In vitro regeneration protocol for Anupam and Chini Champa: two banana (Musa sapientum) cultivars of Bangladesh.
Banana and plantains (Musa spp.) are ranked the number one and one of the most important fruit crops in Bangladesh and adapted to grow in most of the ecological regions in the country. Its sweet taste, easy growing in every homestead and year round availability has made it a popular food item among people. In the country, banana contributes to 41% of the total fruit production and it shares 21% in area on an average yield of 15 t/ha . The edible parts (fruits, pseudostem and inflorescence) of the plant are rich enough in carbohydrates, minerals and vitamins. More importantly, the fruit of this most common wild edible plant possesses medicinal uses . Musa sp. fruits have been reported to prevent anemia, regulate blood pressure , solve the problem of constipation, cure heart burns stress, strokes, ulcers and many other ailments .
Traditional propagation system decreases expected yield due to transmission of viral diseases like banana streak virus and as well produce inadequate number of suckers from mother plants within a certain period. To improve and develop banana variety, in vitro culture is one of the most useful tools in biotechnology that benefits in product uniformity with uniform growth and harvesting time and disease free planting materials . Tissue culture of Musa sp. has been demonstrated by several researchers [2,5,9,11,12,16,17] as a successful method of in vitro clonal propagation.
Among the varieties of banana in Bangladesh, Chini Champa and Anupam are two popular cultivars of Musa sapientum because of their good taste and flavor. Anupam (AAB, syn: Malbhog, Martaman) is a medium sized, sweet and tasty table banana with a tall and yellowish green pseudostem. The average bunch weight is about 10 kg, which contains 85-120 fingers. This variety is widely grown in the north and western areas of Bangladesh. Chini Champa (AAB) is a hardiest tall cultivar grown all over in Bangladesh but mostly cultivated in Chittagong and Chittagong Hill Tracts Districts. Merits of cultivating this cultivar are it can withstand under water-logging, resistant to Fusarium wilt and fairly resistant to bunchy top disease. Small sized fruits with thin peel, creamy pulp and sub-acid taste have a good shelflife. Fruits turn golden yellow when ripe and have excellent keeping quality. The bunch contains 150250 fingers and weighs about 16 kg .
To meet the demand of the farmers and for commercialization of good varieties like Anupam and Chini Champa and increase the total yield, large number of plantlets is needed within a specific time period. Thus in vitro clonal propagation could play a role to reduce this scarcity of plantlets and can serve to develop in vitro techniques on other cultivars of banana.
Materials and Methods
Explant preparation. Newly sprouted suckers (2-3 weeks old) were collected from different banana farmlands of Natore district, Bangladesh. The mother plants for each cultivar were carefully inspected to make sure that they were healthy and phenotypically true-to-type and then these suckers were moved to Dhaka. The experiment was conducted at Plant Cell and Tissue Culture Laboratory at the University of Development Alternative. The meristemic part from each pseudostems were trimmed from the sucker and used to prepare explant. Then the shoot tips were decapitated and a block of tissue (1.5 cm x 1.5 cm x 1.5 cm) was excised and washed under running tap water for 15-20 minutes. The explants were soaked in Tween 80 solution for 7 minutes and then washed thrice with distilled water. The tissues were then surface sterilized under laminar air flow by immersion in 0.1% Hg[Cl.sub.2] solution for 15 minutes and washed thoroughly four times with distilled autoclaved water to remove any traces of Hg[Cl.sub.2]. Explants were implanted onto Murashige and[right arrow] Skoog's (MS) medium  containing different phytohormones (6-benzylaminopurine (BAP), indole-3-acetic acid (IAA), Kinetin (Kn) and Indole-3-butyric acid (IBA) supplemented with 3% sucrose and 0.8% Difco Bacto-agar in test tubes and culture vessels. The pH of the medium was adjusted to 5.8 before autoclaving and the culture condition was maintained at 24 [+ or -] 2[degrees]C under 12 h cool white fluorescent tube light (4000 lux) .
