In vitro multiplication of “Kepok” banana (Musa acuminata x Musa balbisiana) using different concentration of BAP and NAA in MS media

This study aims to determine the effect of different composition of BAP and NAA concentration added to Murashige and Skoog (MS) basic media on the in vitro multiplication of Indonesian “Kepok” banana variety. The research was conducted at the Tissue Culture Laboratory of Bonto-Bonto Horticultural Plant Seed Center. A completely randomized design (CRD) was employed with two factors tested. Four concentrations of each BAP and NAA used were 0 ppm, 4.5 ppm, 5 ppm, and 5.5 ppm for BAP, and 0 ppm, 2 ppm, 2.5 ppm, and 3 ppm for NAA resulted in 15 composition of BAP:NAA in addition to control (without the hormones). The results show no interaction between the BAP and NAA treatments, BAP concentration of 5 ppm gave the best result on parameter time to shoots emergence (7.50 DAP). NAA concentration of 2.5 ppm gave the best results on root emergence time (8.61 DAP), time to plantlet formation (11.72 DAP), number of roots (4.81 roots) and root length (5.93 cm).


Introduction
Banana (Musa paradisiaca) is a type of plant from Southeast Asia which has spread widely throughout the world, including Indonesia.Bananas have the potential as an agribusiness commodity where this commodity is cultivated in almost all countries with tropical and subtropical climates.Bananas are Indonesia's leading export commodities which are always encouraged in international cooperation.The need for horticultural commodities will continue to increase, including bananas.The development of agro-industrial processing of agricultural products including banana products will increase so that the need for raw materials also increases [1].
According to the Ministry of Agriculture in BPSP [2], banana production in Indonesia reached 8.18 million tons in 2020.The increase occurred in total banana production by 12.39% from 7.28 million tons in 2019.Based on the production level in 2018, Bananas are the fruit that is the biggest contributor to Indonesia's superior fruit commodities, amounting to 7.26 million tons.In addition, bananas are also the second largest foreign exchange earner after mangosteen with an export value of 30.38 thousand tonnes [3].
Indonesia as a banana-producing country has various local varieties of bananas, such as: pisang Ambon, Raja Sere, Kepok, Emas, and also non-local banana varieties, such as Cavendish bananas.Besides being consumed fresh, several banana cultivars are also used as raw materials for the banana processing industry, such as chips, sale, and flour.Currently kepok bananas are widely used as raw material for making banana chips so that they have the potential to be developed.On the other hand, the need for bananas for domestic needs has increased so that special attention is needed on the productivity of each cultivated banana plant [4].
Propagation of banana plants is generally done by planting saplings, or parts of the buds with buds.Procurement of conventional banana seeds produces a small number of tillers, so that the needs of banana plants cannot be met optimally.In addition, banana seeds originating from saplings and humps have the potential to carry pathogenic inoculums that cause various diseases, such as the fungus Fusarium oxysporum, Mycospharella fijiensis or bacteria that cause wilt.Another weakness that can be found from conventional banana seeds is non-uniform growth and the difficulty of providing healthy seeds in large quantities [5].
Bananas can be propagated by tissue culture techniques and this technique is expected to solve the problem of procuring banana plant seeds [6].In vitro plant culture is a fast method for plant propagation in an aseptic environment.This method can produce plants that are of high quality, have characters consistent with their parents, and are free from pests and diseases [7].However, constraints often encountered in efforts to cultivate kepok bananas in vitro are the slow process of growth of buds and changes in color to brown (browning) or black (blackening) and subsequently experiencing tissue death.
The process of slow bud growth can be overcome by adding appropriate plant growth regulators (PGR).The addition of PGR is an important factor, especially at the multiplication stage [8].Addition of cytokinin and auxins in the culture media can stimulate cell division and morphogenesis.Cytokinin such as Benzyl Amino Purine (BAP) has function to induce cell division, promote shoot proliferation and adventitious shoot differentiation from callus.The addition of auxins such as Naphthalene Acetic Acid (NAA) affected cell development and induced the formation of axillary roots and plant stem growth [9].In addition to adding PGR to tissue culture media, activated charcoal or carbon can also be added.Activated charcoal functions to absorb toxic compounds in the media secreted by plantlets, stabilizes the pH of the media, and can stimulate root growth by reducing the amount of light entering the media [10].
Study conducted by Utami [11] showed that the highest multiplication of Ambon Green banana shoots resulted from the 4.6 mg/L BAP treatment without NAA of 4.76 shoots per explant.Shoot multiplication ability tended to increase up to the sixth subculture, with an average number of shoots produced up to the sixth subculture of 39.8 shoots on MS + 2 mg/L BAP medium.On the other hand, a research conducted by Pamungkas [12] showed that application of 3 ppm BAP in the MS Medium resulted in the highest number of roots, namely 22.7.
Based on the description above, it is necessary to conduct research to study the effect of concentrations of BAP and NAA in kapok banana in vitro multiplication and to obtain the appropriate concentration for shoot and root growth.

