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Dr. Y. S. Parmar University of Horticulture & Forestry, Solan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, was established on 1st December, 1985 with the objective to promote education, research and extension education in the fields of Horticulture, Forestry and allied disciplines. Late Dr. Yashwant Singh Parmar, the first Chief Minister and the architect of Himachal Pradesh perceived the importance of Horticulture and Forestry to develop and improve the State economy which led to the establishment of this University. Its history lies in erstwhile Himachal Agricultural College, Solan, established in 1962 and affiliated to the Panjab University. It became one of the campuses of Agriculture Complex of Himachal Pradesh University on its formation in 1970. Consequent upon the establishment of Himachal Pradesh Krishi Vishvavidyalaya in 1978, this campus became its Horticulture Complex and finally in 1985, assumed the status of a State University, being the only University in the country engaged exclusively in teaching, research and extension in Horticulture and Forestry.
The University is located at Nauni in Solan District of Himachal Pradesh, 13 km from Solan on Solan-Rajgarh Road, at an elevation of 1300 metres above mean sea level. Solan town is situated on national highway (NH-22) and is well connected by train and bus services.
The University has four constituent colleges, out of which, two are located at the main campus Nauni, one for horticulture and the other for forestry, having 9 and 7 departments, respectively. The third College i.e., College of Horticulture & Forestry is located at Neri in Hamirpur District on Nadaun-Hamirpur state highway, about 6 Km from Hamirpur town and is well connected with bus service. The college offers three Undergraduate Degree Programmes i.e. BSc (Hons.) Horticulture, BSc (Hons.) Forestry and B. Tech. Biotechnology and MSc degree programme in a few subjects. The fourth college i.e. College of Horticulture and Forestry, Thunag (Mandi) is located at Thunag District Mandi. This college offer BSc (Hons.) Horticulture and BSc (Hons.) Forestry degree programme. In addition, there are five Regional Research Stations, 12 Satellite Stations and five Krishi Vigyan Kendras (KVKs) situated in different zones of the State.
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ThesisItem Open Access Studies on construction of frame work genetic linkage map of Stevia rebaudiana Bertoni using molecular markers(YSPU, 2013) Sharma, Neha; Kaur, RajinderThe present investigation on Stevia rebaudiana was carried out with the objective to construct frame work genetic linkage map of stevia by employing multiple marker systems. Before starting the work on linkage map construction, genetic diversity was analyzed amongst the available genotypes/ clones/ accessions/ morphotypes of Stevia rebaudiana to confirm two parents with differences in rebaudioside-A and stevioside content. This study represents the first genetic linkage study of Stevia rebaudiana comprising of multiple marker systems together. Among the collected 16 accessions polymorphism was studied using 27 RAPD, 26 ISSR and 50 EST-SSR. Results were analyzed in the form of dendrograms, similarity, disimmilarity matrices and polymorphism information content. For linkage map construction, F2 population was used as a mapping population. To survey the polymorphism among contrasting parents 170 RAPD, 26 ISSR and 89 EST SSR were employed and it was observed that 36 RAPD, 10 ISSR and 33 EST – SSR primers were found to be polymorphic. These primers were then used for the genotyping of the mapping population. Phenotyping was carried out by using High Performance Liquid Chromatography (HPLC) of parents as well as segregating population. Both genotypic and phenotypic data were used to construct a linkage map using MAPMAKER/EXP ver 3.0b. A total of four linkage groups were constructed spanning a distance of 927.3 cM with an average distance between loci as 16.29 cM. First linkage group (LG1) comprised of 33 markers, second linkage group (LG2) contained 6 markers and third (LG3) and fourth group (LG4) had 16 and two markers, respectively. On QTL identification, a total of 53 QTL locations were found for both trait 1 (rebaudioside-A) and trait 2 (stevioside). Among 53 QTL locations of trait 1, two major QTLs were found for rebaudioside-A viz., in the marker interval of L67- L71 (ISSR HB-11) - (IISRS-3-L) in LG1 and in the marker interval L38 - L40 (Sigma-5383-027)- (Sigma-5383-029) in LG3 at LOD of 2.5 and 2.7, respectively.ThesisItem Open Access Studies on Agrobacteriummediated insect resistance gene transfer in Cabbage (Brassica oleracea L. var. capitata) and molecular analysis of regenerated plantlets(YSPU, 2013) Gambhir, Geetika; Srivastava, D.K.Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India). Agrobacterium tumefaciens strain containing npt-II and cryIAa genes in binary vector pBin-1Aa was used for genetic transformation studies. Plant regeneration studies were carried out using four different types of explants viz. cotyledon, hypocotyl, leaf and petiole Cotyledon and hypocotyl explants were used from seven to nine days old aseptically grown seedlings whereas, leaf and petiole explants were procured from glass house 20-25 days old grown seedlings of cabbage. Hypocotyl explants showed better shoot regeneration as compared to other explants. High efficiency shoot regeneration was obtained in cotyledon (91.11 %), hypocotyl (94.44 %), leaf (91.11 %) and petiole (88.88%) explants on MS medium supplemented with 0.33mg/l TDZ + 79.7 mg/l Adenine, 0.22mg/l TDZ + 0.088mg/l IAA, 0.22mg/l TDZ + 0.02mg/l NAA and 0.33mg/l TDZ + 0.02mg/l NAA, respectively.MS medium supplemented with 0.10mg/l NAA was found best for root regeneration from in vitro developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of leaf and petiole tissues and to select transgenic shoots during transformation experiment. Kanamycin sensitivity (10-60mg/l) was checked by fresh weight of the explants which was measured at the interval of 7 days. From the relative growth of the explants it was found that the concentration as low as 10mg/l is toxic to the explants. Effect of different concentrations of cefotaxime was studied on the regeneration potential in cotyledon and hypocotyl explants of cabbage and found no much effect of cefotaxime on regeneration potential. Effect of different concentrations of cefotaxime and kanamycin (50mg/l) were studied on the growth of agrobacterial cells and regeneration potential of cotyledon and hypocotyl tissues after cocultivation. In both the explants the growth of agrobacterial cells were controlled at concentration of 400mg/l cefotaxime and maximum per cent shoot regeneration in cotyledon (35.55 %) and hypocotyl (48.15 %) was obtained on MS medium supplemented with 400mg/l cefotaxime, respectively. Effect of preculturing and co-cultivation was studied on the transformation frequency. Preculturing of cotyledon and hypocotyl explants for 72 hours and co-cultivation with agrobacterial cells for 48 hours worked out to be the best treatment as it gave the highest transformation frequency (4.66 %) and (14.50 %) in respective explants. Effect of different concentrations of acetosyringone was studied in cotyledon and hypocotyl explants to enhance the transformation frequency. The maximum percent shoot regeneration (18.66 %) and (32.00 %) was obtained from cotyledon and hypocotyl explants cultured on shoot regeneration medium containing 100μM acetosyringone at standardized preculturing and cocultivation time interval i.e. 72 hours and 48 hours. The presence/integration of transgene (cryIAa) into the genome of cabbage was confirmed by PCR using gene specific primers and Southern blot analysis using radioactive labelled DNA probe. The Southern blot analysis has also been used to confirm copy number of transgene into the genome of cabbage. For PCR analysis, 40 putative transgenic shoots were randomly selected and out of 40 putative transgenic shoots/plantlets, 20 shoots were found to be +ve for the presence/integration of transgene i.e. cryIAa into the genome of cabbage. For Southern blot analysis 10 RT-PCR +ve shoots were selected. Out of the 10 PCR +ve shoots, 5 shoots were confirmed +ve for integration of transgene cryIAa into the genome of cabbage with 1 to 3 copies of gene insertion. The confirmation of expression of the transgene cryIAa into the genome of cabbage at transcriptional level was confirmed by Reverse Transcriptase-PCR and Real Time-PCR and at translational level by Bioassay. A protocol for high frequency plant regeneration and insect resistance gene transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India) has