South Pacific Studies Vol.29, No.1, 2008

Eradication of Banana Bunchy Top Virus (BBTV) andBanana Mosaic Virus (BMV) from Infected Plant of Banana
cv. Amritasagar Through Meristem Culture
M. H. Kabir SHIRAGI1*, M. A. BAQUE2 and K. M. NASIRUDDIN3

1:Department of Horticulture, Bangladesh Agricultural University,
Mymensingh-2202, Bangladesh
2:Department of Agronomy, Sher-e-Bangla Agricultural University, Dhaka-1207,
Bangladesh
3:Department of Biotechnology, Bangladesh Agricultural University, Bangladesh

Abstract
An experiment on virus free in vitro banana plantlets production by the meristem cultured from virus
infected four month old banana suckers cv. Amritasagar were used as explants. Four levels of BAP (0, 3, 4
and 5 mg/l), 5 levels of NAA (0.0, 1.0, 1.5, 2.0 and 2.5 mg/l) were used as treatment for shoot proliferationin experiment-I and Four levels of IBA (0, 1, 2 and 3 mg/l), 4 levels of NAA (0, 2, 3 and 4 mg/l) were used
for root formation in experiment-II. ELISA test was conducted for the confirmation of virus eradication rateof regenerated plants and survival rate of regenerated plants in experiment-III. A significant variation was
found among the treatments. Studies on the different concentrations of BAP + NAA on shoot proliferationand differentiation, reveals that 4 mg/l BAP + 1.5 mg/l NAA produced the greater number of shoots (4.58/
explant), the longest shoot (3.56 cm) and the highest numbers of leaves (3.11/explant). On the contrary,plantlets were cultured on half strength of MS media in different levels of IBA and NAA.The highest
number of vigorous roots (6.98/explant) and maximum root length (5 cm) were recorded from the treatment
combination 2 mg/l IBA+2 mg/l NAA and 3 mg/l IBA+ 3 mg/l NAA, respectively. While less vigorous root
was found in control treatments. The rate of virus free plant regeneration from banana bunchy top virus(BBTV) infected plants was 57.14% and from banana mosaic virus (BMV) infected sources was 64.28%.
The highest plant survival rate 83.33% was recorded in a mixture of sand, soil and cow dung (1:1:1). On theother hand, 67.67% plant survival rate was found in a mixture of coir and soil (1:2). Eradication of banana
viruses from infected plants by meristem cultured is a useful tactic that will be helpful for commercial
exploitation and virus free plant production for use of farmer’s level.
Key words: Plants growth regulators, meristem culture, shoot & root proliferation, ELISA and Amritasagar

Introduction
The banana and plantains (Musa spp.) belonging to the family Musaceae are oneof the world’s most important subsistence crops. In Bangladesh, banana is popular for
its year round availability, abundant production as well as high acceptability to theconsumers. 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*mhkshiragi@yahoo.com
Received: 26 September 2007
Accepted: 19 December 2007

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South Pacific Studies Vol.29, No.1, 2008

contains significant amounts of several other vitamins (INIBAP 1987). In addition, it
has importance for tannin, latex and fiber production. Banana ranks first in terms of
production and second in terms of area, among the fruit crops and so has commercial
value in Bangladesh. It is comprising nearly 42% of the total fruit production of thecountry. It occupies an area of 43 thousand hectares of land with total production of
606 thousand metric tonnes with an average yield of 14.16 t/ha (BBS 2003). This yieldis quite low compared to other banana growing countries of the world like Argentina (34t/ha) and Costa Rica (33t/ha) (FAO 2002).
Many natural calamities, such as abiotic and biotic factors cause devastating
losses in banana production. Some major problems are also caused by viruses. Virusdiseases are serious, as insect vectors are abundant and there are many alternate hosts.
It is affected by four known relatively well characterized viruses (Diekmann and
Putter, 1996) namely, banana bunchy top virus (BBTV), banana streak virus (BSV),
banana mosaic virus (BMV) and banana bract mosaic virus (BBrMV). Banana bunchy
top and banana mosaic virus are the most important virus diseases of banana in thisregion. At present, there are no known banana varieties resistant to BBTV & BMV
and no chemicals to control it (MAGEE 1927, DALE 1987). However, there are obvious
advantages in ensuring that only virus free plants are used for propagation by meristem
culture.
Tissue culture is now a standard practice in banana propagation. Conventional
non-molecular control means such as quarantine, eradication and certified virus freestock have successfully been used to reduce crop losses caused by these viruses. But
those were not effective enough for controlling BBTV and BMV. Recently, molecular
genetic engineering techniques have become available and new approaches havebeen suggested to control banana virus diseases. Meristem culture offers an efficientmethod for rapid clonal propagation, production of virus free materials and germplasm
preservation in plants (C RONAUER and KRIKORIAN, 1984, HWANG et al. 1994 andHELLOIT et al. 2002). As regards yield performance, tissue cultured plants have beenreported to produce 39% higher yield than plants from sword suckers (PRADEEP etal. 1992). Under Bangladesh conditions, tissue culture derived plantlets of banana
performed better than the conventional sword suckers (FAISAL et al. 1998).
Plant growth regulators are the essential part for in vitro regeneration of crop
plants grown in any artificial medium. Generally, cytokinin helps in shoot proliferation
and auxins helps in rooting of proliferated shoots. However, the requirement ofcytokinin and auxins depends on the variety of banana and culture conditions(CRONAUER and KRIKORIAN 1984). To obtain virus and disease free healthy planting
materials, development of a protocol for meristem culture of banana are of prime
importance. Therefore, considering the above facts the present study was undertakenwith a view to i) determine the appropriate hormone concentration for shoot proliferation
and rooting, ii) standardize different hormonal concentration for banana meristem
culture for virus free plantlet production and iii) evaluate BBTV and BMV resistance inthe banana plants under green house and field conditions.

