CHAPTER-3 MATERIALS AND METHODS

CHAPTER-3 MATERIALS AND METHODS The plants of Stevia rebaudiana cv. CIM madhu and CIM mithi were collected from the Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow (U.P.). In vitro cultures were established using nodal and leaves segments collected from the mature mother plants maintained under glass house conditions (Plate 4.1). 3.1 Media preparation All the glassware i.e. beakers, glass bottles with polypropylene lids, conical flasks, pipettes, test tubes, petri plates etc. were procured from Borosil company. Throughout the course of investigation, care was taken to use chemicals of high purity. All the chemicals used for the preparation of basal media including vitamins, growth regulators and all the organic reagents of analytical grade were procured from Hi-Media Laboratories pvt. Limited, Mumbai- India. All the glassware were washed with laboratory detergents (teepol) followed by sufficient washing with running tap water to remove the detergents residue. All the cultural vessels used during the experiment were dried in hot air oven at 120-130ᵒC. The prepared media, glassware, forceps, scalpels, cotton, flakes, tissue papers, glass plates, double-distilled water in air tight bottles were wrapped/covered with aluminum foil and carefully autoclaved at 121 C for 20 minutes in a vertical autoclaved at 1.05 kg/cm 2 (15 psi). MS basal medium was used for the present study (Table 3.1). The MS medium was supplemented with different concentration of growth regulators namely, ά-naphthalene acetic acid (NAA), indole-3-butric-acid (IBA), 6-benzylaminopurine (BAP), kinetin (Kn), 2,4- dichlorophenoxyacetic acid (2,4-D) and adenine sulphate (ADS) and their combinations as specified. 44

Table 3.1 Composition of Murashige and Skoog (1962) medium. S.No. Chemical Name 1 X (mg/l) X th Concentration (mg/l) A Macro 20 X 1 NH 4 NO 3 1650.0 33000 2 KNO 3 1900.0 38000.0 3 MgSO 4.7H 2 O 370.0 7400 4 KH 2 PO 4 170.0 3400 B. Calcium 50X 5 CaCl 2.2H 2 O 440.0 22000 C. Micro I 100X 6 H 3 BO 3 6.20 620 7 MnSO 4.4H 2 O 22.3 2230 8 ZnSO 4.7H 2 O 8.6 860 9 KI 0.83 83 10 NaMoO 4.2H 2 O 0.25 25 D. Micro II 100X 11 FeSO 4.7H 2 O 27.8 2780 12 Na 2 EDTA.2H 2 O 37.3 3730 E. Micro III 1000X 13 CuSO 4.5H 2 O 0.025 25 14 CoCl 2.6H 2 O 0.025 25 F. Organics 100X 15 Nicotinic acid 0.5 50 16 Pyridoxine-HCl 0.5 50 17 Thymine-HCl 0.1 10 18 m-inositol 100 100 (1X) 19 Glycine 200 200 (1X) 20 Sucrose 30000 30000 45

The stock solutions of different chemicals were prepared in double distilled water and stored in reagent bottles in a refrigerator. 6-benzylaminopurine (BAP), gibberellic acid (GA 3 ) and auxins viz. ά-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were dissolved first in few drops of 1N KOH solution and then, the required amount of double distilled water was added to make the volume of the stock solution. All the stock solutions were kept in reagent bottles (Schott Duran s) and stored in a refrigerator at 4ᵒC until use. The required amounts of macro and micro-nutrients, organic salts, vitamins, growth regulators and sucrose were added to double distilled water in a vessel. The final volume was made by adding double distilled water. The ph of the solution was adjusted to 5.8 using 0.1N HCI or 0.1N NaOH. For solidification of the medium, agar powder (tissue culture grade; agar-agar type-i) 0.8% w/v was added to the luke warm solution and then boiled up to the proper dissolving and melting of agar powder. Then the medium was dispensed immediately in the conical glass vessels or test tubes. After plugging (using non-absorbent cotton) the vessels were covered with aluminum foil. The contents were then sterilized in a vertical autoclave at 121 C for 20 minutes (15 lbs/inch 2 ). The media containing vessels were then stored at room temperature under dust free condition for further use. The cultures were incubated in culture room and provided with a photoperiod of 16/8 hours light/dark cycle at 25±2 C temperature. The relative humidity in the culture room was maintained at 70-80%. 3.2 Aseptic culture establishment and multiplication All the recommended cultural practices were followed with regular spraying of plant protection chemicals to avoid disease and pests. Fully expanded leaves (3 rd leaf from top) with petiole were collected from healthy and disease-free plants. The petioles were separated from leaves and leaf lamina was transversely cut in square pieces. Shoots were dissected to remove outer leaves without disturbing the nodal segments. Well prepared explants as mentioned above, were washed thoroughly under tap water, then washed with teepol solution for 5 minutes followed by thorough washing under running tap water for 5-10 minutes to remove any residue of the detergents. These explants were then given different treatments before inoculation to minimize the contamination in the culture. The following pre-treatment s were used to minimize contamination. 46

