Tissue culture of tobacco Seed sterilization ( Gamborg and Phillips 1996)

Seeds of tobacco (Nicotiana xanthi) were gifted by Prof KC Upadhayaya; Centre for Plant Molecular Biology, JNU-New Delhi. Seeds were submerged in water in a flask and shaken. Rinsed 2-3 times with distilled water. Water was removed and 70% ethanol was added. It was shaken for one minute and ethanol was decanted. Seeds were transferred to another flask containing 2% sodium hypo-chloride and 2-3 drops of tween 20. It was shaken well _time to time and left for 15 minutes at room temperature. In a sterile laminar airflow chamber, solution was decanted and seeds were washed 4-5 times thoroughly with sterile · double distilled water.

Seed germination

Sterilized seeds were transferred on 1/z strength Murashige and Skoog (1962) medium (composition of the medium is given in Appendix I) with 1% sucrose, in a petriplate (90 mm in diameter) for germination. Plates were kept in dark in culture room, at 24±2 °C.

Callus initiation and maintenance (Phillips et al 1996)

Germinated seedlings were collected aseptically when the cotyledons were fully expanded and the epicotyl was beginning to emerge. Each seedling was placed on a sterile petriplate, one at a time to prepare explants. Two cotyledons were excised from half of the seedlin§s and cultured ~baxial side up, ~r. ~ps~de down on MS ~edia supplemented. with 0.5mgl BAP and 2mgl NAA for callus Initiation. Hypocotyl sections from the decapitated seedlings were excised and cultured on MS media supplemented with 0.5mgr1 BAP and 2mgr1 NAA for callus initiation in culture room at 24±2 °C and 2000 lux fluorescent light for 24 h. Shoot apices from another half of the seedlings were excised, half without cotyledons (from earlier step) and half with cotyledons and inserted at the stem base into MS medium fortified· with 0.5mgr1 BAP and 2mgr1 NAA. Two shoots, one with and one without cotyledons were inoculated per culture tube/flask. This established the stock shoot culture for future use. Cultures were observed weekly. Small pieces of calli (0.5 g fresh weight approx.) were excised and sub-cultured on the fresh medium of the same composition every month to maintain a callus stock.

23

Induction of shoots/ multiple shoots from the calli

Callus obtained by the above procedure were cut into pieces, approximately 0.5 g. One piece was added in each culture tube containing MS media fortified with 0.5mgr1 BAP and 2mgr1 NAA and 0.8mgr1 BAP and O.lmgr1 NAA for shoot/ multiple shoot proliferation. Culture tubes were incubated in culture room the conditions were same as mentioned above.

Biological hardening of tobacco plantlets Culture of P. indica

Minimal agar medium was prepared as per the ingredients given in Appendix I (Verma 1996). pH of the medium was adjusted to 4.8 and sterilized using autoclave at 15 lbs for 15 min. One disc agar base hyphae of P. indica was placed in a petriplate (90 mm disposable) containing modified minimal agar medium. Inoculated petriplates were incubated for 7 days at 30 °C in an incubator in dark. After incubation, a portion of hypha! mat was transferred onto a fresh medium in triplicate and incubated as above.

Broth medium was prepared as per the jngredients given in Appendix I, ·except that the agar was not added. After adjusting the pH at 4.8, medium was transferred in culture tubes and autoclaved. Glass tubes containing fresh minimal broth were inoculated with-one agar disc (about 1 em in diameter) infested with fungal hyphae and spores. In routine, the fungus culture is made on a solid agar medium in the petriplates. This usually takes 10-15 days incubation at 30 °C. One disc was removed from the agar plate with the help of a broad end of the sterile pasture pipette and tubes were incubated for 30 days at 30 °C in dark. Re-transfer process was carried out as per the requirements of the inoculum.

Pot preparation

Thermocol pots of 10 x 6 em size were thoroughly washed and then wiped with 70% ethanol. Garden soil was sterilized by autoclaving at 121 °C, 15 lbs pressure for three times at an interval of 48 h. Sand was acid treated in 10% HCI overnight and washed in running tap water till the pH becomes neutral. Sterile soil and acid washed sand were dried in a hot air oven. Soil and sand were mixed in the ratio of 3: 1 for filling the pots.

