Banana (Musa spp. AAA) is an important fruit crop of the Musaceae family, widely grown in developing countries, and is the second largest fruit crop in the world after citrus [10] Banana originated from the South East Asian region, where the greatest diversity of edible bananas are found [16]. Ethiopia is among the tropical countries where its vast areas are suitable for banana cultivation and has also the opportunity for exporting fresh banana fruits. Banana production in the country ranges from homesteads to large commercial plantations under rainfed and/or with supplementary irrigation conditions [3] The materials used for conventional propagation are corms, suckers, and sword suckers. Since on average only 5 to 10 suckers can be obtained per plant per year, the traditional clonal propagation method appears to be unable to supply the increasing demand for healthy planting materials of banana. Conventional vegetative multiplication of bananas has been found to express several negative impacts including, low production, the transmission of diseases, and poor preservation of original plant genetic material [12]. To augment conventional propagation and to avoid constraints imposed by some pathogens, in vitro approach has been considered due to its potential to provide genetically uniform, pest- and disease-free planting materials  . Propagation of bananas through in Vitro techniques has been reported by several authors using different explant sources as well as regeneration pathways [2]  [8], [13]  [7]. Shoot tip culture has been routinely used for the rapid clonal propagation of banana genetic resources since 1985. Micro propagated plants establish more quickly, grow more vigorously, have a shorter and more uniform production cycle, and produce higher yields than conventional propagules [15] Even though many reports are available on banana micropropagation through shoot tip culture, plants could exhibit great variation under in vitro conditions in terms of shoot establishment, shoot proliferation, and regeneration of shoots and roots because of several factors such as genotype, explant type, culture media composition, plant growth regulators (PGR) and culture environment [18] [20]. 

Therefore, this study was started to optimize quick and reproducible in vitro micropropagation protocol for three banana varieties grown in Ethiopia. The protocols mainly the composition of media that include concentrations and combinations of plant growth regulators are largely plant genotype-dependent and are also influenced by water quality and techniques used in different laboratories. For this purpose, the effects of different cytokinins and auxins at various concentrations on shoot initiation, multiplication and elongation, and in vitro rooting were studied. The effect of an acclimatization media mix on banana plantlets growth was also studied.

   II.            MATERIALS AND METHODS

1. Plant Materials

Three cultivated banana varieties in Ethiopia, namely Poyo, Grand naine andButuza were used as experimental materials. Sword explants were obtained from healthy-looking, field-grown varieties from Adami Tulu Agricultural Research Center (ATARC) banana propagation nursery. The pseudo stems at the lower parts of the suckers containing meristems were used as explants. The shoot tips were the starting materials. This study was conducted at the Plant Tissue CultureLaboratory of ATARC, Oromia, Ethiopia.

2)    Culture Media Preparation and Sterilization

The culture medium used for this study was a modified Murashige and Skoog (MS, 1962) basal medium. The required amount of macronutrients, micronutrients, and vitamins from respective stock solutions were dissolved in double distilled water along with 30 g/L sucrose and 7g/l agar-agar type-I. The final volume was made up to the required level with double distilled water and then divided into the required volume of treatments, to which amount of PGRs from stock solution was added in combinations at different concentrations. The pH was adjusted in all cases to 5.8 by using 1N NaOH and/or 1N HCL before autoclave and gelling with agar. The media was poured into washed and dried culture jars of 60 ml volume and then, capped and labeled properly. The medium was autoclaved at 1.2 KPa and 121ºC for 20 min and then cooled at room temperature before use. The autoclaved medium was kept on a shelf for three days (72hr)  to make sure that there is no microbial contamination

• Explant Preparation and Surface Sterilization

Explants were excised from young suckers of the three banana varieties. The superfluous corm tissue, roots, and leaf sheathes were trimmed and removed by a sharp knife. Explants were washed carefully in running tap water for 15min with a detergent solution (largo) to remove adherent soils. The leaf sheaths near the bases were again removed leaving the young leaves around the meristem until the shoot tip became about 1.5 cm in length. Then the explants were dipped in a 3gm/l solution of mancozeb fungicide for 10min and rinsed three times with autoclaved distilled water. The explants were then briefly sterilized with 2% sodium hypochlorite solution for 15min followed 70% ethanol for one minute. After rinsing three times with autoclaved distilled water, the explants were excised into final size (about 5 mm) under a laminar air flow hood

• Shoot Tip Culture Establishment

Surface sterilized explants were placed on an MS medium containing a combination of N6 benzyl aminopurine (BAP: 1.5 and 2.5 mg/l) and indole-3-acetic acid (IAA: 0.0 and 0.5 mg/l) for shoot tip initiation. The factorial treatment was 3x2x2 combination arrangements. Thirty culture jars with each jar having one shoot tip explant were cultured for each of the three varieties (30 shoot tips per treatment). The initiated cultures were incubated for 8 weeks aseptically at 25±2ºC under 16/8hr cool white, fluorescent lights. Data of shoot initiation were recorded four weeks after culture.

