Biotin
IAA 0.1 Folic acid
2, 4-D 0.1 Calcium pantothenate
NAA 0.1 Niacinamide
IBA 0.1 Growth regulators*
Kinetin 0.1 IAA 0.1
BAP 0.1 2, 4-D 0.1
GA3 0.1 NAA 0.1
IBA 0.1
Carbon source Kinetin 0.1
Sucrose 30 (g) BAP 0.1
GA3 0.1
Solidifying agent Carbon source
Agar - Agar 8(g) White refined sugar (Table 30 (g)
sugar)
Solidifying agent
Agar Agar (AR) 8 (g)
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Table: -2 Comparative cost analysis of low cost Banana tissue culture medium (LBTM) and conventional
medium composition as price quotation of 2015-16.
Conventional MS medium Amount Cost in Low cost Banana tissue Amount Cost in % cost
(Murashige & Skoog (mg/l) Rupees culture medium (mg/l) Rupees reduction
composition, 1962) (Rs.) composition (LBTM) (Rs.)
Macro nutrients Macro nutrients
Ammonium Nitrate 1650 7.2 Ammonium nitrate 2200 0.1 98.6
(NH4NO3) fertilizer
Calcium chloride (CaCl2) 440 1.9 Calcium Chloride 460 0.01 99.4
PotassiumNitrate (KNO3) 1900 1.1 fertilizer
Magnesium Sulphate Potassium Nitrate 2100 0.6 45.4
(MgSO4) 370 1.3 fertilizer
Potassium dihydrogen Magnesium 420 0.02 98.4
Phosphate (KH2PO4) 170 1.8 Sulphate fertilizer
Micro nutrients Single super Phosphate 210 0.8 55.5
Potassium iodide (KI) 0.83 11.2 Micro nutrients
Boric oxide (H3BO3) 6.2 3.7 Potassium Iodide(LR) 0.1 1.9 83.0
Manganese Sulphate Power B-boran, Boric 15.0 2.8 24.3
(MnSO4.4H2O) 22.3 1.4 powder
Zinc Sulphate Manganese Sulphate 27.0 4.0 66.6
(ZnSO4.7H2O) 8.6 4.2 fertilizer
Sodium Molybdate Zinc Sulphate fertilizer 10.0 1.42 72.7
(Na2 MOO4.2H2O) 0.25 1.1 Adbor powder 0.50 0.30 75
Copper Sulphate (CuSO4. Chelated fertilizer 0.025 0.01 70
5H2O) 0.025 0.04 Grandular/ powder 0.025 0.06 63.7
Cobalt chloride (COCl2) 0.025 0.2 Iron Nutrient
Iron Nutrient Ethylene diamine tetra
Ethylene diamine tetra acetic 1.9 5.8 acetic acid (EDTA) 2.1 0.09 99.8
acid (EDTA) Ferrous Sulphate 1.6 0.002 93.1
Ferrous Sulphate 1.39 1.1 fertilizer
(FeSO4.7H2O) Vitamins
Vitamins Becosules B-complex 15.0 1.0 92
Myo Inositol 100 1.4 Tablets containing
Glycine 2 4.8 Thiamine
ThiamineHcl 0.1 1.7 Riboflavin
Nicotinic acid 0.5 1.5 Pyridoxine HCl
Pyridoxine Hcl 0.5 5.3 Ascorbic acid
Growth regulators * Biotin
IAA 0.1 1.27 Folic acid
2, 4-D 0.1 0.85 Calcium pantothenate
NAA 0.1 2.16 Niacinamide
IBA 0.1 1.9 Growth regulators*
Kinetin 0.1 4.8 IAA 0.1 0.10 97.6
BAP 0.1 6.3 2, 4-D 0.1 0.02 95.2
GA3 0.1 1.2 NAA 0.1 0.09 99.9
Carbon source IBA 0.1 0.01 99.6
Sucrose 30 (g) 14.46 Kinetin 0.1 0.004 99.4
BAP 0.1 0.023 75
Solidifying agent GA3 0.1 0.007
Agar - Agar 8(g) 64.80 Carbon source
White refined sugar 30 (g) 3.50 45.9
(Table sugar)
Solidifying agent
Agar Agar (AR) 8 (g) 24.0

Total 152.48 40.86 73.20%

*(This will reduce depends upon the requirement)

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high cost vs low cost instruments for banana tissue thousand and six hundred rupees ) was required. The
culture propagation using conventional (High cost) overall cost percentage reduction for setting banana
Rs.17,95,000 (Seventeen lakes ninety-five thousand tissue culture laboratory 84.31 % respectively (Table
rupees) was required. Similarly in comparison of low –3 ).
cost instruments Rs. 2,81,600 (Two lakes eighty-one

Table -3: Comparative cost analysis of low cost and conventional instruments used for Banana tissue culture as
per the price quotation of 2015-16.

