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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME. Agrotechnology of Department of Agriculture – Wijaya Kusuma University of Surabaya Email: sdwiretna@gmail.com ABSTRACT Sorgum is one kind of plant from the family of grass in the Tropical and Subtropical regions. Sorgum bicolor is a species commonly used for molase, syrup and seeds productions. Furthermore, sorgum is also used as a biofuel material. The purpose of this research is to investigate which type of medium is best for the growth and development of sorgum explants. This research were implemented by using the Complete Random Design with 3 mediums treatment: M1 = Murashige and Skoog; M2 = Vacin and Went; and M3 = Nagata and Takabe. Repititions were done 9 times and each were using 10 test samples. Changes observed were of: qualities, quantities, and contents of phenol in the Sorgum calluses.The results of this research were:quantity of callus in general had shown real differences and most callus found were on the MS medium treatment;quality of callus formed had shown real differences in each mediums and tended to be compact callus, while in MS medium to become friable;NT medium tended to produce most contents of phenol, which was 0.25% . Keywords: In Vitro, Medium, Phenol and Sorgum 1. INTRODUCTION Plants are the main sources of chemical compounds genereally used for pharmaceutical industries, food additives, and fragrances. Most of those compounds are the products of secondary metabolism processes extracted from the plants species (Arijanti, Retna, Ribkahwati, 2006). One plant of the Grass family from the Tropical and Subtropical regions is commonly planted and cultivated by mankind for the production of molase, seeds, biofuel, and more, which is Sorgum. Sorgum is the source of nutrients, because it contains Niacin, Riboflavin and Thiamine, and minerals such as Magnesium, Iron, copper, Calcium, Phosphor, and Kalium (Annonymous, 2016). Sorgum seed has functional food nutrients such as anti-oxydant (polyphend), Fe, fibers, oligosaccharides, and B-glucose which is a type of non-starch carbohydrates polysaccharides. High content of Fe mineral is really helpful for the production of erythrocytes. Some benefits of sorgum for human health are: cancer deterrent, diabetes control, bones health, and increasing the circulation and production of erythocytes, as well as energy sources (Yuwono, 2015). The agrobusiness development of sorgum commodities has a good prospect, because in the year of 2012, the Department of State-Owned Companies (BUMN) has committed to develop sorgums planting in state-owned plantations to reduce the imports of wheat and assigned targets to the Institute of National Science of Indonesia (LIPI), National Board of Nuclear and the Research Center of Sugarcane Plantation of Indonesia (Annonymous, 2015). Based on the above, then as an effort to fulfil the needs of sorgum seeds, one of the most promising way is by using the tissue-cultures (in-vitro) technique, which is expected to be able to 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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | produce more quantities of seeds, better and cheaper, in short period of time and also to investigate the contents of its secondary metabolites (Ali, 2015). Results of this research were hoped to gain informations of the reproduction of sorgum seeds through tissue-cultures technique in 3 (three) types of mediums (Murashige-Skooq, Vacin- Went and Nagata-Takabe) and to know the best contents of secondary metabolites produced by treatment of those 3 mediums. 2. METHOD OF RESEARCH Research was conducted in the laboratory of tissue-cultures in the Department of Agriculture – Wijaya Kusuma University of Surabaya, through June to December 2017 time period. Materials and equipments used were Explants of Shoot Sorgum, basic Mediums of MS, VW and NT (with compositions as on Table 1, 2 and 3), growth regulator substances of NAA and BAP. Coconut water, Glucose, Alcohol 70% and 96%, Chlorox, Betadine, and laboratorium equipments. In this research, the method used is complete random design with one factor, of 3 levels : M1 (Murashige and Skoog); M2 (Vacin and Went); and M3 (Nakata-Takabe). Each treatment were repeated 9 times with 10 samples. 2.1. Implementation 2.1.1. Sterilisation of Equipments The equiments used were wrapped in brown-paper, then sterilised in the oven in temperature of 121oC for 30 minutes. While the cultures tubes were sterilised in Autoclave 17psi for 30 minutes. 