chapter 2 materials and methods - shodhganga :...

CHAPTER 2

MATERIALS AND METHODS

32

CHAPTER 2: MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Plant material

In the present study, Jatropha curcas CP-9 cultivar was used for optimization of

regeneration, transformation and development of transgenic plants. Seeds were stored at

room temperature for further use.

2.1.2 Bacterial Strains

Escherichia coli DH5α: F-, Lambda

-, recA1, endA1

-, hsdR17 (rK

-, mK

+), (lacZYA-

argF), supE44, U169, Φ80dlacZΔM15, thi-1, gyrA96, relA1 (Hanahan, 1983) was used

for cloning gene construct.

A. tumefaciens strain EHA105: (RifR) (Hood et al., 1993) was used for Agrobacterium

mediated gene transfer.

2.1.3 Plasmid and construct

The Binary plasmid vector pCAMBIA1301 (CAMBIA, Australia), a member of

versatile pPZP family of Agrobacterium binary vectors for plant transformation

(Hajdukiewicz et al., 1994) is used in this study. pCAMBIA1301 is comprised of T-

DNA borders flanked by chimeric hygromycin phosphotransferase (hpt) gene under the

control of CaMV35S promoter (as a selection marker for transformed plant cells), a

gusA gene with a catalase intron under the control of CaMV35S promoter (as a reporter

gene) and kanamycin resistance gene as a bacterial selection marker (Figure 2.1). It was

used for cloning of SbNHX1 for transformation in J. curcas and construct was named as

pCAMBIA1301-SbNHX1 (Figure 2.2).

Materials and Methods

33

Figure: 2.1 Schematic map of binary vector pCAMBIA1301. Binary vector

pCAMBIA1301 contains the selection marker gene for hygromycin resistance and the

reporter gene, gus, both driven by the CaMV 35S promoter independently.

Figure: 2.2 Schematic map of plant expression gene construct pCAMBIA1301-

SbNHX1. Expression of SbNHX1 is driven by the cauliflower mosaic virus 35S

promoter.

35SP

Hygr gene

35SP35SP

GUS gene

Nos ANosA NosA

KpnI XbaI

LB RB

PstI PstI

SbNHX1


34

2.1.4 Chemicals and consumables

The chemicals used in this study were purchased from the following companies: Sigma-

Aldrich (St. Louis, USA), Roche Applied Science (Germany), Merck (India), Himedia

(India), Fluka (Germany), MBI Fermentas (USA), Bangalore Genei (India) and

Qualigens (Mumbai, India).

The consumables were purchased from: Sigma-Aldrich (St. Louis, USA), Eppendorf

(Germany), Millipore (USA), Whatman (Maidstone, UK), Amersham Biosciences

(UK), Kodak (USA), Axygen (USA) and Tarsons (Kolkata, India).

2.1.5 Enzymes and reaction kits

Restriction enzymes from MBI Fermentas (USA) were used for genomic DNA

digestion. Taq DNA polymerase from Sigma-Aldrich (St. Louis, USA) was used for

routine PCR. Following kits were used for different purposes in the study:

QIAquick plasmid isolation kit Qiagen (Germany)

QIAquick PCR purification kit Qiagen (Germany)

Biolistic optimization kit BioRad (USA)

GUS staining kit Sigma (USA)

DIG Southern hybridization kit Roche Applied Science (Germany)

QuantiFast SYBR Green PCR reaction kit Qiagen (USA)

2.1.6 Oligonucleotides used in the study

Oligonucleotides were outsourced from Sigma-Aldrich (Mumbai, India) and dissolved

in sterilized nuclease-free milli-Q water. Oligonucleotide stocks were prepared with 100

μM concentration and stored at -20 °C. Working concentration of oligonucleotides was

prepared according to experimental needs.


