AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Faculty of Agriculture, Wijaya Kusuma University of Surabaya Email: arijantiprakoeswa@gmail.com ABSTRACT Plants of papaya were have the main sources of chemical compounds that were generally used in pharmaceuticals and food-additive industries. One of the effort to increase the content of Papain in papaya is through tissue-culture and elicitation technique.The purposes of this research are *To investigate the content of Papain in calluses of papaya leaves with treatment of elicitor Saccharomyces cerevisiae in MS and VW mediums. This research was conducted in the month of November 2017 to January 2018, using Complete Random Design (CRD) with factorial using two factors, repeated four times and each repitition contained 4 samples. The main results of this research had shown that The treatment of VW medium and elicitor Saccharomyces cerevisiae 25 mg Dry Matter/L had produced the highest content of Papain. Keywords : Elicitor Saccharomyces cerevisiae; Medium; Papain; Papaya leaves callus. 1. INTRODCTION Papaya (Carica papaya L.) is a type of tropical plant that can grow well in all region of the world with various characteristics depended on the condition and climate of the region. ( Ludwig- Muller, J. 2000) Papaya plant was originated from Central America and was spread throughout the world by the Spanish traders. Countries that are best known as producers of Papaya are Republic of Dominica, Puerto Rico, Costa Rica, also Brazil, India, and Indonesia (Warisno, 2003). Papaya fruit is a fruit with high nutritious content and high fiber, so it is good for health and digestion, with affordable price to be enjoyed by all people in society(Nisak, Pratiwi, & Ali, 2017). The content of anti-oxidant in Papaya fruit is carotenoids in Vitamin A and Vitamin C (Maisarah et all, 2013) . Papaya fruit helps to lower the risk of heart diseases and stroke caused by the blockage in blood vessels. Almost all parts of Papaya plant can be used, aside from the fruit itself. The enzyme of Papain is known as an anti-inflammation agent that can reduce inflammation for asthma, arthritis, and other diseases that caused by inflammation.( Dalimartha, S. 2003. ) The enzyme is a protein bio-molecule that functions as a catalyst to hasten anorgano- chemical reaction. Papaya produced the enzyme Papain that is a proteolitic enzyme secreted through the isolation of Papaya’s sap. The sap from Papaya plant can be secreted through the fruits, stems, and leaves (Akujobi CN, Ofodame CN, Enweani CA. ,2010). AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | One way to increase the production of Papain contents in Papaya plant is through the use of tissue culture technique (Yusnita, 2004). Through this tissue culture process, the contents of Papain that is a secondary metabolite can be increased with the addition of elisitor Saccharomyces cereviceae (Flora, 2010). One of the determinant factor in the success of reproduction is the use of culture mediums. Some culture mediums used had went through various developments in the formula of the medium compositition for better optimization of growth and development of cultured plants (Nugroho, 2006). Polysaccharides, proteins, lipids, and nucleic acids are the main components of plants, which are the primary metabolites. In the plant cells, beside the obvious photosynthesis process, there are also other biochemical reactions to breakup the primary metabolites (Nurhidayati, 2003). Compounds that are the results of the breakup of these primary metabolites are known as the secondary metabolites. Some of these secondary metabolites have economical values and have been intensively used by the food and pharmaceutical industries as the sources of natural anti- oxidants (Vickery, 1981). Secondary metabolites produced by the culture of plants cells can be increased in content by elicitation technique. The elicitation technique to increase the contents of these secondary metabolites through tissue culture is an induction process that aimed to increase the formation process of secondary metabolites in tissues or calluses by using an elisitor. According to Rahmawati (2006), elisitors are divided to two types Abiotic elisitors and Biotic elisitors. Abiotic elisitors were formed from the anorganic chemical compounds, i,e : ultraviolet, heavy metal, etc. While abiotic elisitors came from organic compounds such as : carbohydrates, proteins, volatile compounds, etc. Mediums that were widely used are the Murashige and Skoog (MS) & Vacin and Went (VW). These mediums were oftenly used in the tissue culture process because these mediums were considered qualified because of the contents