been standardized.ThesisItem Open Access Studies on isolation and characterization of trypsin inhibitor (TI) gene from Dolichos biflorus L. (Kulth)(DYSPU, 2013) Reena Kumari; Nath, A.K.Protease inhibitors are one of the most promising agents that confer resistance in plants against insect pests by inhibiting larval midgut proteases. Maximum extraction of trypsin inhibitor protein from seed flour of Dolichos biflorusL. was in 0.1 M phosphate buffer (pH 7.6) after four hours of extraction. Screening of Dolichos biflorus L. cultivars for trypsin inhibitor activity revealed maximum activity in HPK4 cultivar and further studies were conducted in this cultivar. Crude trypsin inhibitor of all cultivars inhibited midgut protease of P. brassicaelarvae. Inhibitor activity was detected at early stages of seed development (3 days after flowering (DAF)) and it increased progressively with seed development (21 DAF to 60 DAF). Trypsin inhibitor activity decreased during seed germination as compared to dry seeds. Crude trypsin inhibitor extracted from developing and germinating seeds also inhibited larval midgut protease of S. littoralis. Neonate larvae of P. brassicae fed on cabbage leaf discs coated with 0.025-2.50 mg crude trypsin inhibitor caused 10–80 % larval mortality. The calculated LC 50 value was 1.05 mg crude trypsin inhibitor and for 2.5 mg crude trypsin inhibitor the calculated LT 50 value was 3.2 days. Leaf area eaten and faecal matter produced by treated larvae were significantly lower as compared to untreated controls. Larvae fed on leaf discs coatedwith 2.5 mg crude trypsin inhibitor for 5 days had significantly less total soluble protein in faecal matter and midgut trypsin activity as compared to untreated control. Significant reduction in egg hatching (75%) was observed in egg mass treated with 5.3mg of crude trypsin inhibitor of mature seeds. Trypsin inhibitor gene (309 bp) was amplified from cDNA synthesized from mature seeds of Dolichos biflorus L. HPK4 cultivar using designed primers. The amplified PCR product was cloned and sequenced. Sequence of Dolichos biflorusL. HPK4 cultivar trypsin inhibitor (DbTI) gene hasbeen submitted to NCBI with Accession No. JQ259858. DbTI gene and its deduced amino acid sequence showed homology with Bowman-Birk inhibitors of Dolichos spp., Phaeolus spp., Vigna spp. and Glycine spp. The predicted molecular weight of deduced amino acid sequence was ~11.5 KDa and it had N terminal signal peptide of 19 amino acid residues. The secondary structure of deduced amino acid sequence of DbTI showed dominance of coils and sheets over alpha helix. Homology modelling was employed to predict the three dimensional structure of DbTI. Docking of trypsin enzyme and DbTI showed the inhibitor to be of non- competitive typeThesisItem Open Access Bioprospecting of bacteria for production and purification of laccase enzyme(YSPU, 2013) Dhiman, Karuna; Shirkot, PoonamLaccase enzyme has acquired the status of ‘green catalyst’ as it possesses remarkable bioremediation potential along with numerous applications in effluent detoxification, degradation of textile dyes, herbicide and insecticide degradation, wine clarification, enzymatic conversion of chemical intermediates, biosensors and organic synthesis. In the present study, significant high diversity of laccase producing bacteria from rhizosphere of rice plants from paddy fields of H. P. was assessed whereas medium diversity was obtained from the samples of paper mills of H.P. A total of 449 bacterial isolates were obtained from 198 samples using M162 and TY media containing 5mM guaiacol and 40 mg/l CuSO4. These were rescreened on the basis of their ability to oxidise tannic acid and dimethoxyphenol leading to selection of 67 bacterial isolates which were characterized both morphologically and biochemically alongwith the laccase activity and 14 bacterial isolates exhibiting maximum laccase activity of 10-19 U/l were selected. Molecular characterization of the selected isolates was carried out using RAPD-PCR and 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to identification of these bacterial isolates as Pseudomonas putida strain LUA15.1 and LHB7.1, Pseudomonas umsongenesis strain LHB9.1., Pseudomonas mohnii strain LHN12.2, Pseudomonas chlororaphis strain LUD7.1, Pseudomonas jessenii strain LHN9.1, Pseudomonas lurida strain LB6.2, Pseudomonas