Key findings:

The study identified that Kepok Kuning and Mas Kirana banana varieties responded best to tissue culture with 5 ml/l and 3 ml/l foliar fertilizer + 100 ml/l coconut water, respectively, showing significant growth in terms of leaf number, root development, and plantlet length, compared to other treatments.

What is known and what is new?

Indonesian local bananas are less visible in supermarkets, necessitating efforts to ensure their supply. Tissue culture is a common method for producing banana seedlings, typically using Murashige and Skoog media. This study introduces the use of foliar fertilizer and coconut water as substitutes for Murashige and Skoog media in tissue culture. It identifies optimal growth responses in banana plant varieties Kepok Kuning and Mas Kirana when using these substitutes, demonstrating significant improvements in leaf number, root development, and plantlet length.

What is the implication, and what should change now?

The findings suggest that using foliar fertilizer and coconut water as substitutes for traditional Murashige and Skoog media in tissue culture can enhance the growth of local banana varieties. This approach could lead to more efficient and cost-effective banana seedling production, promoting the availability of Indonesian local bananas in supermarkets. Agricultural practices should incorporate foliar fertilizer and coconut water in tissue culture protocols for banana seedling production. 

Banana plants are superior fruit plants in Indonesia. There are several types of bananas that are planted, one of which is the Kepok Kuning and Mas Kirana bananas which are generally developed by conventional Indonesian farmers. The availability of Kepok Kuning and Mas Kirana bananas in supermarkets is less visible, so it is necessary to make efforts to provide bananas. One of the methods that can be used to meet the needs of banana seedlings is tissue culture using leaf fertilizer and coconut water as a substitute for MS media (Murashige and Skoog). Tissue culture is a technique for growing plant parts such as cells, tissues and organs in culture media with complete nutritional content as well as controlled and aseptic culture conditions [1]. Tissue culture techniques have been widely developed and provide advantages in multiplying plants that are difficult to carry out conventional cultivation in a relatively short time and do not depend on the season.

The success of plant propagation through tissue culture is related to several factors. One of the factors that can influence the success of tissue culture is the condition of explants, growth media, and growth regulators [2]. The explants used are important in the continuity of plantlet growth by paying attention to the parts used, plant age, plant health, plant cleanliness, size and method of explant sterilization [3]. MS media (Murashige and Skoog) is a type of media that is often used in tissue culture which contains complete nutrients. However, the use of MS media is too expensive, so it is difficult for the community to obtain. So an attempt was made to replace some of the MS media components with cheaper components by taking into account the basic principles in tissue culture media [4].

Fulfillment of compositions such as macro and micro nutrients can be found in foliar fertilizers. Foliar manure is an alternative source of inorganic salts for plantlet growth in in vitro culture. Macro nutrient content N = 11%, P2O5 = 10%, and K2O = 6% while micro nutrients such as Fe, Mn, Zn, Cu, Co, B, Mo, Mg [5]. Sumihar et al (2021) [6], reported that the use of Growmore and Bayfolan fertilizers can be used as a culture medium in the propagation of feather plantains. The dosage used for Growmore fertilizer is 2 mg/l while for Bayfolan fertilizer it is 2 ml/l.

Growth hormone requirement in in vitro culture is an important component in tissue culture media. The use of growth hormone which is commonly used from chemicals is quite expensive and rarely found on the market, so it is done by utilizing organic materials which are relatively cheap and not difficult to find. Organic materials have the ability to stimulate the growth of explants [7]. Coconut water contains natural hormones such as cytokinins, auxins and several other compounds that can stimulate germination and growth [8]. According to Yunita (2016) [9] the content in coconut water is zeatin, zeatin gluoxide, zeatin riboside, glucose, protein, minerals, carbohydrates, vitamins, Ca and P. Mardhikasari, et al., (2019) [10], stated that the use of combined use with coconut water in MS culture media gave good results at concentrations of 50 – 100 ml/l. A single treatment of young coconut water with a concentration of 250 ml/l can produce leaves and roots faster in in vitro orchid culture (Phalaenopsis amabilis BL.).