of macro and micro nutrients, as well as vitamins, for the plants’ growth that needed nutrients of organic salts and subtances to support their growth (Inglett, G.E. and Charalambous, G. 1979). The plant’s medium should contain all the nutrients needed to ensure the growth of explants. The nutrients were mixed to contain mineral salts, macro and micro nutrients, sugar, proteins, vitamins and hormones (Arijanti, 2008). These are the types of medium widely used in the tissue culture process, according to Widiyana (2013) :In this research, there were used explants of the meristems of Papaya leaves. The apical meristem cultures were explants cultured from the tip of meristem to tissues of 0,3 to 1 cm below. AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | Based on those explanations above, this research was hoping to give informations on the growth and development as well as the contents of calluses of Carica papaya L. with treatment of elisitor Saccharomycescerevisiae in MS and VW mediums.( Wohing, P., et al. 2012) The purpose was to gain a better growth and development of calluses so that can be used as based for vegetative reproduction, as well as to investigate the best contents of Papain to be used as materials for pharmaceutical industries (Yuniarti, T. 2008). 2. MATERIALS AND METHOD 2.1. Place and Time This research was conducted in the Laboratory of Tissue-Culture in the Department of Agriculture of Wijaya Kusuma University of Surabaya. This research was conducted in the month of November 2017 to January 2018. While the analysis of the contents of Papain were conducted in the Laboratory of Industrial Research and Consultation of Surabaya – East Java. 2.2. Materials : Materials needed during this research were explants of Papaya’s young leaves taken from the researcher’s home garden, Basic mediums (MS and VW), growth control nutrients of 20 ml/L NAA and 30 ml/L BAA, Glucose 30 gr, Saccharomyces cereviseae (as treatments), coconut water 150 ml/L. Alcohol 70% and 96%, tween betadine, aluminium foils, and plastic wraps. 2.3. Equipments : Equipments used during this research were analytic scales, autoclave, oven, Laminar Air Flow (LAF), pH-meter, pinset, scalpel, Erlenmeyer, measuring cups, drop pippette, measuring pippette, petridish, spatula, cultures tubes, hotplate magnetic stirrer. 2.4. Method of Research : This research were conducted directly at the laboratory. The method used was Complete Random Design (CRD) with six treatments repeated four times and each repitition contained four samples. Factor 1: Saccharomyces cerevisiae : Factor 2: Type of mediums : S1 : 0 mg Dry Matter/L M1 : MS medium S2 : 25 mg Dry Matter/L M2 : VW medium S3 : 50 mg Dry Matter/L 2.5. Implementation of Research Glasses and metal equipments could be sterilised in the oven on temperature of 160oC for 2-4 hours after wrapped in brown-paper or aluminium-foil. After sterilised, those equipments were AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | taken out and brought inside the LAF, then sterilised further using ultraviolet ray for 30 minutes. Before actually used, these equipments were unwrapped from the brown-paper and then soaked in the alcohol 96% then burned. Every time they would be used, they were soaked in alcohol and then burned again before used. These were implemented during the research. Making of medium Composition of mediums for tissue cultures varied depended on the commodities that would be used. Basically almost every commodity were using MS medium modified with vitamins and hormones as per needed by the commodities. For efficiency, there were made a stock of solutions, so every making of mediums would need only taken some volume from the stock. Planting Explants that were used in this research were of young leaves of Papaya, that would be forming the callus. Medium used in this research were MS and VW medium. Parameters Quantities of Callus Observed visually in 1-week interval with scoring as follows : 1 = no calluss 2 = few calluss (<1 time size of the explant) 3 = medium calluss (1-2 times size of the explant) 4 = many calluss (>2 times size of the explant) Qualities of Calluss Observed visually in 1-week interval with scoring as follows : 1 = no calluss 2 = compact calluss 3 = friable calluss Data Analysis Datas gained from observations and research would be compared using random test analysis to determined whether there was a real differences, with multiple tests (BNT 5%), processed using application of SPSS 18.0. AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | 3. RESULTS AND DISCUSSION 3.1. Quantities of Calluss Results of random analysis had shown interactions between treatments on the parameters