Methodology
This research was carried out in the Tissue Culture Laboratory of UPT Horticultural Plant Seed Center Bonto-Bonto, Gowa Regency, South Sulawesi Indonesia from April to July 2022.Four concentration of BAP were used as the first factor consisted of Control, 4.5 ppm BAP, 5 ppm BAP, and 5.5 ppm, while second factor was the concentration of NAA consisted of Control, 2 ppm NAA, 2.5 ppm NAA, 3 ppm NAA.A completely randomized design (CRD) was used using 3 replications.

Source of plant explant
The planting material used was a banana weevil from the third multiplication which was then split into several parts with a size of ± 1 cm (figure 1).The planting material obtained from the the Tissue Culture Laboratory of UPT Horticultural Plant Seed Center Bonto-Bonto.

Sterilization
Tools and media were sterilized using an autoclave at 121ºC.Sterilization lasts 30 minutes for tools and 15 minutes for media.Planting tools such as tweezers and scalpels are sterilized again in laminar air flow using the combustion method using 96% alcohol.

Media preparation
Media used in the recent study was Murashige and Skoog (MS) media.The media was prepared using 16 treatment combinations, then the 1000 ml MS media was divided into 4 parts so that it became 250 ml.In the preparation of 7.5 g of sugar media, 1.75 g of agar and 0.5 g of activated charcoal were weighed using an analytical balance.MS media stock solutions (stocks A, B, C, D, E, F, and vitamins) with each treatment concentration then sugar, agar and activated charcoal were put into a beaker.
The media was then homogenized using a magic stirrer and placed on a hot plate until homogeneous and then heated to boiling, after which the pH of the media was measured.NaOH solution is added to the media if the pH of the media is too acidic and HCL solution is added to the media if the pH is too alkaline until it reaches a pH of 5.6-5.8.The media was removed and poured into culture bottles that had been prepared beforehand.The media was sterilized using an autoclave at 121ºC for 15 minutes.The sterile media was then placed in the media incubation room for 7 days to ensure that the media was not contaminated.

Planting
The explants were planted in laminar air flow (LAF) under aseptic conditions.Prior to planting, the LAF and planting tools placed was sterilized by spraying 70% alcohol and using UV light for 30 minutes.The explants used were removed from the bottle, after which the outer layer was removed in several layers, as well as the lower stump and shoots and split in 2 with a size of ± 1 cm then planted in the media that had been prepared previously, then the bottle was covered with plastic and tied with a rubber band and tightened with wrap.Following planting, the culture bottles were placed in the incubation room.

Data analysis
The data were analysed to determine the significance of the treatment using Analysis of Variance (ANOVA) for two factors factorial design.Whether there is a significant effect of the treatments followed by a Tukey's test at confidence level of 95%.

Effect of BAP and NAA on shoots and roots emergence
Analysis of variance showed that the BAP (A) and NAA (B) treatments had a significant effect on the time of shoot emergence, while the time of root emergence and plantlet formation were significantly affected by NAA.No significant interaction was found from the combined treatment of the two growth regulators.Time to shoot emergence on the kapok banana explants varied between BAP and NAA concentration (table 1).Addition of 5 ppm BAP and 2 ppm NAA resulted in the earliest shoot emergence of 7.50 days and 7.69 days, respectively.Root emergence was affected significantly by the NAA growth regulator than BAP.Similarly, plantlet formation also affected by this type of growth regulator.Use of 2.5 ppm NAA resulted in earliest root emergence and plantlet formation of 8.61 days and 9.75 days, respectively.

Effect of BAP and NAA on shoots growth
Analysis of variance showed that the concentrations of BAP (A), NAA (B) and their combinations had no significant effect on the shoot growth.No significant difference was found between treatments either in number of shoots formed or shoot height of the kapok banana (figure 2 and 3).   Figure 2 shows that the average number of shoots of the kepok banana plant in the combined treatment of BAP 5 ppm and NAA 2.5 ppm, tends to show the highest yields with an average number of shoots of 2.38.Meanwhile, the combination of 5.5 ppm BAP and 3 ppm NAA tended to produce the lowest average number of shoots, namely 1.33 shoots.Similarly, Figure 3 also shows that the average height of the kepok banana plant in the combined treatment of BAP 5 ppm and NAA 2.5 ppm, tends to show the highest yield with an average shoot height of 7.33 cm.Meanwhile, the combination of 5.5 ppm BAP and 3 ppm NAA treatment tended to show the lowest average shoot height with an average value of 6.27 cm (figure 4).