Effect of cytokinins. The excised cormlets were transferred aseptically in MS medium supplemented with different concentrations (1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50 and 5.00 mg/l) of BAP for single shoot regeneration.
Effect of cytokinins-auxin combinations. The synergistic effect of auxin-cytokinin (BAP + IAA, Kn + IAA) was observed with the sterile explants cultured in MS medium containing different combinations of auxin and cytokinins.
Root induction phase. In vitro regenerated shootlets were inoculated in MS medium fortified with IBA (0.1 to 0.8 mg/l) for root induction.
Establishment into soil. After root induction, good and healthy plantlets were properly hardened and transplanted in soil composed of humus and garden soil (1:1) into plastic pots and kept in the shade house at a relative humidity of 80-90%.
Data assembly and scrutiny. Weekly growth observations were made up to six weeks after setup of each treatment and experimental data were recorded. The data collected were: a) percentage of response (%); b) shoot length in cm and number of leaves/shoot; c) number of roots/shoot and d) days required for response. The data then were analyzed as mean [+ or -] SEM according to Mian and Miyan .
Surface sterilization. Excised explants were surface sterilized with 0.1% Hg[Cl.sub.2] to avoid contamination where survival rate increased according to the increase of treatment period from 10 minutes to 15 minutes. The explants were observed regularly to obtain any sign of contamination after the 6th week of inoculation. Explants treated for 15 minutes showed the best survival rate (90%) and prolonged treatment showed tissue blackening.
Single shoot regeneration. The sterilized shoot tips were examined to show regeneration capacity when cultured on MS medium supplemented with different concentration of BAP for both cultivars. In case of Chini Champa, explant sprouting was observed at 4th week of inoculation (Figure A) and highest percentage of response (50%) was obtained in MS + BAP [4.5 mg/l] after 6th week of culture where the shoot proliferated up to 3.15 [+ or -] 0.15 cm (Table 1). Frequency of response was increased with increases in BAP level but failed to improve above BAP concentration of 4.5 mg/l (rate <30% was not presented in the table). Explants of Anupam when cultured showed 75% shoot regeneration rate on 6th week in MS + BAP [2.0 mg/l] (Figure 2). The highest average shoot length (3.45 [+ or -] 0.10 cm) for Anupam was obtained in the same medium composition with an average of 5 [+ or -] 0.019 leaves/explant. The concentrations of BAP (0.50, 1.0, 1.50 mg/l) showed low percentage of growth responses (40, 45 and 50%, respectively) and it dropped to 58.37% at BAP [2.5 mg/l] in MS medium. Increased concentration of this cytokinin exhibited reduced rate of bud breaks and shoot growth for both cultivars. BAP played effective role on foliation of Anupam better than Chini Champa in optimum concentration.
Effect of BAP-IAA combination. In this present study for the cultivar Chini Champa, variable concentrations of BAP (1.5, 2.5, 3.5, 4.5, 5.0 and 6.0 mg/l) in combination with variable concentrations of IAA (0.25, 0.50, 1.0, 1.5, 2.0 and 2.5 mg/l) was used to enrich MS basal media and in vitro regenerated shootlets from the previous sets of experiments were used as explants. Among the tested combinations, the percentage of explant responses was below 35% when BAP was raised from 1.50 mg/l up to 3.50 mg/l. The sprouting of explants was from 35% to 45% when various concentrations of IAA were combined with BAP (3.5 mg/l to 4.5 mg/l). The morphology of the explants was stunted, and no leaf was observed (not presented in the Tables). Shoot proliferation was improved when the concentration of BAP and as well as IAA was gradually increased above 4.5 mg/l and 1.5 mg/l, respectively. Highest percentage of shoot proliferation (85%) with an average length of 1.90 [+ or -] 0.09 cm and average number of leaves/shoot (5 [+ or -] 0.086) were observed in MS + 5.0 mg/l BAP + 2.5 mg/l IAA at 6th week of culture (Table 2, Figure C). Rest of the tested combinations of BAP + IAA (5.0 + 0.50, 5.0 + 1.0 and 6.0 + 1.0 mg/l) gave low percentage of (45, 50 and 42.12, respectively) shoots proliferation, except BAP [5.0 mg/l] + IAA [2.0 mg/l] in MS medium, which showed 75.8%. Our investigation from this set of combinations revealed that the Chini Champa shoot proliferation depended on high BAP to low auxin level.