SI. No. Pre-treatment Duration (min.) 1. Control (distilled water water shake) 10 2. Carbendazim (0.1%) 10 3. Ridomil (0.1%) 10 (Metalaxil 8%+ Mancosal 64%) 4. Car + Rido (0.1%) 10 The pre-treated explants were surface-sterilized using 0.1% mercuric chloride for 45, 55 and 70 seconds and subsequently washed 4-5 times with sterilized double distilled water to remove traces of HgCl 2. Each treatment was replicated thrice (these treatments were performed inside the laminar air flow chamber). The sterilized explants were then inoculated on culture medium. The cultures were maintained at 25±1ᵒC temperature under fluorescent white light (47 µmol/m 2 /S) at a period of 16/8 hours light and dark cycles. All the experimental manipulations were carried out under aseptic conditions in an inoculation chamber (Laminar air flow) fitted with ultraviolet tube (15W). The floor of chamber was thoroughly scrubbed with cotton dipped in absolute alcohol and all the vessels, forceps and scalpels were placed in the chamber. Then the chamber was sterilized by U.V. light for half an hour. Hands were properly washed with soap and rinsed with absolute alcohol before working in the chamber. The surface sterilized explants were trimmed aseptically to smaller segments (1.0-1.5 cm) in laminar air flow chamber. The cotton plug removed from test tube near the flame of spirit lamp to cease the entry of micro-organism, and then the forceps were dipped in alcohol and heated over the flame of spirit lamp, then after the cooling of forceps the explants were picked and inoculate on MS medium in the test tube. After inoculation, the test tubes were again flamed over the lamp and plugged with cotton plugs. The following concentrations of auxins (2,4-D, IBA) and cytokinins (BAP) were used for callus induction from different explants: 1. MS devoid of hormones 2. MS + 2,4-D (1.0 mg/l) + BAP (0.2 mg/l) 3. MS + 2,4-D (2.0 mg/l) + BAP (0.2 mg/l) 4. MS + 2,4-D (3.0 mg/l) + BAP (0.2 mg/l) 5. MS + 2,4-D (4.0 mg/l) + BAP (0.2 mg/l) 6. MS + IBA (1.0 mg/l) + BAP (0.2 mg/l) 7. MS + IBA (2.0 mg/l) + BAP (0.2 mg/l) 47

8. MS + IBA (3.0 mg/l) + BAP (0.2 mg/l) 9. MS + IBA (4.0 mg/l) + BAP (0.2 mg/l) The calli derived from various explants were transferred to regeneration medium having various growth regulators i.e. BAP, Kinetin, Adenine sulphate and NAA. Different concentrations of sucrose (3, 4, 5 and 6%) were also used. 1. MS devoid of hormones 2. MS + BAP (1.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 3. MS + BAP (2.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 4. MS + BAP (3.0 mg/l) + NAA(0.2 mg/l) + ADS (40 mg/l) 5. MS + Kn (1.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 6. MS + Kn (2.0 mg/l) + NAA(0.2 mg/l) + ADS (40 mg/l) 7. MS + Kn (1.5 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 8. MS + Kn (3.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 9. MS + BAP (0.5 mg/l) + Kn (0.5 mg/l) + ADS (40 mg/l) For direct regeneration, the nodal segments were cultured on MS medium supplemented with various growth regulators. The following concentrations of BAP and Kinetin were used in different combination along with NAA for bud break from. 1. MS devoid of hormones (Control) 2. MS + BAP (0.5 mg/l) + NAA (0.2 mg/l) 3. MS + BAP (1.0 mg/l) + NAA (0.2 mg/l) 4. MS + BAP (1.5 mg/l) + NAA (0.2 mg/l) 5. MS + BAP (2.0 mg/l) + NAA (0.2 mg/l) 6. MS + Kn (0.5 mg/l) + NAA (0.2 mg/l) 7. MS + Kn (1.0 mg/l) + NAA (0.2 mg/l) 8. MS + Kn (1.5 mg/l) + NAA (0.2 mg/l) 9. MS + Kn (2.0 mg/l) + NAA (0.2 mg/l) Sub culturing of regenerants derived through direct or in direct mode of regeneration was done on those media which support the maximum response. To assess the multiplication potential of the regenerated shoots they were transferred on differentiating media as mentioned below. 1. MS devoid of hormones 2. MS + BAP (1.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 48