The use of tissue culture media with substitute media for foliar fertilizer and coconut water at the right concentration is expected to result in good growth of Kepok Kuning and Mas Kirana banana plantlets at a relatively lower cost. This study aims to determine the optimal growth response of banana plantlets of the Kepok Kuning and Mas Kirana varieties on substitution media using leaf fertilizer and coconut water. 

The research was carried out by the Biotechnology Laboratory, Faculty of Agriculture, East Java "Veteran" National Development University from September to November 2022. The tools used in this study were LAF (Laminar Air Flow), autoclave, stove, tweezers, scissors, scalpel, measuring cup, measuring bottles, beakers, petri dishes, bunsen, pH meters, analytical balances, hot plates, magnetic stirrers, heating pots, stirrers, refrigerators, and culture racks. The materials used in this study were the Kepok Kuning and Mas Kirana banana plantlets, collections from CV. Megah Persada Nusantara, MS medium, sugar, aquadest, agar-agar, young coconut water, liquid leaf fertilizer (Bayfolan), 96% alcohol, 0.1 N HCL solution, 0.1 N NaOH solution, spiritus, betadine, stationery, tissue, plastic, rubber, and labels.

The design used in this study was a Completely Randomized Design (CRD) factorial with 2 treatment factors repeated 3 times. The first factor is the banana variety which consists of 2 varieties, namely Kepok Kuning and Mas Kirana. The second factor of substitution media consisted of 4 levels, namely Murashige and Skoog (M0), 1 ml/l foliar fertilizer + 100 ml/l coconut water (M1), 3 ml/l foliar fertilizer + 100 ml/l coconut water (M2), 5 ml/l foliar fertilizer + 100 ml/l coconut water (M3). So that there were 8 combination treatments. Parameters observed were number of leaves, number of roots and length of plantlets. Data were analyzed using variance (ANOVA). If the results of the analysis show significantly different treatments, then proceed with the Tukey test or Honestly Significant Difference (HSD) at the 5% level.

Total Leaves

The mean results in (Table 1) show that the banana variety Kepok Kuning aged 49 DAP grown on MS media and 1 ml/l substituted media and 3 ml/l foliar fertilizer + 100 ml/l coconut water was not significantly different on the number of leaves parameter, but on the media substitution of 5 ml/l foliar fertilizer + 100 ml/l coconut water was significantly different from other treatments. Bananas of the Mas Kirana variety grown on MS media were significantly different from other substituted media treatments in the number of leaves parameter, but were quite good in 3 ml/l foliar fertilizer + 100 ml/l coconut water media. It can be concluded that the Kepok Kuning variety is well grown on 5 ml/l foliar fertilizer + 100 ml/l coconut water media, while the Mas Kirana variety is well grown on Murashige and Skoog and 3 ml/l foliar fertilizer + 100 ml/l coconut water media. 

Substitution medium 3 ml/l foliar fertilizer + 100 ml/l coconut water aged 49 DAP used in the two varieties did not differ significantly in producing the number of roots. Meanwhile, the Murashige and Skoog treatment when compared with the 1 ml/l and 5 ml/l foliar fertilizer + 100 ml/l coconut water substitution media was significantly different in producing the number of leaves on the two banana varieties. It can be concluded that using Murashige and Skoog media, 1 ml/l, or 5 ml/l foliar fertilizer + 100 ml/l coconut water produced a good number of leaves for both banana varieties.

The Kepok Kuning variety aged 56 DAP grown on MS media when compared with 1 ml/l foliar fertilizer and 5 ml/l foliar fertilizer + 100 ml/l coconut water substitute media did not differ significantly in the number of leaves parameter. Meanwhile the Kepok Kuning variety grown on 3 ml/l foliar fertilizer + 100 ml/l coconut water media was significantly different from that grown on Murashige and Skoog media and 5 ml/l foliar fertilizer + 100 ml/l coconut water. The Mas Kirana variety grown on 1 ml/l, 3 ml/l, and 5 ml/l foliar fertilizer + 100 ml/l coconut water substitution media when compared to Murashige and Skoog media had a significant difference in the number of leaves parameter. It can be concluded that the Kepok Kuning variety is well grown on 3 ml/l foliar fertilizer + 100 ml/l coconut water media, while the Mas Kirana variety is well grown on Murashige and Skoog and 3 ml/l foliar fertilizer + 100 ml/l coconut water media.