Name of the instruments

Cost in Cost in (2015-16)
Conventional Equivalent Low cost % cost
S.No (2015-16)price price Quotation
( High cost) instruments instrument reduction
Quotation (Rs.) (Rs.)
1 Laminar air flow chamber 1,0,0000
Laminar air flow champer
with all accessories 50,000 50
with minimum accessories
2 pH meter in (Digital) 20,000
pH meter ordinary
10,000 50
3 Autoclave 1.Small size 25,000
Pressure cooker (10 L)
2. Medium size 50,000 5,000 80
3. Large size 75,000

4 Micro oven 20,000

Induction stove
1,500 92.5
5 Microbalance (0.01mg) 1,25,000
Microbalance (0.1 mg)
(Sartorious) 10,000 92

6 Culture rack with light and 40,000

Ordinary culture rack
timer 10,000 75
with light
7 Double Distillation unit 25,000
Single distillation unit
10,000 60
8 1.Deep freezer (-200C) 40,000
Refrigerator
2.Double Door Refrigerator 25,000 10,000 75

9 Hot air oven with all 25,000

Hot air oven with minimum
accessories 10,000 60
accessories
10 Plant growth chamber with 10,00,000
Shade net with humidity
all accessories 50,000 95

11 Working table with all racks 50,000

Ordinary table with
and accessories 50,000 -
accessories
12 Air conditioner (spilit AC) 30,000
Used spilt or window air
15,000 50
conditioner
13 Glass bead sterilizer 10,000
Spirit lamb
100 99
14 Further with all necessary 2,00,000
Minimum miscellaneous
tissue culture room 50,000 75
amenities
miscellaneous amenities
Total 17,95,000 Total 2,81,600 84.31 %

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Comparative cost analysis of low cost Glassware’s banana tissue culture propagation low cost glasswares
Rs. 2790 (Two thousand seven hundred ninety rupees)
For the Banana tissue culture laboratory purpose was required. Similarly in high cost glassware Rs.
several high cost and low cost glasswares were 43,205 (Forty three thousand and two hundred five
analyzed. As per the comparative price list 2015-16, rupees) was required. The overall cost reduction was
the high cost vs low cost glasswares (Table-4) for the 93.4 % respectively (Table - 4).

Table 4: Comparative cost analysis of low cost and conventional glass ware used for Banana tissue culture as
per the price quotation of 2015-16.
Name of the Glassware’s
S.No Conventional Cost in Equivalent Low cost Cost in % cost
( High cost) (2015-16) instrument (2015-16) reduction
instruments price Quotation price Quotation
(Rs.) (Rs.)
1 Test tube (Borosil) 20.0 Test tube from local 10.0 50
Conical flasks (Borosil) blowers
2 1.250 ml 150.0
2. 500ml 200.0 Horlicks and Jam bottles 5.00 96.6
Measuring cylinders
3 (Borosil) 1. 10 ml 250 Measuring Cylinder Plastic*
2.100 ml 500 1. 10 ml 50 80
3.500 ml 1000 2.100 ml 200 60
4.1000ml 1500 3.500 ml 250 75
Beakers 4.1000ml 250 83.3
4 1. 50ml 250 Beakers plastic
2.100 ml 500 1. 50ml 50 80
3. 500 ml 1000 2. 100 ml 200 60
4. 1000 ml 1500 3. 500 ml 250 75
Culture bottle (Borosil) 60 4. 1000 ml 250 83.3
with cap Soda glass bottle with cap 25.0 58.3
Hospital used glucose saline 20.0
5 Micropipette 0.1 ml 10,000 bottle 66.6
various size 0.5 ml 25,000

Glass pipettes *
6 Vessels for micro oven 1. 0.5ml 250 97.5
Aluminum foil roll 250 2. 1ml 250 97.5
3. 2.5ml 250 97.5
4.5ml 200 98
Parafilm roll 500 5. 10ml 200 98

Forceps 500 Used news paper 5.0 98

7
Surgical blade 25 News paper
5.0 99

8 Forceps 500 0

Normal blade with holder 25 0

10

11

Total 43,205 Total 2790 93.4%

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Effect of different growth regulators on Shoot growth regulators BAP and KIN (0.1, 0.2, 0.3, 0.4 and
induction frequency of var. Monthan 0.5 mg/l) with 0.1 mg/l IBA and IAA in the LBTM
composition. The high shoot induction frequency was
The trimmed surface sterilized sword sucker explant obtained in the combination of BAP 0.2 mg/l + IAA
of Musa paradisiaca L. var. monthan were subjected 0.1 mg/l (68.1±4.2) followed by KIN 0.2 mg/l + IAA
to LBTM composition with different growth 0.1 mg (65.2±2.8) respectively (Table - 5)
regulators. Among the different concentration of

Table : 5 Effect of BAP and KIN in combination of (0.1 mg/l) IAA and IBA supplemented with LBTM (low
cost banana tissue culture medium) on shoot induction of Musa paradisiacaL. Var. Monthan using sword
suckers explants.