5)    In vitro shoot proliferation

The same MS medium supplemented with a combination of BAP (2.0, 2.5 3.0, 3.5, and 4.0 mg/l) and IBA (0.0, 0.2, 0.3, and 0.5 mg/l) was used for shoot proliferation. The experiment was laid out inCRD with three factorial combinations of five levels of BAP; four levels of IBA  and three levels of banana varieties (Poyo, Grandnaine, and Butuza) resulting in 5x4x3 factorial treatment structure. After 8 weeks of the initiation stage contamination-free shoots were decapitated and split longitudinally into two or more parts depending on vigor and thickness. Then, they were transferred to a multiplication medium. However, smaller shoots were not split; rather they were put three together in a culture vessel. The multiplication rate was studied by subculturing the shoots on MS media every 3 weeks for 5 cycles. Data on the number of shoots per explants, the number of leaves, and the length of shoots were carefully recorded during each sub culturing.

6)    In Vitro Rooting of Shoots

In the root development stage, well-grown shoots with expanded leaves were separated
and transferred singly to fresh rooting half MS medium with different concentrations of indole butyric acid (IBA) (0.5, 1.0, 1.5, and 2.0 mg/l). After 6 weeks of culture, and root growth,data on the number of roots per shoot, and length of roots (cm) were recorded. For each treatment, fourshoots in each jar (12 shoots per treatment) were lined up randomly in CRD with three replications.

7)    Acclimatization and Hardening of Plantlets

In vitro rooted plantlets were taken out of the culture jars and the roots were carefully
washed with water to remove all the traces of the rooting medium. Plantlets were disinfected with Ridomil (2.0 g/l) for 3min to prevent fungal infections before transplanting. Individual plantlets were then transferred into small polybags filled with sterile soil mixtures top forest soil, farm yard manure, and sand soil in 2:1:1/2 ratio. The soil mixtures were sterilized by drying oven at 180^oC for an hour.  Then, plants were later transferred to bigger polybags that were filled with top forest soil and manure in a ratio of 2:1. The hardened plants were finally transferred to the field and successfully established and evaluated for it fruit.

8)    Experimental Design and Data Collection

The treatments were arranged in a Completely Randomized Design (CRD) with factorial arrangements. In the initiation stage, the number of explants that survived and initiated shoots was recorded was expressed as a percentage. Explants were subcultured five times and after each subculture, the number of shoots per explant, number of leaves per shoot and shoot length for each Variety were counted.  In the rooting stage, leaf numbers (determined by counting all leaves per plant), and root numbers (determined by counting all roots per plant), were examined. In the acclimatization and hardening stage, the survival of transplanted plants (determined by counting all live plants), plant height, pseudo stem diameter, and leaf numbers was also examined.

• Data Analysis

In the present study, the treatments were the plant growth regulators (the cytokinin – BAP; and the auxins – IBA) with various concentration levels and the banana varieties at each micropropagation stage. Furthermore, survival percentages were calculated for acclimatization and hardening plantlets.. Experimental data were analyzed By R software using Multiple Analyses of Variance at 95% of confidence level. When F-Test showed statistical significance at p < 0.05 level, means were separated according to Duncan Multiple Range Test (DMRT) Procedure.