of quantities of Papaya calluss (Carica papaya L.) through visually observed within 1-week interval could be found in Table 1. Table 1. Average Results of the Observations of Quantities of Calluss Formed in Various Treatments from Time-Periods 1-10 MST (Weeks After Planting) Treatments Time Periods (weeks after planting) 1 2 3 4 5 6 7 8 9 10 Saccharomyces 0 mg/L MS medium (S1M1) 1.00 1.00 1.00 1.00 1.78 1.88 2.57 2.72 2.63 2.63 Saccharomyces 0 mg/L VW medium (S1M2) 1.00 1.00 1.00 1.00 1.66 1.75 1.91 2.35 2.41 2.41 Saccharomyces 25 mg/L MS medium (S2M1) 1.00 1.00 1.00 1.00 1.72 1.88 2.13 2.69 2.75 2.75 Saccharomyces 25 mg/L MS medium (S2M2) 1.00 1.00 1.00 1.00 1.63 1.75 1.85 2.28 2.03 2.03 Saccharomyces 50 mg/L MS medium (S3M1) 1.00 1.00 1.00 1.00 1.66 1.75 1.88 2.28 2.13 2.13 Saccharomyces 50 mg/L MS medium (S3M2) 1.00 1.00 1.00 1.00 1.61 1.85 1.91 2.32 2.19 2.19 LSD 5% NS NS NS NS NS 0.13 0.23 0.28 0.44 0.44 Note: S = Significant, NS = Not Significant, (1) no calluss, (2) few calluss (<1 times of explant size), (3) medium calluss (1-2 times explant size), (4) many calluss (>2 times explant size). Table 2. Single Factor of Quantities of Calluss Within 1-Week Interval Treatment Periods (weeks after planting) 1 2 3 4 5 S1 1.00 1.00 1.00 1.00 1.72 S2 1.00 1.00 1.00 1.00 1.69 S3 1.00 1.00 1.00 1.00 1.63 LSD 5% NS NS NS NS 0.004 M1 1.00 1.00 1.00 1.00 1.72 M2 1.00 1.00 1.00 1.00 1.64 LSD 5% NS NS NS NS 0.004 From the random analysis shown in Table 1, in week 1 to week 5, there were no interactions yet. In week 6, there were interactions. These were because in week 1 to 5 MST, the Papaya leaves explants might still adapted to the treatments of mediums and Saccharomyces cerevisiae. While in time periods of 5 to 10 MST, the results of random analysis had shown real differences, because the calluss of the Papaya leaves had grown and started disassembled their food reserves as responses to the treatments of mediums and elisitor of Saccaharomyces cerevisiae AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | in the mediums. The observations’ results above had shown that the explants of Carica papaya L.’s leaves in MS medium with treatments of elisitor Saccharomyces cerevisiae 25 mg BK/L had produced more calluses than other treatments. The response of growth were varied because of the effect of mediums and treatments to the commodities of calluses were very specific (Arijanti, 2008). On the Table 1, the treatment of 25 mg Dry Matter/L of Saccharomyces cerevisiae in MS medium had better effect on the growth and development of the explants than other treatments. This was postulated due that the effect of Saccharomyces cerevisiae were more optimal in quantity of 25 mg Dry Matter /L and the more complex composition of MS medium had caused better growth and development for the calluss. Generally the growth and development of explants’ cells in-vitro would be better to reach the optimum when the nutrients in the medium’s composition were more complex. In this instances, the MS mediums had the optimum composition for growth. Thus, the MS mediums were more suitable for the commodities of Papaya leaves’ calluses (Widya, 2014). On Table 2 were shown the results of single factor of quantities of calluses within 1-week interval. In week 5, it was appeared that on week 5 had shown real differences. As according of Arijanti (2008), the factor of medium’s composition is very affecting to the growth of calluss. 3.2. Qualities of Calluss Results of random analysis had shown interactions between treatments on the parameters of qualities of calluss through visually observations within 1-week interval could be found in Table 3. Table 3. Average Results of the Observations of Qualities of Calluss Formed in Various Treatments from Time-Periods 1-10 MST (Weeks After Planting) Treatments Time Periods (weeks after planting) 1 2 3 4 5 6 7 8 9 10 Saccharomyces 0 mg/L MS medium (S1M1) 1.00 1.00 1.00 1.00 1.50 1.57 1.57 1.97 1.85 1.85 Saccharomyces 0 mg/L VW medium (S1M2) 1.00 1.00 1.00 1.00 1.57 1.69 1.69 1.69 1.69 1.69 Saccharomyces 25 mg/L MS medium (S2M1) 1.00 1.00 1.00 1.00 1.53 1.72 1.72 1.97 2.00 2.00 Saccharomyces 25 mg/L MS medium (S2M2) 1.00 1.00 1.00 1.00 1.32 1.41 1.41 1.78 1.82 1.82 Saccharomyces 50 mg/L MS medium (S3M1) 1.00 1.00 1.00 1.00 1.35 1.47 1.47 1.79 1.72 1.72 Saccharomyces 50 mg/L MS medium (S3M2) 1.00 1.00 1.00 1.00 1.19 1.26 1.26 1.91 2.00 2.00 LSD 5% NS NS NS NS 0.15 0.20 0.20 0.23 0.27 0.27 Note: S= Significant, NS = Not Significant, (1) no calluss, (2) compact calluss, (3) friable calluss. AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | Table 4. Single Factor of Qualities of Calluss Within 1-Week Interval Treatment