Concentration of growth regulators

(mg/l) % of response Shoot induction frequency

BAP+ IAA
0.1+0.1 78.1±1.0 42.1±1.0
0.2+0.1 85.6±6.1 60.6±6.8
0.3+0.1 82.4±5.2 58.4±5.0
0.4 +0.1 81.1±6.4 45.1±2.8
0.5+0.1 72.5±2.8 40.5±2.8
BAP+ IBA
0.1+0.1 70.0±6.0 60.0±2.0
0.2+0.1 75.6±3.8 40.6±3.2
0.3+0.1 77.1±4.0 68.1±4.2
0.4 +0.1 68.2±3.1 58.2±3.0
0.5+0.1 64.8±2.8 38.8±2.4
KIN +IAA
0.1+0.1 80.4±1.2 35.4±1.6
0.2+0.1 95.6±6.0 54.6±6.2
0.3+0.1 84.8±1.4 42.8±1.0
0.4 +0.1 78.8±3.2 38.8±3.2
0.5+0.1 82.6±2.0 32.6±2.8
KIN +IBA
0.1+0.1 88.8±6.7 28.8±6.4
0.2+0.1 72.4±7.2 55.8±7.0
0.3+0.1 87.5±1.9 32.4±1.8
0.4 +0.1 90.2±3.4 65.2±2.8
0.5+0.1 80.2±3.1 52.6±1.9

Data presented as the mean value ±standard error after 30 days of culture from four independent experiments
each with 10 replicates..

Effect of different growth regulators on multiple shoot induction. Among the different growth
shoot induction of var. Monthan regulators in the BAP 2.0 mg/l + IAA 0.5mg/l
combination produced high frequency of shoots
After 30 days old elongated shoot tip of banana are (18.1±3.6) followed by the combination of KIN 2.0
sub cultured in LBTM supplemented with different mg/l + IBA 0.5 mg/l (13.2±0.8) respectively
concentration of BAP and KIN (1.0, 1.5, 2.0, 2.5 and (Table -6).
3.0 mg/l) with IAA and IBA 0.5 mg/l for multiple

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Table : 6- Effect of BAP and KIN (0.5mg/l) in combination of (0.5mg/l) IAA and IBA supplemented with
LBTM (low cost banana tissue culture medium) on multiple shoot induction of Musa paradisiaca L. Var.
Monthan using sword suckers explants.

Concentration of Growth
% of response No. of Shoots
regulators (mg/l)

BAP+IAA
1.0+0.5 85.8±0.6 12.8±0.3
1.5+0.5 86.8±3.8 12.5±1.2
2.0+0.5 94.8±3.1 15.5±0.8
2.5 +0.5 75.4±4.1 10.8±0.8
3.0+0.5 81.6±3.6 8.4±1.2
BAP +IBA
1.0+0.5 82.9±0.8 12.7±0.8
1.5+0.5 85.8±3.1 14.2±1.2
2.0+0.5 91.6±4.1 18.1±3.6
2.5 +0.5 87.4±3.6 10.4±1.8
3.0+0.5 78.3±6.1 7.2±0.7
KIN +IAA
1.0+0.5 81.9±0.2 12.1±0.2
1.5+0.5 90.8±0.7 13.2±0.8
2.0+0.5 84.2±3.3 8.2±1.6
2.5 +0.5 87.4±3.6 8.31±2.1
3.0+0.5 74.1±2.8 14.1±0.8
KIN +IBA
1.0+0.5 82.3±0.2 12.9±0.3
1.5+0.5 71.3±1.8 13.1±0.8
2.0+0.5 88.2±2.7 10.4±1.3
2.5 +0.5 86.2±3.0 8.4±3.6
3.0+0.5 85.37±0 3.7±1.8

Data presented as the mean value ±standard error after 30 days of culture from four independent experiments
each with 10 replicates..