III.            RESULTS AND DISCUSSION

1.      Shoot Initiation

In the present study, in vitro culture of banana shoot tips resulted in hard meristematic ball-like structure in initiation media containing different concentrations of BAP and IBA. The cultured shoot tip turned brown in color from the initial creamy white a few days after inoculation. Four weeks later, the external leaf primordia of explants turned green and a globular hard coat mass grew from which adventitious plantlets were developed. Among the treatment combinations considered, the maximum shoot tip initiation response of 100% sprout was obtained from explants cultured on MS medium supplemented with 3.0mg/l BAP the Grandnaine banana variety. Likewise, poyo and Butuza varieties gave maximum shoot imitation on MS medium supplemented with 2.5mg/l BAP and 2.5mg/l BAP respectively (Table 1). A similar result has been reported by  replace references by number system like [1] [2] etcusing MS medium supplemented with 2.0 to 3.0 mg/l BAP. Cronauer and Krikorian (1984) and Vuylsteke (1998) also reported BAP as the most commonly preferred cytokinin used in banana tissue culture. Furthermore, Al-Amin et al. (2009) observed the color change of culture meristems to brown in 4 to 5 days and development of a green hard ball-like structure after 30 to 35 days of inoculation. Azam et al. 2009 found that cultured shoot tips were visible as a swelling and greenish color after 10–15 days of inoculation in MS media supplemented with different concentrations of BAP. Al-amin et al. 2010 observed a meristematic ball like structure in regeneration media containing different concentrations of BAP and NAA.

Table 1:  Effect of different concentrations of BAP along with IBA and BAP alone on shoot initiation of three banana varieties

2.      Invitro Shoot Multiplication

After 8 weeks of culture initiation, when shoots with at least one leaf emerged, shoots were transferred to multiplication medium (Figure 4). It has been reported that multiple shoots could be produced from sliced shoot tips of bananas and plantain [5]). Analysis of variance revealed that the interaction effects of Variety, BAP, and IBAwas significant (Variety * BAP * IBA = p < 0.05) on the number ofshoots per explant, average shoot length (cm), and the number of leaves per shoot. The interaction of genotype, BAP, and IBA indicated that all three factors are dependent on each other for in vitro shoot proliferation of banana varieties. The maximum number of shoot per explants were obtained on MS medium supplemented with a combination of BAP and IBA at concentrations of 3.5/0.0, 3.0/0.2, and 3.5/0.3 mg/l for Grand Naine, Poyo andButuza respectively(Table-3, figure 4,5 and 6). Adenine-based cytokinin particularly BAP is the most commonly preferred cytokinin to affect shoot multiplication rate in several Musa spp. [11]; [18] [23] stated the highest shoot number per explant (3.4) with 5.0 mg/L BAP+2.0 mg/L IBA. The formation of multiple shoots and buds in banana varieties were promoted by supplementing the reported with relatively high concentrations of cytokinins. With the increase in BAP concentration from 2mg/l to 3.5mg/l numbers of shoots/ explant increased were increased from 4.67 to 12.67, 5.0 to 7.3 and 6.67 to 8.0 inGrand Naine, Poyo andButuza respectively. The number ofshoots increased with the increase of BAPconcentration up to 3.5 mg/l and then decreased. [6] reported an increase of shoots with an increase of BAP hormone concentration up to 5mg/l and the decrease. It was experimental that all the explants of banana varieties did not behave similarly in vitro in terms of multiplication.

Table 2: ANOVA summary of the Effect of BAP and IBA on invitro Shoot Multiplication

 DF =Degree of freedom, SE=Standard Error, BAP = 6- Benzylaminopurine, IBA = Indole butyric acid,CV = Coefficient of variation, *=p≤0.05, **=p≤0.01,***=p≤0.001

Table 3: The effect of BAP and BAP on number of shoots per explant, Shoot
length and number of leaves per shoot

Figure 1: Grand naine shoot initiation on 3mg/l BAP          Figure 2: Poyo shoot initiation on 2.5mg/l BAP

Figure 3: Butuza Shoot Initiation on 2mg/l BAP

Figure 4:                Grand naine shoot proliferation on 3.5mg/l BAP        Figure 5 Poyo shoot multiplication on 3mg/l BAP and 0.2mg/l IBA

Figure 6 Butuza Shoot multiplication on 3mg/l BAP and 0.3mg/lIBA

3.     In Vitro Rooting of Shoots

ANOVA showed a significant (p≤0.05) effect of all main and interaction effects of varieties and, IBA on the number of roots per shoot, number of leaves per shoot, and shoot length in three Varieties indicating the interdependence of these factors in vitro root induction. Fine roots began to be induced from the basal portion of the shoots after 9-11 days in Grandnaine, while 10-13 days in Poyo and Butuza cultivars on treatments fortified with different concentrations. Rooting can be stimulated when individual shoots are transferred to a basal medium without any PGR
[15], [18]. However, auxins induce further root initiation in bananas [18].