24

Measurement of water holding capacity: (Neeraj 1992; Dogra and John 1995)

The above soil sand mix1ure was finely powdered. A thermocol pot of 10 x 6 em size was weighed separately (A). Few small holes were made at the base of the pot. A circular piece of Whatman no. 1 filter paper (equal to the bottom of the pot) was placed inside the pot and weighed (B). The pot having filter paper at the base was filled with soil and sand mixture and weight was taken (C). These pots were placed in a plastic tray containing water, upto 2/3 height of the tray. Pots were left as such for half an hour and then was removed from the tray and placed on a thick filter/blotting paper. The pots were left in this condition for 20h to allow the extra gravitational water to come out. Then again the weight was taken (D). Pots were kept in an oven at 60 °C for 48 h to evaporate the water and the weight was recorded.

To determine the amount of water absorbed by the filter paper present in the pot, paper of the pot base size was made, soaked in water and then weighed separately. The difference in weight gives the amount of water soaked by filter paper.

[wt. in g] wt. of empty thermocol pot = A

-wt. of empty pot + filter paper = B wt. of pot + filter paper + powdered soil = C wt. of pot + filter paper + powdered soil + water = D wt. of pot + filter paper + oven dried soil = E wt. of dry filter paper = (B - A) wt. of wet filter paper = F water absorbed by filter paper = F - (B - A) = G

D-A-G water holding capacity of the soil = ----------------- x 1 00

E-A

Soil pH was determined immediately after this. An aqueous slurry of 10 g soil ( 1:5 w/v) was made, allowed to decant for a few minutes and then pH was recorded electrometrically using control dynamic digital pH meter.

Inoculum placement in the pot

Live inoculum of Glomus mosseae from the pot culture of maize was taken. This contained soil with spores, colonized root and fungal hyphae, etc. In the pot, soil base was added first upto l/3rd of the length of pot. Then a layer of live inoculum was layered over

25

it. Above this layer, one layer of soil base was added to sandwich the inoculum in between the layers of soil base. This is shown in schematic diagram in Fig. 4. For the inoculation of Piriformospora indica, mycelium was mixed in small amount of sterile soil and then spread as above 'sandwich model'. Both the inocula were added at the rate of one per cent.

Pots prepared by the above method were sentilized by the ll10th strength Hoagland solution in a plastic tray for 20 minutes to absorb the nutrients and water. Composition of the Hoagland solution is given in Appendix I. Pots were removed from the Hoagland solution and kept over a thick blotting sheet for 10 -15 minutes to remove the extra­gravitational water. One plant each was placed into the center of the pot with the help of a plastic stick. Roots of the plant were adjusted in such a manner that they were in constant touch with the inocula.

Biological hardening technique-I

Pots prepared, inoculated and transplanted as above were kept in the mist chamber, maintained at 900/o relative humidity, 25±2 °C temperature and I 000 lux of diffused light. Morphological condition of the whole plant such as colour and nature of leaf, tensile strength and colour of stem, etc., was recorded at regular intervals. Rate of plantlet survival was recorded after 8 weeks.

Biological hardening technique-II

Pots with plantlets were covered tightly with a plastic bag and kept in a mist chamber, maintained at 900/o relative humidity, 25±2 °C temperature and 1000 lux of diffused light. After one week, a cut was made in the plastic bag for the exchange of air and after a week the plastic bag was removed. Morphological conditions of the whole plant such as colour and nature of leaf, tensile strength and colour of stem, etc., were recorded at regular interval. Rate of plantlet survival was counted after 8 weeks.

Tissue cui ture of brinjal Seed sterilization (Sharma and Rajam 1995)

Seeds ofbrinjal (Solanum melongena L var. pusa purple cluster) were obtained from IARI, New Delhi. Seeds were submerged in water in a flask and shaken. Rinsed for 2-3 times with sterile distilled water. Water was removed and 700/o ethanol was added. It was shaken for one minute and ethanol was decanted and washed several times. Seeds was transferred to another flask containing 2% sodium hypo-chloride and 2-3 drops of tween 20. It was shaken well and left for 15 minutes. In a sterile lam~nar airflow chamber, solution

76

Fig.4: Pot preparation for the biological hardening of tissue culture raised plantlets (sandwich-model)

substratum

step I

step II

Plastic pots were half-filled with prepared soil substratum ( 1) consisting of sterile garden soil-sand mixture (3 : 1 ). About one em layer of inoculum (spores, hyphae, and colonized root segments) was placed on the surface (2). Top of the inoculum about 2-3 em upper area was covered with the same substratum (3). Micropropagated plantlets were inserted upto the 2 nd layer in the upward direction. Little water was gently sprinkled to wet the upper layer ofthe soil.

was decanted and seeds were washed 4-5 times thoroughly with sterile double distilled water.

