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Chapter 3 MATERIALS AND METHODS

3.1. Plant material

Seeds of Solanum tuberosum (kufri jyoti) were obtained from Government

Breeding Garden, Kashipur, Uttarakhand and grown in pots. All the explants were taken

from these donor plants for the present research work.

3.2. Source of chemicals

All the chemicals and solvents used in the study were of analytical and molecular

grade. Chemicals were purchased from Himedia, Duchefa, Merck, Fermentas.

3.3. Equipments

The equipments used in the present study are listed below-

1. Air conditioner (LG)

2. Air purifier (Hi-media)

3. Autoclave (Metrex)

4. Bead sterilizer (Hi-media)

5. Centrifuges (SIGMA , Eppendorf)

6. Deep freezer (Vestfrost, BSF-345, Denmark)

7. Digital pH Meter (Inolab, Eutech)

8. Electrical balance (Sartorius)

9. Gel documentation system (BioRad)

10. Gel electrophoresis assembly (Thermo)

11. Hot air oven (Sanco)

12. Laminar air flow (Unitech)

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13. Micropipette (Eppendorf, Germany)

14. Microwave oven (LG, India )

15. Photoperiodic timer, Tissue culture Castor racks, PAR Lamps (Vista Biocell)

16. Polyhouse (Indo-dutch Technologies, Bhimtal)

17. Refrigerator (Samsung)

18. Shaker Incubator (Innova 4230) (New Brunswick Scientific)

19. Spectrophotometer (Thermoscientific)

20. Thermocycler (Eppendorf)

21. UV transilluminator (Vilber Lourmat)

22. Water bath shaker (REMI,Thermoscientific)

23. Water purification system (Rions, India Ltd.)

3.4. Glassware

All glassware and plastic wares were supplied by Borosil, Tarson, Schott Duran,

qualigens.

3.4.1. Washing and sterilization of glasswares

The glasswares were washed with detergent (labolene) solution, rinsed in tap

water to remove the detergent and finally rinsed in double distilled water to remove the

last traces of the detergent. The previously used and contaminated glassware were first

autoclaved, rinsed in water and then soaked overnight in potassium dichromate and acid

solution so as to remove the stains. Washed glassware were oven dried for 3-5 h. Forceps,

scalpels, scissors, pipette tips and non absorbent cotton plugs were sterilized by

autoclaving at 121ºC and 15 lbs pressure for 15-20 min. Instruments used during

inoculation viz. forceps, scalpel, scissors etc. were surface sterilized by dipping in

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rectified spirit and flamed on burner in laminar air flow or by putting it into bead

sterilizer before each inoculation.

3.5. Media preparation

3.5.1. Preparation of macro and micronutrient stock solutions

Stock solutions of macro-and micronutrients, vitamins and growth regulators were

prepared in double distilled water. Stock solutions of macro and micronutrients were

prepared as 10 and 100x concentrations as shown in (Appendix 1). For macronutrient

stock solution, calcium chloride was dissolved separately in water and then added to the

rest of the solution to avoid precipitation. All hormones, vitamins and other constituents

were dissolved separately in double distilled water to form a stock solution and were

stored at 40C. FeSO4 and Na2EDTA were dissolved separately and stored in amber bottle

and then added to the basal medium so as to avoid precipitation of the salts (Appendix I).

3.5.2. Preparation of vitamin stock solutions

Stock solutions of vitamins of MS medium were prepared as shown in

(Appendix I).

3.5.3. PGR (Plant Growth Regulators) preparation

All the growth regulators are not soluble in water and vary in their solubility.

Hence, growth regulators were dissolved in different solvents.

3.5.3.1. Preparation of stock solution

Auxins

5μM stock of auxins (2,4-D, IAA, IBA) was prepared in 50 ml by dissolving

suitable amount of each hormone in minimum amount of absolute EtOH and then made

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up the volume 50 ml with sterilized distilled water and then used or stored at 40C for

further use (Appendix- I).