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

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Materials and Methods
The experiment on virus free banana plantlets production by meristem culture
and establishment of virus free in vitro plantlets into rooting media was conducted at
the Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural
University, Mymensingh, during September 2003 to March 2004. Banana cv.
Amritasagar was used as planting materials. MURASHIGE and SKOOG (1962) medium
supplemented with different phytohormones as per treatments were used as culture
medium. For the preparation of media, different stock solutions e.g. Stock solution I(Macronutrients), Stock solution II (Micronutrients), Stock solution III (Iron source),
Stock solution IV (Vitamins and amino acids), Stock solution V (Growth regulators),
hormone stock solution VI were prepared at the beginning and stored at 4±1°C. The
culture vessels with media, glassware and instruments were then autoclaved forsterilization at 1.16-kg/cm2 pressure at 121°C for 20 minutes. The medium was thencooled at room temperature before use. Initially, the culture room was cleaned bygently washing all floors and walls with detergent or Lysol (germicide). This wasfollowed by carefully wiping them with 70% ethyl alcohol. The transfer area was also
sterilized twice a month by 70% ethyl alcohol. Laminar Airflow Cabinet was usuallysterilized by switching on the cabinet and wiping the working surface with 70% ethyl
alcohol for 30 minutes before starting the transfer work.

Culture techniques
Explant culture
The meristem was the starting material. It was obtained form developing
infected suckers (about four months of age) of banana cv. Amritasagar grown underfield conditions and was brought to the preparation room. The suckers were washedthoroughly 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. Thenthe initial explant was prepared under stereomicroscope by removal of outer tissue ofmeristem with the help of sterile scalpel, which was about (5×5 mm) in size.
Surface sterilization of explants
The pale white tissue block (1.0×2.0 cm) containing meristem and rhizomatousbase were taken in a beaker. Surface sterilization was done under Laminar Airflow
Cabinet with 70% ethanol for one minute. After sterilization with 70% ethanol, theexplants were surface sterilized with 0.1% mercuric chloride (HgCl2) and a few dropsof Tween-20 for 15 minutes. After that, the explants were then rinsed three to fourtimes with sterile dw. Finally, the explants were surface sterilized by renamycil capsuledissolved solution.

20South Pacific Studies Vol.29, No.1, 2008

Culture explantation
The isolated and surface sterilized meristem tissue blocks were prepared carefully
under (the stereomicroscope through maintaining) aseptic condition inside thelaminar airflow cabinet, to use those as explants. The individual meristematic tissue
was directly inoculated to each of the culture tube containing 20 ml of MS medium
supplemented with different concentration of hormones required as per treatment,
covered with plastic cap and were sealed with parafilm.
Incubation
The culture tubes were transferred to growth room and were allowed to grow incontrolled environment. The temperature of the growth room was maintained within 25 ±2°C by an air conditioner. A 16-hour light period was maintained with lightintensity of 2000 lux of the growth and development of culture.
Browning of the explantSome explants become black or brown in colour within 6-7 days after inoculation.
To control blackening or browning after about one week the blackish or browning
tissues on the explants were removed and the meristematic tissues were transferred tosimilar fresh medium. It was repeated each of 10 days interval for about one month tominimize further blackening or browning of the tissues.

Subculture or Transfer
Maintenance of proliferating shoots
Initial sub culturing was done when the explants had produced some shoots. For
subculturing, in vitro shoot were cut into small pieces so that each piece would contain
about one shoot. Leaf and blackish or browned basal tissues were removed to expose
the meristem. Each piece was inoculated into a similar fresh medium. It was practiced
at the interval of every one month. Through subculturing, the numbers of shoots wereincreased.
Regeneration of plants from in vitro proliferated buds
In vitro proliferated micro shoots were separated and each of the micro-shootwas placed on culture medium, which supplemented with different concentration of
hormone for shoot differentiation.
Root induction of regenerated shoots
When the shoots grown about 3-5 cm in length with 2-3 well developed leaves
they were rescued aseptically from the culture vessels and were separated from eachother and again cultured on freshly prepared medium containing different combinationsof hormonal supplements for root induction. The produced shoot obtained from virus

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus21

infected plants and also confirmed by ELISA test.
The meristem cultured banana cv. Amritasagar plantlets were used as explant. Fourlevels of BAP (0, 3, 4 and 5 mg/l) and 5 levels of NAA (0.0, 1.0, 1.5, 2.0 and 2.5 mg/l) were used as treatment for shoot proliferation in experiment-I and four levels of IBA(0, 1, 2 and 3 mg/l) and 4 levels of NAA (0, 2, 3 and 4 mg/l) were used as treatment
for root formation in experiment-II. ELISA test was conducted for the confirmation ofvirus eradication rate of regenerated plants and survival rate of regenerated plants inexperiment-III.

