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Natural growth substances has effective role in callus culture of Banana (Musa spp.) cultivar 'Anupam'.

Introduction

More than 120 countries worldwide cultivate banana and plantain (Musa sp.) with an annual production of around 104 million tons. The edible fruit is the fourth major crop after rice, wheat and maize and it is termed in tropical and subtropical countries as a poor man's crop (Swennen et al., 2000; Jain and Swennen, 2004). Most of the edible fruits are seedless and propagate through suckers and it is cheap to grow and capable of bearing fruits under a wide range of environmental conditions. As it is parthenocarpic, conventional breeding failed or showed limited success; cultivars are found to be polyploidy and available information on genetic and genomics are still limited (Chopra, 2005; Capdeville et al., 2009). The major obstacles in banana fruit production are several major pests and diseases, low reproductive fertility and slow propagation rate but demands huge plant for plantation every year. These problems had been mitigated from plant cell and tissue culture which has contributed greatly to plant regeneration of banana through clonal propagation, micropropagation (Cronauer and Krikorian, 1986; Diniz et al., 1999; Nauyen and Kozai, 2001; Kagera et al., 2004; Roels et al., 2005; Parveen, et al., 2008; Azam et al., 2010), somatic embryogenesis (Afza et al., 1996; Strosse et al., 2003) and protoplasts (Assani et al., 2001, 2002, 2006; Xiao et al., 2007). Different banana plant parts like meristems, rhizome tissue, leaf bases, immature zygotic embryos and male flowers have been shown to regenerate somatic embryos as well as clonal propagation (Strosse et al., 2003).

The cost of production from micropropagated banana plants is high due to several factors, including technical grade sugar, agar, electricity and high labor cost (Kodym and Zapata- Arias, 2001). For mass propagation, mutation induced breeding, somatic embryo genesis, protoplast culture, and cell suspension culture, callus induction is the most vital phase among the in vitro studies on banana. Considering this issue, we tried to investigate the effect of organic supplements and activated charcoal for in vitro morphogenic responses of calluses of banana. It is evident that organic supplemented medium tends to show morphogenic responses as they are the sources of vitamins, amino acids, organic acids, alternative carbohydrates and growth substances (George et al., 2008).

In our study we chose Anupam (genome AAB), one of the popular cultivars of banana (Musa sapientum, Family: Musaceae) in Bangladesh. Locally it is also called Sabri or Malbhog. The plant is tall and bears bunch with an average weight of 10 kg. A bunch contains 85-120 fingers, fruits are medium-sized, firm in texture, sweet and tasty. The cultivar is widely grown in the north and western areas of Bangladesh. Objectives of our study were to select the suitable media composition for callus induction through using organic additives and activated charcoal.

Materials And Methods

Explant source and preparation. The experimental material was in vitro generated calluses of Musa sapientum cv. Anupam, which was induced from leaf and stem segments (0.8-1.0cm) cultured onto MS (Murashige and Skoog, 1962) medium containing 2,4-D (3.0 mg/l) and NAA (0.5 mg/l). In vitro grown microcallus were sub-cultured in Murashige-Skoog (MS) medium (Murashige, T. and F. Skoog, 1962) containing different concentrations (see detail in Results section) of organic supplements including biotin (w/v), peptone (w/v), casein hydrolysate (w/v), yeast extract (w/v), coconut water (v/v), activated charcoal (w/v), sucrose (3%) and agar (7%). Twenty replicates were inoculated for each treatment. The pH of all media was adjusted to 5.8 before autoclaving and media was sterilized by autoclaving for 20 min at 121[degrees]C and 1.05 kg/cm2.

Culture of explants. Cultures were maintained at 26 [+ or -] 2[degrees]C with a photoperiod of 16/8 h under an illumination of 20 imol [m.sup.-2][s.sup.-1] provided by cool white fluorescence lamps.

Data recording and analysis. Weekly visual observation of culture was made and data of callus growth were recorded as callus index number (CIN); its structure, morphology, color and texture was determined for 4 weeks and tabulated for detailed study. The callus index number refers to the product value of length (cm) and width (cm) of callus which indicates the area ([cm.sup.2]) of the callus and is expressed as Mean [+ or -] SEM.