Cytokinins

5μM stock of cytokinin (Kinetin, Zeatin, GA3, BAP) was prepared in 50 ml by

dissolving suitable amount of each in minimum amount of 1N NaOH/DMSO according

to the solubility and then made up the volume to 50 ml with sterilized distilled water and

then used or stored at 40C for further use (Appendix I). GA3 was stored at room

temperature.

3.5.4. Composition of MS medium

MS (Murashige and Skoog, 1962) medium with supplements of vitamins and

combination of various PGRs was used as basal media for callus induction, shoot

regeneration, microtubers formation and root induction (Appendix II ).

3.5.5. Culture medium and conditions

3.5.5.1. Sterilization of medium

The medium was prepared by adding required amount of salts, vitamins, and

growth regulators from respective stock solutions in flask (1L) and final volume was

made by double distilled water. Sucrose 3% (w/v) was added as carbon source. The pH

was adjusted to 5.8 by 1N NaOH or 1N HCl. Agar 0.7% or clarigel 0.24% was added as a

gelling agent in jam bottles, flasks or test tubes after transfering medium (40-50 mL) in

them. The jam bottles, flasks or test tubes containing medium were autoclaved at 121ºC

and 15 psi for 20 min.

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3.5.5.2. Incubation Conditions

All the cultures were maintained at 25 ± 20C and 60 ± 5 relative humidity in the

culture room conditions under 16/8 h (light/dark) photoperiod with photosynthetic photon

flux density of 40 μ mol m-2

s-1

fluorescent lamps.

3.6. In vitro macropropagation of Solanum tuberosum

3.6.1. Explant preparation

Explants such as leaf, nodes and internode were initially washed with Tween-20

and then with distilled water 3 to 4 times to remove the traces of the chemical applied.

Thereafter they were treated with bavistin (fungicide) solution (0.5%, 15 min) to avoid

fungal contamination and rinsed with distilled water 4 to 5 times. For surface sterilization

explants were subjected to HgCl2 (0.1%, 1 min) and thoroughly washed with distilled

water 2 to 3 times. Leaves were dissected into small pieces and treamed, nodes and

internodes were cut into small pieces (approx 5 mm). After a quick dip in 70% alcohol

explants were then washed with sterile distilled water and inoculation was done.

All operations including transfer of plant material were carried out under aseptic

conditions preferably under the hood of a laminar air flow.

3.6.2. Callus induction and shoot regeneration

For callus induction juvenile leaf sections and internodes with cut ends were

placed on full strength MS medium with different concentrations of PGR like 2,4-D (4.53

to 18.12 µM) alone and 2,4-D (0 to 18.12 µM) with Kinetin (1.16 µM). Callus initiated

after 15-20 days of incubation. Calli were subcultured in every 15 days on the same

medium composition. Well differentiated calli were placed on MS medium supplemented

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with various combinations of BAP (4.44 to 13.22 µM) and GA3 (1 µM) for shoot

regeneration. All cultures were maintained at 25 ± 20C with 16/8 h (light/dark)

photoperiod. Shoot regeneration initiated in seven days.

3.6.3. Direct regeneration of shoots

For direct regeneration of shoots explants taken were nodes. Explants were cut

into small sections of 2-5 mm size and inoculated in the full strength MS medium

supplemented with different concentrations of Zeatin, IAA, and GA3 for shoot

multiplication. Cultures were kept at 25 ± 2°C with 16/8 h (light/dark) photoperiod.

Shoot induction initiated in three to four days of incubation.

3.6.4. Production of microtubers

Well grown plantlets obtained from direct regeneration of nodes were maintained

in culture room at 16/8 h light/dark condition and observed for the production of

microtubers.

3.6.5. Regeneration of roots and development of elite plantlets

When shoots grew up to a height of 3-4 cm, they were aseptically separated from

each other and subcultured on half and full strength MS medium with varying

concentrations of IBA for root induction. Root development initiated after 4-5 days of

incubation. The completely rooted plants (2-3 weeks) were taken out carefully and gently

washed under running water to remove excess clarigel. They were then potted in

thermocole cups (12 x 8 cm) containing soil and farmyard manure (3:1, v/v); covered

with transparent polythene bags with small holes to maintain humidity. These plants were

placed inside growth chamber under 16 h photoperiod at 25 ± 2°C temperature. Plants

were watered regularly and gradually acclimatized over a period of one month. The

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polythene bags were then removed and the established plantlets were subsequently

transplanted to earthen pots and kept in a polyhouse.