ELISA Test
The ELISA test was conducted to confirm the virus free banana plants throughmeristem culture at the Microbiology Laboratory, Department of Plant Pathology,
Bangabundho Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur during

F8
H11

G10
H11
Plate 1. ELISA test for detection of virus in banana cv. AmritasagarA) well of the micro titer plate after ELISA test (F8 to H11 indicates BBTV sample)B) well of micro titer plate after ELISA test (G10 to H11 indicates BMV sample).

22South Pacific Studies Vol.29, No.1, 2008

March 16 to 23, 2004. The comparative results obtained in the investigation have beenpresented in Plate -1.

Steps in ELISA (Utilizing commercial kit: Agdia, USA)
Sample extraction buffer was prepared as per instruction supplied with the kit.
Amount needed: 3000 ml (for 288 well or three 96 well micro titer plate) for safety
allowance prepared about 500 ml. Disease sample (leaf, tissue etc.) and extraction
buffer was mixed in 1:10 ratio eg. take 0.1 g tissue, extract with 1 ml extraction buffer
in a mortar with pestle. Grinded it to make sap (discarding the debris) and dispensed
in an append drop tube mark the tube. A loading diagram of the test was made (i.e.
in what well which sample is to be placed) in a piece of paper.100 µl of the extracted
sample in each well of the micro titer plate (pre coated with antibody) was dispensed.
Also similar amount of solution was put for positive control and extraction bufferin two wells as check. The plate was incubated for 2 hrs in a humid box at room
temperature or overnight at 4oC. PBST (washing buffer) [Amount needed: 2 liter (fortwo time washing for 3 micro titer plate)], ECL buffer (conjugate buffer), enzymeconjugate [Amount needed: 30-50 ml kit (to be prepared about just before beginningof washing the plates)] was prepared as per instruction supplied with the kit. The microtiter plate was washed with PBST (washing buffer): During washing the well was
filled with buffer and quickly turned the plate upside down in whipping fashion on thesink and repeated the process several times (4 to 8) and finally dried on few sheets ofpaper towel or newsprint paper or blotting paper by repeated knocking. Dispensed 100µl enzyme conjugate per well and the plate was incubated for 2 hrs in a humid box atroom temperature. PNP (substrate for enzyme) buffer [Amount needed 30-50 ml] andPNP solution was prepared as per instruction supplied with the kit. The micro titerplate was washed with PBST again like previous system. 100 µl of PNP solution wasdispensed per well and incubated in the humid box for 30-60 minutes.

Lay out and design of the experiment
The experiments were arranged in Completely Randomized Design (CRD) with 4
replications. Each of replications consisted of 5 culture vessels.
Number of shoots per explant
Number of shoots per explant was recorded at 10-15 days interval up to 40 days ofculture and mean number of shoots per explant was calculated by using the following
Σ×iformula.
X=
Where, X = Mean number of shoots/explant,n×i = Number of shoots/explant, n= Number of observations and Σ =Summation

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

23

Rate of Plantlets to Establishment
The percentage of plantlets established in the pot mixture of soil, sand, cowdung
and coir were calculated using following formula:
Survival (%) = (Number of plantlets established in substrate / Total number of plantlets
placed in substrate) × 100
Analysis of Data
The experiment was laid out in Completely Randomized Design (CRD) with four
replications. The data recorded for different parameters were statistically analyzed
using the “MSTAT” program. The differences between the treatment means werecompared by the Least Significant Difference (LSD) test at 5% level of significanceafter performing ANOVA (GOMEZ and GOMEZ, 1983) for the interpretation of results.

Results and Discussion
Experiment-I: Effects of different concentrations and combinations of BAP and
NAA on meristem cultured shoot proliferation and differentiation of banana cv.
Amritasagar
To obtain plant regeneration and subsequent shoot proliferation from meristem
cultured plantlets of banana cv. Amritasagar, MS medium supplemented with different
concentrations and combinations of BAP and NAA were used in this experiment.
Healthy and vigorous suckers were collected from the meristem cultured plantlets andcut into pieces having half stem and half rhizome. Moreover, a piece of 2.5-3.5 cm
long and about 2 cm diameter plantlets were used as primary culture (explant) for shoot

Plate2. Half shoot and rhizomatous explant for multiple shooting of banana cv.
Amritasagar on MS media containing 4 mg/l BAP +2.5 mg/l NAA

24South Pacific Studies Vol.29, No.1, 2008

proliferation (Plate 2).
Number of shoots per explant
The number of shoots produced per explant varied on MS media supplemented
with different concentrations and combinations of BAP and NAA. The main effectof BAP on shoot proliferation was found significant. The results showed that 4 mg/
l BAP gave the highest number of shoots at 15, 30 and 40 DAE. The highest numberof shoots proliferated (3.76/explant at 40 DAE) with 4 mg/l BAP followed by 5 mg/
l BAP (2.96/explant at 40 DAE) and the lowest number (1.52/explant at 40 DAE) was
obtained from hormone free media (Fig. 1). The effect of NAA showed that the highest
number of shoots (3.06/explant) obtained from MS medium supplemented with 1.5
mg/l NAA whereas, the lowest (2.20/explant) from MS medium without any NAA
supplementation at 40 DAE. The results indicated that the increasing trend of shootnumber was faster in 1.5 mg/l NAA than others (Fig 1).