2.1.2. Making of Mediums Mediums used were prepared as the treatments and modified by the addition of ZPT. 2.1.3. Planting The sorgum explants were sterilised with Chlorox 25% + 1 drop of Tween for 5 minutes, Chlorox 15% + 1 drop of Tween for 10 minutes, and Chlorox 5% + 1 drop of Tween for 20 minutes, then rinsed with sterilised water. After sterilised, then the explants were cut into approximately 1 cm2. Then planted in the cultures tubes in the mediums as treatments. 2.1.4. Incubation After the planting, the cultures tubes were put in the incubation racks and observed for the forming of calluses. 2.2. Variables The observation variables were as follows : 2.2.1. Quality of Calluses Visually observed once in 1-week interval by scoring of : 1 = no calluses 2 = compact calluses 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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | 3 = friable calluses 2.2.2. Quantity of Calluses Visually observed once in 1-week interval by scoring of : 2 = no calluses 3 = low quantity of calluses (<1 of explant size) 4 = medium quantity of calluses (1-2 of explant size) 5 = high quantity of calluses (>2 of explant size) 2.2.3. Contents of Polyphenol on Calluses Observed after the calluses were 8-weeks old, destructively through analysis by Spectrophotometer. 2.3. Data Analysis Results were analised by using One-way Anova through SPSS and, if there were real differences, further tested with LSD 5%. 3. RESULTS AND DISCUSSIONS 3.1. Quality of Calluses Results of type analysis had shown real differences in between the treatments of the mediums on the qualities of sorgum leaves’ calluses on week 5 to 10. Table 1. Results of the Observation on Quality of Callus in Sorgum Leaves Treatment Average / Week after planting 1 2 3 4 5 6 7 8 9 10 M1 1.00 1.00 1.00 1.19 1.41a 1.60a 1.76a 1.93a 2.07a 2.27a M2 1.00 1.00 1.00 1.13 1.21b 1.26b 1.42b 1.66b 1.70b 1.98b M3 1.00 1.00 1.00 1.13 1.11c 1.16c 1.21c 1.54c 1.62c 1.79c LSD 5% NS NS NS NS 0 0 0.02 0.4 0 0.4 Table 1. had shown the effect of MS medium in forming the quality of callus tended to be compact to become friable, while VW and NT medium formed the callus to be compact. This has matched with the research by Prasetyo (2006). The callus were brownish-yellow and formed nodules which were the embryonic callus that have the ability of regeneration. The embryonic callus were callus that had grown and developed to form the structures to become embryo. While the organogenic callus were callus that their morphogenesis growth needed a medium with different concentration of auxin and cytokines in forming shoots and roots. In medium by Murashige and Skoog (MS), there were enough macro and micro nutrients, as well as vitamins for plants growth. Explants of a plant needs to be compatible with their medium to be able to grow callus (Hendrayono and Wijayanti, 1994). 3.2. Quantity of Callus Results of type analysis had shown real differences in between the treatments of the mediums on the qualities of sorgum leaves’ callus on week 4 to 10. 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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | Table 2. Results of the Observation on Quantity of Callus in Sorgum Leaves Treatment Average / Week after planting 1 2 3 4 5 6 7 8 9 10 M1 1.00 1.00 1.00 1.26a 1.58a 1.69a 1.89a 2.39a 2.58a 2.76a M2 1.00 1.00 1.00 1.21b 1.44b 1.54b 1.70b 1.91b 1.18b 1.37b M3 1.00 1.00 1.00 1.17c 1.27c 1.36c 1.47c 1.54c 1.68c 1.86c LSD 5% NS NS NS 0 0 0.03 0 0.03 0.04 0.04 Table 2. had shown the effect of MS medium in producing the most quantities of callus than the other mediums. This has matched with the opinions of Hendrayono and Wijayani (1994), that medium by Murashige and Skoog (MS) has enough macro and micro nutrients as well as vitamins for a plant growth. Explants of a plant needs to be compatible with their medium to be able to grow callus. The success of reproduction of tissue-cultures were affected by many factors, such as nutrients and explants (Ali, 2015). Additional nutrients into plant’s mediums were very affecting to the growth and development of explants (Yusnita, 2003). 