Seed germination

Surface sterilized seeds were transferred on Y2 strength MS medium with 1% sucrose in a petriplate for germination. Plates were kept in dark in culture room at 24±2 °C.

Callus initiation and maintenance (Sharma and Rajam 1995; Phillips et al1996)

Germinated seedlings were collected aseptically when the cotyledons were fully expanded and the epicotyl was beginning to emerge. Each seedling was placed on a sterile solid MS medium in petriplate, one at a time to prepare explants. Two cotyledons were excised from half of the seedlings and cultured abaxial side up, or upside down on MS media supplemented with 2mgl-1 BAP and 0.1 mgr1 NAA for callus initiation. Hypocotyl sections from ~he dec~pitated seedlings were excised an? . ~ul~ure? on MS media supplemented With 2mgl BAP and 0.1mgr1 NAA for callus InitiatiOn tn culture room at 24±2 °C and 2000 lux fluorescent light for 24 h. Shoot apices from another half of the seedlings were excised half without cotyledons (from earlier step) and half with cotyledons and inserted at the stem base in to MS medium fortified with 2mgl-1 BAP and 0.1 mgl-1

NAA. Two shoots, one with and one without cotyleaons, were inoculated per culture tube/flask. This stabilizes the stock shoot culture for future use. Cultures were observed weekly. Small pieces of calli (0.5 g fresh weight approx.) were excised and sub-cultured on the fresh medium of the same composition every month to maintain a callus stock.

Induction of shoots from the calli

Callus obtained by above procedure was cut into pieces approximately 0.5 g. One piece was added to each culture tube containing MS medium fortified with 2mgr1 BAP and 0.1mgr1 NAA. Culture tubes were incubated in the culture room in conditions as mentioned above.

Rooting

Shoots were cut from the base in aseptic condition and placed on basal MS medium to encourage shoot elongation and/or spontaneous rooting. Upto three shoots were transferred in each culture tube. Growth tubes were kept in culture room provided with 16 h photoperiod, 2000 lux fluorescent light and 24±2 °C temperature.

27

Establishment of host with P. indica

Shoots were cut from the base in aseptic condition and placed on basal MS medium to encourage shoot elongation and/or spontaneous rooting. Upto three shoots were transferred in each culture tube. Tubes were kept in culture room provided with 16h photoperiod, 2000 lux fluorescent light and 24±2 °C temperature.

Treatment of regenerated shoot with P. indica

P. indica was cultured in an agar base minimal medium as described above and regenerated shoots of tobacco were transferred into this culture tube, one each to see the effect on root formation and plantlet establishment. Tubes were kept in culture room set at 24±2 °C, 16h photoperiod and 2000 lux fluorescent light. Diagramatic representation of the technique is shown in Fig. 5.

Plant establishment

Six-weeks-old, well-rooted plantlets were removed from the culture vessel, keeping the root intact. Plantlet with roots encased in agar media was transferred to a container of warm but not hot water and gently rinsed to remove the adhering agar media off the roots.

Interaction of P. indica with tobacco in suspension culture

Dual culture of tobacco (Nicotiana xanthi L.) callus tissue and P. indica was developed for studies of colonization pattern and invasive property of fungus . This system is excellent for interaction studies, due to the relative ease of manipulation of both tobacco and P. indica in culture.

Establishment of tobacco suspension culture (Phillips et al1996)

The orifice of 125 ml Erlenmeyer flask containing 15 ml liquid MS media supplemented with 0.5mgr1 BAP and 2mgr1 NAA, was sterilized under the flame. Callus stock was collected and gently sliced into pieces of 0.5mg size in a sterile petridish using forceps. A small piece of callus was transferred into the liquid medium using sterile forceps . Again orifice of the flask was heat sterilized and then allowed to cool. The flask was plugged and labelled. Five replicates were prepared and the flask was kept on a gyratory shaker set at 125 rpm and 24±2 °C. Culture was grown for a week.