Effect of different growth regulators on Root and 3.0 mg/l) with GA3 0.5 mg/l for root induction.
induction of var. Monthan Among the different concentration of IBA + GA3
combination the 2.0 mg/l IBA + 0.5 mg/l GA3
After 41 days of old multiple shoot culture were produced more number of roots (12.5 ± 2.2) followed
trimmed into single shoot tips using laminar air flow by other combination. In all the IBA combination
champers. This shoot tips were transferred to rooting root was significantly produced in sub cultured banana
medium containing LBTM supplemented with shoot tips var. monthan (Table- 7).
different concentration of IBA (0.5, 1.0, 1.5, 2.0, 2.5

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Table :7. Effect of LBTM supplemented with different concentration of IBA with GA3 (0.5 mg/l) on root
induction frequency of Musa paradisiacal L. Var. Monthan

Concentration of growth
Root induction frequency of
regulators No. of roots
root
(mg/l) (Mean ±SD)
(Mean ±SD
IBA + GA3
0.5+ 0.5 10.8±1.7 9.5±3.2
1.0+0.5 15.8±2.5 8.3±2.1
1.5+0.5 14.6±2.1 9.6±1.8
2.0+0.5 10.4± 3.6 8.9±2.6
2.5+0.5 16.8±1.7 12.5±2.2
3.0+0.5 10.8±2.5 6.3±2.1

Data indicate mean ± standard deviation. Ten replicates were used per treatment and experiment was repeated
trice.

Hardening mixture and maintained in the green house conditions.

Among the different low cost autoclaved compost
The plantlets having sufficient root and shoot system mixture were also tried for hardening (Table – 8).
were taken out from the culture were washed under Among the different composition mixture autoclaved
running tap water to remove the solidifying agent. The sand + garden soil + vermi culture showed high
plantlets were transferred to poly cups containing survival percentage 78 ± 1.2 respectively (Table – 8).
autoclaved sand, soil and vermi compost (1:1:1)
Table –8. Effect of different low cost compost mixture for hardening on Musa paradisiaca L.

Compost mixture Number of Survival (%) Average shoot Average Leaves

plantlets weight (cm) plantlet
transferred
Autoclaved sand,
Vermicompost 10 65 ±0.16 5.2 ±1.0 2.8 ±0.5
+Vermiculate (1:1:1)

Autoclaved sand
Garden soil
+Vermiculate 10 78 ±1.2 7.3 ±0.6 3.2 ± 0.4
(1:1:1)

Autoclaved sand
Humus +
Vermiculate 10 85 ± 1.71 8.1 ±0.2 3.8 ±0.3
(1:1:1)

Data presented as the mean value ±standard error after 30 days of transfer.

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(5): 240-253

Figure – 1 : Effect of LBTM supplemented with different concentration of growth regulators on

micropropagation of Musa paradisica L. var. Monthan using sword sucker explants.

a b

c d

e f

a- Shoots induction
b- Multiple shoot
c- Shoot elongation
d- Root induction
e, f- Hardening

Low cost banana tissue culture Package cost of tissue culture raised banana plants. In this
several factors are identified for the production of
In the present study main aim was to produce low cost tissue culture raised banana. They are cost of the
micro propagation package in banana instruments, chemicals glassware etc., Among the cost
micropropagation using var. Monthan using sword wise the low cost instruments, chemicals and
sucker explants. To evaluate the factors contributing glassware were standardized and this was used.

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(5): 240-253

To evaluate the factors contributing medium In the present investigation, stated the effect of
composition for tissue culture raised propagation different concentrations of BAP+IAA on shoot
suitable alternative low cost nutrients were selected. induction, and shoot multiplication were investigated
Among the several instruments, high cost vs low cost using LBTM. Similar results achieved in Adenine
was analyzed the following low-cost instruments are based cytokinins are used in several Musa sp. For in
used throughout the study. They are 1. Laminar air vitro propagation, BAP is the most commonly
flow chamber with minimum accessories 2.pH meter prepared cytokine by Vulsteke, 1989. A part from the
3. Pressure cooker, 4. Induction stove, 5. Ordinary influence of genotypes, shoot proliferation rate and
culture rack with light, 6.Single distillation unit 7. Hot elongation are affected by cytokinin types and their
air oven etc. (Table -3).Likewise, glasswares (Table - concentration. The concentration of exogenous
4) and medium alternative components were listed in cytokine appears to be the main factor affecting
(Table -2). multiplication of shoot.

