Role of benzyladenine in regeneration of mulu bebe banana planlets in vitro

Banana is a type of local fruit that has the potential to become a leading commodity. Mulu Bebe bananas are local bananas in Indonesia originating from North Maluku. The lack of readily available sources of seed sources restricts banana cultivation. Tissue culture techniques are expected to produce seeds en masse relatively quickly. The study aims to determine the effect of various concentration levels of appropriate BA growth regulators in regenerating mulu bebe banana plantlets in vitro. The research was conducted at the Laboratory of Bio-Science and Plant Reproductive Biotechnology, Teaching Industry, Faculty of Agriculture, Hasanuddin University, Makassar. The study was designed using a completely randomized design (CRD) which consisted of 3 treatment levels, namely BA (0 ppm, 3.5 ppm, and 7 ppm). Further tests were carried out with the Honest Significant Difference (HSD)/ Tuckey 0.05 test. The results showed that treatment with BA administration at various concentrations had a significant effect on the number of shoots and number of leaves but had no significant effect on root number.


Introduction
Banana is a very popular tropical fruit in Indonesia and has high social, economic, and cultural values in various regions of the world [1].Indonesia is one of the countries that have a fairly high diversity of banana varieties so many types of wild bananas are found which are one of the richest genetic resources (Sutanto et al., 2000 in [2]).The potential for abundant germplasm is well-developed for banana cultivation, especially local bananas.Mulu bebe bananas are local bananas originating from North Maluku, precisely in West Halmahera.This banana has a characteristic that is a large fruit section at the base and gets smaller or conical towards the end which resembles a duck's mouth [3].
This banana is widely known by the people of North Maluku and based on data this banana has not been found in other areas of Indonesia.This indicates that the Mulu Bebe banana is a typical banana originating from North Maluku.However, the availability of Mulu Bebe banana seeds still has problems with the supply of seeds and the quality of the seeds is not sufficient to meet the needs of the community the cultivation method is still conventional so the number of seeds produced is limited.An alternative to providing high-quality banana seeds in large quantities, consistently and in a short period of time is in vitro culture technology [2,4].
The micropropagation technique, which allows banana seeds to be turned into intact plants in vitro and planted in the field, takes 5-8 months, depending on the vitality of the plant [5].Tissue culture is a fast vegetative propagation method and genetically the characteristics of the plants produced will be the 1230 (2023) 012128 IOP Publishing doi:10.1088/1755-1315/1230/1/012128 2 same or identical to the parent [6].Today, plant tissue culture technology has become a powerful tool for researching and solving fundamental and applied problems in plant biotechnology [7,8].In vitro propagation, both exogenous and endogenous growth regulators are very influential at the multiplication stage.In the multiplication stage, exogenous growth regulators belonging to the cytokinin group play a role in the processes of cell division, organogenesis, and sprout formation [9].
In tissue culture, different explant responses to media formulations are often found [10].Growth regulatory substances are very important to control the processes of organogenesis and morphogenesis [11].Cytokinins that are often used to accelerate shoot growth are BAP/BA (6-benzyl amino purine/6benzyladenine) and kinetin (6-furfurylaminopurine) which are growth regulators of the cytokinin class which have been widely used in tissue culture.The study aims to determine effect of the concentration of growth regulator BA (Benzyladenine) concentration on shoot number, leaf number, and root number in Mulu Bebe banana using tissue culture techniques or in vitro.

