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Abstract
The present study was to investigate the best plant growth regulators for shoot proliferation and
multiplication; and to determine the optimum concentrations of phytohormones for shoot tip culture of
banana cv. Agishwar by observing the effect of different culture conditions during induction. A micropropagation protocol for banana (Musa sp.) cv. Agnishwar was established by using shoot tip culture.
Shoot tips obtained by removing leaf sheaths from sucker were cultured aseptically in MS (Murashige
and Skoog) medium supplemented with different concentrations of cytokines viz. 6-benzylaminopurine
(BAP), kinetin (kin), N6 - (2-isopentyl) adenine (2iP) for multiplication of shoot and auxins viz. Indole-3butyric acid (IBA), -naphthalene acetic acid (NAA) for induction of root. Maximum multiplication (95%)
was obtained in MS medium containing 4.0 mg/1 BAP. The highest average number of shoots for each
explant (5.9) was found in MS medium fortified with 4.0 mg/l BAP while maximum elongation of shoot
(4.9cm) was observed in MS medium having 5.0 mg/l BAP. IBA at a concentration of 1.0 mg/l was found
most suitable for rooting of shoot. The rooted shoots were acclimatized and successfully transferred to
plastic pots. After hardening, they were transferred to the main field and the survival rate was around
90%.This protocol might be used for the massive in vitro production of the plantlets of banana cv.
Agnishwar.
Keywords: Micro propagation, shoot tips, fresh sucker, tissue culture, MS medium.
INTRODUCTION
Banana is one of the most important fruits in the world,
both as a staple food as well as a major export
commodity for many tropical and subtropical countries.
The extensive basic work on the in vitro propagation of
banana (Kodym and Zapata, 1999; Nandwani et al.,
2000) had led to the technological development of in vitro
mass production of different cultivars. Plant tissue culture
techniques can potentially overcome some of the factors
*Corresponding
Author's
E-mail: nirupamgene@gmail.com
Rahman et al. 85
Table1. Effects of different concentrations of cytokinins for shoot proliferation and shoot multiplication in banana cv. Agnishwar
Cytokinin
BAP
2iP
Kin
Conc.
(mg/l)
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Shoot
proliferation (%)
69
67
80
95
88
56
62
74
79
83
46
66
68
60
57
No of
Shoot
No of
Shoot/explants
Length(cm) Shoot/explants
2.60.28
2.80.53
3.30.47
2.60.37
2.80.53
3.50.45
5.90.45
4.80.64
5.70.63
4.90.76
2.80.42
2.20.32
2.60.27
2.60.27
3.40.34
3.80.66
4.10.52
3.50.43
4.80.37
3.60.69
1.60.22
1.90.43
3.20.36
3.50.37
3.40.38
2.30.18
2.90.19
2.70.39
2.60.34
2.40.25
1.730.18
0.990.24
1.490.32
2.610.17
2.460.44
0.870.29
2.690.38
1.490.47
1.950.46
1.150.19
0.440.24
2.320.34
1.580.19
1.320.28
0.740.15
while the lowest shoot length (1.9 cm) was found in 1.0
mg/l Kin. Although the shoot length increased gradually
with time in all treatments, the rate of increase was faster
in BAP compared to 2iP and Kin (Figure 1C).
The number of leaves produced in different
concentrations of BAP, 2iP and Kin is as shown in Table
1. The average number of leaves produced in different
concentrations of BAP, 2iP and Kin ranged from 0.44 to
2.69. The maximum number of leaves (2.69) was
produced (P<0.05) under 2.0 mg/l 2iP while the lowest
number was found under 1.0 mg/l Kin. Though the
number of leaves increased with time in all treatments,
the increasing rate was faster in BAP compared to 2iP
and Kin (Figure 1D).
The regenerated shoots were cultured in half strength
of
MS
medium
supplemented
with
different
concentrations of auxin(Table 2) and 3% sucrose for
healthy root development. Two auxins namely IBA and
NAA were supplied in half strength of MS medium.
Although few roots developed from multiple shoots during
culture, but these roots are not so healthy for future
growth and development of plantlets. So multiple shoots
were separated and were allowed to grow on half MS
medium supplemented with 0.5, 1.0 and 2.0 mg/l each of
IBA and NAA for production of healthy as well as enough
roots for proper growth of plantlets. All the treatments
responded differently to produce rooting. IBA was found
better than NAA in rooting of shoot in banana cv.
Agnishwar.
The percentage of shoots responded to produce
rooting ranged from 67 to 96 in various concentrations of
IBA and NAA (Table 2). IBA at a concentration of 1.0 mg/l
showed highest response (96%) in rooting (Figure 1E)
while NAA at a concentration of 0.5 mg/l produced lowest
response (67%) in rooting of shoot.
Regenerated shoots were cultured in half strength of
MS medium supplemented with different concentrations
Figure1. In vitro shoot tip culture of banana cv. Agnishwar. (A) Initiation of proliferations of shoot tip (explants) in banana
cv.Agnishwar cultured in MS medium fortified with 4 mg/l BAP. (B) The best response for shoot formation was observed in
MS medium fortified with 4 mg/l BAP (C) The best length of multiple shoot from shoot tip explants in banana cv.
Agnishwar culture was found in MS medium supplemented with 4 mg/l BAP.(D) Production of multiple shoot in banana cv.
Agnishwar cultured in MS medium fortified with 4 mg/l BAP.(E) Rooting of in vitro regenerated shoots was higher in
banana cv. Agnishwar cultured in MS medium supplemented with 1.0 mg/l IBA.(F)Acclimatized plantlets in polythene bag
containing normal soil, sand and compost.
Table2. Effects of different concentrations of IBA and NAA on root induction in in vitro regenerated shoots.
Auxin
IBA
NAA
Conc.(mg/ml)
0.5
1.0
2.0
0.5
1.0
2.0
% of Shoot rooted
No of roots/Shoot*
83
96
88
67
76
79
7.40.67
8.61.16
5.41.65
3.81.35
5.80.67
5.20.51
Root length(cm)*
2.260.59
3.690.34
2.290.39
1.610.26
2.380.45
2.790.19
* Data were collected after 5 weeks of culture. Average values of 12 replicates and std. error ().
of IBA and NAA (0.5, 1.0 and 2.0 mg/l). All the media
showed satisfactory root induction. 1.0 mg/l IBA produced
highest average number of roots per explant was
recorded to be 8.6.The lowest number of roots per
explant was recorded in medium having 0.5 mg/l NAA.
Moreover, it was recorded that root length ranged from
1.61 cm to 3.69 cm under above three concentrations of
Rahman et al. 87