Murashige and Skoog medium and 3 ml/l foliar fertilizer + 100 ml/l coconut water aged 56 DAP used in the two varieties were not significantly different in producing the number of leaves. Meanwhile, the 1 ml/l and 5 ml/l foliar fertilizer + 100 ml/l coconut water substitution media used in the two varieties were significantly different in producing the number of leaves. It can be concluded that using substitution media 1 ml/l and 5 ml/l foliar fertilizer + 100 ml/l coconut water produced the best number of leaves for both banana varieties.

Table 1. Mean Total Leaves from the Combination Treatment of Banana Varieties and Substitute Media

Note :     Numbers followed by the same letter in the same column are not significantly different in the HSD test     of 5%; MS = Murashige and Skoog; PD = foliar fertilizer; AK = coconut water; DAP = days after 

planting.

The large number of leaves in each treatment and the increase in each week is thought to be due to differences in the ability of the explants to absorb nutrients [11]. Culture media has an important role in the ongoing growth of banana plantlets. This is because the availability of the main macronutrients (N, P, and K) in foliar fertilizers can meet the needs of young plants that are actively compiling cell constituents. Nitrogen is needed by young plants [5]. Bayfolan foliar fertilizer contains 11% N. The availability of N in sufficient quantities can facilitate plant metabolism and ultimately affect the growth of other organs for the better [12]. Coconut water added to the culture media also contributed to plantlet growth. The addition of 100 ml/l of coconut water to the culture medium is the optimum concentration for the growth of shoots and leaves. Maninggolang, et al., (2018) [13] states that coconut water contains active substances for embryo development such as cytokinins. Cytokinins affect nucleic acid metabolism and protein synthesis which affect cell division and plant differentiation.

Number of Roots

The mean results in (Table 2) show that the treatment of banana varieties and substitution media gave a real interaction with the number of banana plantlet roots. Treatment combination interactions occurred at the age of 42 DAP to 56 DAP. Bananas of the Kepok Kuning variety aged 42 DAP grown on Murashige and Skoog media and substituted media were not significantly different in terms of the number of roots, however they were quite good when planted on 3 ml/l foliar fertilizer + 100 ml/l coconut water media. Bananas of the Mas Kirana variety grown on Murashige and Skoog media were significantly different from other substituted media treatments in terms of number of roots, but were quite good in 3 ml/l foliar fertilizer + 100 ml/l coconut water media. It can be concluded that the banana variety Kepok Kuning is good for planting on 3 ml/l foliar fertilizer + 100 ml/l coconut water media, while the banana variety Mas Kirana is good for planting on Murashige and Skoog media and 3 ml/l foliar fertilizer + 100 ml/l coconut water. Substitution media 1 ml/l, 3 ml/l, and 5 ml/l foliar fertilizer + 100 ml/l coconut water aged 42 DAP used in the two varieties were not significantly different in producing the number of roots. Meanwhile, the Murashige and Skoog media treatment used in the two banana varieties was significantly different in producing the number of roots. It can be concluded that the use of Murashige and Skoog media and 3 ml/l foliar fertilizer + 100 ml/l coconut water produced a good number of roots for both banana varieties. 

Bananas of the Kepok Kuning variety aged 49 DAP grown on Murashige and Skoog media and substituted media did not differ significantly in the number of roots, but were quite good when planted on 5 ml/l foliar fertilizer + 100 ml/l coconut water media. Bananas of the Mas Kirana variety grown on Murashige and Skoog media were significantly different from those treated with 3 ml/l foliar fertilizer + 100 ml/l coconut water substitution medium in terms of the number of roots. It can be concluded that the banana variety Kepok Kuning is good for planting on 5 ml/l foliar fertilizer + 100 ml/l coconut water media, while the banana variety Mas Kirana is good for planting on Murashige and Skoog media and 3 ml/l foliar fertilizer + 100 ml/l coconut water. Substitution media 1 ml/l, 3 ml/l, and 5 ml/l foliar fertilizer + 100 ml/l coconut water aged 49 DAP used in the two banana varieties were not significantly different in producing the number of roots. Meanwhile, the Murashige and Skoog media treatment used for the two varieties was significantly different in terms of the number of roots produced. It can be concluded that the use of Murashige and Skoog media and 3 ml/l foliar fertilizer + 100 ml/l coconut water produced a good number of roots for both banana varieties. 