Fig. 1. Effect of different concentrations of BAP and NAA on shoot number of banana cv.
Amritasagar at different days after explantation.

The combined effect of different concentrations and combinations of BAP and
NAA on shoot proliferation has been presented in Table 1. Among the concentrations
and combinations of MS media, BAP 4 mg/l + NAA 1.5 mg/l showed the highest shootproliferation (4.58/explant) followed by BAP 5 mg/l treatment (3.30/explant) at 40
DAE (Plate 3). These results are in partial support of Rabbani et al. (1996) where theyfound that highest number of shoots per explants at 28 DAE was 3.11 ± 0.66 with 5
mg/l each of BAP and Kinetin. This variation might be due to the differences of species
and inoculated explant. The explants cultured on MS medium without growth regulator
did not produce any shoot. Similar results also reported by KHANOM et al. (1996) and
REHANA (1999).

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus25

Table 1. Combined effect of different concentrations of BAP and NAA on shoot proliferation of banana
plantlet cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)
NAA (mg/l)
0
0
1.0
1.5
2.0
2.5
0
3
1.0
1.5
2.0
2.5
0
4
1.0
1.5
2.0
2.5
0
5
1.0
1.5
2.0
2.5
LSD (0.05)
CV (%)

Shoot number per plantlet at DAE
15
30
40
0.00
0.00
0.00
0.90
1.50
1.90
1.14
1.65
1.98
1.05
1.65
2.00
1.00
1.50
1.71
1.00
2.10
2.55
1.25
2.15
2.55
1.40
2.25
2.75
1.50
2.30
2.82
1.25
2.10
2.48
1.30
2.55
2.95
1.51
2.80
3.55
2.10
4.00
4.58
1.83
3.65
4.05
1.65
3.10
3.70
1.55
3.00
3.30
1.53
2.65
3.20
1.45
2.60
2.95
1.61
2.58
2.88
1.42
2.10
2.45
0.049
0.048
0.169
2.5
1.09
4.03

Plate 3. Multiple shoots of banana cv. Amritasagar produced on MS medium containing 4 mg/l BAP +1.5
mg/l NAA (left) and containing 2.5 mg/l NAA (right) at 40 days after explantation

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South Pacific Studies Vol.29, No.1, 2008

Shoot length
The MS medium supplemented with BAP and NAA showed different results forincreasing shoot length. Shoot length was significantly influenced by different BAP

Fig. 2. Effect of different concentrations of BAP and NAA on shoot length of banana plantlet cv.
Amritasagar at different days after explantation.

Table 2. Combined effect of different concentrations of BAP and NAA on mean shoot length of banana
plantlet cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)
NAA (mg/l)
0
0
1.0
1.5
2.0
2.5
0
3
1.0
1.5
2.0
2.5
0
4
1.0
1.5
2.0
2.5
0
5
1.0
1.5
2.0
2.5
LSD (0.05)
CV (%)

15
0.00
1.06
1.22
0.96
0.85
1.25
1.48
1.72
1.90
1.90
1.90
1.68
2.03
1.36
1.30
1.72
1.40
1.65
1.32
1.28
0.049
2.22

Mean shoot length (cm) at DAE
30
40
0.00
0.00
1.48
1.86
1.65
1.98
1.88
2.12
1.20
1.46
1.75
2.25
1.78
2.42
2.12
2.41
2.20
2.65
2.25
2.78
2.92
3.22
2.48
3.04
2.98
3.56
2.10
2.58
2.10
2.42
2.93
3.35
2.63
2.74
2.95
3.12
1.94
3.42
1.88
2.25
0.138
0.048
4.34
1.03

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

27

levels at 15, 30 and 40 DAE. At 15 DAE, the longest shoot (1.65 cm) was obtained from
4 mg/l BAP followed by 3 mg/l BAP (1.65 cm) supplemented MS medium. Finally, at
40 DAE the longest shoot (2.96 cm) was found from 4 mg/l BAP that was identical withthe shoot length obtained from 5 mg/l BAP (2.77 cm) supplemented MS medium (Fig.
2). At 15, 30 and 40 DAE, similar result was found in case of NAA where highest shootlength (2.77 cm at 40 DAE) was obtained from 1.5 mg/l NAA supplemented MS media.
Moreover, fluctuated results were observed when the media was supplemented with
1.0, 2.0 and 2.5 mg/l NAA (Fig. 2). The smallest shoot (2.21 cm) was recorded in themedium supplemented without NAA.
The combined effect of different concentration of BAP and NAA on the incrementof shoot length of banana cv. Amritasagar have been presented in Table 2. Various
concentrations and combinations of supplements showed significant variation in shootlength of banana plantlets. Among the combinations used, 4 mg/l BAP + 1.5 mg/l NAA
showed the longest shoot (3.56 cm) followed by 5 mg/l BAP (3.35 cm) treatment.
Rabbani et al. (1996) found similar results from 5.0 mg/l BAP supplemented MS media.
During the culture period, it was also noted that there was fluctuation in increment ofshoot length with all the media supplemented with different concentrations of BAP and
NAA alone or in combination. Therefore, the rate of increase was faster in 4 mg/l BAP +1.5 mg/l NAA supplemented media (Table 2). The results of present experiment agreed
with the findings of KHANAM et al. (1996) who obtained the longest shoot in banana on
MS medium supplemented with 25 µM/l BAP treatments.
Leaf Number per plantlet
The number of leaves produced per plantlet was also influenced by the different
concentrations and combinations of BAP and NAA. The effect of BAP showed significant
variation in leaf number of banana plantlets. The maximum number of leaves (2.78/
plantlet) counted from the medium supplemented with 3 mg/l BAP which was identicalwith that of explant cultured on the MS medium containing 5 mg/l BAP at 40 DAE (Fig.

