In vitro multiplication of Pothos tener shoots

South Sulawesi-endemic Pothos tener can be used as a raw material for medicine because it contains antimicrobial compounds. To date, plants have been collected directly from the wild, without any cultivation or propagation measures. This will threaten its continued existence. Micropropagation is an efficient method for propagating plants. Multiplication of shoots is an essential step in micropropagation. Explants can be cultured on media containing cytokinins to increase the number of shoots that serve as new plant material. This study investigated the effect of cytokinins on the in vitro multiplication of Pothos tener shoots. The growth medium utilized was MS media (1962), which was added with BAP, Kinetin, 2iP at concentrations ranging from 0 to 2.5 mg/l. Every treatment was administered 10 times. This research indicates that the rate of shoot multiplication increases as cytokinin concentrations rise. However, the best results were obtained on MS media added with 2 mg/l BA, as shoot height, number of shoots, and number of leaves were all increased. At a concentration of 2.5 mg/l, fewer shoots were produced, and their growth was stunted.


Introduction
Indonesia is one of the countries with the greatest Araceae diversity.Araceae species are distributed as follows: 297 in Kalimantan, 159 in Sumatra, 49 in Sulawesi, 22 in Bali and Nusa Tenggara, 67 in Java, 35 in Maluku, and 114 in Papua.The species diversity of the Araceae tribe is distributed across diverse habitats, including land and water.Locals consume aquatic plants of the genus Pothos from the family Araceae for food.According to ethnobotanical research conducted in India, all parts of the plant P. scandens L. are used for medicinal purposes.It is utilized by the people of Bangladesh to treat skin diseases, snake venom, diarrhea, cancer, and measles.Individually or in combination, the Kani/Kanikaran people of India use the leaves of P. scandens Linnaeus to treat wounds.In the interior of Sri Lanka, injured areas were treated with the leaves of Pothos sp.The leaves' decoction is used as an anticonvulsant and antiepileptic.The root is used as a treatment for asthma.[1] [2].
The study's findings demonstrated that the ethanol extract of P. scandens promoted wound healing [3].Aromatic ester isolates of hemiterpene glucosides, pothobanosides A, B, and C and phenylisobutanoids, pothobanol from Pothos chinensis have estrogenic/anti-estrogenic, antitumor, and antihistamine properties against two breast cancer cell lines.The P. scandens ethanol extract has anti-inflammatory properties.The 50% ethanol extract and water extract of P. scandens exhibited antiasthmatic potential [4}.The lipophilic extract of P. scandens has anti-inflammatory properties.The extract of n-hexane, 50% chloroform, and 50% ethyl acetate from the fruit of P. officinalis contains flavonoid and phenolic compounds that are responsible as antioxidants.It is believed that the steroids, flavonoids, and terpenoids in the fruit of P. officinalis are responsible for the antiinflammatory and analgesic activity of the ethanol extract.P. scandens is composed of carbohydrate, mineral oil, protein, alkaloids, catechins, coumarins, tannins, saponins, flavonoids, phenols, and xanthoprotin.P. chinensis contains flavonoids, alkaloids, coumarins, terpenes, lignanoids, and organic acids [5].
This resource is extracted directly from the natural environment, threatening its sustainability.To preserve the viability of plants, an efficient propagation technique is required.The technique for propagation is in vitro culture.This technique can shorten the time required to produce plants of sufficient quality and quantity to meet the needs without harming the environment.
This technology can be used to produce uniform and high-quality seeds, and plants can be propagated at any time of year, regardless of the season.The recommended regeneration system for producing plantlets through in vitro culture is direct formation from plant organs (also known as "direct organogenesis").In vitro propagation consisted of the phases of shoot induction, shoot multiplication, shoot elongation, and root induction.The multiplication of shoots is a crucial step in plant micropropagation because it determines the number of plants that will be produced.Several factors influence the rate of shoot multiplication in vitro, including the composition of the medium, the concentration and type of growth regulator, the type and size of explants, and the explants themselves.[6][7].
The presence of growth regulators in the medium is a significant factor in determining the success of each stage of shoot propagation.Growth regulators play a crucial role in regulating the biological processes of plant tissues [8] [9].Its role, among others, regulates the growth rate of each tissue and unites the parts that make up the plant.The activity of growth regulators depends on the type, chemical structure, concentration, plant genotype and plant physiological phase [10].
Cytokinins are growth regulators that stimulate cell division and organ formation [11].Cytokinins like BA, Kinetin, and 2iP are frequently used as growth regulators for shoot multiplication.This research aims to identify the optimal cytokinin types and concentrations for Pothos tener shoot multiplication.

Materials and Methods
This study was conducted at Genomics Laboratory, National Research and Innovation Agency.The plant material utilized in this study was a one-week-old MS culture of Pothos tener.The used parts were in vitro shoots with four leaves and two internodules.This study's experimental design was a factorial Completely Randomized Design (CRD).The first variable is the cytokinin type (BA, Kinetin, 2-iP), and the second is the auxin concentration (0; 0.5; 1; 1.5; 2 and 2.5 mg/l).The obtained combinations consisted of 18 experimental units with ten replications.MS media with 3% sucrose and 0.8% agar was used as the basic medium, and the pH was adjusted to 5.8 with 0.1 N KOH or HCl.The observed parameters were number of shoots, length of shoots, number of leaves, and number of roots.Four weeks after planting, the parameters of the culture were observed.The cultures were incubated in a culture room for 16 hours per day under bright conditions (with a light intensity of 1000-1400 lux) and a temperature of +25 o C.