Bananas of the Kepok Kuning variety aged 56 DAP grown on Murashige and Skoog media and substituted media were not significantly different in terms of the number of roots, however they were quite good when planted on 3 ml/l foliar fertilizer + 100 ml/l coconut water media. Bananas of the Mas Kirana variety grown on Murashige and Skoog media were significantly different from those treated with 3 ml/l foliar fertilizer + 100 ml/l coconut water substitution medium in terms of the number of roots. It can be concluded that the banana variety Kepok Kuning is good for planting on 3 ml/l foliar fertilizer + 100 ml/l coconut water media, while the banana variety Mas Kirana is good for planting on Murashige and Skoog media and 3 ml/l foliar fertilizer + 100 ml/l coconut water. Substitution media 1 ml/l, 3 ml/l, and 5 ml/l foliar fertilizer + 100 ml/l coconut water aged 56 DAP used in the two varieties were not significantly different in producing the number of roots. Meanwhile, the MS treatment used for the two varieties was significantly different in terms of the number of roots produced. It can be concluded that the use of Murashige and Skoog media and 3 ml/l foliar fertilizer + 100 ml/l coconut water produced a good number of roots for both banana varieties. 

Table 2. Average Number of Roots from the Combination Treatment of Banana Varieties and Substitute Media

Note :     Numbers followed by the same letter in the same column are not significantly different in the HSD test 

of 5%; ns = not significant; MS = Murashige and Skoog; PD = foliar fertilizer; AK = coconut water; 

DAP = days after planting.

The increase in the number of local banana plantlet roots occurred after the appearance of the leaves. Plantlets whose roots have emerged will be more optimal in absorbing nutrients from the media. Argues that the existence of roots for plant growth plays an important role as the absorption of nutrients and water, besides that it also plays a role as a support for the upright growth of a plant. Culture media in tissue culture also has an important role in the ongoing growth of banana plantlets. The content of Bayfolan foliar fertilizer itself has a N content of 11%. The availability of N in sufficient quantities can facilitate plant metabolism and ultimately affect the growth of other organs for the better [12]. Mardiana (2019), stated that the treatment of using super grow fertilizer on Barangan banana plants with a concentration of 1.50 ml/l was able to bring out the best root start.

Coconut water added to the culture media contributes to plantlet growth. The addition of coconut water to the substitution medium of 100 ml/l is the optimum concentration for the growth of shoots and leaves. This is due to the addition of sucrose into the media (Djajanegara, 2010) [14]. Kristina dan Syahid (2012) [15] stated that young coconut water also contained 14.11 mg/100 ml of potassium, 4.67 mg/100 ml of calcium, and 43.00 mg/100 ml of nitrogen. Coconut water contains ZPT which is used in tissue culture to increase callus initiation and root development [16]. Menurut Darlina et al., (2016) [17] Hormone analysis showed that young coconut water contains the hormones gibberellin (0.460 ppm GA3; 0.255 ppm GA5; 0.053 ppm GA7), cytokinins (0.441 ppm kinetin; 0.247 ppm zetin), and auxin (0.237 ppm IAA).

Figure 4.2. Kepok Kuning Banana Planlet Roots (a – d) and Mas Kirana (e – h) Subculture I on 

a) Murashige and Skoog media; b) 1 ml/l Foliar Fertilizer + 100 ml/l Coconut Water; c) 3 ml/l 

Foliar Fertilizer + 100 ml/l Coconut Water; d) 5 ml/l Foliar Fertilizer + 100 ml/l Coconut 

Water.