The highest numbers of roots (8.0, 7.67, and 8.0) were observed on the medium supplemented with 1mg/l IBA, 1.5mg/l IBA, and 2mg/l IBA, for Grandnaine, Poyo, and Butuza respectively. While in the control medium produced 4.33, 3.67, and 4.0 roots per shoot for Grandnaine, Poyo and Butuza respectively. These results are in agreement with the findings of [14] who obtained 11 maximum numbers of roots per shoot on 1mg/l IBA for red banana variety. Similarly, [9] reported a maximum number of roots (7.80) in BARI Kola-4 on ½MS medium supplemented with 0.5 mg/l IBA. IBA is known to plays an important role in the formation and development of rooting. Root formation and plant regeneration with IBA has been reported by [1] and [11].

Table 4: Effect of IBA different concentration on in vitro rooting and root growth on half MS media

Figure 7: Grand naine rooting on 1mg/l IBA            Figure 8; Poyo shoot rooting on 1.5mg/l IBA

Figure 9: Butuza shoot rooting on 2mg/l IBA

4.      Acclimatization of three banana varieties

After the sufficient shoot and root development, the small plantlets were taken out from the culture vessel carefully without damaging any roots. Excess media around the root was washed off by running tap water to prevent further microbial infection. In vitro rooted plantlets were transferred into a plastic tray filled with sterile soil mixtures top forest soil, farm yard manure, and sand soil in 2:1:1/2 ratio. Then, plants were later transferred to bigger polybags containing top forest soil and manure in a ratio of 2:1.  The hardened plants were finally transferred to the field and successfully established for further evaluation.

1. CONCLUSIONS AND RECOMMENDATIONS
2. Conclusion

From the result obtained in the present study, it is concluded that the developed protocol
is helpful for rapid in vitro propagation of the banana planting materials and hence enhances the availability of healthy and true-to-type planting materials accordingly, the information below was obtained

For shoot tip culture initiation and establishment Grand nainePoyo and Butuza  varieties showed best performance on 3.0 mg/L BAP, 2.5 mg/L BAP and 2.0 mg/L BAP respectively, without IBA concentration

A combination of 3.0mg/LBAP + 0.2mg/LIBA was the best combination forshoot multiplication of Poyowhile 3.5mg/l BAP without IBA and 4.0mg/L BAP + 0.3 mg/l IBAwere the most selected combinations for Grand naineand Butuza respectively.

For in vitro rooting, half-strength MS semi-solid medium fortified with 1.0 mg/l IBA
alone was the best concentration for genotype Grandnaine, while half MS medium supplemented with 1.5 mg/L and 2.0mg/lIBA were the best growth regulator concentration for poyo and Butuza respectively.

• Recommendations

Based on the results of the present study, the following recommendations were made

In the future, it will be better to determine the optimum volume of liquid medium per a
given jar or flasks to develop efficient protocols for the above-mentioned varieties
using a Bioreactor system.

It is also recommended to optimize protocols for these varieties using another type
of plant growth hormone concentration and combination to get the best multiplication.

It is best to use an Automatic greenhouse for the primary acclimatization of banana plantlets under strictly controlled Relative humidity and temperature.