3. MS + BAP (2.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 4. MS + BAP (3.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 5. MS + Kn (1.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 6. MS + Kn (2.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 7. MS + Kn (1.5 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 8. MS + Kn (3.0 mg/l) + NAA (0.2 mg/l) + ADS (40 mg/l) 9. MS + BAP (0.5 mg/l) + Kn (0.5 mg/l) + ADS (40 mg/l) The in vitro proliferated shoots were used to study the effect of NaCl. Two-node leafy microcuttings were excised from initial cultures and were sub-cultured on previously standardized shoot proliferation medium with different concentrations of sodium chloride (NaCl) ranging from 0-125 mm. Following concentrations of NaCl were used: 1. Without NaCl (Control) 2. 25 mm NaCl 3. 50 mm NaCl 4. 75 mm NaCl 5. 100 mm NaCl 6. 125 mm NaCl 3.3 Root Development The proliferated shoots were excised and transferred to rooting media consisting of half MS basal medium supplemented with (3, 4, 5, 6 and 7%) sucrose and different concentration of IBA as given below: 1. ½ MS devoid of hormones 2. ½ MS + IBA (0.2 mg/l) 3. ½ MS + IBA (0.5 mg/l) 4. ½ MS + IBA (0.2 mg/l) + AC (100 mg/l) 5. ½ MS + IBA (0.5 mg/l) + AC (100 mg/l) The in vitro proliferated shoots were excised and transferred on to previously standardized root development medium containing different concentrations of sodium chloride (NaCl) salt ranging from 0-125 mm. The different salt concentrations used are as follows: 1. Without NaCl (Control) 2. 25 mm NaCl 49

3. 50 mm NaCl 4. 75 mm NaCl 5. 100 mm NaCl 6. 125 mm NaCl 3.4 Acclimatization This is the last step of micropropagation in which the plantlets were taken out from the medium after the proper development of sufficient roots. The rooted plantlets were gradually pulled out from the cultured test tubes and immersed in sterilized double distilled water to remove agar particles sticking to the root system. The roots were dipped in to 0.1% ridomil solution for few seconds and then transferred to plastic pots or glass jars consisting of sterilized vermiculite: soil: sand (1:2:1) or vermicompost: peatmoss: sand (1:2:1). The hardening mixture was sterilized at a pressure of 1.03kg cm -2 for 30 min. Each pot was covered properly to maintain high humidity around the plants. The potting mixture was moistened with ½ strength MS salt (devoid of organics and sucrose). Caps of the jars were kept closed for a week in order to maintain high humidity and then gradual hardening of plants was done by loosening the caps of glass jars in order to allow the moisture to escape and then finally removing the polypropylene caps after two weeks of planting. The plastic pots were covered with polythene covers and these cover were pierced at two days interval to allow the moisture to escape, the plantlets were then exposed to the laboratory environment by removing the covers. After 15 days of their acclimatization under laboratory conditions, the plants were transferred to glass house. 3.5 Stevioside quantification Dried leaves were extracted by using methanol and water according to Nishiyama (1991). Methanol and water were added to ground leaves (4:1 v/w) and left for 7 hour then filtered through filter paper (Whatman No. 4). The filtrate was further concentrated in rotary flash evaporator at 60ᵒC and to this dried extract 500 ml of distilled water was added. Extract was separated using separatory funnel with 100 ml of diethyl ether to remove green colour of the extract. Lower transparent layer was collected and treated with butanol. Finally the butanol layer was collected and refrigerated at 4ºC to get fine crystals. Crystals were resuspended in methanol and used for further purification of stevioside. Chromatographic analysis High performance liquid chromatography, being more sensitive and accurate, was used for the estimation of stevioside content in the samples. The identification and 50

quantification of stevioside content in the samples was done by comparing the retention time and peak area of sample with that of the standard. All the other chemicals and solvents used were of analytical grade. Operating conditions for HPLC includes C18 column, separation was performed with a methanol: water (63:35 v/v) as the elusion solvent at flow rate of 0.5 ml/min and the detection wavelength was 245 nm. 3.6 Biochemical parameters Total chlorophyll The chlorophyll content of the leaves was analyzed following the method of Arnon (1949). Fully mature open leaves were chosen as the experimental sample for chlorophyll estimation. For extraction, 50mg of plant sample was homogenized with about 10 ml of ice cold 80% acetone. A pinch of CaCO 3 was added to avoid the destruction of chlorophyll and other pigments. Extraction has to be carried out under dim light to avoid photo oxidation of the pigments. It was centrifuged in a centrifuge at low speed 5000 rpm for about 20 min. Ice cold pestle and mortar were used for grinding the samples. After centrifugation cell wall debris were settle down and was discarded. Only supernatant was taken out and raised to a volume of 10 ml with ice cold 80% acetone. Absorbance was recorded soon after extraction was over with the help of UV- vis spectrophotometer (Specord-205 Analytic Jena, Germany) at wavelengths of 663 and 645 nm. The amount of chlorophyll a, b and total chlorophyll was calculated by the formula of Arnon (1949). 12.3 A663 0.86 A645 V Chlorophylla (mg/g fresh wt.) = α 1000 W 19.3A645 3.6 A663 V Chlorophyll b (mg/g fresh wt.) = α 1000 W Total Chlorophyll (mg/g fresh wt.) = Chlorophyll a + Chlorophyll b Where, α = Light path in cm (1 cm), W= Fresh weight in grams, V= Volume of extract in ml. Total soluble sugars The amount of total soluble sugars was estimated using Anthrone reagents as given by Thimmaiah (2004). One hundred mg leaves were taken in a boiling tube and hydrolyzed with 5 ml 2.5 N HCl in a water bath for 3 h. It was then neutralized with solid sodium carbonate until effervescence ceased. The volume was made to 100 ml followed by centrifuge at 5000 rpm for 10 min. The supernatant was collected and 1 ml sample was taken for analysis. Four 51

