The Effect Of 2,4-D (2,4-Dichlorophenoxyacetic Acid) and Kinetin (6-Furfuryl Amino Purine) Concentrations on The Induction of Embryogenic Callus In Porang (Amorphophallus muelleri Blume) In Vitro

is being explored to induce embryogenic callus, offering a solution for large-scale cultivation of high-quality Porang within a shorter timeframe. This research adopts an experimental approach employing a completely randomized design (CRD) with two factors. The first factor is the auxin 2,4-D (2,4-Dichlorophenoxyacetic acid), and the second factor is the cytokinin kinetin. The optimal concentration for inducing embryogenic callus in Porang is found to be 2,4-D at 1 mg/L and kinetin at 0 mg/L, resulting in intermediate callus with a yellowish-brown color and a wet weight of 2.43 grams.


INTRODUCTION
Porang is a type of tuberous plant with potential and opportunities for cultivation in Indonesia [1].Porang contains a high level of glucomannan in its tubers.Porang tubers contain more than 50% glucomannan, depending on the location, soil, climate, and age of the tubers.Porang is commonly commercialized as flour and chips derived from processed Porang tubers.Porang has been widely used in the food industry, cosmetics, chemicals, petroleum, pharmaceuticals, and coating industries [2].
The propagation of Porang plants naturally can occur through vegetative organs, namely leaf bulbs (bulbils) and stem tubers, while generative propagation employs seeds.Propagation through bulbils, when directly planted in a seedling medium, undergoes a dormancy period of approximately 5-6 months, thus failing to meet the demand for Porang seedlings in a short time [3].The supply of bulbils is further constrained because on average, Porang plants produce 1 bulbil in the first growth phase, 4 -7 in the second phase, and 10 -20 in the third phase.The use of stem tubers is also not recommended because harvested tubers, when used for reseeding, may reduce Porang tuber production [4].Tuber seedlings typically require about one year before they can be harvested [5].Additionally, Porang tubers experience shrinkage and a decrease in glucomannan content (degradation) after Porang flowers bloom, so Porang tubers are usually harvested before flowering [6].
One solution to obtain a large quantity of high-quality Porang seedlings in a short time is through in vitro culture.One of the in vitro culture techniques that can be utilized is callus culture.Callus culture is an initial stage of in vitro culture technique that produces and multiplies callus cells massively, where each callus cell has the ability to form new individuals through embryogenesis and organogenesis stages [7].Callus as a collection of amorphous cells (undifferentiated cells) derived from continuously dividing cells in culture bottles.
[8].The advantage of callus culture is its ability to generate new plants with superior quality that are free from diseases, and it can produce higher levels of secondary metabolites compared to the original plants [9].Factors such as the type of media used, the type of explants utilized, and growth regulators are some factors that can influence callus growth during the callus propagation process [10].
Previous research on the induction of embryogenic callus has been conducted.Induced callus from embryo explants of maize seeds (Zea mays L.) with a concentration of 1.5 mg/L 2,4-D and 0.3 mg/L kinetin, resulting in embryogenic callus with a friable texture and whitish-yellow color, with the highest percentage of callus formation achieved within 15 days [11].Induced callus from barley plant (Hordeum vulgare L.) caryopsis explants, showing that the primary callus gradually transformed into a homogeneous embryogenic mass, with a solid and brittle texture ranging from white to yellowish on media containing 2 mg/L 2,4-D and 2.5 mg/L kinetin [12].

Tools
The tools used in this research includes Petri dishes, tissue paper, label paper, pH meter, autoclave, plastic measuring cups, micropipette, glass beakers, analytical balance, hot plate and magnetic stir bar, culture bottles, rubber bands, forceps, blade no.22, scalpel, laminar air flow (LAF), lighter, Bunsen burner, aluminum foil, incubation rack, and oven.

Porang Callus Induction
Porang callus (Amorphophallus muelleri Blume) resulting from three subcultures on NAA 2 mg/l media were removed from the bottle.They were then cut on a Petri dish to a size of 1 cm and soaked in sterile distilled water previously treated with betadine, before being planted in each treatment media.Explant initiation was conducted inside a Laminar Air Flow (LAF).After initiation, the culture bottles were tightly closed and placed in the incubation chamber.