28

Fig.5: Schematic diagram for in vitro establishment of regenerated shoots of tobacco

~..___ regenerated shoot

mycelia

1 2 3

Tube 1, shows the hyphal disc inoculated on the minimal medium. Tube 2, illustrates the growth of P. indica after 7 days of incubation. Tube 3, depicting the transfer of regenerated shoot into the P. indica grown culture tube

Cell suspension was sub-cultured every week. Flask was handled using the heat sterilization method described above. For the first few sub-cultures, a portion of the spent medium was removed using a sterile, large-bore pipette and replaced with fresh medium. When the cell mass became double, the culture was divided into two flasks with an equal volume of fresh medium and the inoculum cycle was repeated.

When the suspension culture became a finely dispersed mass of cell cultures and aggregated, a dilution ratio of 1:4 to I: 10 of the old culture was added to the fresh medium to maintain the cell line. Transfer was made into heat sterilized flasks using sterilized, large­bore pipettes.

P. indica inoculation

One disc of agar base culture of Piriformospora indica was added to each flask during the fresh transfer of 1:4 dilution of cell suspension culture of tobacco in 15 ml of minimal media.

Colonization study (Phillip and Hayman 1970)

Sample of suspension culture with P. indica was collected every day/week by using a sterile large-bore pipette and stained with trypan blue (0.05 w/v in lactophenol) and observed under the inverted microscope. Infected cells-were photographed at various stages using an Orthoplane phase contrast microscope to analyse the colonization pattern.

Root organ culture of brinjal (Becard and Piche 1994) Seed sterilization

Similar to tissue culture ofbrinjal as described above.

Callus initiation

Similar to tissue culture ofbrinjal as described above.

Root initiation

15 days old calli were transferred on MS medium containing growth regulators NAA at the rate of 0.2mgr1

. Plates were incubated at 24±2 °C for another 15 days. When the root was differentiated from the calli, a clonal culture was established from single root tip by repeated transfer of the root tip on a fresh medium.

29

Culture media

Routine maintenance of root was done on MS medium solidified with 0. 7% agar containing 0.2mgr1 of NAA.

Fungal inoculum

Culture procedure for P. indica was similar to described for biological hardening of tobacco plantlets.

Minimal agar medium

Composition is given in Appendix I

Establishment of dual culture

Regenerated clonal roots of brinjal were transferred onto a fresh minimal agar and Whites media (Appendix I). Plates were incubated for one week and then in the next week, an agar based hyphal disc of P. indica was inoculated in these plates. The two partners were thereafter treated as a single biological experimental unit.

Germination paper bridge technique

Seeds of brinjal pusa purple long (obtained from IARI, New Delhi) were surface sterilized with 0.1% HgCh for 3 m and washed 6 times with sterilized double distilled water. These seeds were germinated in a petriplate containing Y2 strength MS media.

Mycelium of P. indica was grown in a culture tube containing 15 ml broth minimal media for 15 days in an incubator at 30 °C. For making bridge germination paper, first soaked in ethanol 95% and washed with sterile double distilled water, dried and then autoclaved in a plastic (autoclavable) bag, three times at 121 °C, 15 lbs pressure for 15m at the interval of 48 h.

Two-week-old germinated seedlings of brinjal were transferred into the culture tube containing 15 days old mycelium. Plants were supported in the culture tube by germination paper bridge. Schematic diagram of the technique is shown in Fig 6. These plants were kept in culture room at 24±2 °C and 14 h light/ I 0 h dark photoperiod.

30

Fig.6: Stylized diagram of the germination paper bridge technique

a b

~-germination paper +--minimal medium (broth) +---root

,___ growing hyphal disc

P. indica was allowed to grow in nutrient broth. After one week, sterile germination paper disc was laid on the surface. A hole was made at the center of the paper and an aseptically germinated seedling of Solanum melongana L. was inserted through the hole into the medium 1 em below. Tubes were incubated in the culture room maintained at 24±2 °C, and light intensity 1000 lux. a) indicates the initial and b) the final stage of growth

Root colonization Staining of colonized root (Phillip and Hayman 1970)

Plants were uprooted after 45 and 60 days of treatment. Roots were washed thoroughly in running tap water and cut into I em pieces. Root samples were placed in a small beaker and covered with 10% KOH solution. They were left overnight on the laboratory bench and if necessary, heated for 3-5 minutes to release the yellow pigments. KOH solution was decanted and root pieces were washed 3-5 times with double sterile distilled water and placed in a beaker containing 1% HCl for 3-4 minutes to acidify the sample.