Periods (weeks after planting) 1 2 3 4 S1 1.00 1.00 1.00 1.00 S2 1.00 1.00 1.00 1.00 S3 1.00 1.00 1.00 1.00 LSD5% TNS NS NS NS M1 1.00 1.00 1.00 1.00 M2 1.00 1.00 1.00 1.00 LSD 5% NS NS NS NS On Table 3 were shown that on week 1 to 4 MST, the Papaya callusS were still adapting, thus there were no real differences and the callusS still in the form of Papaya leaves’ explants, as can be seen in Figure 1. While in the week 5 to 10 MST, by random analysis there were shown real differences as the callusS tended to have qualities of compact calluss with organogenesis characteristics.The organogenesis calluss were calluss that their morphogenesis growth had needed mediums with different concentrates of auxin and cytokinen for growing shoots and roots. The forming of compact calluss in this research were due to the specific characteristics of Papaya commodity itself. When planted, the organogenesis calluss would form their own plants’ organs (Ribkhawati, 2012). There were shown analysis of single factor of qualities of calluses within 1-week interval, from week 1 to 4, in Table 4. The treatments of mediums and Saccharomyces cerevisiae had not shown any interactions as the Papaya leaves’ calluss were still adapting (Ribkhawati, 2012). 3.3. Analysis of Secondary Metabolites Results of analysis by spectrophotometer using wavelength of 225 µ m to investigate the contents of Papain in the calluss of Papaya leaves of time period of 10 weeks. Table 5. Contents of Papain in calluss of Papaya leaves on Various Treatments in the Week 10 MST Treatment Contents of Papain enzyme in % S1M1 (Saccharomyces c. 0 mg Dry Matter /L, MS medium) 0.126 S1M2 (Saccharomyces c. 0 mg Dry Matter/L, VW medium) 0.129 S2M1 (Saccharomyces c. 25 mg Dry Matter /L, MS medium) 0.146 S2M2 (Saccharomyces c. 25 mg Dry Matter /L, VW medium) 0.148 S3M1 (Saccharomyces c. 50 mg Dry Matter /L, MS medium) 0.144 S3M2 (Saccharomyces c. 50 mg Dry Matter /L, VW medium) 0.139 AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | In Table 5, the results of observations on the contents of Papain with all treatments of Saccharomyces cerevisiae had shownthat, based on the laboratory analysis, the treatments of addition of Saccharomyces cerevisiae25 mg Dry Matter/L tended to caused higher contents of Papain. This was due to the Papain response in Papaya plant to the medium and elisitor of Saccharomyces cerevisiae. According to Ribkhawati (2012), the additionof Saccharomyces cerevisiaeconcentrates must be in optimum quantity, because it would affect the production of the secondary metabolites. Calluss that had grown in lower quantities tended to produce better contents of secondary metabolites. In this research the treatments of VW medium tended to produce lower quantities. And every plant commodity responses were specific, thus the varied production of secondary metabolites. The optimum addition of elisitor Saccharomyces cerevisiae concentratesin best medium for Papaya plant was 25 mg Dry Matter /L and had produced the highest contents of secondary metabolites than any other treatments. Figure 2 showed the comparison of production of secondary metabolites, the best was of S2M2 (Saccharomyces cerevisiae 25 mg Dry Matter /L in VW medium). 4. CONCLUSIONS From the observations on the quantities, qualities, and analysis of contents of Papain, with different treatments on mediums and addition of elisitor Saccharomyces cerevisiae, on young leaves explants of Papaya (Carica papaya L.) could be concluded as follow. Treatment of MS medium and elisitor Saccharomyces cerevisiae25 mg Dry Matter /L had produced the highest quantity of calluss than other treatments. All treatments of medium and elisitor Saccharomyces cerevisiaehad produced compact calluss and Treatment of VW medium and elisitor Saccharomyces cerevisiae 25 mg Dry Matter /L had produced the highest contents of Papain. Pictures of Calluss from the Research AGRICULTURAL Journal Of Agricultural Science And Agriculture ISSN : 2597-8713 Available on http://agris c cientific -work.org/inde x.php/agris c This is Under CC BY SA Discussion of Growth And Content of Papain In Papaya Callus (Carica Papaya L.) By Tissue Culture Technique With Elicitor Saccharomyces Cerevisiae Treatment In Ms And Vw Sri Arijanti , Dwie Retna Suryaningsih, Arief Eryanto Page | Figure 1. Kuantitas Kalus Carica papaya L. yang terbaik pada media MS dengan elisitor Saccharomyces serevisiae 25 mg Dry Matter/L Figure 2. Kalus Carica papaya L. yang menghasilkan metabolite sekunder terbanyak pada media VW dengan elisitor Saccharomyces serevisiae 25 mg Dry Matter/L REFERENCES Akujobi CN, Ofodame CN, Enweani CA. (2010). 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