3.7. Agrobacterium tumefaciens strain and plasmid vector

The plasmid pBinAR carrying the desired osmotin gene regulated by CaMV 35s

promoter and nptII gene as a marker gene was immobilized in Agrobacterium

tumefaciens strain GV3101 which was further used in the genetic transformation process

of potato (Appendix III).

3.7.1. Antibiotic sensitivity test

To determine optimum concentration of kanamycin for the selection of

transformed plants, explants of wild type were inoculated on pre-cultured medium

supplemented with different concentrations of kanamycin (50-300 mg l-1

). The cultures

were regularly and carefully observed to determine the minimum concentration at which

the explant starts bleaching. In a similar manner, cephotaxime concentration (50-400 mg

l-1

) was optimized to check the overgrowth of Agrobacterium tumefaciens.

3.7.2. Transformation of Agrobacterium strain GV3101 with the plasmid vector

This was done using Freeze Thaw method as described below:

3.7.2.1. Preparation of competent cells

Starter culture was grown by inoculating a single colony of the Agrobacterium

strain GV3101 in 5ml of YEM medium with antibiotics viz. gentamycin (30 µg ml-1

) and

rifampicin (10 µg ml-1

) at 28°C. One ml of the above culture was added to 100 ml YEM

medium in culture vials and shaken vigorously at 200 rpm for 6 h at 28°C. The above

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grown cell suspension was centrifuged at 6,000 rpm for 10 min at 4°C in Oakridge tubes.

The pellet was suspended in 400 µl of ice-cold solution of 20 mM CaCl2.

3.7.2.2. Freeze thaw method of plasmid uptake

About 50 ng of plasmid DNA was added to the competent cells and mixed well

by tapping. The cells were kept for 30 min on ice followed by freezing in Liquid N2 for

few seconds. Immediately after freezing, the cells were kept at 37°C in water bath for 5

min for thawing. 800 µl of YEM was added to the tube and incubated at 28°C for 4-6 h

with gentle shaking at 150 rpm. 200-300 µl of the above grown culture was poured on

YEMA plates containing kanamycin (50 mg l-1

) to select the transformed colonies. The

plates were incubated at 28°C. The transformed cells of bacteria showed colonies within

36-48 h of incubation.

3.7.2.3. Plant transformation and regeneration

Agrobacterium cultures harboring osmotin and nptII gene were grown overnight

separately in Yeast Extract Mannitol (YEM) medium (Appendix IV) containing

kanamycin (50 µg ml-1

), gentamycin (30 µg ml-1

) and rifampicin (10 µg ml-1

) at 280C.

Bacterial cells were pelleted and suspended in liquid MS medium. The cultures were used

to infect the explants after diluting to the optical density 0.15-0.20 at λ590 nm. The

explants were submerged in the bacterial suspension for 15-20 min, blotted dry on sterile

tissue paper and transferred to the co-cultivation medium (basal MS medium without

hormones and antibiotics).

The infected explants were kept in dark for 48 h at 250C. Following the co-

cultivation, the explants were washed twice in distilled water containing 250 µg ml-1

cephotaxime, blot dried and cultured on a selection medium (the callus induction medium

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and direct shoot regeneration medium supplemented with 50 µg ml-1

kanamycin and 250

µg ml-1

cephotaxime). Explants showing callus formation were subcultured in every 15-

20 days on fresh selection medium. Callus showing proper growth were transferred on

shoot regeneration medium and elongated shoots (3-5 cm in height) were then cultured

on root induction medium. Selection pressure was maintained at each step for stringent

selection and to minimize the chances of escapes. Well rooted plants were processed for

hardening in a mixture of soil and farmyard manure (3:1, v/v) in thermocole cups covered

with transparent polythene bags with small holes to maintain humidity. These plants were

placed inside growth chamber under 16 h photoperiod at 25 ± 2°C temperature. Plants

were watered regularly and gradually acclimatized over a period of one month. The

polythene bags were then removed and the established plantlets were subsequently

transplanted to earthen pots.