Materials and methods
The research was conducted at the Laboratory of Bio-Science and Plant Reproduction Biotechnology, Faculty of Agriculture, Hasanuddin University, Makassar.The time for conducting this research starts from May 2021 to March 2022.The planting material used as a source of explants was young shoots on healthy Mulu Bebe banana weevils with a height of about 15 to 20 cm.Planting material was obtained from Ternate, North Maluku.The buds as explants were removed from the stump using a knife and cleaned off the remaining adhering soil then peeled and scaled down to 1 x 5 cm in size.then washed using detergent and rinsed with running water.After cleaning the explants, the explants were peeled and cut several times, then soaked in sterile distilled water for one hour.Then the explant material was successively sterilized in fungicide (Dithane-M45) 2 g/l for 1 hour, bactericidal (Agrept) 2 g/l for 1 hour, 70% alcohol for 1 minute, Bayclin (NaOCl) 5.25%) 30% for 15 minutes and 20% bayclin for 10 minutes.Rinse with sterile water after each stage of the sterilization.The explants were then isolated from the plant material, then the midrib was peeled to reveal a white apical bud with a size of 1 cm.The sterilized explants were planted in preconditioned media for 4 weeks.After 4 weeks of planting in the preconditioning medium, young shoots will grow which will be used as new planting material for shoot multiplication, after which the explants can be subcultured to the treatment medium.Explant cultures were maintained in an incubation room at 25 °C under TL (tube luminescent) light with a light intensity of 1000 to 2000 lux and a photoperiod of 16 hours per day.
The growth medium used was the composition of MS media (Murashige & Skoog, 1962), 8 g/l agar, and 30 g/l sucrose.The acidity is regulated using NaOH or HCl up to a Ph of 5.8 and a growth regulator of the cytokinin BA (Benzyladenine).The experiment was carried out using a completely randomized design (CRD) which consisted of 3 treatment levels and was repeated 3 times, viz Benzyladenine (BA) (0 ppm, 3.5 ppm, 7 ppm).The observed variables included the number of shoots, the number of leaves, and the number of roots.Obtained observational data were analyzed based on the F test (ANOVA) with a confidence level of 0.05% and 0.01%.If there is a significant difference in the BA concentration treatment in terms of variance, then proceed with the 5% BNJ test.

Number of shoots
Mulu bebe banana sterile shoot explants initiated from weevils showed a response in the form of changes after incubation in media with BA treatment in vitro.At first, the base of the explants seemed enlarged and changed color to green.The concentration of BA given to the explants was able to trigger adventitious shoot induction of the cultured Mulu Bebe banana explants.The growth of mulu bebe banana shoots at various concentrations of BA is shown in Figure 1.
Based on observational data and variance, it shows that the BA treatment had a significantly different effect on the number of shoots of mulu bebe bananas (Table 1)..39 a Note: The mean value followed by the letters (a,b,c) in the same column shows a significant difference according to the BNJ test at the 5% level.The results of variance are in Table 1.Shows that the treatment of different concentrations of Benzyladenine (BA) in MS basal medium has been shown to affect the development of shoot number, showing different responses to Mulu Bebe banana shoot formation.BA 7 ppm produced the highest number of shoots compared to BA concentration levels of 3.5 ppm and 0 ppm (Figure 1).This shows that BA is included in the cytokinin class which can encourage shoot proliferation.According to Wattimena (1992) in [12], the physiological role of cytokinins such as BAP and BA is to promote cell division, morphogenesis, budding, and chloroplast formation.
The results of research by Apriani et al. (2016) on "kusto" bananas, namely the highest shoot proliferation were obtained in media with the addition of 7 mg L -1 BA [13].Sipen and Davey (2012) also reported that 7 mg L -1 BA maximized the average number of shoots in 'Awak' bananas [14].This shows that the administration of BA treatment with high concentrations was also followed by an increase in the number of shots.The higher the concentration of cytokinins administered to the plant, the higher number of shoots [5].The large number of shoots formed on plantlets is due to a balance between exogenous growth regulators and explants to stimulate the emergence of new shoots and produce large amounts of shoots.In the treatment without BA, shoots formed could be caused by stimulation from the media [15].
The Number of different shoots may also be influenced by the nutrient uptake capacity of explants in MS medium, certain growth regulators and sufficient levels of endogenous cytokinins within plant cells.MS medium has macro and micronutrients as well as carbohydrates and vitamins.The specialty of MS medium is its high nitrate, potassium, and ammonium content so that nutrients for plants can be fulfilled [16].The need for the type and concentration of cytokinins to stimulate shoots is specific for certain varieties.Gahan and George (2008) state that the most suitable growth regulators for in vitro propagation are often specific to a particular species or cultivar within a species [17].