Effect of BAP and NAA on roots growth
Growth of plantlet roots were significantly affected by the growth regulator treatment added to the basic MS media.Analysis of variance showed that the concentrations of BAP (A) and NAA (B) had a significant effect on the number of roots, while root length was only affected significantly by NAA.No significant effect on the interaction of both growth regulation treatments on root growth.Number of root and root length varied between the concentration of BAP and NAA (table 2).A .
B .1.29 1.17 Values followed by the same letter are not significantly different based on Tukey's α=0.05; ns= not significant.The Tukey's test (α=0.05) in table 2 shows that the NAA treatment of 2.5 ppm resulted in highest average number of roots (4.81 roots), which was not significantly different from the treatment with NAA concentrations of 2 ppm and 3 ppm but significantly different compared to the control treatment (without NAA).Longest root obtained from the NAA treatment of 2.5 ppm (5.93 cm) which was not significantly different from the other treatments with NAA concentrations except from control treatment.Absence of BAP or NAA in the media can reduce the number of roots formed ad the length of root during in vitro multiplication.

Discussion
The results showed that the treatment with a combination of BAP and NAA had no significant effect on the growth of shoots and roots in the multiplication of kepok banana explants.This is thought to occur because the concentration of hormones given to the culture medium of banana kepok shoots is not optimal in spurring the growth of buds.In addition, the BAP and NAA given did not affect each other on the increase in the number of kepok banana shoots.The interaction and balance between growth regulators administered in the media and those produced by cells endogenously and the types of species A .