In later experiments for Anupam variety, moderate to high concentrations of BAP were added with moderate concentration of IAA in MS medium as we found lower combinations of BAP did not play effective role in shoot proliferation of Chini Champa. Gradual increase of both BAP and IAA showed increased rate of proliferation, while BAP [>5.0 mg/l] with IAA [2.0 mg/l] gave rapid decrease in shoot length (Table 3). Such synergism showed a moderate percentage of response (50%) for shoot regeneration where length of shoot was 2.9 [+ or -] 0.11 cm in MS + BAP [5.0 mg/l] + IAA [1.50 mg/l] (Figure D). In this cultivar, we found the same effect that high cytokinin to low auxin ratio was suitable for shoot proliferation. In addition, the micro-shoots commenced roots during the culture in these compositions.
Effect of Kn-IAA combination. The combinations of Kinetin (Kn) and IAA were shown to have positive role in shoot regeneration only for Anupam cultivar. Moderate Kn [2.0 mg/l] to low IAA [0.25 mg/l] in MS medium resulted good shoot length (3.5 [+ or -] 0.09 cm) where 85% explants responded in the composition with an average of 5.0 leaves per shoot (Table 4, Figure E). Increase of Kn was found to have reduced bud sprouting response and shoot length as well. Rooted shootlets were obtained in these combinations too. All the tested combinations were not successful for improving shoot proliferation response more than 25% in Chini Champa.
Root Induction Stage. After the phase of direct shoot regeneration, healthier micro-shoots were recovered and then introduced in 1/2 MS medium enriched with IBA for inducing in vitro roots. Effectiveness of auxin for root initiation was evaluated for formulating rooting media composition for both the cultivars. Root was observed and unmasked at the basal tufts within 10-12 days after inoculating in the medium for both cultivars. The rate of rooting was calculated and found that the response was cultivar independent as 100% response was obtained in every medium composition. Depending on the treatment, highest rooting composition for both cultivars were achieved in MS + IBA [1.0 mg/l] and mean number of roots were 5.2 [+ or -] 0.36 and 4.9 [+ or -] 0.41 roots per shoot for Chini Champa and Anupam, respectively (Figure F(a) and F(b), respectively; Table 5). Hairy roots emerged out in plenty from the medium after three weeks. Acclimatization and field observation. The in vitro rooted plantlets were transferred to hardening for seven days and then acclimatized into soil media (garden soil:cowdung at 1:1) in polybags. Survival rate 95% was achieved after eight weeks in field condition where morphologically no distinguishable feature was observed with the donors for both the cultivars.
Our current study showed that BAP was successful for bud initiation and proliferation which was similar to that reported by Azam et al.  for in vitro cormlets formation in BARI-I banana variety within 2-3 weeks of culture in semi-solid MS media fortified with BAP [2.0 mg/l]. In addition, Mollah, et al.  also reported in shoot tip culture of banana in MS media supplemented with BAP [5.0 mg/l] the highest range of shoot proliferation. In agreement with our study, Cronauer and Krikorian , Jarret et al.  and Vuyslteke  found optimal BAP concentrations to be effective in shoot regeneration of banana in their studies. Moreover, Wong (1986) has reported that increased BAP declined shoot growth, which was similar to our present result. Jafari et al.  produced a regeneration protocol of Musa acuminata (banana) cv. Berangan where both BAP and IAA were supplemented in MS media to enhance regeneration in vitro which was similar to our current findings on shoot proliferation. Combinations of plant growth hormones like Kn and IAA were also found effective for 'Malbhog' (Musa paradisiaca, AAB group), a banana cultivar, by Roy et al.,  as observed in the present study. Auxin induced rooting was reported by Vuyslteke  and the role of IBA was investigated as an effective phytohormone for root formation in banana by Vuyslteke and De Langhe  that is also supportive of the results obtained from the current research.
