In Vitro Propagation of Banana: Ezaul Arim Alek Ajia Ahman MIN AND Uhul MIN

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ISSN 0258-7122

Bangladesh J. Agril. Res. 34(2) : 269-278, June 2009


IN VITRO PROPAGATION OF BANANA

M REZAUL KARIM1, M. A. MALEK2, SAJIA RAHMAN3


M. AL-AMIN4 AND M. RUHUL AMIN5
Abstract
An in vitro technique for plant regeneration using meristem-derived plantlets of
banana cv. BARI-l (Musa sp.) has been developed. Highest number of shoot
regeneration was noticed on basal media supplemented with 7.5 mgL-1 BAP +
0.5 mgL-1 NAA at 30 days after inoculation (DAI). The mean number of shoots
significantly reduced when the concentrations of BAP and NAA in the medium
was high. Regenerated shoots were rooted on half strength MS medium
containing 0.5 mgL-1 IAA + 0.5 mgL-1 IBA at 30 DAI. In vitro raised plantlets
were transferred to poly bags containing ground soil and cowdung mixture (1:1)
for acclimatization and hardening in room temperature (28-30°C) and the
established plantlets are ready for planting in the field.
Key Words: In vitro propagation, banana (Musa sp.).

Introduction
The banana and plantains (Musa spp.) belonging to the family Musaceae is one
of the world’s most important subsistence crops. It is originated in Malaysia
through a complex hybridization process (Novak, 1992). It is widely grown in the
tropics and subtropic regions of the world. In Bangladesh, banana is popular for
its year round availability, abundant production as well as high acceptability to
the consumers. It is a rich source of carbohydrate and also rich in some minerals,
notably phosphorus, calcium, and potassium. Banana is particularly rich in
vitamin C and also contains significant amounts of several other vitamins
(INIBAP, 1987). In addition, it has importance for tannin, latex and fiber
production. Banana occupies an area of 45 thousand hectares of land with total
production of 909 thousand metric tones with an average yield of 20.20 t/ha
(BBS, 2006). This yield is quite low compared to other banana growing countries
of the world like Argentina (34 t/ha) and Costa Rica (33 t/ha) (FAO, 2002). The
low yield and production of banana is influenced by many biotic and abiotic
stresses. Among the biotic stress, virus disease is one of the important factors.
The traditional clonal propagation method appears unable to satisfy the increase
in demand for disease free and healthy planting materials of banana. The
productivity of vegetative propagated crops, such as banana and plantain is
greatly reduced by virus disease (Lepoivre, 2000).

1&4
Biotechnology Divn., BARI, Joydebpur, Gazipur, 2&3Plant Genetic Resources Centre,
BARI, Joydebpur, Gazipur, 1701 5Shere-Bangla Agricultural University, Dhaka- 1207,
Bangladesh.
270 KARIM et al.

Meristem culture offers an efficient method for rapid clonal propagation,


production of virus free materials and germplasm preservation in plants
(Cronauer and Krikorian, 1984a; Hwang et al., 2000 and Helloit et al., 2002). As
regards yield performance, tissue cultured plants have been reported to produce
39% higher yield than plants from sword suckers (Pradeep et al., 1992). Under
Bangladesh conditions, tissue culture derived plantlets of banana performed
better than the conventional sword suckers (Faisal et al., 1998). So, the
present work was conducted at the laboratory of Biotechnology Division,
Bangladesh Agricultural Research Institute (BARI), Gazipur.

Materials and Method


The plant materials of banana cv. BARI-l were collected from Biotechnology
Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur,
Gazipur. The meristem used for establishment of culture was prepared under the
microscope from the collected suckers through dissection and removal of leaf
sheath. The meristem was obtained from developing suckers (about four months
of age) of banana cv. BARI- 1 grown under field conditions and was brought to
the preparation room. The suckers were washed thoroughly under running tap
water. The roots and outer tissues of the suckers were removed with the help of a
sharp knife. A number of outer leaves were removed until the shoot measured
about 1.0-2.0 cm in length and 1.0 cm width at the base. The initial explant was
prepared under stereomicroscope by removal of outer tissue of meristem with the
help of sterile scalpel, which was about 5x5 mm in size and the excised explants
were surface sterilized with 0.1% HgCl2 for 15 minutes and then placed on MS
media supplemented with different concentrations of BAP (0.0, 2.5, 5.0, 7.5, and
10.0 mgL-1) and NAA (0.0, 0.5, 1.0, 1.5, and 2.0 mgL-1) for in vitro shoot
regeneration. The culture tubes with media were autoclaved at 1.06 kg/cm2
pressure at 121°C for 25 minutes. The medium was then cooled at room
temperature before use. Sterilization of media was done after adding hormone.