Number of shoots
Four weeks after cultivation, the number of shoots was determined through observation According to statistical analysis, the interaction between treatments significantly affected the number of shoots.As an indicator of the activity's success, the number of produced shoots can be counted.Increasing the concentration of cytokinins led to an increase in the number of shoots, as shown in Table 1.The best treatment was 2 mg/l BA, which resulted in 4.4 shoots.This is a greater sum than the other treatments, significantly dissimilar to the effects of other treatments.Table 1 also demonstrates that 3 BA is capable of producing more shoots than Kinetin and 2-iP.BA, Cytokinin is that affects the formation and multiplication of shoots.It has a stronger effect than cytokinins such as kinetin and 2-iP [12].BA is a synthetic cytokinin derivative of adenine that is highly effective at promoting shoot development.BA plays a crucial role in the induction of physiological responses, including the regulation of cell division, tissue and organ differentiation, and chlorophyll biosynthesis [13]  Increasing the BA concentration from 2 mg/l to 2.5 mg/l resulted in 3.21 fewer shoots than the 2 mg/l BA treatment.Similarly, the 2 mg/l kinetin treatment resulted in 3.48 shoots, whereas the 2.5 mg/l kinetin treatment resulted in fewer shoots, namely 2.2.The 2-iP treatment produced the same number of shoots as the 2 mg/l BA treatment, 3.29, while an increase in 2-iP to 2.5 mg/l produced 2.35 shoots.This indicates that the use of growth regulators at higher concentrations causes stunted shoot growth because the use of cytokinins at high concentrations can impede nutrient absorption and inhibit explant growth [14] [15].

Shoot height
Due to the availability of nutrients in the medium, rapid shoot development is indicated by rapid shoot growth.The height of the shoots is observed as an indicator of plant growth in response to the addition of a treatment.The pattern of growth rate during the in vitro culture period can be described by the growth of the shoot height.The statistical analysis revealed that the interaction between treatments had no significant impact on shoot height.However, in comparison to other treatments, the BA treatment had the greatest effect.Due to the synergistic effect between these growth regulators, using cytokines in a single treatment medium can stimulate shoot proliferation [16].The 2 mg/l concentration of BA had the greatest effect on shoot height.The 2 mg/l BA treatment produced a shoot height of 2.78 cm, which was not significantly different from the kinetin or 2-IP treatments.Cytokinins can stimulate shoot growth and cell division while inhibiting elongation, making shoot elongation comparable to the three types of cytokines.[17].

Number of leaves
On explants, the formation of leaves occurs more rapidly than the formation of shoots.Because new shoots will emerge from the axils of the leaves, the formation of this leaf is crucial.Upon observation four weeks after cultivation, all organisms are able to form leaves.The best treatment was 2 mg/l BA because it produced the greatest number of leaves, 8.2 leaves.The same concentration of kinetin can result in the production of 6.2 leaves.This also occurred in coffee culture, where the number of leaves produced by the BA treatment was greater than that of the kinetin treatment [18].
The 2 mg/l BA treatment yielded the greatest number of leaves, while the other treatments yielded greater quantities.Increasing the concentration of BAP to 2.5 mg/l decreased the number of leaves produced.8.2 leaves were produced from the 2 mg/l BA treatment, as shown in Figure 1.Similarly, the Kinetin treatment demonstrated the same effect.The number of leaves in the 2 mg/l kinetin treatment was 6.8, while increasing the kinetin concentration to 2.5 mg/l decreased the number of leaves to 6.2.The same thing occurred with the 2-iP treatment.High concentrations of growth regulators inhibit leaf development [14][15]. .

Number of roots
In in vitro plant micropropagation, the success of the acclimatization phase will depend on the root formation process that produces high-quality roots.Figure 2 demonstrates that as cytokinin concentration increases, the number of roots decreases.The greatest number of roots were found in the absence of cytokines.Increasing the concentration of BA causes the number of roots to decrease.The same thing occurred with treatment with kinetin and 2ip.An increase in the concentration of kinetin and 2iP reduced the number of roots.Root formation is determined by the correct ratio of auxins to nutrients.In addition to being influenced by the administration of exogenous auxin, it is also influenced by genetic differences in the explants used and their endogenous cytokinin content [19].

Conclusion
It has been determined through research that the addition of cytokinins can increase shoot multiplication.The 2 mg/l BA treatment produced the greatest number of shoots, shoot height, and number of leaves compared to the other treatments.High concentrations of cytokinins (> 2 mg/l) will inhibit the multiplication of Pothos tener shoots.This method can be used to calculate the number of tillers to be produced in propagation using tissue culture techniques.

Figure 1 .
Figure 1.Influence of cytokinin type and concentration on the number of leaves of Pothos tener.

Figure 2 .
Figure 2. Influence of cytokinin type and concentration on the number of roots of Pothos tener.

Table 1 .
Influence of cytokinin type and concentration on the number of Pothos tener shoots.
Remarks: Values followed by the same letter in the same row and column are not significantly different at the 5% level, according to Duncan's multiple-range test

Table 2 .
Influence of cytokinin type and concentration on the height (cm) of Pothos tener shoots.