Fig. 3. Effect of different concentration of BAP and NAA on number of leaves of banana plantlet cv.
Amritasagar at different days after explantation.

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3). At 40 DAE significant influence was found with different concentrations of NAA.
The highest number of leaves (2.55/plantlet) obtained from 1.5 mg/l NAA, whereas thelowest (2.20/plantlet) without NAA.
The combined effect of different concentrations of BAP and N AA has beenpresented in Table 3. The results showed that the maximum number of leaves (3.11/
plantlet) produced with 4 mg/l B AP+1.5mg/l N AA, which was identical with thetreatment of 5 mg/l BAP. The results of the present experiment also agreed with thefindings of Rabbani et al. (1996) who obtained the same results from 5.0 mg/l BAP. The
lowest number of leaves (1.78/plantlet) obtained from 1.0 mg/l NAA supplemented MS
media (Table 3).
Longest leaf length
A significant variation in leaf length was noted among the different concentrations
and combinations of BAP and NAA at different DAE. It was observed that the media
containing 3 mg/l BAP produced the longest leaves (2.12 cm), which was identicalwith the explant cultured on the medium containing 4 mg/l BAP (2.10 cm) at 40 DAE.
During the culture period, there was a sharp increase trends in leaf length for the main
Table 3. Combined effect of different concentrations of BAP and NAA on mean leaf number banana plantlet
cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)
NAA (mg/l)
0
0
1.0
1.5
2.0
2.5
0
3
1.0
1.5
2.0
2.5
0
4
1.0
1.5
2.0
2.5
0
5
1.0
1.5
2.0
2.5
LSD (0.05)
CV (%)

Mean leaf number per plantlet at DAE
15
30
40
0.00
0.00
0.00
1.15
1.48
1.78
1.10
1.48
1.96
1.09
1.62
1.89
1.00
1.58
1.84
1.24
2.49
2.83
1.08
2.44
2.75
1.12
2.38
2.57
1.22
2.60
2.92
1.20
2.53
2.84
1.63
2.40
2.88
1.40
1.98
2.49
1.60
2.47
3.12
1.36
1.88
2.38
1.27
1.78
2.0
1.70
2.68
3.10
1.52
2.18
2.80
1.39
2.29
2.56
1.24
1.80
2.33
1.22
1.78
2.20
0.049
0.109
0.154
2.13
3.52
4.18

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

29

Fig. 4. Effect of different concentrations of BAP and NAA on length of longest leaves of banana cv.
Amritasagar at different days after explantation.
Table 4. Combined effect of different concentrations of BAP and NAA on mean length of longest leaf ofbanana plantlet cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)
NAA (mg/l)
0
0
1.0
1.5
2.0
2.5
0
3
1.0
1.5
2.0
2.5
0
4
1.0
1.5
2.0
2.5
0
5
1.0
1.5
2.0
2.5
LSD (0.05)
CV (%)

Mean length of longest leaf (cm) at DAE
15
30
40
0.00
0.00
0.00
0.99
1.23
1.38
0.89
1.16
1.41
0.82
1.15
1.33
0.91
1.35
1.46
1.23
1.78
2.11
1.18
1.62
1.91
1.22
1.87
2.15
1.41
1.98
2.35
1.28
1.81
2.10
1.48
1.98
2.09
1.35
1.81
2.09
1.52
2.12
2.29
1.29
1.89
2.18
1.18
1.65
1.90
1.60
2.14
2.22
1.38
1.67
2.05
1.40
1.86
2.12
1.40
1.81
1.98
1.31
1.68
1.91
0.049
0.048
0.048
1.96
1.39
1.33

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effect of BAP and NAA (Fig. 4). Highest leaf length (1.99 cm) was obtained from 1.5
mg/l NAA supplemented MS media and smallest length (1.61cm) was recorded in the
media supplemented without NAA at 40 DAE.
The combined effect of BAP and NAA on leaf length on banana plantlets has beenpresented in Table 4. Among the combinations, BAP 3 mg/l + NAA 2.0 mg/l containing
MS media produced the longest leaf (2.35 cm) followed by 4 mg/l BAP+1.5 mg/l
NAA (2.29 cm) at 40 DAE. Therefore, the lowest leaf length (1.33 cm at 40 DAE) wasrecorded from the treatment of 2.0 mg/l NAA. REHANA (1999) also reported the similar
result.
Number of roots per explant
The effect of BAP showed that there was negative relationship between rooting
and BAP application, the highest number (2.82/explant) of roots produced on themedium without BAP supplementation, which was identical with the treatment of 5 mg/
l BAP at 40 DAE. Therefore, the number of roots was decreased with the increase of
BAP concentration at different DAE (Fig. 5). The effect of NAA was found significant
at different DAE. The highest number of roots (2.78/explant at 40 DAE) was obtainedby 2.0 mg/l NAA. The lowest number of roots (1.92/explant) was recorded with 1.0
mg/l NAA, where no response was taken in control treatment. The results showed thatthe number of roots gradually increased with the increase of NAA concentrations at
different DAE (Fig. 5).
The combined effect of BAP and NAA on root number, a small number of roots
was produced by different concentrations at 15 DAE. Moreover, control with the othertreatments showed absence of root at 15 DAE. At 30 and 40 DAE similar result was
found, where the highest root number (4.19/explant) was obtained with 2.0 mg/l NAA
at 40 DAE, which was significantly higher over other treatments (Plate 4 and Table

Fig. 5. Effect of different concentrations of BAP and NAA on number of roots of banana plantlet cv.
Amritasagar at different days after explantation. Control treatment (without NAA) did not show
any root formation.