Number of leaves
In tissue culture, the emergence of leaves occurs after the formation of shoots on explants.Based on the test of variance, it was found that the BA treatment had a significantly different effect on number of leaves mulu bebe banana plantlets (Table 2).The results of the variance in Table 2 show that the BA treatment with a concentration of 7 ppm produced a higher number of leaves, viz 4.39 leaves compared to the 3.5 ppm treatment which produced 3.11 leaves, and the treatment without BA gave an average number of leaves 2.72 leaves (Figure 2).The number of Mulu Bebe banana leaves is directly proportional to the number of shoots (Table 2), where the more shoots the greater number of leaves.Tilaar and Sompotan (2007), Banana 'Barangan' reported that non-BA treatments at 7.5 ppm and 10 ppm produced most leaves, but no leaves were formed at 12.5 ppm treatment because the concentration was too high [15].While Hossain et al. (2016), the 'Grand Naine' and 'Amritasagar' bananas yielded the highest number of leaves with in vitro multiplication when using 5.0 mg/l BA and decreased when using 6.0 mg/l BA [18].The application of BA is considered very good for the propagation and growth of various bananas in vitro at certain opti-mum concentrations, but the application of BA for the propagation is different for each type of banana because it depends on the need for different types and concentrations for each variety.Shirani et al. (2009) found that the shoot proliferation rate was greatly affected by the type and concentration of cytokinins and the type of banana cultivar [19].The addition of 7 ppm BA in MS media made a good contribution to the number of mulu bebe banana leaves related to the function of BA itself in encouraging cell division and the process of organogenesis in micropropagation.Roots can absorb nutrients in the culture media so that they can be used for plant growth, including leaf formation.Lakitan (1996) reported that cytokinins translocated from roots could stimulate leaf growth so, high concentrations of cytokinins could increase leaf growth [20].

Number of Roots
Based on observational data and variance, it was shown that the BA treatment had no significantly different effect on number of roots mulu bebe bananas (Table 3).Variance results showed that the BA treatment at a concentration of 7 ppm (Figure 3) yielded a higher number of roots, namely 14.44 roots compared to the 3.5 ppm treatment and the 0 ppm treatment.According to Yatim (2016), the more roots there are, the greater the surface area for uptake of nutrients from the medium, so the higher the number of roots, the better the uptake of nutrients from the medium.In this case, it is known that the cytokinin treatment given is capable of producing roots even without the addition of a growth regulator in the form of exogenous auxin to the treatment medium [21].This is presumably because the explants of mulu bebe bananas already contain sufficient endogenous auxin so that they can stimulate the emergence of roots.Endogenous auxin has a role in root elongation and also MS media supports root growth and development.In Table 3.Each treatment tested showed a tendency for the ability of the explants to form roots and experienced an increase in each treatment given.It is known that explants that have formed shoots are mostly capable of producing roots.
According to Santosa and Nursandi (2004), the emergence of roots often occurs after the explants or tissues form shoots, then the shoots that are formed will stimulate root formation [22].Cytokinins can stimulate ethylene production under certain conditions, namely, ethylene stimulates the formation of adventitious roots by synthesizing injured plant parts and making them a place for the formation of adventitious roots on injured parts or tissues due to explant cutting activities [23].The results of Yatim's research (2016) gave the highest number of roots of the 'raja bulu' banana at a BAP concentration of 3.0 ppm with an average number of roots of 15.33 [21].Meanwhile, research from Khatun et.al (2017) on 'Sabri' bananas reported obtaining several roots of 5.20 in explants with a concentration of 3.0 mg/l BA and the addition of exogenous auxin in the form of IBA 1.0 mg/l, while at BA concentration of 5.0 mg/l with a combination IBA 0.5 mg/l obtained the lowest number of roots [24].In addition, shallot plantlets respond to root growth at BAP concentrations of 2.5-7.5 ppm [25].The success of the appli-cation of growth regulators is largely determined by the concentration used and the optimum concen-tration will vary between species, growth phases, and environmental conditions [21].

Conclusion
The addition of Benzyladenine (BA) at various concentrations had a significant effect on the regeneration of mulu bebe banana plantlets on several shoots and some leaves but had no significant effect on number of roots.A concentration of 7 ppm BA gave a good effect and was able to produce the highest number of shoots 1.39 shoots, 4.39 leaves, and 14.44 roots formed per plantlet.

Table 2 .
Effect of the role of BA cytokinins on the number of Mulu Bebe banana leaves in vitro.mean value followed by the letters (a,b,c) in the same column shows a significant difference according to the BNJ test at the 5% level.

Figure 3 .
Figure 3.The number of roots in the 7 ppm BA treatment.

Table 1 .
Effect of the role of BA Cytokinins on the number of shoots of Mulu Bebe Bananas in Vitro.

Table 3 .
Effect of the role of BA cytokinins on the number of Roots of Mulu Bebe Bananas in Vitro.Note: The mean value followed by the same letter (a), in the column shows that it is not significantly different according to the BNJ test at the 5% level.