The overall cost analysis stated that in vitro Banana Wong (1986) stated that when 11.1 µM BAP is
propagation package was cost wise in instruments. supplemented in the medium, each of the explants
Rs. 2,41,600 (Two lakes forty one thousand and 600 produces as average of shoots, while increasing BAP
hundred only) was needed and the percentage of 84.31 concentration of 65.6 shoot induction frequency in per
% respectively. For the glasswares, Rs.3745 (Three explants respectively. However, the optimum
thousand seven hundred forty five rupees only), was recommended BAP concentration is 20µM for banana
needed and the comparative percentage cost reduction micro propagation.
was 93.4 % respectively. In the medium
components per litre production cost was Rs.40.86 Root initiation and development was observed in all
(Fifty rupees and fifty six paisa only) the Overall the media containing different concentrations of NAA,
comparative cost reduction analysis percentage was and also in the conventional medium and low cost
73.20 % for LBTM medium preparation. media containing a combination of NAA and GA3.
The superiority of NAA and IAA and IBA in the in
Discussion vitro rooting of banana plantlets cv. William, Grande
Naine by while Ahsan et al., 1998 reported that the
Tissue culture plants are the major source of planting best response was achieved in hormone free MS media
material, however the cost of production is higher than for table banana (Musa sapientum). In the present
the conventional method of propagation by sword finding of root induction in hormonal combinations
suckers in banana. In tissue culture industry the cost confirms the findings of Baby et al.,1997 who
of media preparation chemicals and carbon source can achieved 100% rooting in low cost media
account to 15-20 percent and the cost of energy can supplemented with various combination of NAA and
account up to 60 per cent of the production cost GA3.
(Prakash and Savangikar, 2002).
The overall cost analysis stated that in vitro Banana
The need for low-cost plant tissue culture systems, propagation package was cost wise in instruments.
applicable for micro propagation and in vitro Rs. 2,41,600 (Two lakes forty one thousand and 600
conservation of plant genetic resources, has been hundred only) was needed and the percentage of 84.31
emphasized to allow the large-scale application and % respectively. For the glasswares, Rs.3745 (Three
adaptability of such technology in developing thousand seven hundred forty five rupees only), was
countries (IAEA, 2004). Low cost option should lower needed and the comparative percentage cost reduction
the cost of production without compromising the was 93.4 % respectively. In the medium
quality of the micro propagation (Stephan and components per litre production cost was Rs.40.86
Dhanalakshmi, 2014). This problem has been (Fifty rupees and fifty six paisa only) the Overall
addressed by inventing reliable cost effective tissue comparative cost reduction analysis percentage was
culture methods without compromising on quality of 73.20 % for LBTM medium preparation.
plants. Cost of chemicals, media, energy, labor and
capital affects the production cost.

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(5): 240-253

Similar results achieved by Majuju Rakshi, et al., Kadota, M. And Niimi, Y. 2004. Improvement of
2017, reported 90% resource cost reduction in tissue micropropagation of Japanese Yam using liquid
culture of banana was achieved by replacing tissue and semi-solid medium culture.Sci. Hortic., 102:
culture grade sucrose and Gelrite in the medium with 461-466.
locally available commercial sugar and a starch/Gelrite Kodym, A. and Zapata-Arias, F.J. 2001. Low cost
mixture or sago 39.9% of agar cost, cotton fiber alternatives for the micropropagation of banana. Pl.
support 60.22% of agar cost, Starches of corn or Cell Tis. Org. Cult. 66: 67-71.
potato could partially substitute for Gelrite and agar. MajujaRakshi, K, VenkataSubbaiah G,
Sugars of table sugar were suitable. AR grade sucrose Prabhuling, GSK Swamy and Praveen Jholgiker
by rock sugar 95.85% of sucrose cost and distilled 2017.A review on low cost micro propagation
water by aqua guard water 86.60% of double distilled techniques in banana. Journal of pharmacognosy
water cost and by using sun light instead of artificial and phytochemistry 357-359.
light. Piatezak, E., Wielanek M. and Wysokinska, H.
2005. Liquid culture system for shoot
Acknowledgments multiplication and secoiridoid production in
micropropagated plants of Centaurium erythraea
Rafn. Plant Sci., 168:431-437.
The authors are grateful to thank University Grants
Prakash, S., Hoque, MI., Brinks, T. 2001.Culture
Commission, New Delhi for providing financial
media and container.In Low Cost Options for
Assistance (File No. 41/461/2012(SR)) to carry out
Tissue Culture Technology in Developing
this study.
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organized by the joint FAO/IAEA Division of
Nuclear Techniques in Food and agricultural held
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How to cite this article:

S. Dhanalakshmi and R. Stephan. (2016). Low cost micropropagation package for Banana
(Musa paradisiaca L.). Int. J. Adv. Res. Biol. Sci. 3(5): 240-253.

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