REFERENCES

1. Agastian P, Williams L, Ignacimuthu S(2006).. In vitro propagation of Justicia gendarussa   Burmf.–A medicinal plant. Indian J. Biotechnol.; 5(2): 246–248
2. Alango Kelta, Sunil T. Hajare and Akilu Banjaw(2018) . Studies on in vitro Micropropagation inBanana. Int.J.Curr.Microbiol.App.Sci 7(7): 3366-3375
3. Asmare Dagnew, Surafel Shibru, Abel Debebe, Alemshet Lemma, Lemma Dessalegn, BehailuBerhanu, Worku Beyene and Yulien Miguelez Sierra(2012). Micropropagation of Banana Varieties (Musa spp.) Using Shoot-Tip Culture.Ethiop. J. Agric. Sci. 22:14-25.
4. Bhosale U.P., Dubhashi S.V., Mali N.S. and Rathod H.P. (2011) In vitro shoot multiplication indifferent species of Banana. Asian Journal of Plant Science and Research, 1 (3):23-27.
5. Cronauer, S.S. and A.D. Krikorian. 1984. Multiplication of Musa from excised stemtips. Annals of Botany, 53:321-328.
6. Ferdous, M.H., Billah, A.A.M., Mehraj, H., Taufique, T. and Uddin, A.F.M.J(2015). BAP anIBA pulsing for in vitro multiplication of banana cultivars through shoot-tip culture. Biosci. Agric. Res., 3(2): 87-95.
7. J. Lohidas and D. Sujin(2015). Effect of Growth Hormones in the Micropropagation of Bananacv.Matti Plant Archives 15 (1), 307-314.
8. Kagera, A.G., G.R. Kagera, C.B.M. Kagera, I. Van den Houwe and R. Swennen. (2004).Rapid mass propagation and diffusion of new banana verities among small scale
   farmers in north-western Tanzania. African Crop Science Journal, 12(1):7-17
9. M.M.H. Molla1*, D. Khanam1, Z.A. Firoz2, S.P. Chakma3 S.C. Halder1 and M.A. Salam(2017). In Vitro Production of Banana Plantlets and their validation at Hill Valleys. SAARC J. Agri.,15(1): 67-76.
10. Madhulatha P et al (2004) Influence of liquid pulse treatment with growth regulators on in vitropropagation of banana (Musa spp. AAA). Plant Cell Tissue Organ Cult 76:189–192
11. Naika HR, Krishna V(2008).. Plant regeneration from callus culture of Clematis gourianaRoxb.–A rare medicinal plant. Turk. J. Biol.; 32(2):99–103.
12. Ngomuo, M., Mneney, E. and Ndakidemi, P (2014). The in vitro Propagation Techniques forProducing Banana Using Shoot Tip Cultures. American J. of Plant Sciences, 5, 1614
13. Philip Sipen and Michael R Davey(2012).. Effects of N6-benzylaminopurine and Indole AceticAcid on In Vitro Shoot Multiplication, Nodule-like Meristem Proliferation and Plant Regeneration of Malaysian Bananas (Musa spp.) Tropical Life Sciences Research, 23(2), 67–80
14. Preeti Rajoriya, Vivek Kumar Singh, Nancy Jaiswal, and Rohit Lall (2018). Optimizing the effectof plant growth regulators on in vitro micropropagation of Indian red banana (Musa acuminata). Journal of Pharmacognosy and Phytochemistry. 628-634
15. Robinson, J.C., C. Fraser and K. Eckstein. (1993). A field comparison of conventionalsuckers with tissue culture banana planting material over three crop cycles.
    Journal of Horticultural Science, 68:831-836.
16. Stover RH, Simmonds NW. Bananas. Longman, London. Vuylsteke D, De Langhe EA(1985). Feasibility of in vitro propagation of bananas and plantains. Trop. Agric. (Trinidad). 62: 323-328.
17. Tripathi, L. (2003). Genetic engineering for improvement of Musa production in Africa.African Journal of Biotechnology, 2(12):503-508.
18. Vulysteke, D.R. 1998. Shoot-tip culture for the propagation, conservation and distribution ofMusa germplasm. International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.
19. Y.A. Kaçar,, B. Biçen, İ. Varol, Y.Y. Mendi1,, S. Serçe3 and S. Çetiner. Gelling agents andculture vessels affect invitro multiplication of banana plantlets ((2010). Genetics and Molecular Research 9 (1): 416-424.
20. Yapa Mudiyanselage A.M. Wijerathna, Herath Mudiyanselage P.C. Kumarihami. Effects ofDifferent Hormonal Concentrations and Culture Medium on Multiplication and Rooting of Stage II Banana (Musa cavendishii) (2016). Not Sci Biol, , 8(1):69-72. DOI: 10.15835/nsb.8.1.9749.
21. Al-amin M, Karim MR, Amin MR, Rahman S, Mamun ANM. In vitro micropropagation ofBanana (Musa sp.)(2009). Bangladesh J. Agril. Res.; 34(4):645-659.
22. Azam FMS, Islam S, Rahmatullah M, Zaman A. Clonal propagation of Banana (Musa sp.) cultivar ‘BARI-1’ (AAA genome, sapientum subgroup)( 2010). Acta Hort.; 879.
23. Fahima Khatun, M. E. Hoque, Homayra Huq, Md. Adil,Kh. Ashraf-Uz-Zamanand MominulHaque Rabin. Effect of BAP and IBA on in vitro Regeneration of Local Banana Variety of Sabri(2017).. BJI, 18(1): 1-10