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

31

Table 5. Combined effect of different concentrations of BAP and NAA on mean root number of banana
plantlet cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)
NAA (mg/l)
0
0
1.0
1.5
2.0
2.5
0
3
1.0
1.5
2.0
2.5
0
4
1.0
1.5
2.0
2.5
0
5
1.0
1.5
2.0
2.5
LSD (0.05)
CV (%)

Mean root number per plantlet at DAE
15
30
40
0.00
0.00
0.00
1.26
2.35
2.95
1.31
2.75
3.33
1.50
2.90
4.19
1.50
2.83
3.65
0.00
0.00
0.00
0.00
0.83
1.32
0.51
1.21
1.88
0.98
1.67
2.11
1.10
1.90
2.08
0.00
0.00
0.00
0.38
0.91
1.44
0.58
1.33
2.05
0.65
1.08
2.32
0.95
1.37
2.65
0.00
0.00
0.00
0.00
0.90
1.99
0.31
1.08
2.40
0.61
1.28
2.51
0.71
1.49
2.71
0.049
0.048
0.049
5.89
2.86
1.31

5). The lowest root number (1.32/ plantlet) was gained with 3 mg/l BAP +1.0mg/l
NAA. The present results indicated that treatment 2.0 mg/l NAA was suitable for root
formation, which is similar with the findings of GUBBUK and PEKMEZCI (2001).

Plate 4. Root formation of banana plantlets cv.
Amritasagar on MS medium containing 0
mg/l BAP+2.0 mg/l NAA at 40 days after
explantation.

32

South Pacific Studies Vol.29, No.1, 2008

Experiment-II: Effects of different concentrations and combinations of IBA and
NAA on rooting of meristem cultured banana plantlet cv. Amritasagar
The meristem cultured shoots were collected from in vitro grown plants (used in
expt.-I). When those were subcultured on half strength of MS medium supplemented
with different levels of IBA (0, 1, 2 and 3 mg/l) and NAA (0, 2, 3 and 4 mg/l) in order
to allow root formation. Some roots were developed from the proliferated shoots. Root
numbers varied with different concentrations and combinations of IBA and NAA.
Number of roots
The prolific shoot of banana cv. Amritasagar was cultured on half strength of MS
media supplemented with different levels of IBA (0, 1, 2 and 3 mg/l) and NAA (0, 2,
3 and 4 mg/l). The effect of IBA was found significant at different DAE. At 30 DAE,
the highest number of roots (5.10/explant) of banana cv. Amritasagar was found from
2 mg/l IBA, followed by 1 mg/l IBA. The lowest number of roots (1.13/explant) at 30
DAE was produced by MS medium without IBA supplementation. Moreover, at 10
and 20 DAE, the root number was increased with the increase of IBA concentration up
to 2 mg/l (Fig. 6). The effect of NAA showed significant result at 10, 20 and 30 DAE,
whereas 3 mg/l NAA represented highest number of roots (5.66 /explant) at 30 DAE
and lowest number of roots (2.75/explant) at 30 DAE was produced by MS media
without NAA supplementation. So the present result reveals that NAA is superior to
IBA in respect to root formation.
At different DAE, the combined effect of IBA and NAA also showed significant
variation in root number. The highest number of roots (6.98/explant) was obtained
from 2 mg/l IBA + 2 mg/l NAA and the lowest (1.65/explant) from control at 30 DAE(Table 6 and Plate 5). DOMINGUES et al., (1995) reported that rooting was stimulated ina nutrient solution containing 0.1 mg/l NAA or IBA. The result of the present study
was in consisted with the findings of KHANAM et al. (1996).

Fig. 6. Effect of different concentrations of IBA and NAA on number of roots of banana cv.
Amritasagar at different days after explantation.

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

33

Table 6. Combined effect of different concentrations of IBA and NAA on root number of proliferated shootof banana cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)
NAA (mg/l)
0
0
2
3
4
0
3
2
3
4
0
4
2
3
4
0
5
2
3
4
LSD (0.05)
CV (%)

Root number per plantlet at DAE
10
20
30
0.45
0.98
1.65
0.90
2.93
4.68
0.99
3.09
4.85
1.10
3.12
5.35
0.77
1.60
2.90
1.20
3.25
5.05
1.49
4.50
6.60
1.65
4.72
5.65
0.92
1.98
3.05
1.35
4.26
6.98
1.40
3.37
5.37
1.21
3.29
5.01
0.88
2.01
3.42
1.10
2.91
3.99
1.45
3.41
5.81
1.20
2.81
4.92
0.051
0.049
0.050
2.52
0.91
0.43

Plate 5. In vitro Plantlet with vigorous Roots of banana cv.
Amritasagar grown on MS medium supplemented with
2 mg/l IBA + 2 mg/l NAA at 30 days after explantation.