Results:

Effect of different concentrations of Coconut Water (CW). In the present study, freshly collected coconut water (v/v) was added to MS media before autoclaving and sterile calluses were sub-cultured in MS media with different concentrations (5%, 10%, 15%, 20%) of coconut water. Highest average CIN (2.65) was observed with 15% coconut water after 28 days [Table 1] where maximum calluses were greenish in color, 100% fragile in texture, nearly 60% non-embryonic and with 70% root formation [Figure A and Figure B]. Induction of root from callus was increased with increased concentration of CW.

Effect of different concentrations of Casein Hydrolysate (CH). In this part of our experiment, induced micro-calluses were sub-cultured in MS media with different concentrations (100mg/L, 200mg/L, 300mg/L) of CH. Among them the highest average CIN (1.065) was observed at 200mg/L of CH after 28 days [Table 1]. Callus found in this composition were brownish in color (89%), loose in texture (100%), non-embryonic (100%) and 60% of the calluses showed root formation [Figure C]. All the cultured calluses were found to contain root when MS was supplemented with 300mg/l of CH [Figure D].

Effect of different concentrations of Biotin (Bn). To obtain the effect of Bn in callus culture, we subcultured the microcalluses in MS medium containing different concentrations of Bn (0.25mg/l, 0.5mg/l, 1.0mg/l, 5.0mg/l). After the 4th week of inoculation, Bn at 0.5mg/l showed that the average CIN was 1.225 where mostly the color was greenish [Figure E]. This composition yielded 60% fragile and all non-embryonic calluses. 60% of the calluses commenced roots at 0.25mg/l and 1.0mg/l of Bn [Table 1].

Effect of different concentration of Peptone (Pn). Highest average CIN (0.85) was observed at (0.1gm/L) peptone after 28 days when in vitro calluses were sub-cultured in MS media with different concentration (0.1gm/L, 0.3gm/L, 0.5gm/L) of peptone. In our experiment, the lowest concentration of Pn showed 50% greenish callus and 40% blackish in color, loose in texture (90%), non-embryonic (90%) along with 70% of root formation [Table 1]. Cultured calluses showed 100% and 70 % roots formation at 0.3gm/l and 0.5mg/l of peptone, respectively [Figure F].

Effect of different concentrations of Yeast extract (YE). YE was supplemented in different concentrations (100mg/L, 300mg/L, 500mg/L and 800mg/L) with MS medium for culturing the microcalluses. Among them, the highest average CIN (1.0) was observed at 300mg/L where 80% were non- embryonic, brownish in color (60%), loose in texture (60%) and 100% rhizogenesis occurred in this composition [Table 1, Figure G]. 100% loose in texture and non-embryonic structure was observed at 800mg/L yeast extract.

Effect of different concentrations of activated charcoal (AC). From previous reports, AC is added to culture medium so that it can either eliminate or decrease undesirable compounds and thus improve anticipated morphogenic responses of explants. In the present study it was observed that when AC was added at 0.20% and 0.50% the CIN was 0.85 and 0.94, respectively [Table 1] after 4 weeks of sub- culturing, and 20%-30% calluses were embryonic along with greenish color in these moderate concentrations of AC [Figure H]. The texture of the calluses was loose and no root formation was observed from the addition of AC in the medium.

Discussion:

Coconut water was shown to have cytokinin activity by Kuraishi and Okumura (1961). Cytokinins are a class of plant growth substances active in promoting cell division. The present study showed that CW has influence on morphogenic responses on callus of Musa sp. This additive not only increased CIN but also played role in rooting from calluses. Al-Khayri (2010) also demonstrated that adding CW stimulated date palm callus growth, enhanced and expedited somatic embryogenesis, and promoted growth of somatic embryos.

