Effect of Media Types on the Growth of Callus Culture in Kumis Kucing Orthosiphon aristatus (Blume) Miq

The lack of conventional availability of plant Kumis Kucing makes tissue culture techniques used as a solution to overcome this problem. In tissue culture, media is a major factor in producing a good crop of plantlets. Media Murashige & Skoog (MS), Lloyd & McCown Woody Plant (WPM) media was used in the induction of Orthosiphon aristatus (Blume) Miq cat whiskers callus culture, in this study also used growth regulators in the form of 2,4-D added to each-individual media. The results showed the best callus growth occurred in Murashige & Skoog (MS) media compared to the Lloyd & McCown Woody Plant (WPM) media, where the callus produced was 3.28 g on MS media.

the quality and quantity of the products produced [13]. Intercropping system results in the quality of raw materials obtained still not meeting the standards of the quality of a pharmaceutical industry [14]. Therefore, tissue culture techniques can be applied to overcome the existing problems. Tissue culture techniques have the several advantages, namely in tissue culture techniques to produce metabolites are big and fast, can produce plantlet in large quantities and does not require large amounts of land, but it also can be used directly in the research of genetic level [15].
Studies in plant tissue culture cannot be separated from media use. The culture medium contains macro and micronutrient elements used for the process of growth of an explant into a plantlet or callus. Callus is an initial stage for making other cultures such as cell suspension culture, root hair culture, and embryo culture used for the production of compounds in the field of pharmaceutical biotechnology [15]. There are many media available and have their respective functions in their uses. MS media used generally for all species of plants, media WPM used in woody plants, B5 media to crop legume species, media Vacin and Went for orchids [15].
The development of kumis kucing plants in the field of tissue culture is still very little and has not been widely carried out regarding the exploration of media used, previous research is only based on the use of one type, MS media [16][17][18][19][20][21]. On the other hand research in the field of culture proves that the use of various types of media produces different growth responses such as research on acacia plants that have been explored using a variety of media (¾ MS, WPM, B5) and produce different responses for the formation of callus, root & embryos from acacia plants [22]. In addition to the other types of plants such as blueberries have been cultured using media WPM, MS, and MW and produce different shoots of growth of any media used [23]. Therefore the use of the right media for the production of a plant is very important to be investigated further to find media that is suitable for a plant.
The focus of this study is to compare the use of MS media with WPM media to see how the callus growth response from explants of kumis kucing plants, so that good media is obtained in a culture of kumis kucing plants.

Explant Preparation
Explat preparation was carried out at the Mulawarman University Faculty of Pharmacy Tissue Culture Laboratory, by taking young leaves at the top of the leaves, then the leaves washed with running water and cleaned from dirt, after that the leaves were placed into culture bottles and explants were ready to be sterilized.

Explant Sterilization
Explant sterilization is carried out aseptically, by immersing the explants into several sterilizing solutions, the solution used consists of a 10-minute soap solution; bactericidal 0.05% 5 minutes; fungicide 0.1% 5 minutes; 70% alcohol 2 minutes; Sodium hypochlorite solution (5.25% NaOCl + Tween 80 0.1 mL) 5 minutes then the explants were rinsed with sterile aquades three times, then cut the explants with a size of ± 0.25-5 cm 2 , then placed on the surface of the basal media, then incubated at 22°C temperature conditions, 4000 Lux, and photoperiod phase 8/16. [24].
Preparation of stock solution of 2,4-D As much as 0.05 g 2,4-D, put it in a beaker glass, add a few drops of KOH 1 N until dissolved, then transfer it to a 100 ml volumetric flask, enough to mark the limit, then homogenize, store the stock of solution in bottle storage.

Media Preparation
The MS and WPM media used were sucrose with a concentration of 3%, pH of the medium 5, 8. If the pH is not appropriate add a few drops of HCl/KOH solution (1N HCl to reduce pH and 1N KOH to increase pH). Then the media added a solidifying agent in the form of 0.7% while stirring and heated to dissolve, add ZPT 2,4 D with a variety of concentrations (0.5; 1; 1.5; 2; and 3) ppm in each media, then the media is divided into culture bottles. Sterilize with Autoclave 121ºC ± 15 minutes, remove and store the media in the incubation room before use.

Induction of Callus Culture
The explants were sterilized was added to each medium with various concentrations of PGR (0.5, 1, 1.5, 2, and 3) ppm, conducted three replication for each of the various concentrations of PGR. In one bottle the media contains 3 leaf explants. Incubation of explants for 28 days, with conditions of 22 °C, lighting 4000 Lux, and photoperiod 8/16.