This protocol for clonal propagation can serve as a convenient method for in vitro plantlets production of M. sapientum cv. Chini Champa and M. sapientum cv. Anupam to meet the demand for disease free, healthy plants and ultimately it can enhance the economic benefit of the cultivators. This study brought out the selection of suitable phythormone level and media composition for bud sprouting, shoot proliferation and consequently, root formation of microshoots in vitro for Chini Champa and Anupam.
Published Online 2014 February 30.
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Rabab Mahdi, Md. Jahidul Islam, Md. Ataur Rahman, Anup Biswas, F.M. Safiul Azam, Mohammed Rahmatullah
Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1209, Bangladesh.
Received: 21 January 2013; Received: 18 February 2014; Accepted: 20 March 2014; Available online: 4 April 2014
Corresponding Author: Professor Dr. Mohammed Rahmatullah, Pro-Vice Chancellor, University of Development Alternative, House No. 78, Road No. 11A (new), Dhanmondi R/A, Dhaka-1205, Bangladesh. Phone: 88-02-9136285 Fax: 88-02-8157339 E-mail: email@example.com
Table 1: Effect of BAP (mg/l) on explants of Chini Champa and Anupam in MS medium. MS + BAP Percentage of explants (mg/l) responded (%) Chini Champa Anupam 0.5 -- 45.0 1.0 -- 40.0 1.5 -- 50.0 2.00 30.0 75.0 2.50 -- 58.75 3.00 33.3 35.0 3.50 30.0 -- 4.00 46.6 -- 4.50 50.0 -- 5.00 33.3 -- MS + BAP Average of shoot length (cm) (mg/l) (Mean [+ or -] SEM) Chini Champa Anupam 0.5 -- 1.90 [+ or -] 0.10 1.0 -- 1.50 [+ or -] 0.10 1.5 -- 2.50 [+ or -] 0.10 2.00 1.80 [+ or -] 0.11 3.45 [+ or -] 0.10 2.50 1.87 [+ or -] 0.20 2.75 [+ or -] 0.10 3.00 2.24 [+ or -] 0.15 2.80 [+ or -] 0.23 3.50 1.87 [+ or -] 0.13 -- 4.00 2.72 [+ or -] 0.17 -- 4.50 3.15 [+ or -] 0.15 -- 5.00 2.16 [+ or -] 0.20 -- MS + BAP Average number of leaves/shoot (mg/l) (Mean [+ or -] SEM) Chini Champa Anupam 0.5 -- 1.0 [+ or -] 0 1.0 -- 2.0 [+ or -] 0 1.5 -- 3.0 [+ or -] 0.33 2.00 2.1 [+ or -] 0.11 5.0 [+ or -] 0.02 2.50 1.9 [+ or -] 0.12 4.0 [+ or -] 0 3.00 2.1 [+ or -] 0.18 3.0 [+ or -] 0.38 3.50 2.2 [+ or -] 0.15 -- 4.00 2.4 [+ or -] 0.13 -- 4.50 2.5 [+ or -] 0.13 -- 5.00 2.0 [+ or -] 0.00 -- Table 2: Effect of BAP and IAA on explant sprouting for Chini Champa in MS medium. MS + BAP + Percentage of Average of shoot Average number of IAA (mg/l) explants length (cm) (Mean leaves/shoot (Mean responded (%) [+ or -] SEM) [+ or -] SEM) 5.00 + 0.50 45.0 1.4 [+ or -] 0.20 3.0 [+ or -] 0.17 5.00 + 