The ability of P. indica to colonize the host was checked in 4- and 6-week- old roots. A portion of the rootlet was harvested and stained with 0.05% trypan blue in lactic acid (Phillips and Hayman 1970). Infected root pieces were selected under the dissecting microscope and mounted on a glass slide and morphology was examined using compound microscope. The development of extramatrical phases and sporulation were monitored using inverted microscope.

A concentration of 0.05% w/v in lactoglycerolllactophenol was used to stain the cleaned root. Staining quality was subsequently improved by destaining the root in 50% glycerol/lactophenol for 1-2 h prior to observation to allow excess stain to leach out from roots. Semi-permanent slide of root was made in glycerolllactophenol and a cover slip was mounted over it with the help of a needle.

Quantification of fungal colonization

Stained slides were observed under a compound microscope. Root pieces were randomly collected and observed. Presence and absence of mycelium, hyphae, vesicle and arbuscules were recorded in each root piece at 1 0 different points. In this way all randomly selected root pieces were examined. Hyphae, vesicles, arbuscule etc., were taken as an index for the presence and absence of colonization. Per cent colonization was determined by the following formula:

Number of colonized segments % Colonization = X 100

Total number of segments examined

31

Scanning electron microscopy Reagents

0.5% saline solution (NaCI)~ 0.1 molar phosphate buffer, pH 7.2~ 2.5% glutaraldehyde in 0.1 molar phosphate buffer~ acetone series 30-95% and 100% (dry acetone).

Procedure

Cleared roots were washed 3-4 times with saline solution, fixed in 2.5% glutaraldehyde for 2h at 4 °C and again washed three times with buffer, each for 2h. Samples were dehydrated in acetone series 30-95% each for 30 m at 4 °C. Finally dehydrated in dry acetone (I 00%) with 2 changes each of I h duration, once at 4 °C and another at room temperature. Samples were dried in liquid C02 in a critical point drying apparatus and mounted on SEM stubs with silver paint and coated with metal (Au) approximately at 400 A0 (40 nm) by sputter coater (Balzers). Specimens were examined in Phillips SEM-50 1 B at 15 k Volts. SEM was done using the facilities of All India Institute of Medical Sciences, New Delhi.

Effect of culture filtrate a. Test tube experiments

Aseptically germinated seedlings of brinjal were transferred into culture tubes containing slant of minimal media. 50 Ill of culture filtrate was applied on the agar slants, and equivalent amount of minimal media and/or de-ionized water served as the control. These culture tubes were kept in a culture room equipped with 2000 lux fluorescent light, 25±2 °C temperature and 14 h photoperiod.

b. Pot experiment

I5 days old germinated seedlings of brinjal were transferred to disposable coffee pots containing vermiculite (autoclaved) and sand (acid washed) in a ratio of 3:1. Experiments were designed to see the effect of culture filtrate of P. indica on plants growth. 15 ml of cultUre filtrate was applied for this purpose, equivalent amount of minimal medium and/or de-ionized water served as control. 5 replicates were made. Pots were kept under environmentally controlled conditions (I 000 lux diffused light, 70% relative humidity and 25±2 °C temperature)

32

Physiological and biochemical studies Hydrolytic enzymes Reagents

Tris-HCl buffer (pH 7.0), 0.03 per cent sodium azide, 1 per cent carboxy methyl cellulose, 0.5 per cent oat spelts xylan, potassium phosphate buffer (pH 7.0), 0.02 N NaOH, sodium polypectate, pectin, sodium acetate buffer (pH 5.2), 1M sodium carbonate (Na2C03), O.IN iodine, 2M sulfuric acid (H2S04), O.IN sodium sulfide (Na2S203), DNSA reagent (Appendix I).

Procedure Preparation of crude extract

Freshly harvested, thoroughly washed and dried samples were homogenized in a pre­chilled mortar and ·pestle, in cold Tris-HCl buffer (pH 7.0) and 0.03 per cent sodium azide at 4 °C. Homogenized mixture was taken out in I ml eppendorftube and centrifuged at I4,000 rpm for 10 minutes at 4 oc in the microfuge. Supernatant was taken in a fresh eppendorf tube and made the volume upto 1 ml with buffer. Extract was stored at -20 °C temperature until it was used to check the activity of CMCase, xylanase, and polygalacturonase.