Data analysis
Analysis of the data in this study was in the form of an assessment of the ideal callus criteria by looking at the callus morphology (color and texture) and callus growth obtained from the callus mass for 28 days. The results of the data presented in the form of images and graphics to determine the media that are good in the culture callus of kumis kucing explant.  [19,20,26].
Callus formation (Figure 1) average occurred on the 7th day on media MS and WPM medium, characterized by there is a set of cells that grow at the edge of the explant with irregular shape, it is consistent with other studies [20] using kumis kucing leaf explants, mention the callus can be induced on the 7th day. The difference in the concentration of 2.4D from MS media and WPM media which can induce the best callus is due to the content of the media that can affect callus cell growth.

Effect of MS and WPM media on callus characteristics at each 2.4 D Growth Regulating Substance concentration Color Callus
Color is the main indicator in the success of callus cell growth, callus has a variety of colors but in general the color of the callus is white to bright yellow, bright colors that have a callus indicate that the callus condition is still quite good [27] and has not undergone stationary phase [28] The color of callus produced from MS media and WPM media with variations in the concentration of growth regulator 2.4 D resulted in different callus colors.
The initial color of callus on MS media ( Callus color on MS media (Figure 2), with variations in the concentration of regulated substances growing 2.4 D (0.5 ppm; 1 ppm; 1.5 ppm; 2 ppm; and 3 ppm), on the 7-21 day is a good callus. While on WPM medium (Figure 3), callus was good only at a concentration of 0.5 ppm 2,4 D, therefore it can be said MS medium good medium in growing callus from kumis kucing leaf explants, because of the results obtained initial color callus on MS medium entry in the indicator as a good callus.
The brown color of the callus found in the explants of the Kumis Kucing leaves shows that the callus undergoes a stationary process, wherein this phase the callus does not carry out cell division [27]. Browning is a natural event that can occur in callus caused by the influence of other biochemical compounds that often occur in parts of plants [16]. Brown color on the callus indicates that the growth of callus decreased [20], so that good callus has the colors tend to be bright.

Callus Texture
In addition to color, callus texture is used as an indicator in ideal callus assessment. Callus texture is divided into two types, namely compact and crumbs. This study produced a compact callus texture from both media used, namely MS media and WPM media. The compact callus texture shows that the callus contains more secondary metabolites than the crumb texture [25]. Nutritional elements from the media play a role in the formation of callus textures. Callus texture of Kumis Kucing leaf explants (Orthosiphon aristatus (Blume) Miq) produced in this study is different from previous studies [29], where the results of other studies state that kumis kucing leaf explants have crumby callus texture, this difference is caused by callus texture influenced by the type of plants used, the composition of nutrient media, growth regulators and environmental conditions of the culture [30].    Callus mass measured in weight units is used as an indicator of the effect of the successful use of a culture medium. Obtaining a large callus mass can increase the production of secondary metabolites and plantlets from an explant. The callus mass generated from this study (Figure 4), MS media is higher than the WPM media. Callus mass on MS media is 3.28 g and WPM media is 2.30 g. Optimal growth of callus mass from MS media and WPM media occurred at week 3 and began to decline at week 4, this was caused by nutritional factors from different types of media can affect the mass of callus produced.
The nutritional composition of MS and WPM media is very different. Sources of producing nitrogen elements in MS media are higher than WPM media, Nitrogen plays a role in forming glutamine [28] which is used for energy metabolism and cell proliferation [31], if the higher the proliferation, then the callus period formed is also greater. Then the potassium ion (K + ), MS media has a higher potassium (K + ) ion content compared to WPM media, potassium ions have a role in the process of diffusion between cells [32], cell turgor, and stomata movement [28], which play a role in callus cell growth process. From the composition of nutrition media, MS media is a medium that can be used to increase the mass gain of callus from kumis kucing leaf explants.

■ Conclusion
Orthosiphon aristatus (Blume) Miq Kumis Kucing callus culture from two types of media used, namely MS media and WPM media, seen from the morphology and weight of the callus obtained, that the use of MS media produced better callus growth compared to WPM media.

■ Acknowledgements
My gratitude, to the pharmacy faculty who have provided research funding through the grants of beginner lecturers (Hibah Dosen Pemula) in 2016 to develop their potential and train the skills of lecturers and researchers ■ References