Results and Discussion


Regeneration of shoot from meristem explants
In vitro culture of meristem (Plate 3) showed hard meristematic ball like structure
in regeneration media containing different concentrations of BAP and NAA
(Plate 4). The culture meristems first turned brown in colour in 4-5 days and after
30-50 days a green globular hard coat mass grow round in shape producing a ball
like structure. From this ball like structure, adventitious plantlets were developed
(Plate 5). Rahaman et al. (2004) observed that hard ball like structure developed
from meristem explant in MS media supplemented with 5.0 mgL-1 BAP. They
also noticed that single shoot regeneration from meristem explant was thinner
than shoot derived from shoot tip. Rabbani et al. (1996) denoted that 2ip have the
IN VITRO PROPAGATION OF BANANA 271

more effective in producing ball like structure. Similar results were also obtained
by Habib (1994) and Ali (1996) in their experiments. They observed that some
ball like structures formed from the base of the shoot during shoot multiplication.
These balls like structures are suitable for in vitro germplasm conservation.
Plant regeneration and subsequent shoot multiplication from meristem
derived plantlets of banana cv. BARI-1 were observed in MS medium
supplemented with different concentrations of BAP and NAA. The results
obtained from this study are presented in Table 1-4 and discussed under the
following sub-heads:
Table 1. Effect of different concentrations of BAP and NAA on shoot multiplication
of banana at different days after inoculation.
Treatments Shoot number Shoot length (cm)
BAP NAA 10 DAI 20 DAI 30 DAI 10 DAI 20 DAI 30 DAI
(mgL-1) (mgL-1)
0 0.0b 1.00c 1.00f 0.00i 0.00j 0.00j
0.5 1.0a 1.50bc 2.25def 0.30gh 1.03ghi 1.88defgh
0 1.0 0.5 ab 1.25 bc 2.50 de 0.30 gh 1.28 defgh 1.95 bcdefg
1.5 1.0 a 1.50 bc 1.50 ef 0.23 h 1.68 bcde 2.33 b
2.0 0.75 a 1.25 bc 2.00 def 0.20h 1.40 cdefg 1.78 fgh
0 1.0 a 1.00c l.50ef 0.23h 1.23 efgh l.9ocdefg
0.5 1.0 a 1.25 bc 1.75 ef 0.30 gh 1.48 cdefg 2.08 bcdefg
2.5 1.0 a 1.0 a 1.25 bc 2.50 de 0.45 feg 1.28 defgh 1.80 efgh
1.5 1.0a 1.75abc 1.75ef 0.35fgh 1.lofghi 1.83efgh
2.0 l.0a 1.50bc 2.25def 0.45efg 0.70i l.50hi
0 1.0a 1.25bc 1.50ef 0.25h 0.88hi 2.28bc
0.5 1.0 a 1.75 abc 2.50 de 0.48 ef 2.43 a 3.13 a
5.0 1.0 1.0a 2.25ab 2.50de 0.88abc 2.05ab 2.l3bcdef
1.5 1.0 a 1.50 bc 2.50 de 0.58 de 1.53 cdef 1.78 fgh
2.0 0.75a 1.50bc l.75ef 0.55e 1.28defgh 1.70gh
0 0.75 a 1.75 abc 2.50 de 0.58 de 1.20 efgh 1.25 i
0.5 0.75 a 2.75 a 6.25 a 1.03 a 2.45 a 3.38 a
7.5 1.0 0.75 a 2.75 a 5.25 ab 0.95 ab 1.75 bcd 2.23 bcd
1.5 1.0a 1.75 abc 4.25bc 0.88 abc 1.88bc 2.l0bcdef
2.0 l.0a 1.75 abc 2.25 def 0.78c 1.3odefgh 2.05 bcdefg
0 1.0a 1.25bc 2.25def 0.75c 1.05 fghi 1.93 cdefg
0.5 0.75a 2.00abc 2.50de 0.73cd 1.60bcde 2.l8bcde
10.0 1.0 0.75 a 2.00 abc 2.50 de 0.80 bc 1.48 cdefg 2.28 bc
1.5 0.5ab 1.50bc 3.25cd 0.73cd 1.35defgh 1.98bcdefg
2.0 0.75 a 1.50 bc 2.50 de 0.73 cd 1.23 efgh 1.90 cdefg
LSD value (0.Ol) 0.61 1.05 1.26 0.16 050 0.39
CV (%) 38.88 34.55 26.71 16.06 19.27 10.49
272 KARIM et al.