CMCase and Xylanase (Mandels et al 1976; Rajoka and Malik 1984)

1 ml substrate that contained I per cent carboxy methyl cellulase or 0.5 per cent oat spelts xylan was added in 0.1 ml of the extract for CMCase or xylanase assay, respectively. For enzyme blank, one ml of the potassium phosphate buffer (pH 7.0) was used in place of the substrate. Reaction mixture was incubated at 3 7 °C for 30 minutes in a water bath shaker. 1.5 ml of DNSA was added and heated for 5 m in a boiling water bath. Colour appeared in the reaction mixture. Glucose and xylose was used for the standard caliberation of CMCase and xylanase, respectively. Absorbance was taken against the enzyme blank at 540 nm on Shimadzu UV -I60 spectrophotometer.

Polygalacturonase (PG) and polymethylgalacturonase (PMG) (Jansen and Me Donnell1986)

PG and PMG were assayed by using quantitative estimation of galacturonic acid method. Sodium polypectate and pectin (mixed in 0.714 per cent sodium acetate buffer, pH 5.2) were used as a substrate for PG and PMG, respectively. 233 J.tl of the respective substrates were added to 0.2 ml root extract and incubated at 37 °C in a water bath shaker.

33

After every 20, 40 and 80 minutes, 33.3 11! of the aliquot was removed and added 6 11! of IM. sodium carbonate and 33.3 111 of 0.1N iodine. Reaction mixture was acidified with 13.3 11! 2M H2S04. The residue was titrated with 0.1N Na2S20J. The liberated reducing group was compared with the simultaneously run standard of glucose. The enzyme activity was expressed in terms of Jlmol/mllmin.

Acid and Alkaline Phosphatase (Bessey et al1946) Reagents

0.15 M acetate buffer, pH 5.0, 0.1 M Tris-buffer, pH 9.1, 5.5 mM p-nitrophenyl phosphate (PNPP) disodium salt, and 1M sodium hydroxide.

Procedure Preparation of extract

Acid and alkaline phosphatase activ-ities were determined in the fresh root, shoot and leaf tissue extract. All three plant tissues (root, shoot, !eat) were extracted separately in chilled 0.05 M potassium phosphate buffer (pH 7.2, 1: 1 w/v). One gram of the respective tissues were homogenized in pre-chilled mortar and- pestle kept on ice, and then homogenized samples were centrifuged in micro-centrifuge at a speed of 12000 rpm for 30 m at 4 °C. Supernatant was taken out ( 1 ml) and used for estimations.

Acid phosphatase

The reaction mixture containing 1. 5 ml acetate buffer, 200 111 sample (diluted to 1 ml with deionized water) and 0.5 ml PNPP was incubated at 37 °C for 30 minutes. The reaction was stopped by adding 1.0 ml of IM NaOH and the absorbance read at 400 nm on Shimadzu UV -160 spectrophotometer.

Quantification

One enzyme unit= Absorbance of0.362 at 400 nm Enzyme activity = Awo x 2. 76

Alkaline phosphatase

The estimation procedure for the alkaline phosphatase was similar to acid phosphatase. Only pH of the reaction mixture was changed to 9.1 substituting acetate buffer with Tris buffer.

34

Quantification

One enzyme unit= Absorbance of0.084 at 400 nm Enzyme activity = Awo x 11.82

Total Phosphorus (Paze et al1982) Reagents

60 per cent perchloric acid (HC104), hydrogen peroxide (H202), nitric acid (HNOJ), ammonium paramolybdate-vandate reagent.

Procedure

20 mg of the fresh root sample was taken in a 50 ml volume of glass conical flask. 60 per cent perchloric acid was added and the mixture was digested inside a fume hood at 150 °C for 10 m on an electric hot plate. After the root tissues were completely dissolved and produced the black fumes with pungent smell, 0.5 ml hydrogen peroxide was added and heated for another 10 m at the boiling temperature. At this stage, heavy white fumes of perchloric acid appeared. 0.1 ml of nitric acid was added and again heated for next I 0 m to oxidize the sample. Yellow fumes were produced. Then 0.1 ml hydrogen peroxide was added followed by 0.05 ml hydrogen nitrite after another I 0 minutes of continuous heating. The whole reaction mixture underwent digestion process for 30 minutes until all white fumes disappeared. To obtain a clear solution with a volume of about less than one ml, the mixture was cooled at room temperature and allowed to settle before taking an aliquot in the eppendorftubes. Final volume of the aliquot was noted in each sample.