3.8. Molecular analysis of transformed plants

3.8.1. Plasmid DNA isolation

10 ml of saturated bacterial culture grown overnight in liquid YEM containing 50

µg ml-1

kanamycin, 10 µg ml-1

rifampicin and 30 µg ml-1

gentamycin was taken in

centrifuge tubes. The tubes were centrifuged at 12000 rpm for 1 min. at 40C and

supernatant was discarded. A 200 µl of ice cold solution I (Appendix V) was added to cell

pellet and resuspended the cells. 400 µl of solution II (Appendix V) was added and the

content was mixed by gently inverting the tubes for 5 min. 300 µl ice cold solution III

(Appendix V) was added and mixed with the viscous bacterial lysate by inverting the

tube several times and the tubes were kept on ice for 3-5 min. The tubes were then

centrifuged for 5 min at 40C and 600 µl of the supernatant was transferred to a fresh

microcentrifuge tube. This fractionation step separated the plasmid DNA from the

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cellular debris and chromosomal DNA in the pellet. An equal volume of isopropanol was

added to the supernatant. The tubes were kept at room temperature for 2 min and again

centrifuged for 5 min. Supernatant was discarded without disturbing the pellet. This

fractionation step further purified the plasmid DNA from contaminants. 1 ml of ice cold

70% ethanol was added and centrifuged for 2 min. The tubes were dried for 5 min at

room temperature. Finally 50 µl of TE was added to the tubes and stored at -200C.

3.8.2. Genomic DNA isolation

DNA was isolated from plants using CTAB method (Doyle and Doyle, 1990).

The reagents required in the process are described in Appendix VI. Steps involved in the

isolation are described below:

1. 0.5 gm leaf samples were ground to a fine powder in liquid nitrogen in pre-chilled

pestle mortar

2. Powder was transferred to centrifuge tubes containing 10.0 ml extraction buffer pre-

warmed to 650C and incubated for 1 h in water-bath maintained at 65

0C with

occasional mixing.

3. Equal volume (10 ml) of chloroform: isoamylalcohol (24:1, v/v) was added and

vortexed gently for proper mixing of buffer.

4. Tubes were centrifuged at 10,000 rpm for 8 min at room temperature.

5. The upper aqueous phase was transferred to a new sterile centrifuge tube.

6. Equal volume (6 ml) of chloroform: isoamylalcohol (24:1, v/v) was added and

vortexed gently for proper mixing.

7. Tubes were centrifuged at 10,000 rpm for 6 min at room temperature.

8. The upper aqueous phase (about 7 ml) was transferred to a new sterile tube.

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9. Equal volume (7 ml) of ice cold isopropanol was added and mixed gently by

inversion.

10. Tubes were incubated at -200C overnight.

11. Tubes were centrifuged at 12,000 rpm for 8-10 min at room temperature.

12. Supernatant was discarded and DNA pellets were washed with 1ml 70% ethanol and

pellet was allowed to dry.

13. DNA pellet was dissolved in 200 µl 1X TE buffer.

14. 5 µl RNase (10 mg/ml stock) was added to each tube and tapped gently for mixing.

15. Tubes were incubated at 370C for one hour.

16. DNA was stored at 40C.

3.8.3. PCR analysis

DNA was isolated from young leaf samples of transformed and wild-type plants,

using the cetyl trimethyl ammonium bromide (CTAB) method of Doyle and Doyle

(1990). The transgene integration was confirmed by amplification using gene-specific

primers in thermocycler. About 100 ng of plant DNA from each sample was used for the

25 µl PCR reaction mixture containing 1.0 µl DNA, 1.0 µl each of 20 pmol forward and

reverse primers, 0.5 µl of 10 mM dNTP mix, 2.5 µl of 10X PCR buffer, and 1.0 U Taq

polymerase (Fermantas). PCR was carried out by amplifying the coding regions of nptII

and osmotin gene using gene specific oligonucleotide primers (Appendix VII).