Casein Hydrolysate is produced from the acid hydrolysis of casein protein found in milk which is an excellent source of amino nitrogen and free amino acids (Bister-Mieletal., 1985). Addition of CH in medium has always been found to be a cheap source of amino acids (George et al., 2008) and is used in plant tissue culture. For instance, Won et al. (2011) established a suitable protocol for Elymus dahuricus L. where CH along with growth regulators showed good effects on callus induction. In the present study, it was found that this supplement plays a positive effect on callus growth and root organ development. Similarly, Abdel-Rahim et al. (1998) confirmed the promoting activity of CH on growth of date palm callus; in pea, Pisum sativum, Cardi and Monti (1990) found CH was important for callus production and Deeks et al. (2002) also observed that CH enhanced callus induction and proliferation from seeds of milestone, Arceuthobium tsugense. Biotin is also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin, discovered by Bateman in 1916 and which also plays a role as nitrogen source. EL-Shiaty et al. (2004) found that the most active medium for callus initiation and growth for Phoenix dactylifera L. Sewy cultivar was MS medium enriched with biotin. In a similar fashion, addition of Bn in this study was found be a good organic source for callus growth in Anupam. As peptone is the source of organic nitrogen we tried to examine its effect on callus growth of banana and found it gives moderate response, whereas in tobacco callus culture, addition of peptone in media resulted in a high stimulation in cell proliferation and biomass increase of callus (Parc, et al., 2007).

Like other natural complex additives in culture media yeast extract exerts variable influence on in vitro growth and morphogenesis in numerous plant species (George et al., 2008; Al- Khayri, 2011) as this is a source of vitamins, nitrogen, amino acids, peptides and carbohydrates. For example, Matsubara (1975) in cowpea, Vigna sinensis, showed that 1.0 g/l YE enhanced callus induction from hypocotyl and root explants. During investigating effect of YE, our results revealed that this complex nutrient source was effective for improving callus mass. However, the calluses were brownish in color, which might be the result of producing excess terpenoids and alkaloids. It has been reported that YE elicited phytoallexin accumulation in several plant species in vitro (Molnar et al., 2011)

Activated charcoal provides many advantages as shown in in vitro studies and reported to be supplemented during in vitro culture protocols for several plant species (Pan and van Staden, 1998; Hassan et al., 2009) for promoting embryogenesis (Chee and Tricoli, 1988), enhancing rooting (Dumas and Monteuuis, 1995), and to avoid or retard browning (Verdeil and Buffard-Morel, 1995). Our medium composition utilizing AC also demonstrated that it enhanced callus growth of banana. Moreover, Moller et al. (2006) in Pinus radiata demonstrated that differentiation rate of callus was also influenced by the addition of AC in the medium.

Conclusion:

It is concluded that organic supplements and activated charcoal has enormous and diverse effect on cell growth and organogenic response on callus of banana (M. sapientum cv. Anupam). The results of current study can play a role for in vitro research on organogenesis and cellular morphology because we found notable points from our experiment, as outlined below.

1. Callus growth varied with nutrient supplements.

2. Features of callus (color, texture and morphology) depended on culture medium.

3. Rhizogenesis can be achieved from addition of additives.

4. This protocol would be a fundamental step towards artificial seed production, breeding programs and for genetic manipulation.

5. Supplementations of organic growth adjuncts in culture medium is a simple, practical, and beneficial method to improve media used for commercial production.

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Mst. Rabeya Pervin, F. M. Safiul Azam, Md. Tanvir Morshed, Shiblur Rahman, Md. Khairul Anam Hero, Asaduzzaman, Mohammed Rahmatullah

Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1209, Bangladesh.