Data Collection Technique
Data were obtained from final observations, which were conducted 45 days after explantation.The observed parameters include: callus color, callus texture, callus weight, and callus anatomy.The observation parameters are as follows: a. Wet weight observation of the callus was conducted on the final day, by weighing the callus using an analytical balance.b.Callus color observation was based on the color that appeared on each callus.c.Callus texture was visually observed to classify the formed callus as friable callus, intermediate callus, or compact callus.Callus anatomy was observed using an Olympus binocular microscope

Callus Weight
The results of the analysis of variance (ANOVA) for the addition of growth regulators 2,4-D and kinetin in the induction of embryogenic callus of Porang (Amorphophallus muelleri Blume) showed a significant effect on the variable of callus wet weight in the induction of Porang embryogenic callus.Therefore, further testing was conducted using Duncan Multiple Range Test (DMRT) with a significance level of 5%.The results of the DMRT calculation are presented in Table 1.

Table 1. The Effect of 2,4-D and Kinetin
Combination on the Wet Weight of Porang Callus (Amorphophallus muelleri Blume) Based on Table 1, it is known that the best combination of 2,4-D and Kinetin for callus induction is a concentration of 1 mg/l 2,4-D + 0 mg/l Kinetin, with an average wet weight of callus of 2.43 g.From the observations based on Figure 1, the addition of growth regulators into the media is crucial in determining the direction of growth of an explant.At a concentration of 0 mg/l 2,4-D + 0 mg/l Kinetin, no embryogenic callus formation was observed for all observation variables.The absence of embryogenic callus formation in samples without the addition of 2,4-D and kinetin is attributed to the presence of two crucial types of growth regulators in vitro culture, namely auxin and cytokinin, which influence the growth and morphogenesis of explants [13].Auxin and cytokinin added to the media will interact with endogenous auxin and cytokinin hormones, thus The combination of 2,4-D and Kinetin also affects the wet weight of the callus.At a concentration of 1 mg/l 2,4-D + 0.1 mg/l Kinetin, it was able to produce a callus weighing 1.80 g.The wet weight of the callus indicates growth related to an increase in the number and volume of cells.The combination of 2,4-D and Kinetin can enhance cell division, thus increasing the wet weight of the callus.The provision of these growth regulators likely occurs in a balanced concentration with endogenous auxin and cytokinin, thereby interacting in cell enlargement and division.The presence of auxin will decrease the pH in the cell wall, causing the cell wall to soften and cell enlargement to occur.Furthermore, with the presence of cytokinin, cell division in the meristematic tissue is stimulated, which is related to the process of RNA transcription and translation in protein synthesis during the interphase stage.With the interaction of hormones in this balanced concentration, it can increase the speed of enlargement, division, and proliferation of cells, thereby increasing the weight of the callus [15].
Based on the research results, the addition of 2,4-D and Kinetin at both the lowest and highest concentrations resulted in poor callus formation.At these concentrations, many calluses exhibited browning and entered a phase of death due to decreased or halted growth.This is suspected to be a sign of decreased callus growth due to either a deficiency or excess of exogenous hormones, leading to an imbalance between exogenous and endogenous hormones.A concentration that is too low does not stimulate growth significantly, while a concentration that is too high can lead to growth inhibition because high concentrations of exogenous hormones can be toxic to explants/plants [16].Browning in explants is related to the oxidation of phenolic compounds.Polyphenol oxidase (PPO) catalyzes the reaction between phenolic compounds and oxygen to produce quinone.Quinone, which is highly reactive, reacts to polymerize proteins and other cellular components, resulting in the formation of dark amorphous melanin pigments [17].