graminis strain LHN8.1, Pseudomonas veronii strain LUA14.1, Pseudomonas fulva strain LR5.1, Lysnibacillus fusiformis strain LKM7.1, Lysnibacillus sphaericus strain LH3.4 and Bacillus subtilis strain LB6.1 and LR6.3 . On the basis of maximum laccase enzyme activity Pseudomonas putida strain LUA15.1 was selected for production and purification of the laccase enzyme. Maximum extracellular enzyme production was achieved at 28°C, pH 7 (24 hrs incubation) with 5mM guaiacol, 50 mg/l CuSO4, 5% tryptone and 3% yeast extract in combination as nitrogen source in Tryptone Yeast medium. The laccase crude extracellular enzyme preparation was purified by ammonium salt precipitation (50-90%) followed by gel filtration and ion exchange chromatography which showed 10.74 yield and 61.36 fold purification. The purified enzyme had optimal activity at pH 7.0 and 40°C and 0.80 mM Km value. The molecular weight of laccase in the present study was found to be 42.5 kDa. The activity was inhibited by sodium azide and DTT. Strain LUA15.1 as well as its enzyme preparations were studied for their ability to decolourize dyes which are the potential contributors of water pollution. All six different synthetic dyes were decolourized RBBR (48%), congo red (35%), indigo carmine (80%), brilliant green (97%), bromophenol blue (78%) and aniline blue (23%) when treated with the culture of Pseudomonas putida strain LUA15.1. However, the crude as well as partially purified enzyme preparation of Pseudomonas putida strain LUA15.1 showed greater decolourization of dyes comparatively congo red (98%), indigo carmine (99%), RBBR ( 96%), aniline blue (37%) bromophenol blue (70%) and brilliant green (60%). The purified enzyme was successfully immobilized using encapsulation method in calcium alginate beads with 76% immobilization percentage and immobilized laccase enzyme beads were studied for their ability to degrade dyes. The stability and reusability of the immobilized enzyme system has the potential to make the entire treatment process inexpensive. An extracellular laccase producing gene has been isolated using degenerate primer based on the copper I and II conserved site of laccase enzyme, from the rice rhizospheric bacteria, Pseudomonas putida strain LUA15.1 followed by determination of the nucleotide sequence of this gene and it showed 91% similarity with Pseudomonas putida strain mt-2 Mn(II)-oxidation-associated multicopper oxidase (cumA) gene, partial cds. This nucleotide sequence of laccase was translated into amino acid and encodes a polypeptide comprised of 113 amino acids which showed 85 % identity with the amino acid sequences of bacterial laccases i.e. Mn (II)-oxidation-associated multicopper oxidase [Pseudomonas putida]. Further multiple sequence alignment using MULTALIN and structure prediction using Phyre 1 & 2 revealed conserved histidine residues.ThesisItem Open Access IN VITRO SELECTION OF CELL LINES IN Punica granatumL. (DARU) AGAINST BACTERIAL BLIGHT(UHF,NAUNI, 2013) GARIMA, KUMARI; KANWAR, KAMLESHABSTRACT The present investigation aims at “In vitro selection of cell lines in Punica granatum L. (Daru) against bacterial blight. Cotyledon and hypocotyl segments of 3 weeks old in vitrogerminated seedlings and mature leaf were used as explants. For leaf explants the sterilization protocol was standardized with 0.2 % bavistin treatment for 10 minutes and 0.5% sodium hypochloride treatment for 5 minutes. Callus induction and plantlet regeneration varied with explant type and growth regulators. The best callus induction medium for all the explants was MS medium supplemented with 2.0 mg/l BA and 4.0 mg/l NAA. Hypocotyl and cotyledon were more responsive explant and showed 96.67 and 85.00 per cent callus induction. The best medium for callus differentiation and shoot bud induction fromcotyledon derived callus was MS medium supplemented with BA (2.0 mg/l), Kinetin (1.5 mg/l) and GA 3 (3.0 mg/l), while best medium for shoot bud induction from leafderived callus was MS medium containing BA (2.0 mg/l) and Kinetin (0.5 mg/l). Cotyledon showed better regeneration (81.67 percent) as compared to leaf explant (48.33per cent). No regeneration was observed in hypocotyl derived callus explants. Rooting of in vitro raised shoots was done on half strength MS medium supplemented with 0.04 % charcoal. The well rooted plantlets were acclimatized in autoclaved