Corresponding Author: 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: rahamatm@hotmail.com

Table 1: Callogenic response of Anupam (Musa sapientum cv. Anupam)
when subcultured in MS medium supplemented with additives

Media
(MS + additives)                Color              Texture

Coconut water        5%         Greenish 90%       Fragile 80%
                     10%        Greenish 100%      Fragile 100%
                     15%        Greenish 100%      Fragile 100%
                     20%        Greenish 100%      Fragile 100%
Casein Hydrosylate   100mg/l    Brownish 63%       Loose 100%
                     200mg/l    Brownish 89%       Loose 100%
                     300mg/l    Greenish 60%       Loose 50%
Biotin               0.25mg/l   Blackish 50%       Loose 100%
                     0.50mg/l   Greenish 80%       Fragile 60%
                     1.00mg/l   Greenish 70%       Loose 70%
                     5.00mg/l   Brownish 40%,      Loose 100%
                                Greenish 60%
Peptone              0.1mg/l    Greenish 50%       Loose 90%
                                Blackish 40%
                     0.3mg/l    Peel yellow 50%    Loose 100%
                                Brownish 40%
                     0.5mg/l    Brownish 50%       Loose 100%
Yeast Extract        100mg/l    Brownish 90%       Fragile 60%
                     300mg/l    Brownish 60%       Loose 60%
                     500mg/l    Brown 80%          Loose 100%
                     800mg/l    Brown 70%          Loose 100%
Activated Charcoal   01.%       Greenish 40%       Fragile 50%,
                                Brownish 40%       Loose 50%
                     0.2%       Greenish 50%       Fragile 60%
                     0.5%       Greenish 60%       Loose 78%
                     1.0%       Peel yellow 56%    Loose 100%

                                Average Callus Index
Media                           Number (CIN)
(MS + additives)                (Mean [+ or -] SEM)

Coconut water        5%         2.05 [+ or -] 0.24
                     10%        1.50 [+ or -] 0.17
                     15%        2.65 [+ or -] 0.25
                     20%        2.38 [+ or -] 0.34
Casein Hydrosylate   100mg/l    0.78 [+ or -] 0.13
                     200mg/l    1.06 [+ or -] 0.12
                     300mg/l    0.84 [+ or -] 0.16
Biotin               0.25mg/l   0.65 [+ or -] 0.08
                     0.50mg/l   1.23 [+ or -] 0.21
                     1.00mg/l   0.90 [+ or -] 0.19
                     5.00mg/l   0.50 [+ or -] 0.08
Peptone              0.1mg/l    0.85 [+ or -] 0.11
                     0.3mg/l    0.65 [+ or -] 0.13
                     0.5mg/l    0.70 [+ or -] 0.10
Yeast Extract        100mg/l    0.68 [+ or -] 0.11
                     300mg/l    1.00 [+ or -] 0.08
                     500mg/l    0.90 [+ or -] 0.07
                     800mg/l    0.65 [+ or -] 0.10
Activated Charcoal   01.%       0.70 [+ or -] 0.08
                     0.2%       0.85 [+ or -] 0.13
                     0.5%       0.94 [+ or -] 0.12
                     1.0%       0 78 [+ or -] 0 08

Media
(MS + additives)                Morphology

Coconut water        5%         NE 100%, R 50%
                     10%        NE 78%, R 67%
                     15%        NE 60%, R 70%
                     20%        NE 100%, R 90%
Casein Hydrosylate   100mg/l    NE 88%, R 88%
                     200mg/l    NE 100%, R 60%
                     300mg/l    NE 100%, R 100%
Biotin               0.25mg/l   NE 100%, R 60%
                     0.50mg/l   NE 100%
                     1.00mg/l   NE 90%, R 60%
                     5.00mg/l   NE 100%, R 30%
Peptone              0.1mg/l    NE 90%, R 70%
                     0.3mg/l    NE 90%, R 100%
                     0.5mg/l    NE 100%, R 70%
Yeast Extract        100mg/l    NE 90%, R 50%
                     300mg/l    NE 80%, R 100%
                     500mg/l    NE 100%, R 40%
                     800mg/l    NE 100%
Activated Charcoal   01.%       NE 100%
                     0.2%       NE 80%
                     0.5%       NE 67%
                     1.0%       NW 100%

NE: non-embryonic; R: root.
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Title Annotation:AAB genome, Sapientum subgroup). (Original Article
Author:Pervin, Mst. Rabeya; Azam, F.M. Safiul; Morshed, Md. Tanvir; Rahman, Shiblur; Hero, Md. Khairul Anam
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9BANG
Date:Apr 1, 2013
Words:3701
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