Morphology of Callus
Qualitative parameters in this research are divided into two categories: morphology and anatomy.The qualitative parameter of callus morphology includes callus color and texture.Callus color is a visual representation of callus growth that indicates whether the callus cells are still actively dividing or not, while callus texture is an indicator to determine the quality of the callus [18].The observation results regarding the effect of adding combinations of 2,4-D and Kinetin on callus morphology are presented in Table 2.The changes in callus color are influenced by pigmentation, nutrient availability in the culture media, and environmental factors such as light [19].Treatment combinations of 2,4-D 1 mg/l + Kinetin 0.1 mg/l and 2,4-D 3 mg/l + Kinetin 0.1 mg/l are the best combinations that can produce whitish to brownish callus, indicating that the callus has embryogenic capacity.This is supported by the literature of Marthani et al [20], which states that whitish callus with intermediate texture has embryogenic potential.Leupin et al [21] added that whitish callus consists of embryonic tissue that does not contain chloroplasts yet but contains plastids filled with high-starch granules, which serve as polysaccharide or food reserves in plants.Whitish callus indicates that the callus is young, gradually changing to brownish as it ages.
Treatment combinations of 2,4-D 0 mg/l + Kinetin 0 mg/l; 2,4-D 3 mg/l + Kinetin 0.3 mg/l; 2,4-D 4 mg/l + Kinetin 0.1 mg/l; and 2,4-D 4 mg/l + Kinetin 0.2 mg/l resulted in dark brownish callus.The dark brownish color of the callus indicates physiological deterioration because the cells have reached maximum growth and are entering the cell death phase, or it may be due to the presence of high levels of phenolic compounds leading to oxidation.This aligns with the literature of Setiawati et al [22] which states that the brownish coloration of callus is caused by several factors such as excessive metabolism of phenolic compounds or plant adaptation processes due to wound stress response.
Treatment combinations of 2,4-D 1 mg/l + Kinetin 0.1 mg/l and 2,4-D 3 mg/l + Kinetin 0.1 mg/l resulted in friable callus texture.Friable texture in this research indicates characteristics such as being brittle and easily detachable due to its low cell density and high water content.According to Thao et al [23] friable callus contains a lot of water because its cell walls have not undergone lignification, making it easy to separate one cell from another.Yelnititis [24] adds that visually, friable callus is easily separable and fragile, and it easily sticks when picked up with forceps.Callus with this friable texture is very useful for cell suspension cultures because undifferentiated cell clusters can grow rapidly in liquid culture media [25].
Most of the treatment combinations of 2,4-D and Kinetin resulted in compact callus texture.Compact callus has characteristics of being hard, solid, and difficult to separate.According to Mahadi et al [26], the compact texture is due to the lignification process (thickening of cell walls), which makes the callus hard and rigid because of the involvement of endogenous and exogenous hormones in nutrient transport processes.High levels of auxin and cytokinin hormones also affect the formation of compact callus because the water potential inside the cells causes increased water absorption from the culture media into the cells, making them more rigid [27].Litz et al [28] also added that the compact callus texture is related to sucrose content because sucrose, as one of the components in cell wall formation, consists of cellulose chains that are not easily separable [29].On the other hand, some treatment combinations resulted in callus with an intermediate texture.Intermediate callus has characteristics where the cell clusters consist of both friable and compact callus textures.

Callus Anatomy
To ensure the type of callus resulting from the treatment combinations of growth regulators 2,4-D and Kinetin is embryogenic callus, anatomical observations were conducted.Three types of callus were observed, namely friable, compact, and intermediate callus.Anatomical observation was carried out using an Olympus binocular microscope with a magnification of 400x.The anatomical observation results of the callus are shown in Figure 2. where friable callus exhibits clustered cells with meristematic properties, characterized by evident intercellular spaces, clearly visible nuclei, dense cytoplasm, and high cell division activity.
Thomy [31] suggests that the friable texture of callus facilitates the detachment and separation of its cells into single units, thereby enhancing oxygenation between cells.Consequently, friable callus texture can be utilized for suspension culture in liquid media.Conversely, compact callus displays cells with smaller nuclei compared to those in friable callus, with fewer nuclei observed overall.The cell walls appear distinct, and the cell arrangement is dense, resulting in the absence of intercellular spaces.Ariati [27] further asserts that compact callus is characterized by its dense and firm texture, typically featuring small and tightly packed cells with dense cytoplasm.The nuclei are often small and may not be easily

CONCLUSION
The combination of growth regulators 2,4-D at 1 mg/L and Kinetin at 0 mg/L significantly influenced the wet weight parameter of the callus, resulting in a wet weight of 2.43 grams.The callus exhibited a yellowish-brown color with an intermediate texture.
2,4-D + 0,3 Kinetin 1,61ab Note: Mean values followed by the same letter notation indicate that the combination of 2,4-D and Kinetin does not significantly differ based on the Duncan Multiple Range Test (DMRT) at a significance level of 5%.

Table 2 .
The Effect of 2,4-D and Kinetin Combination on the Morphology of Porang Callus (Amorphophallus muelleri Blume) discernible, and the cells contain starch granules.In observations of intermediate callus anatomy, both large and small nuclei are visible, with intercellular spaces present and distinct cell walls.According to Rasud & Bustaman [7] intermediate callus comprises a mixture of compact and friable cell masses.