sand. Cell line selection was done by using bacterial culture filtrate of Xanthomonas axonopodis pv. punicae as a selective agent. Resistant lines were selected at 40 per cent level of culture filtrate after two cycles of selection. Multiplication and shoot regeneration from selected calli was obtained on previously standardized medium. Fresh weight of callus increased progressively upto third subculture passage while shoot bud induction from selected calli was observed only after third subculturing. The selected microshoots were rooted on the rooting medium. After in vitro testing of shoots regenerated from selected calli 4 resistant plantlets were obtained.ThesisItem Open Access In vitro PROPAGATION AND CONSERVATION OF Swertia chirayita(UHF,NAUNI, 2013) SHAILJA; KANWAR, KAMLESHAbstract A protocol for in vitro propagation and conservation was developed for Swertia chirayita, an endangered medicinal plant. The sterilized explants (leaves) cultured on MS medium supplemented with 0.1 mg/l NAA and 3.0 mg/l BA gave best results for in vitro callus induction. Shoot regeneration was obtained from the callus on the same medium. The in vitroshoots cultured on MS medium supplemented with 2.5mg/l BA and 0.1 mg/l Kinetin gave best results for in vitro shoot multiplication. The MS medium supplemented with 0.1 mg/l NAA and 3.0 mg/l BA medium was found to be thebest for direct shoot regeneration from in vitroleaves. 80.30% root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 400 mg/l activated charcoal and 0.1 mg/l NAA. In vitro conservation was carried out by using two different approaches namely slow growth by changing media composition (sucrose and abscisic acid), at low temperature and cryopreservation following vitrification.With increase in concentration of sucrose and ABA decrease in growth of in vitroshoots was observed. No shoot multiplication with average leaf size of 0.35 cm and shoot length 0.67 cm was observed on half MS containing 90 g/l sucrose. Similarily, in case of media containing half strength MS salts and 3.0 mg/l ABA showed no shoot multiplication,0.83 cm average leaf size and 0.83 cm shoot length. At low temperature the in vitro shoots incubated at 4 o C, showed 100% retrieval, with 1.00 cm average number of shoots, 0.86 cm shoot length and 0.34 cm leaf size. In vitro shoots incubated at 10 o C, showed 100% retrieval, with 1.00 cm average number of shoots, 0.76 cm shoot length and 0.23 cm leaf size. During studies the vitrified shoot gave retrieval of 42.33% when precooled at 4 o C while only 22.37% vitrified shoots were retrieved from those precooled at 10 o C.ThesisItem Open Access GENETIC TRANSFORMATION STUDIES IN LETTUCE (Lactuca sativaL.) WITH npt-IIAND gusGENES(UHF,NAUNI, 2014) SHAUNAK, ISHANI; SRIVASTAVA, D.K.ABSTRACT Genetic transformation studies were carried out to standardize a protocol using Agrobacterium-mediated gene transfer technique. High efficiency plant regeneration protocol is a prerequisite of genetic transformation, hence the plant regeneration protocol was also standardized. Two types of explants viz leaf and petiole were used for plant regeneration studies. High percentage shoot regeneration in petiole (70.73%) and leaf (70.10%) explants were obtained on MS medium containing 0.25mg/l BAP+0.10mg/l NAA and MS medium containing 1.00 mg/l BAP+0.10mg/l NAA respectively. MS medium supplemented with 0.50mg/l IBA was found to be best for root regeneration (68.00%). Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of leaf and petiole explant and to select transgenic shoots during genetic transformation experiment. Kanamycin concentration above 30mg/l was toxic to the explants on selective shoot regeneration medium. Disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β- glucuronidase [uidA (gus)] gene in binary vector (pBI 121) system along with kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for genetic transformation studies in lettuce. After co-cultivation only the transformed cells/tissues were able to grow on selective shoot regeneration medium (25mg/lKanamycin and 400mg/l cefotaxime) whereas non-transformed cells/tissues turned brown and died on the selective medium. Transformation experiment could be scored as early as 5-6 weeks after selection. The effect of pre-culturing and cocultivation was studied on transformation frequency. Explants pre-cultured for 48 hours and cocultivated