Root length
A significant variation in the root length developed by the plantlets was influenced
considerably by different concentrations and combinations of IBA and NAA (Table
7). The effect of IBA showed significant variation in root length at different DAE.
The longest roots (4.18 cm) were observed with 3 mg/l IBA followed by 2 mg/l IBA,
whereas the shortest (1.87 cm) was obtained from MS medium without IBA at 30
DAE. On the contrary, the longest root (4.23 cm) was observed with 3 mg/l of NAA

34South Pacific Studies Vol.29, No.1, 2008

Table 7. Combined effect of different concentrations of IBA and NAA on root length and vigour ofregenerated root of proliferated shoot of banana cv. Amritasagar at different days after explantation

Treatment combination
BAP (mg/l)

NAA (mg/l)

0

0
2
3
4
0
2
3
4
0
2
3
4
0
2
3
4

3

4

5

LSD (0.05)
CV (%)

Root length (cm) at DAE
10
0.30
0.50
1.03
0.71
0.38
0.65
1.05
0.68
0.75
0.77
1.01
0.83
0.66
0.87
1.11
0.92
0.050
2.08
Legend:

20
1.00
1.98
2.25
1.49
1.28
1.90
2.23
1.80
2.65
2.09
2.98
2.55
2.95
2.72
3.45
2.41
0.160
0.73

30
1.95
2.55
4.01
2.99
1.60
3.11
3.90
2.91
3.80
3.29
4.01
3.83
3.80
4.02
5.00
3.89
0.016
0.54

Vigour ofregenerated root
+
+
+++
++
+
++
+++
++
++
++
+++
+++
++
+++
+++
++
-

+
indicate light green,++ indicate green and+++ indicate dark green

Fig. 7. Effect of different concentration of IBA and NAA on length of root of banana cv.
Amritasagar at different days after explantation.

and the shortest (1.78 cm) was obtained from MS medium without NAA at 30 DAE.
The result indicated that there was a sharp increasing trend in root length at different

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

35

DAE (10, 20 and 30 DAE). Moreover, root length increased with the increase of IBA
and NAA concentration up to 2 and 3 mg/l, but the increasing rate was considerably
faster in case of 2 and 3 mg/l IBA and NAA respectively (Fig. 7).
The combined effect of IBA and NAA also showed significant variation on root
length at different DAE. The length of longest root (5 cm) was obtained from 3 mg/l IBA +3 mg/l NAA and the shortest length of roots (1.6 cm) from 1 mg/l IBA in
combination with control at 30 DAE (Table 7 and plate 6). The present result partiallyagreed with the findings of Gubbuk and Pekmezci (2001) who reported that 1.0 µM/l
IBA in MS medium was sufficient for rooting. The results are similar with the findingsof KHANAM et al. (1996) who found longest roots (4.2 cm) of banana by adding 2.0
mg/l IBA in the media.

Plate 6. Rooted plantlets of banana cv. Amritasagar on MS media containing 2 mg/l IBA (left) and 3 mg/l
IBA + 3 mg/l NAA (right) at 30 days after explantation.

Root vigour
The root vigour was recorded by eye estimation. It was influenced by applicationof different levels of IBA and NAA (Table 7). Root initiated from the 2 and 3 mg/
l concentrations of IBA were more vigorous compared with other treatments. It wasalso observed that more vigorous roots produced from 2, 3, and 4 mg/l NAA treatmentcompared to without NAA (Plate 6). The present result agreed with the findings of
HABIB (1994), who also obtained more vigorous roots with 2.0 mg/l IBA in variety
Sabri, whereas, ALI (1996) was from banana cv. Amritasagar.

36South Pacific Studies Vol.29, No.1, 2008

Experiment –III (a): Eradication rate of virus free banana plants cv. Amritasagar
regenerated from meristem cultured by using ELISA test
MOREL et al. (1952) first obtained virus free Dahlias and Potatoes with the helpof meristem culture. Initially, the health status of explants and regenerated plants
were checked through ELISA. Collected explants were fully virus infected which was
confirmed by ELISA test, because the wells of micro titer plate showed colour, indicate
positive results i.e. explants virus infected. The wells were examined by eye ormeasured on a micro titer reader at 405 nm. On the contrary, wells of micro titer plateof regenerated plants after ELIS test in which colour developed, indicate positive result
i.e. regenerated plants virus infected and in which wells no colour developed, indicate
negative results i.e. regenerated plants was not virus infected or virus free plate-1. Test
result was valid on the basis of positive control well gave a positive colour and buffer
well remained clear. In this experiment, no colour development result was significant
because regenerated plants were virus free. The regeneration rate of banana bunchy top
virus (BBTV) free plants were 57.14 % and for banana mosaic virus (BMV) free plants
were 64.28% (Table 8). The present results agreed with the findings of HELLIOT B. etal. (2002) who also reported on successfully BMV eradication by cryopreservationof highly proliferated meristem of banana (cv. Williams BSJ, ITC 0570, AAA). The
Thirty percent of the regenerated plants were found to be healthy by DAS- ELISA.
BMV eradication rate was also achieved by GUPTA (1986) in approximately 100%of the regenerated plants when using meristem cultured in combination with a two
week heat therapy (38- 40ºc). Similar results also reported by BONDOK et al. (1987).
RAMOS et al. (1990) reported that elimation rate of banana bunchy top virus (Musasp. cv. Lacatan) by heat pre-treatment (40ºc for 16 hrs. photoperiod and 32ºc for 8 hrs.
darkness) with meristem culture, result yielded 59.3% and 68.2% BBTV free plants,
respectively. Results from other studies also showed that regenerated banana plants
by meristem culture eradicated BBTV and BMV to great extent (MORI et al.1977,
BHOJWANI et al. 1983, LOGON et al.1985, ALLAM et al. 2000).
Table 8. Eradication rate of virus free banana cv. Amritasagar by meristem cultured after ELISA test