10 J.d of the aliquot was taken in a new eppendorf tube. 790 f..ll of deionized water and 200 f..ll of ammonium paramolybdate-vanadate reagent were added. It was left for IS m to develop the colour. Absorbance was taken at 400 nm on Shimadzu UV -160 spectrophotometer. Blank had all the chemical reagents but no root sample. For standard caliberation, different concentrations of potassium dihydrogen phosphate was taken in deionized water. I 0 J..ll blank was added in each standard concentration and rest of the steps were similar to described above.

Total phosphorus was calculated by using the formula:

Awo X 11.54 X VI p (Jlg) == ----------------------------

35

p (Jlg) Per cent of P = X 100

Wt

where,

P = Phosphorus A Absorbance at 400 nm V1 = Total volume of aliquot V2 = Volume ofthe aliquot used Wt = Dry weight of the root sample used

Nitrate Reductase (Srivastava 1975) Reagents

1.0 M sodium phosphate buffer (pH 7.4), 2.0 M potassium nitrate (KNOJ), 25 per cent n-propanol, 1 per cent sulphanilamide in 1 N HCl, and 0.2 per cent NEDD (N-(1-naphthyl) ethylenediamine dihydrochloride), sodium nitrite (NaN02)

Procedure

Freshly harvested leaves (0.5 g) without midrib were taken in test tubes, and wrapped with a black paper to protect against light. Leaves were treated with 0.8 ml of 1.0 M sodium phosphate buffer, 1 ml of 2.0 M potassium nitrate and 1 ml of 25 per cent n­propanol. The reaction mixture was incubated in dark at 33 °C for 30m. To the 2 ml of the aliquot, an equal volume of0.2 per cent NEDD prepared in l per cent sulphanilamide in 1 N HCl, was added. Reaction mixture was incubated at 33 °C for 15 m. Pink colour appeared. Absorbance was taken at 540 run on Shimadzu UV -160 spectrophotometer. Sodium nitrite was used for the standard caliberation.

Total sugar and reducing sugar (Spiro 1966) Preparation of extract

Total sugar, reducing sugar and proteins were estimated in the fresh root, shoot and -leaf tissue extracts. All three plant tissues (root, shoot, leaf) were extracted separately in chilled 0.05 M potassium phosphate buffer (pH 7.2, 1: 1 w/v). One gram of the respective tissues were homogenized in pre-chilled mortar and pestle, kept on ice, and then homogenized samples were centrifuged in micro-centrifuge at a speed of 12000 rpm for 30 m at 4 °C. 1 ml supernatant was used for estimations.

36

Total Sugar Reagent

2 per cent anthrone solution in concentrated sulfuric acid (prepared fresh for each use and stored in dark bottle).

Procedure

50 J.ll of aliquot was made upto I ml with deionized water. 4 ml anthrone reagent was added gently to it and then vortexed. The tubes were placed in a boiling water bath for I 0 minutes and cooled in dark. The concentration was determined on Shimadzu UV -160 spectrophotometer by taking absorbance at 625 nm against reagent blank. Minimum exposure to the light was allowed during whole reaction process. Glucose was used for standard calibration.

Reducing Sugar Reagents

Somogyi reagent I, Somogyi reagent II, Nelson HI (arseno-molybdate) reagent. (Appendix I)

Procedure

I 00 111 extract was diluted to I ml with deionized water. I ml of the reagent I and reagent II (25: I v/v) mixture was added. Tubes were placed in boiling water bath for 20 minutes and then cooled down quickly. I ml of arseno-molybdate reagent was added to the above solution and vortexed. Colour was allowed to develop for I5 minutes and the absorbance was taken at 500 nm on calibrated Shimadzu UV -I60 spectrophotometer against the reagent blank. Glucose was used for standard calibration.

Protein a. Quantitative Analysis (Lowry et a/1951) Reagents

Sodium hydroxide, sodium carbonate, sodium potassium tartrate, copper sulphate, and Folin~Ciocalteu reagent.

Alkaline sodium carbonate. 20 g sodium carbonate was dissolved in I: I of O.I N sodium hydroxide.

37

Copper sulphate-Rochelle salt solution. 0.5 g .copper sulfate was dissolved in 100 ml of 1 per cent sodium-potassium-tartarate. Always fresh solution was prepared before use.