Shoot number and shoot length per explant


The number of shoots produced per explant varied in MS media supplemented
with different concentrations of BAP and NAA. Data were recorded at 10, 20,
and 30 days after inoculation (DAI) and results are presented in Table 1. The
effect of different concentrations of BAP and NAA on shoot regeneration and
proliferation were statistically significant at 1% level of significance. Among the
different concentrations, 7.5 mgL-1 BAP + 0.5 mgL-1 NAA showed highest shoot
proliferation (0.75, 2.75, and 6.25 shoots per explant) at 10, 20, and 30 DAI,
respectively. A good number of shoot proliferations was achieved at 7.5 mgL-1
BAP + 1.5 mgL-1 NAA at 30 DAI (4.25), which is superior to the control
treatments (1.00). The regeneration and proliferation of shoots was sequentially
described in plate 6, 7, 8, and 9 from initial sub culture to 4th sub culture.
Rahaman et al. (2004) found highest shoot number at 1.5 mgL-1 BAP + NAA
(4.52 explant) at 30 DAI. The result of current investigation is not fully
supported by Rabbani et al. (1996) where they found highest number of shoots
per explant at 28 DAI (3.11 ± 0.66) with 5.0 mgL-1 of BAP and Kn. This
variation might be due to the different concentrations of NAA and BAP and their
associations.
The MS medium supplemented with BAP and NAA showed different results
for increasing shoot length which was significantly influenced by different
concentrations. The longest shoot was produced by the concentration of 7.5 mgL-
1
BAP + 0.5 mgL-1 NAA (1.03, 2.45 and 3.38 cm) at 10, 20, and 30 DAI,
respectively. Statistically identical shoot length was observed in 5.0 mgL’ BAP +
0.5 mgL-1 NAA at 20 DAI (2.43 cm) and 30 DAI (3.13 cm). Rahaman et al.
(2004) observed the similar result. They obtained longest shoot in 5.0 mgL-1 BAP
(3.62 cm) using BARI Banana-1. They also found shortest leaf in 2.0 mgL-1
BAP. The results of the present study agree with the findings of Khanam et al.
(1996) who obtained longest shoot in banana on MS medium supplemented with
25 µM BAP treatments.

Leaf number and leaf length per explant


The effect of different concentrations of NAA and BAP on leaf number and leaf
length were presented in Table 2. The results showed that the maximum number
of leaves (2.50, 3.25, and 7.00 leaves per explant at 10, 20, and 30 DAI)
produced on the medium supplemented with 7.5 mgL-1 BAP and 0.50 mgL-1
NAA. The second highest number of leaves (2.75, 4.00, and 6.75 leaves/explant
at 10, 20, and 30 DAI, produced on the medium supplemented with 5.0 rngL-1
BAP and 1.0 mgL-1 NAA. Rahman et al. (2004) found the maximum number of
leaves (3.12/plantlet) at 30 DAI produced with 5.0 mgL-1 BAP, which was
identical with the treatment of 4.0 mgL-1 BAP + 1.50 mgL-1 NAA. Rabbani et al.
IN VITRO PROPAGATION OF BANANA 273