for 48 hours resulted in shoot regeneration. Putative transformed shoots obtained from leaf and petiole explants, were able to grow on the selective medium containing 25mg/l Kanamycin. The presence of npt-IIand uid A (gus)was confirmed by PCR using designed primers in thetransgenic shoots. Out of 5 putative transformed shoots, 3 have shown the amplification of npt-IIand 4 shown the amplification of uid A (gus)genes. Expression of uid A (gus) gene was studied in the PCR positive shoots by β-glucuronidase assay using biochemical and histochemical methods. The transformed shoots were GUS positive. A protocol for genetic transformation in lettuce with npt-IIand gusgenes has been standardized.ThesisItem Open Access EVALUATION OF GENETIC RELATEDNESS AMONG TEMPERATE POME FRUIT CROPS OF FAMILY ROSACEAE USING PCR BASED MOLECULAR MARKERS(UHF,NAUNI, 2014) SHARMA, HIMANI; SHARMA, RAJNISHABSTRACT The family Rosaceaeis third most economically important plant family that include temperate pome fruit crops having a wide genetic diversity. For a rationale use of the genetic resources in breeding programs it is necessary to understand relationship within and among related crop varieties and to assess their transferability to the species with little genomic information making them significant for genetic research. Thus, in the present study genetic relatedness and SSRs transferability among fifty genotypes of temperate pome fruit crops were tested using (RAPD, ISSR and SSR) molecular marker analysis. The level of polymorphism across genotypes was 97.29% and 97.45% by RAPD and ISSR markers, respectively. The dendrogram derived from RAPD and ISSR analysis grouped temperate pome fruit crops into two major cluster comprising of 40 (cluster A) and 10 (cluster B) genotypes which branched at similarity value of 0.31 and 0.33, respectively. Estimates of pooled similarity coefficient ranged from 0.30 to 0.94. It was concluded from the pooled analysis of both the molecular marker that 50 genotypes of temperate pome fruit crops were grouped in two clusters A and B comprised 45 and 5 genotypes, respectively which branched at similarity value of 0.30 with the maximum similarity of 94% was found between EC-02520 and EC-024530 quince genotypes. Further, out of 13 and 15 screened apple and pear genomic SSR, 10 (76.92%) and 11 (73.33%) produced amplification in 20 apple and pear genotypes, respectively. Highest transferability of apple and pear SSR, 61.53% and 73.33% was observed in closely related quince and apple genotypes, respectively. This high level of polymorphism and good transferability of apple and pear SSRs to other temperate pome fruit crops indicated their promise for application to future molecular screening, map construction, and comparative genomic studies, etc.ThesisItem Open Access Refinement of technology for micropropagation of carnation (Dianthus caryophyllus L.) cv. ‘Master’(UHF,NAUNI, 2015) THAKUR, KANIKA; KANWAR, KAMLESHABSTRACT The present investigation aims at “Refinement of technology for micropropagation of carnation (Dianthus caryophyllus L.) cv. ‘Master’”. Surface sterilized shoot buds cultured on control (MS + 3% sucrose + 0.8% agar-agar) supplemented with 2.0 mg/l BA which showed 77.77% establishment. Best medium for multiplication was MS medium supplemented with 2.0 mg/l BA producing 4.50 number of microshoots of shoot length of 2.44 cm, but as it resulted in hyperhydricity of shoots so medium supplemented with kinetin (2.0 mg/l) was used which produced 3.67 number of microshoots and 4.55 cm shoot length. Low cost medium containing 30 g/l table sugar, 50 g/l starch and aquaguard water produced 3.52 shoots with 3.01 cm shoot length. It was also observed that subculturing passage affected the average number of shoots per explant and also the shoot length which was highest in case of fifth subculturing resulting in 5.58 and 5.27 number of shoots per explant on control and low cost medium and shoot length of 4.66 cm and 3.53 cm , respectively. 62.49% rooting and 4.50 average roots per shoot with 1.61cm was found on 1/2 MS + 0.04% activated charcoal and 48.58% rooting with 3.50 average roots per shoot and 1.75 cm was observed on 1/2 MS + 50g/l starch + 0.04% activated charcoal + 15 g/l table sugar. The study has resulted in the reduction cost of the medium by substituting agar-agar and sucrose with starch and table sugar.