Planlet length

The mean results in (Table 3) the treatment of banana varieties showed that there were significant differences in the length of local banana plantlets. Based on (Table 3) the Kepok Kuning banana variety was significantly different from the Mas Kirana banana variety in the plantlet length parameter. The Kepok Kuning banana produced a plantlet length of 18.10 cm, while the Mas Kirana variety produced 12.22 cm. In the treatment of substitution media, Murashuge and Skoog media was significantly different from substitution media 1 ml/l, 3 ml/l, or 5 ml/l foliar fertilizer + 100 ml/l coconut water. Meanwhile, the 1 ml/l foliar fertilizer + 100 ml/l coconut water substitution medium was not significantly different from the 3 ml/l or 5 ml/l foliar fertilizer + 100 ml/l coconut water treatment. Murashige and Skoog media produced plantlet lengths of 27.81 cm, whereas substitution media 3 ml/l foliar fertilizer + 100 ml/l coconut water produced plantlet lengths of 12.96 cm.

The treatment of banana varieties has an average yield that is not much different, as well as the treatment of substitution media. Substitution media treatment had average results that were not much different from MS media (Murashige and Skoog) in the plantlet length parameter. This indicates that the substitute media used can be used as an alternative medium to replace MS media (Murashige and Skoog). This is because the availability of the main macronutrients (N, P, and K) in foliar fertilizers can meet the needs of young plants that are actively compiling cell constituents. Nitrogen is needed by young plants [5]. The content of Bayfolan foliar fertilizer itself has a N content of 11%. The availability of N in sufficient quantities can facilitate plant metabolism and can affect the growth of other organs for the better [12].

According to research results of Setiawati, et al., (2018) [18] stated that the application of 2 ml of Bayfolan foliar fertilizer was able to increase plant height in bean plants. In addition, at a concentration of 3 ml of foliar fertilizer, it was able to give the highest yield on the number of leaves of the bean plant. The addition of plantlet length can directly increase the number of leaves containing chlorophyll pigments that can be used in the process of photosynthesis. According to the statement Gustini, et al., (2012) [19] stated that the increase in plant length was thought to be due to the elements contained in Bayfolan fertilizer such as nitrogen which can stimulate the growth of the vegetative parts of the plant. In addition, the addition of organic growth regulator such as coconut water can contribute to the plantlet length parameter.

Coconut water added to the culture media also contributed to plantlet growth. The addition of coconut water to the substitution medium of 100 ml/l is the optimum concentration for leaf growth. Maninggolang, et al., (2018) [13] states that coconut water contains active substances for embryo development such as cytokinins. Cytokinins affect nucleic acid metabolism and protein synthesis which affect cell division and plant differentiation.

Table 3. Average Length of Plantlets Treated With Banana Varieties And Substitute Media

Note :     Numbers followed by the same letter in the same column are not significantly different in the HSD test 

of  5%; MS = Murashige and Skoog; PD = foliar fertilizer; AK = coconut water.

Figure 4.3. Kepok Kuning Banana Planlet Length (a – d) and Mas Kirana (e – h) Subculture I on 

a) Murashige and Skoog media; b) 1 ml/l Foliar Fertilizer + 100 ml/l Coconut Water; c) 3 ml/l 

Foliar Fertilizer + 100 ml/l Coconut Water; d) 5 ml/l Foliar Fertilizer + 100 ml/l Coconut 

Water.

Responses to the use of various varieties of bananas grown on substitution media gave different responses. Bananas of the Kepok Kuning and Mas Kirana varieties grown on substitution media of 3 ml/l of foliar fertilizer combined with 100 ml/l of coconut water can be used as alternative media for MS (Murashige and Skoog) in producing the number of leaves, number of roots, and plantlet length. 

ACKNOWLEDGMENTS

The author would like to thank my lecturers, Mrs. Dr. Ir. Sukendah, M.Sc and Mrs. Dr. Felicitas Deru Dewanti, S.P., M.P who have guided me during my research. The author also thanks Rika Rahmawati and Swakentunk friends who had supported and helped me during my research. 

Conflict of Interest: The authors declare that they have no conflict of interest.

Funding: No funding sources 

Ethical approval: The study was approved by the Institutional Ethics Committee of Universitas Negeri Makassar

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