3.3. Contents of Phenol Results of type analysis had shown real differences in between the treatments of the mediums on the contents of Phenol in sorgum leaves’ callus on week 10. Table 3. Results of the Contents of Phenol in Callus of Sorgum Leaves NO TREATMENT MS Medium % Phenol VW Medium % Phenol NT Medium % Phenol 1 M1U1S1 0.160 M2U2S3 0.195 M3U3S2 0.248 2 M1U2S3 0.158 M2U1S9 0.190 M3U5S6 0.156 3 M1U1S9 0.147 M2U3S4 0.198 M3U6S1 0.172 4 M1U3S4 0.155 M2U5S6 0.192 M3U9S5 0.188 5 M1U5S6 0.157 M2U4S5 0.201 M3U2S6 0.213 6 M1U6S9 0.149 M2U6S3 0.205 M3U1S7 0.231 7 M1U7S2 0.159 M2U5S1 0.196 M3U4S1 0.225 8 M1U4S5 0.162 M2U6S3 0.210 M3U1S6 0.213 9 M1U3S7 0.220 M2U8S5 0.188 M3U5S2 0.209 10 M1U8S5 0.181 M2U5S1 0.190 M3U5S3 0.242 11 M1U4S1 0.179 M2U3S7 0.206 M3U2S6 0.235 12 M1U6S3 0.185 M2U6S9 0.205 M3U2S7 0.242 13 M1U2S4 0.190 M2U7S2 0.213 M3U6S2 0.215 14 M1U5S1 0.185 M2U2S4 0.199 M3U9S5 0.223 15 M1U1S3 0.192 M2U5S6 0.197 M3U8S5 0.208 16 M1U3S2 0.205 M2U1S7 0.209 M3U4S1 0.250 17 M1U5S6 0.212 M2U5S2 0.197 M3U2S4 0.248 18 M1U3S1 0.198 M2U6S9 0.209 M3U4S5 0.240 19 M1U9S5 0.189 M2U3S3 0.190 M3U6S9 0.209 20 M1U6S3 0.205 M2U6S3 0.205 M3U1S9 0.203 21 M1U4S2 0.195 M2U5S6 0.185 M3U4S6 0.205 22 M1U2S6 0.178 M2U2S6 0.192 M3U3S4 0.202 23 M1U6S2 0.202 M2U9S5 0.199 M3U1S3 0.228 24 M1U3S3 0.215 M2U5S3 0.201 M3U8S5 0.232 25 M1U1S7 0.209 M2U6S2 0.193 M3U7S2 0.215 26 M1U5S2 0.186 M2U4S1 0.195 M3U1S1 0.247 27 M1U4S1 0.201 M2U2S6 0.202 M3U1S9 0.225 28 M1U6S9 1.809 M2U6S2 0.191 M3U8S5 0.240 29 M1U1S6 1.946 M2U3S2 0.191 M3U3S4 0.241 30 M1U5S3 0.186 M2U5S6 0.210 M3U7S2 0.250 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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | Treatment Phenolic (%) M1 0.16 M1 0.22 M2 0.19 M2 0.21 M3 0.25 M3 0.23 The highest contents of Phenol in callus were found in the condition of NT medium. This was because the sorgum plant were very responsive to the NT medium. While the highest phenolic contents, which was 0.25%, was found in the NT medium. Then, it was assumed that as NT medium has more simplified nutrients than MS medium, so the explants had stressings and this produced the highest contents of secondary metabolites. This was matched with the opinions of Rahmawati (2006), that suggested that slow growth was needed for the cells to process the maximum secondary metabolites and also a balance of carbon nutrients in the plants’ cells metabolism, where if the nutrients availibility for the plants were in excess than they will be used by the cells to process the secondary metabolites (Pratiwi, Ali, Setiawan, Budiyanto, & Sucahyo, 2017). But each commodity requires a different time of exposure to produce the secondary metabolites. 4. CONCLUSIONS AND SUGGESTIONS 3.4. Conclusions 1. Quantity of callus in general had shown real differences and most callus found were on the MS medium treatment. 2. Quality of callus formed had shown real differences in each mediums and tended to be compact callus, while in MS medium to become friable. 3. NT tended to produce most contents of phenol, which was 0,25% . 3.5. Suggestions Further research were needed to be conducted to investigate the contents of Phenol with the addition of carbohydrates in NT medium. ATTACHMENTS Table 1. Compositions of Medium by Murashige and Skoog Materials Need (mg/L) 1. Macro nutrients : KNO3 NH4NO3 CaCl2.2H2O MgSO4.7H2O KH2PO4 1.900 1.650 440 370 170 2. Micro nutrients : MnSO4.7H2O ZnSO4.7H2O H3BO3 Kl CuSO4.5H2O NaMoO4.2H2O 22.3 8.6 6.2 0.83 0.025 0.25 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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | CaCl2.6H2O FeSO4.7H2O NaEDTA,2H2O 0.025 27.8 37.3 3. Macro nutrients : Myo-inositol Thamin HCl Nicotinic acid Pyridoxine HCl Glycine 100 0.1 0.5 0.5 2 4. Energy source : Sucrose 30.000 Source: Arijanti et al. (2014) Table 2. Compositions of Medium by Vacin and Went Materials Need (mg/L) 1. Macro nutrients : NH4NO3 KNO3 KH2PO4 MgSO4.7H2O (NH4)2SO4 200 mg/L 525 mg/L 250 mg/L 250 mg/L 500 mg/L 2. Micro nutrients : Fe(EDTA) FeSO4.7H2O MnSO4.7H2O 37 mg/L 28 mg/L 7,5 mg/L 3. Energy source : Sucrose 20g/L Source: Arijanti et al. (2014) Table 3. Compositions of Medium by Nagata and Takabe Materials Need (mg/L) 1. Macro nutrients : KNO3 NH4NO3 CaCl2.2H2O MgSO4.7H2O KH2PO4 950 825 220 1.233 680 2. Micro nutrients : MnSO4.7H2O ZnSO4.7H2O H3BO3 Kl CuSO4.5H2O CoSO4.7H2O FeSO4.7H2O Na2EDTA,2H2O 22.3 8.3 6.2 0.83 0.025 0.030 27.8 37.3 3. Organic substances : Myo-inositol Thamin HCl Sucrose D Mannitol 100 1 10.000 12.700 Source: Arijanti et al. (2014) 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 Propagation In Vitro of Sorgum in MS, VW and NT mediums Dwie Retno Suryaningsih, Sri Arijanti Prakoeswa, Maria ME Page | REFERENCES Arijanti, S., Ribkahwati and Adriani. (2006). Analysis of Polyphenol in Rosa-hybride with the Addition of 3 types of Carbohydrates. Report of Fundamental Research DIKTI 2006 ST:241/SP3/PP/DP2M/2/2006. Ali, M. (2015). 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