1.00 50.0 1.4 [+ or -] 0.10 2.0 [+ or -] 0.30 5.00 + 2.00 75.8 1.5 [+ or -] 0.08 5.0 [+ or -] 0.10 5.00 + 2.50 85.0 1.9 [+ or -] 0.09 5.0 [+ or -] 0.07 6.00 + 1.00 42.1 1.1 [+ or -] 0.05 2.0 [+ or -] 0.27 Table 3: Effect of BAP + IAA (mg/l) on explants of Anupam in MS medium. MS + BAP + Percentage of Average of shoot IAA (mg/l) explants length (cm) (Mean responded (%) [+ or -] SEM) 4.00 + 1.50 30.0 1.7 [+ or -] 0.22 4.00 + 2.00 36.7 2.7 [+ or -] 0.26 5.00 + 1.50 50.0 2.9 [+ or -] 0.11 5.00 + 2.00 46.7 2.9 [+ or -] 0.12 6.00 + 1.50 30.0 2.0 [+ or -] 0.09 6.00 + 2.00 33.33 1.1 [+ or -] 0.08 MS + BAP + Average number of Average number of IAA (mg/l) leaves/shoot (Mean roots (Mean [+ or -] SEM) [+ or -] SEM) 4.00 + 1.50 2.6 [+ or -] 0.18 3.0 [+ or -] 0.24 4.00 + 2.00 2.4 [+ or -] 0.15 4.0 [+ or -] 0.30 5.00 + 1.50 2.6 [+ or -] 0.13 6.0 [+ or -] 0.40 5.00 + 2.00 2.4 [+ or -] 0.14 5.0 [+ or -] 0.33 6.00 + 1.50 2.2 [+ or -] 0.15 2.0 [+ or -] 0.33 6.00 + 2.00 2.4 [+ or -] 0.16 1.0 [+ or -] 0.30 Table 4: Effect of KIN + IAA (mg/l) on explants of Anupam. MS + KIN + Percentage of Average of shoot IAA (mg/l) explants length (cm) (Mean responded (%) [+ or -] SEM) 1.50 + 0.25 40.0 1.5 [+ or -] 0.16 2.00 + 0.25 85.0 3.5 [+ or -] 0.09 2.50 + 0.25 50.0 1.9 [+ or -] 0.18 3.00 + 0.25 45.0 0.9 [+ or -] 0.04 3.50 + 0.25 55.0 0.7 [+ or -] 0.04 MS + KIN + Average number of Average number of IAA (mg/l) leaves/shoot (Mean root/shoot (Mean [+ or -] SEM) [+ or -] SEM) 1.50 + 0.25 2.0 [+ or -] 0 00 2.00 + 0.25 5.0 [+ or -] 0 8.0 [+ or -] 0.45 2.50 + 0.25 1.0 [+ or -] 0.26 1.0 [+ or -] 0.30 3.00 + 0.25 00 00 3.50 + 0.25 00 00 Table 5: Effects of IBA (mg/l) on Musa sapientum cv. Anupam. 1/2 MS Percentage Average length + IBA of explants of Shoot (cm) (mg/l) responded (%) (Mean [+ or -] SEM) Anupam Chini Champa 0.50 100 3.17 [+ or -] 0.15 3.24 [+ or -] 0.21 1.00 100 3.46 [+ or -] 0.06 3.30 [+ or -] 0.23 1.50 100 3.26 [+ or -] 0.07 3.53 [+ or -] 0.20 1/2 MS Average No. of + IBA root/explant (mg/l) (Mean [+ or -] SEM) Anupam Chini Champa 0.50 3.0 [+ or -] 0.30 4.1 [+ or -] 0.53 1.00 4.9 [+ or -] 0.41 5.2 [+ or -] 0.36 1.50 3.0 [+ or -] 0.26 3.4 [+ or -] 0.34
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|Title Annotation:||Research Article|
|Author:||Mahdi, Rabab; Islam, Jahidul; Rahman, Ataur; Biswas, Anup; Azam, Safiul; Rahmatullah, Mohammed|
|Publication:||American-Eurasian Journal of Sustainable Agriculture|
|Date:||Jan 1, 2014|
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