ml Anthrone reagent was added to aliquot and heated for one min. in water bath (70ᵒC). The sample was then rapidly cooled and the change of green to dark green colour was read at 630 nm against blank. Reducing sugars Reducing sugars were estimated by the method of Somogyi (1951). One hundred mg leaves were taken and extracted with hot 80 per cent ethanol for twice (5 ml each time). Supernatant was collected and evaporated to dryness by keeping in water bath at 80 0 C. Then, 10 ml distilled water was added to dissolve the sugars. From this, 0.2 ml aliquot was pipetted out to separate test tubes and volume was made to 2 ml with distilled water, 1 ml of alkaline copper tartarate was added followed by keeping in water bath at 100 0 C for 10 minutes. Tubes were cooled and 1 ml of arseno-molybolic acid reagent was added to all the tubes. The volume was then made to 10 ml with distilled water and absorbance of blue colour was read at 620 nm after 10 minutes. Total phenols The method of Malik and Singh (1980) was employed for quantification of total phenols. One hundred mg leaves were taken and homogenized in a mortar by adding 80 per cent ethanol. It is then centrifuged at 10,000 rpm for 20 min. and the supernatant was filtered using filter paper whatman No. 42. The residue was re-extracted (5 times) with 80 per cent ethanol and the supernatant collected were evaporated to dryness. Residue was dissolved in 5 ml of distilled water from which about 0.2 ml was taken and total volume was made upto 3 ml with distilled water. To this, freshly prepared Folin-Ciocalteau reagent (0.5 ml) was added. After 3 min, 2 ml of sodium carbonate solution (20%) was added in each tube, mixed thoroughly and placed in a hot water bath (58ᵒC) exactly for one min. It was then cooled to room temperature and then absorbance was taken at 650 nm against blank. Proline The proline content was estimated according to the method used by Bates et al. (1973). Five hundred homogenized fresh leaves were added in 10 ml of 3% sulfo-salicylic acid. Then it was filtered through Whatman No. 2 filter paper. Two ml of the filterate was taken in a test tube containing 2 ml of acid ninhydrin solution (1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml of 6 M orthophosphoric acid). After this 2 ml of glacial acetic acid was added and heated for 1 h at 100ºC. Test tubes were then shifted in an ice bath to terminate the reaction. Reaction mixture was then extracted with 10 ml toluene and mixed 52

vigorously by passing a continuous air stream for 1-2 minutes. Aqueous phase was separated, warmed at room temperature and absorbance was noted at 520 nm. 3.7 Bio-hardening The in vitro hardened plants were subjected to bio-hardening by using different arbuscular mychorrhizal fungi (AMF) strains to reduce their mortality rate. The different AM fungi strains used were as follows: T0 = Without AMF (control) T1 = Glomus mosseae T2 = Acaulospora laevis T3 = Glomus mosseae + Acaulospora laevis Both of the strains were pure strains maintained on Rhodes grass Chloris gayana (Plate 4.16) grown on sterile potting mixture and maintained in controlled glasshouse conditions. The potting mixture was composed of sand, soil and FYM (1:2:1). AM fungi treatments consisted of approximately 20 g inoculum (containing rhizosphere soil, spores besides hyphae, arbuscules and vesicles). The plants acclimatized under in vitro conditions were inoculated with AM fungi strains. The plantlets were maintained in a glasshouse with day-night temperatures ranging from 27±1 0 C. Day length was extended to 16 hours with cool white fluorescent lights at 630 µmol sec -1 m -2. Humidity was maintained to the level of 85 per cent using humidifier. Plantlets were watered on alternate days with sterile tap water. The different morpho-physioclogical parameters at 30, 45 and 60 days after inoculation (DAI) were studied under glasshouse conditions. Data were analyzed for significance using one-way analysis of variance (ANOVA) and the differences contrasted using a Duncan s multiple range test (DMRT) at p 0.05. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS, version 11.5). 53