Procedure

200 111 of aliquot was diluted to 1 ml with deionized water and 5 ml alkaline copper reagent (freshly prepared mixture of 50 ml alkaline sodium carbonate solution and 1 ml of sulphate-Rochelle salt solution) was added to it, vortexed, allowed to stand for 20 minutes at room temperature. 0.5 ml freshly diluted (I: 1 v/v) Folin-Ciacalteu reagent was added, vortexed and allowed to develop colour for 30 minutes. The concentration was determined colorimetrically at 750 nm on Shimadzu UV-160 spectrophotometer. Bovine serum albumin (BSA) was used for standard caliberation.

b. Qualitative Analysis SDS-Poly Acrylamide Gel Electrophoresis (Chrambach and Rodbard 1988; Neeraj 1992)

Stock solutions

Sample buffer (2%) consisting of0.12 M Tris, 2 mM EDT A, 10%2- ME, 4% SDS, and 20% glycerol, pH 8.2, stored at -20° C in dark Stacking gel buffer, 0.5M Tris, pH 6.8 Resolving gel buffer, 3.0M Tris, pH 8.8 Tank buffer, 25mM Tris, 0.192M glycine, 0.1% SDS, pH 8.3 10% SDS (w/v) 30% Acrylamide mixture 29g acrylamide plus lg bis-acrylamide in 1 liter deionized water (stored in dark at 4° C) 10% freshly prepared APS 2% Agarose (aq. w/v)

Working solutions

100/o resolving gel consisting of 10 ml acrylamide mix., 3.75 ml Tris (3M), 300 Jll SDS, 300 Jll APS, 25 Jll TEMED and 17.75 ml deionized water 4% stacking gel comprising of 1.33 ml acrylamide mix, 2.5 ml Tris (0.5 M), 100 Jll SDS, IOO Jll APS, IO Jll TEMED, 5.96 ml deionized water 2% bromo-phenol-blue solution (aq. w/v) IO% glycerol (aq. v/v) Fixing solution. II% TCA, 3.5% sulfosalicylic acid, 30% methanol in aquous medium Staining-solution. 2.25% commasie brilliant blue R-250, 45% methanol, IO%

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acetic acid in deionized water Destaining solution. 10% methanol, 8% acetic acid in deionized water

Note: resolving and stacking gel solutions were degassed before addition of SDS and TEMED.

Procedure

I 0% poly acrylamide slab gel of I mm thickness was casted in between glass plate pair sealed with hot agarose solution. Over the resolving gel, sample loading wells were casted in 4% stacking gel.

Proteins were concentrated by chilled trichloro acetic acid (TCA) I 0% precipitation at 4 °C, followed by centrifugation using Heraeus Sepatech Biofuge at I 0,000 rpm for 10 min. I 00 J.d of sample was prepared by dissolving the protein pellet in 50 J..Ll sample buffer, I 0 J..Ll BPB, I 0 J..LI glycerol and 40 J..Ll deionized water and subsequently placing in a boiling water bath for 5 min. 50 J..LI of each sample was loaded in separate wells using Hamilton syringe. Suitable protein markers from Pharmacia Fine Chemicals (I4.2 to 97 kDa) were also introduced in one of the wells. Gel was run using LKB 8 Romma 2197 high voltage power supply. To start with, the system was run at I5 mN60V till the BPB front crossed the stacking gel and at 35 mNI20V till the dye reached the end ofthe gel slab. The slab was fixed for an hour in fixing solution, washed with deionized water, submerged in staining solution for 2h and then destained with constant gentle shaking giving 4-5 changes in . destainer. After the bands cleared, electrophoretograms were transferred to preserving solution pending documentation.

Photosynthetic pigments (Hiscox and lsraelstam 1979) Reagent

Dimethyl sulfoxide (DMSO)

Procedure

100 mg fresh leaves were taken in a test tube. 10 ml of dimethyl sulfoxide (DMSO) was added to dip the leaf tissue. All tubes were covered with a black paper to protect the reaction mixture from light and incubated at 60 °C in hot air oven for 2h. Leaf colour was changed to pale yellow. The absorbance of the extract was read on Shimadzu UV-I60 spectrophotometer at 480, 5IO, and 645 nm. The chlorophyll a, chlorophyll b and carotenoids were calculated using formulae given by Arnon ( I949):

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v CW a = 12.7 (~63)- 2.69 (~45) x -----------

W X 1000 v

Chi b = 22.9 (~45)- 4.68 (~63) X ----------Wx 1000 v

Carotenoids = 7.6 (A.80)- 1.49 (A5w) x ------------

where,

A= absorbance at respective wavelengths, nm V =final volume of the extract, ml W = weight of fresh leaf sample, g

40

Wx 1000