Variety

Amritasagar

Name ofdiseases

Numberof plantstestedNumber ofvirus freeplantsNumber ofvirus
infected
plants

% of
virus
free
plantsBananabunchytop virus(BBTV)

14

8

6

57.14

Bananamosaic virus
(BMV)

14

9

5

64.28

SHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

37

Experiment-III (b): Survival rate of regenerated ex vitro hardened plants frommeristem cultured of banana cv. Amritasagar in different pot mixtures
After rooting, the lids of the culture vessels were opened slightly and kept ingrowth room for five days and then transferred to laboratory at room temperature foranother two days for hardening. After that the plantlets with well developed roots werePlate 7. Hardened in vitro grown plantlets of banana cv. Amritasagar (above row) and Sabri (below row)
after 15 days of transfer to polyethylene bag.

Plate 8. Virus free healthy plant of banana cv. Amritasagar established in pot mixture (sand : soil : cow
dung = 1:1:1 and coir : soil = 1: 2) after 40 days of transfer to earthen pot.

38

South Pacific Studies Vol.29, No.1, 2008

Table 9. Survival rate of regenerated plant from meristem cultured of banana cv. Amritasagar transferred tothe pot with different soil mixture

Variety

Amritasagar

Number ofplantlets potted

Number ofplantssurvived

Pot mixture% ofsurvivalplants6

5

Sand : Soil :Cowdung = 1:1:1

83.33

6

4

Coir: Soil = 1:2

66.67

removed from the culture vessels without damaging the roots. The agar was washedaway from the roots with running tap water. After that, the plantlets were treated withfungicide (Ridomil) for few seconds and transferred to small polyethylene bag filledwith soil, sand and well decomposed cow dung (1:1:1) and coir & soil (1:2) as well as
kept in the hardening room for 3-4 days (Plate 7). Then the polyethylene bag containing
plantlets were transferred to net house, where proper care was taken for growth anddevelopment of banana plantlets. After 10-15 days, maximum plants showed vigorous
growth while some were less vigorous (Plate 7). Therefore, the plantlets became wellhardened and ready to replace in the field conditions after 30 days (Plate 8). The
survival rate of plantlets was about 83.33% in sand, soil and cow dung (1:1:1) pot
mixture and 66.67% in coir and soil (1:2) pot mixture (Table 9). The findings are inagreement with the result of DILIP et al. (2002) who reported that more than 25,000
plantlets produced through tissue culture were transferred to the field with survival rate
90%. The well developed plantlets were hardened for the establishment in the field
by JASARI et al. (1999) who showed that average 92% of plantlets survived. Azad andAmin (1999) also mentioned that survival rate of the plantlets under ex vitro condition
was 80% in pot mixture containing sand, soil and compost at 1:1:1 ratio. Similar results
also reported by (CRONAUER and KRIKORIAN 1984, GUPTA 1986).
Based on the findings of this study it may be concluded that for rapid shootproliferation, the highest number of shoots, the longest shoots and the highest numberof leaves were recorded in the media containing 4 mg/l BAP + 1.5 mg/l NAA. On
the contrary, plantlets were cultured on half strength MS media supplement withdifferent levels of IBA and NAA. For rapid root formation, and the highest number ofmore vigorous roots were obtained from 2 mg/l IBA +2 mg/l NAA and the treatment
3 mg/l IBA+3 mg/l NAA produced the longest roots at 30 DAE. The effect of BAP
on rooting was negative. The numbers of roots were decreased with the increase of
BAP concentrations. On the other hands, the number of roots gradually increased
with the increase of NAA concentrations but the effect of IBA was found significant
at different DAE. Shoot excised meristem of regenerated plants of banana cv.
Amritasagar were tested either they were virus free or not by ELISA test. In vitro
meristem culture produced 57.14% BBTV free plants and 64.28% BMV free plants.
The survival rate of regenerated plants were found to be better 83.33% in a pot mixtureof sand, soil and cowdung (1:1:1) compared to 66.67% in pot mixture of coir andSHIRAGI et. al: Eradication of Banana Bunchy Top Virus and Banana Mosaic Virus

39

soil (1:2). In Bangladesh, very few investigations have yet been carried out on thisstudy. The present study was indicated that to find out the suitable concentrations andcombinations of appropriate PGRs for the regenerations of virus free healthy planting
materials of banana cv. Amritasagar for use of farmer’s level through in vitro meristem
culture that may be helpful for commercial exploitation.

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42South Pacific Studies Vol.29, No.1, 2008