(1996) obtained same results from 5.0 mgL-1 BAP. The lowest number of leaves
(0.00, 0.50, and 2.25 at 10, 20, and 30 DAI leaves per explant) were obtained
from control treatment (Table 2).
Table 2. Effect of different concentrations of BAP and NAA on leaf number and leaf
length of banana at different days after inoculation.
Treatment Shoot number Shoot length (cm)
BAP NAA 10 DAI 20 DAI 30 DAI 10 DAI 20 DAI 30 DAI
(mgL-1) (mgL-1)
0 0.00 e 0.5 c 2.25 d 0.50 defg 0.55 h 0.95 i
0.5 1.50 cd 2.25 b 4.75 c 0.58 cdef 1.65 fg 2.03 gh
0 1.0 1.75bcd 2.75ab 4.50c 0.60bcde 1.70efg l.95h
1.5 1.75bcd 3.00ab 5.50bc 0.70abc 1.8obcdefg 2.00gb
2.0 1.50cd 2.25b 5.50bc 0.46defg 1.60g 2.08gh
0 1.50cd 2.50b 4.75c 0.5odefg 1.55g l.95h
0.5 1.75 bcd 3.00 ab 5.00 c 0.63 bcd 2.03 bcd 2.58 efg
2.5 1.0 2.25 abc 3.25 ab 5.50bc 0.600bcdc 1.95 bcdef 2.53 efgh
1.5 1.50cd 2.75ab 5.75abc 0.45efg 2.075bc 2.43fgh
2.0 2.00 abcd 3.25 ab 5.50 bc 0.45 efg 2.05 bcd 2.28 gh
0 1.25d 2.5b 4.75c 0.35g 1.68fg 2.15gh
0.5 2,50 ab 3.25 ab 5.25 c 0.75 ab 2.60 a 3.60 bc
5.0 1.0 2.75 a 4.00 a 6.75 ab 0.70 abc 2.05 bcd 3.10 cde
1.5 2.25 abc 3.25 ab 5.50 bc 0.50 defg 2.03 bcd 2.93 def
2.0 1.75 bcd 2.75 ab 5.25 c 0.50 defg 2.00 bcde 2.98 def
0 2.00 abcd 3.25 ab 5.25 c 0.43 fg 1.65 fg 2.23 gh
0.5 2.5 ab 3.25 ab 7.00 a 0.85 a 2.70 a 4.23 a
7.5 1.0 1.75 bcd 2.75 ab 5.25 c 0.70 abc 2.10 b 3.70 ab
1.5 2.00abcd 3.00ab 5.75abc 0.S5cdef 1.95bcdef 2.58efg
2.0 2.00 abcd 2.5 b 4.50 c 0.55 cdef 1.76 cdefg 2.28 gh
0 1.50cd 3.25ab 4,75c 0.S3def 1.53g 2.15gh
0.5 2.00 abcd 2.5 b 4.50 c 0.50 defg 1.75 defg 2.98 def
10.0 1.0 1.50cd 3.25 ab 5.25 c 0.43 fg 1.93 bcdef 3.23 bcd
1.5 2.25 abc 2.75 ab 4.75 c 0.45 efg 1.95 bcdef 3.23 bcd
2.0 1.75 bcd 2.75 ab 4.75 c 0.45 efg 1.60 g 3.08 cde
L.SD value (0.01) 0.97 1.28 1.39 0.16 0.31 0.58
CV (%) 28.71 24,22 14.46 15.12 8.86 11.86
274 KARIM et al.

The MS medium supplemented with BAP and NAA showed different results
for leaf length, which was significantly influenced by different concentrations.
The longest leaves were produced by the concentration of 7.5 mgL-1 BAP + 0.5
mgL-1 NAA (0.85, 2.70, and 4.23 cm at 10, 20, and 30 DAI, respectively, which
was statistically significant. Statistically identical results was observed in 7.5
mgL-1 BAP + 1.0 mgL-1 NAA at 20 DAI (2.10 cm) and 30 DAI (3.70 cm).
Rahaman et al. (2004) observed the similar result. They obtained longest leaf in
the treatment 5.0 mgL-1 BAP (3.62 cm) followed by 1.5 mgL-1 NAA and 4.0
mgL-1 BAP (3.40 cm) using BARI Banana-I. They also found shortest leaf in 2.0
mgL-1 BAP. The results of present experiment agree with the findings of Khanam
et al. (1996) who obtained longest leaves in banana on MS medium
supplemented with 25 µM BAP treatments.
Table 3. Effect of different concentrations of IAA and IBA on root number of
multiple shoot of banana at different days after inoculation.
Treatment Vigor of Root number
-1
IAA (mgL ) IBA (mgL ) -1 regenerated 10 DAI 20 DAI 30DAI
root
0 + 0.00 e 0.00 e 0.00 f
0.5 + 1.50 cd 2.00 d 3.25 e
0
1.0 ++ 2.25 bc 2.25 d 3.50 de
1.5 ++ 2.25 bc 2.50 cd 3.50 de
0 +++ 2.75 ab 2.25 d 3.25 e
0.5 +++ 3.50 a 4.50 a 6.50 a
0.5
1.0 ++ 3.25 ab 3.50 abc 6.00 ab
1.5 ++ 3.25 ab 4.00 ab 5.00 bc
0 + 1.00 de 2.50 d 3.25 e
0.5 + 2.75 ab 2.75 cd 3.75 de
1.0
1.0 ++ 1.SOcd 3.O0bcd 4.O0cde
1.5 + 1.25 cd 3.50 abc 4.50 cd
LSD value (0.01) 1.03 1.16 1.16
CV(%) 25.36 22.19 15.51

+ = Less vigorous growth, ++ = Good growth and vigor, +++ = Best growth and vigor