3.8.3.1. PCR amplification for osmotin gene

PCR amplification for osmotin gene was carried out using the following cyclic

parameters as an initial denaturation at 94oC for 4 min, followed by 35 cycles of

denaturation (94oC for 30 sec), annealing (52

oC for 35 sec) and primer extention (72

oC

for 2 min), with a final extension step at 72oC for 10 min.

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3.8.3.2. PCR amplification for nptII gene

PCR amplification for nptII gene was carried out using the following cyclic

parameters as an initial denaturation at 94oC for 4 min, followed by 35 cycles of

denaturation (94oC for 30 sec), annealing (65

oC for 35 sec) and primer extention (72

oC

for 2 min), with a final extension step at 72oC for 10 min.

The amplified PCR products were electrophoresed on 1% agarose gel containing

ethidium bromide and the bands were visualized and captured using gel documentation

system.

3.8.4. Agarose gel electrophoresis

Agarose gel (1%) was prepared by dissolving appropriate amount of agarose in

1X TAE buffer [50X TAE, 100 ml: Tris base-24.2 gm; Glacial acetic acid-5.7 ml; and 10

ml EDTA (0.5 M, pH-8)] and allowed to cool to about 500C. Ethidium Bromide to a final

concentration of 0.5 μg ml-1

was added.

3.8.4.1. Casting the gel

1. The open ends of the gel casting plate were sealed with cello tape and it was placed

on a horizontal platform.

2. The comb was placed in the casting tray.

3. Prepared molten agarose (1%) was then poured into the gel casting plate ensuring

that no air bubbles have entrapped underneath the comb.

4. Gel was allowed to solidify for about 30 min.

5. After solidification, the comb and cello tape were removed carefully.

6. The casted gel was placed in the electrophoresis tank with wells towards the

cathode.

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7. Filled the gel tank with sufficient volume of electrophoresis buffer (1X TAE) to

submerge gel.

3.8.4.2. Loading and running the gel

1. DNA samples were prepared in 6X gel loading dye and loaded into the wells with

micro-pipette.

2. Electrophoresis was carried out at a constant voltage of 70 volt until loading dye

has migrated about 75% toward the positive pole.

3. Bands were visualized under UV light.

4. The gel was photographed using gel documentation system and saved.

3.8.5. Sequencing

PCR positive samples for osmotin gene were sent for sequencing (Sinos Biotech

& Consultants, Haldwani, Uttarakhand) and the obtained nucleotide sequence of PCR

positive samples were analysed by BLAST with osmotin gene sequence in the NCBI data

base for further confirmation.

3.9. Bioassay for drought stress tolerance

Selected transgenic plants along with the wild-type plants were subjected to five

days water stress and plants were assayed for comparative analysis under stressed and

non stressed conditions as described below:

3.9.1. Estimation of proline content

Free proline content in the leaf tissues was estimated by the method of Bates et

al., (1973). Leaf tissue (500 mg) was grounded with liquid nitrogen. Fine powder was

homogenized with 3% Sulphosalysilic acid. 2 ml of filtered homogenate was mixed with

equal volume of glacial acetic acid and acid-ninhydrin reagent (Appendix VIII). The

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mixture was incubated in a boiling water bath for 1h. Brick red colour appears. Reaction

mixture was snap cooled on ice to terminate the reaction. Then 4 ml toluene was added

and after thorough mixing the chromospheres containing toluene was separated. Optical

density was recorded at 520 nm in spectrophotometer using toluene as blank. The

concentration of proline (µg g-1

Fresh Weight) was estimated by referring to a standard

curve made from known concentration of proline (Appendix VIII).

3.9.2. Relative water content (RWC) estimation

To determine RWC, 10 fully expanded leaves were weighed immediately after

harvest and placed in autoclaved distilled water for 6 h at room temperature and then

their turgid weight was recorded. After soaking, the leaves were quickly and carefully

blotted dry with tissue paper prior to determination of turgid weight. The samples were

dried in oven at 80oC for overnight to obtain their dry weight. The relative water content

(RWC) of a plant tissue is expressed as follows:

RWC (%) =

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