B .
and cultivars determine the direction of development of a culture [13].The need for growth regulators for the growth and development of banana plants depends on genetic characteristics and the concentration of auxins and cytokinin given [14].Different types of bananas will give different growth responses to the balance of auxin and cytokinin given to the media.
The addition of cytokinin which is higher than auxin in the planting medium will have an impact on the growth of shoots and leaves, if the concentration of cytokinin given is lower than auxin it will affect root growth.In this study it was found that there was no interaction between the treatments with the addition of BAP and NAA.It is suspected that this occurred because the two treatment factors could not produce an appropriate balance to stimulate the growth of kepok banana shoots and roots.In addition, the growth and development of plants in tissue culture is also influenced by interactions between endogenous hormones present in plant tissues and the addition of exogenous hormones.Interaction of auxin and cytokinin in the right balance will spur cell division and enlargement which leads to shoot morphogenesis [15].In addition, there is an interaction between the exogenous growth regulator in the media and endogenous growth regulator produced by cells that determines the direction of development of a culture [16].Application of excessive auxin to the culture media will cause an imbalance in the interaction with endogenous auxin and cannot produce a greater number of roots [17].Similarly, administration of high concentrations of auxin found to affects the formation of many plant roots but can inhibit root elongation [18].Higher administration of exogenous cytokinin hormones can have an impact on inhibition of shoot formation [19] and in turn causes stunted growth in plants [8].
The results showed that the addition of BAP had a significant effect on the time of emergence of shoots and the number of shoots and had no significant effect on the time of emergence of roots, the number of shoots, the height of shoots and the length of roots.The time of emergence of shoots had the best effect on BAP concentrations of 5 ppm (7.50 DAP), while the lowest effect was in the treatment without BAP administration (10.40 DAP).The number of roots gave the best effect on the BAP concentration of 5 ppm (4.67) while the lowest effect was in the treatment without BAP administration (3.31).This is presumably because the growth regulator added to the media can increase explant growth, as well as explant shoot growth which is influenced by the concentration of cytokinin given.This statement is in accordance with the opinion of Kasutjianingati and Boer [20], that one of the growth regulators that play a role in increasing shoots in banana explants is cytokinin.Cytokinin play a role in regulating cell division, organ formation, enlargement of cells and organs, and development of buds and shoots [21].
The time of emergence of shoots and the time of plantlet growth were influenced by the concentration of BAP given to induce shoot growth.The use of 5 ppm BAP in this study showed shoot growth which was classified as faster compared to other concentrations.This statement is in accordance with the opinion of Bella et al. [22] which states that the effect of concentration is the main factor in plant propagation to obtain optimal shoot emergence time.Administration of high concentrations of cytokinin can respond to slow shoot growth because the endogenous cytokinin content is sufficient.
The highest number of roots was found in the treatment without BAP (4.67) while the lowest number of roots was found in the BAP concentration of 5.5 ppm (3.31).Explants cultured on media without the addition of BAP already contain endogenous auxin to support plant root growth.In addition, the provision of cytokinin which are classified as low also affects the formation of plant roots.This is in line with the opinion of Rionaldi [8], stating that low cytokinin content is able to maximize cell division to form roots, and root emergence has occurred in explants that have been cultured and form shoots which will stimulate root formation.
Treatment with the addition of BAP did not significantly affect the number of shoots, shoot height and root length.This is presumably due to the addition of a high enough concentration of the hormone cytokinin in the form of BAP, so that it can produce an average number of shoots that are large.This is in line with the opinion according to Rainiyati et al. [14] that the higher the concentration of cytokinin given, the number of shoots formed will increase, but the formation of each shoot can be hampered.Determining the right concentration really needs to be considered in multiplication of banana shoots.
The results showed that the addition of NAA had a significant effect on the time of shoot emergence, root emergence time, plantlet growth time, number of roots and root length and had no significant effect on the number of shoots and shoot height.Giving NAA concentration of 2 ppm resulted in the fastest shoot emergence time (7.69 DAP) and at a NAA concentration of 2.5 ppm produced the fastest average plantlet growth time (9.75 DAP).This influence is suspected that the endogenous cytokinin in the explants are able to interact in achieving the right balance with the given exogenous auxin in stimulating shoot division and enlargement.So that explants are able to work by themselves in influencing the process of cell division.Relatively low concentrations of exogenous auxin can stimulate the induction of cell division and morphogenesis [23].
Giving NAA concentration of 2.5 ppm on explant growth resulted in the fastest root emergence time (8.61HST), the highest number of roots (4.81 roots) and the longest roots (5.93 cm).This is presumably due to the effect of giving NAA which has an effect on increasing the success of root growth and development.According to Arlianti et al. [24] that NAA is a type of hormone that is widely used in plants in the process of cell division, cell differentiation, influencing the process of growth and development of plant roots, and is a determining factor for success.root induction in explants.
Auxin is a hormone that can affect the growth of plant roots.NAA belongs to the auxin group used in this study.The use of NAA to stimulate root formation and elongation has also been widely used in several previous studies.The optimum concentration of NAA in inducing and elongating roots in each plant is different and is influenced by the balance between endogenous and exogenous hormones.This is in accordance with the opinion of Arlianti et al. [24] and Pamungkas [12] that the use of optimal concentrations is a determining factor for whether or not the explants are planted.The auxin concentration will reach the optimal point for the longest root growth in explants.The higher the addition of auxin to the culture medium will increase the number of roots but will inhibit explant root elongation.
Treatment with the addition of NAA did not significantly affect the number of shoots and shoot height.The addition of concentrations with various levels is thought to have not been able to increase the height of the shoots because the concentrations given were still not balanced with the needs of the explants.This is in line with the opinion of Saifuddin [25] that at the right concentration, growth regulators will have a good effect on shoot growth.Growth regulators given in concentrations that are too low, show results that are not so good.While giving concentrations that are too high will result in poisoning of plants and growth will be stunted, and can even cause death.
The average number and height of the shoots produced varied as shown in figure 2 and 4.This is presumably because the addition of growth regulators with various concentrations has not been able to stimulate the growth of plant shoots.At low to high concentrations, various plant heights were produced, presumably because the response of each explant had a different sensitivity.This is in accordance with the opinion of Pranata et al. [26] which states that data variations can occur because each explant has a different cell sensitivity to a given stimulus, such as a given exogenous hormone stimulus.

Conclusion
Based on the results of the research that has been done, it can be concluded that hormone treatment with a combination of BAP and NAA did not have a significant effect on the growth of shoots and roots of kepok banana explants; the 5 ppm BAP hormone treatment gave the best results on time to shoots appearance (7.50 DAP), and treatment of 2.5 ppm NAA hormone gave the best results on root emergence (8.61 DAP), plantlet growth time (9.75 DAP), number of roots (4.81 roots) and root length (5.93 cm).

Figure 1 .
Figure 1.Planting material A. Source of explants B. Splitting of explant sources C. Explants ready for planting.

Table 1 .
Average of time to shoot and root emergence and plantlets formation (days after planting) of Kepok banana.

Table 2 .
Average of number of root (roots) and Root length (cm) of Kepok banana.