Root number per explants


The effect of IAA and IBA on number of roots per explant produced by different
combinations at 10, 20, and 30 DAI (Table 3) showed significant variation. The
highest number of roots was produced by 0.5 mgL-1 IAA + 0.5 mgL-1 IBA (3.50,
4.50, and 6.50 per explant, respectively), which was statistically significant than
IN VITRO PROPAGATION OF BANANA 275

other treatments (Table 5). The treatment 0.5 mgL-1 IAA + 1.0 mgL-1 IBA
produced 6.0 roots per explant at 30 DAI, but at 20 DAI, 3.50 roots was
produced per explant. Vigorous roots of in vitro grown plantlet on MS media
supplemented with 0.5 mgL-1 IAA + 0.5 mgL-1 IBA was shown in plate 10 (a &
b). The present results are similar with the findings of Gubbuk and Pekmezci
(2001). Molla et al. (2004) obtained 8.28 roots per plantlet on 0.5 mgL-1 IBA
followed by 6.33 roots in 0.6 mgL-1 BAP that was similar to the present work.
The results were also similar with the findings of Khanam et al. (1996) but
Rahman et al. (2004) obtained only 2.88 roots per explant at 30
DAI by 3.0 mgL-1 NAA.
Table 4. Effect of different concentrations of IAA and IBA on root length of
multiple shoot of banana at different days after inoculation.
Treatment Root number
-1 -1
IAA (mgL ) IBA (mgL ) 10 DAI 20 DAI 30DAI
0 0.00 e 2.00 ef 2.00 f
0.5 1.08 d 1.88 f 2.30 e
0
1.0 1.08d 2.3Ode 3.15d
1.5 1.13d 2.45d 3.08d
0 l.23d 1.60f 2.08e
0.5 2.93 a 4.63 a 5.88 a
05
1.0 2.55 ab 3.88 b 4.83 b
1.5 3.03 a 3.85 b 4.88 b
0 1.80 c 2.33 de 3.45 cd
0.5 1.80 c 2.35 de 3.48 cd
10
1.0 2.O8bc 3.15c 3.75c
1.5 2.lSbc 2.70d 3.70c
LSD value (0.01) 0.49 0.42 0.55
CV(%) 16.28 7.95 7.57

Root length per explant


The length of roots developed by the plantlets was influenced considerably by
different concentrations of IAA and IBA and the results were presented in Table
4. The result indicated that there was an increasing trend in root length at
different DAI (10, 20, and 30 DAI), which is significant at 1% level. The root
length of plantlets after 30 DAI was remarkably highest by auxin, which was
essential for successful root induction of banana and has been reported by Raut
and Lokhand (1989). The highest length was observed at 10, 20, and 30 DAI in
concentration 0.5 mgL-1 IAA and IBA (2.93, 4.63, and 5.88 cm), which was
276 KARIM et al.

statistically significant. The second highest result (3.03, 3.85, and 4.88 cm at 10,
20, and 30 DAI) was observed with 0.5 mgL-1 IAA and 1.5 mgL-1 IBA. A similar
result was obtained by Molla et al. (2004) where they got 2.60-5.67 cm root
length in 0.5 mgL-1 IBA. Habiba (2002), Khanam et al. (1996) and Ali (1996)
also got more or less same observation.

Establishment of plantlet
Meristem derived plantlets were transferred to poly bags containing ground
soil:cow dung mixture (1:1) for acclimatization and hardening in room
temperature (28-30°C) and a good number of established plant (95%) is ready for
planting in the field.

A. Excised meristem of B. Shoot initiation from green globular hard


banana cv. BARI-1. coat meristematic ball like structure.

C. Multiple shoots with 7.5 D. Multiple shoots with 7.5 mgL-1 BAP+0.5
mgL-1 BAP+0.5 mgL-1 NAA mgL-1 NAA at 2nd subculture.
at 30 days after inoculation.
IN VITRO PROPAGATION OF BANANA 277

E. Multiple shoots with 7.5 F. Multiple shoots with 7.5 mgL-1 BAP+0.5
mgL-1 BAP+0.5 mgL-1 mgL-1 NAA at 4th subculture.
rd
NAA at 3 subculture.
Fig. 1. Shoot regeneration from meristem derived of banana cv. BARI-1

G. Vigorous roots with 0.5 H. Well established meristern derived banana


mgL-1 IAA + 0.5 mgL-1 plants in poly bags.
IBA.
Fig. 2. Plantlet developed from meristern derived banana plants in poly bags.

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