35

Table 2.1: List of oligonucleotides used in the study

S.N. Primer code Primer Sequence Primers

1. SbNHX1 TF: 5-TGCGGTACCA TGTGGTCACAGTTGAGCTC-3 Forward

2. SbNHX1 TR: 5-TCGTCTAGACTATGTTCTGTCTAGCAAATTG -3 Reverse

3. Gus F: 5-GATCGCGAAAACTGTGGAAT-3 Forward

4. Gus R: 5-TGAGCGTCGCAGAACATTAC-3 Reverse

5. hptII F: 5-TTCTTTGCCCTCGGACGAGTG-3 Forward

6. hptII R: 5-ACAGCGTCTCCGACCTGATG-3 Reverse

7. RT-NHX1F: 5’- ATGGTGTTTGGGTTGCTGA -3’

Forward

8. RT-NHX1R: 5’-CTGCTTCGTCTTGGTTGTCC-3’

Reverse

9. gusRTF2: 5’-CGACTGGGCAGATGAACAT-3’ Forward

10. gusRTR2: 5’-CTGTAAGTGCGCTTGCTGAG-3’ Reverse

11. JcKAS1F: 5’-GCACTTGGCTGCAAAACAAAT-3’ Forward

12. JcKAS1R: 5’-CGTCCAGTCAACATATCGAG-3’ Reverse

2.1.7 Media, buffers and solutions

Most of the buffers, media and solutions were prepared as described by Sambrook and

Russel (2001) and Bhojwani and Razdan (1996) unless supplied with the kits. The pH

was adjusted with 1 N NaOH or 1 N HCl. Media for cultivating bacteria and tissue

culture were sterilized by autoclaving (121 °C, 15 psi and 20 min). All the thermolabile

solutions and components, such as antibiotics, were prepared as stock solutions by filter

sterilization (0.2 μm) and added to the medium after cooling to 50 °C. Compositions of

solutions or buffers are given in respective method section.


36

2.1.8 MS Basal Medium

MS basal medium (Murashige and Skoog, 1962) [For 1000 ml, Macronutrients-

NH4NO3 1650 mg, KNO3 1900 mg, CaCl2.2H2O 440 mg, MgSO4.7H2O 370 mg,

KH2PO4 170 mg; Micronutrients - H3BO3 6.2 mg, MnSO4.4H2O 22.3 mg, ZnSO4.7H2O

8.6 mg, KI 0.83 mg, Na2MoO4.2H2O 0.25 mg, CuSO4.5H2O 0.025 mg, CoCl2.6H2O

0.025 mg; Iron stock - Na2EDTA 37.3 mg, FeSO4.7H2O 27.8 mg; Organic nutrients -

Nicotinic acid 0.5 mg, Pyridoxine-HCl 0.5 mg, Thiamine-HCl 0.1 mg, Glycine 2.0 mg,

Inositol 100 mg; Distilled water as required] supplemented with 3% (w/v) sucrose and

0.8% (w/v) agar and pH 5.8 was used for tissue culture studies.

2.1.9 Bacterial Growth Media

The medium used for E. coli was Luria-Bertani (Yeast extract 0.5%, Tryptone 1%, NaCl

1%, Bacteriological agar 1.5%, Distilled water upto 100 ml, pH 7.2). LB medium was

supplemented with kanamycin (50 mg/l) when used for growth of transformed E. coli

cells. LB medium supplemented with rifampicin (25 mg/l) was used for growth of non-

transformed A. tumefaciens strain EHA105 while for transformed A. tumefaciens strain,

kanamycin (50 mg/l) was also used along with rifampicin.

2.1.10 Equipments

Following equipments were used in this study:

Autoclave: Hiclave HV-85 (Hirayama, Japan), Systec VX-100 (Germany), Tomy SS-

325 (Tomy Seiko, Japan); Balance: Shimadzu AUW 220D (Shimadzu, Japan), AB204

(Mettler Toledo, Switzerland), Anamed MX-7301A (Anamed Instruments P. Ltd,

India); Centrifuges: 3K30 (Sigma, USA), Centrifuge 5415R Model (Eppendorf,

Germany); Biolistic gene gun PDS 1000/ He (Bio-Rad, USA); DNA gel electrophoresis

apparatus: Midi and mini cells for DNA agarose electrophoresis and power supplies


37

(Bio-Rad, USA and Bangalore Genei, India); Gel documentation System: BIOVIS

(BioVision Technologies, Inc., USA), UVP GelDocIt (UVP, UK); Hybridization Oven:

Problot L12-2 (Labnet, USA); Iceflecker: F80C (Icematic, Castel Mac SpA, Italy);

Incubator shaker: Lab-Therm LT-X (Kuhner, Switzerland); Intensifying Screen

(Cassette): Kodak BioMax MS (Eastman Kodak, USA); Laminar Air Flow Cabinet:

Streamline SCV-4A1 (Streamline, Singapore), LCB-1501V (Daihan Labtech, Korea);

Microscope: Olympus SZX16 and 1X70 S8F (Olympus, Japan); Camera: DSC-W200

(Sony, Japan); Microwave: Intellowave MS-255R (LG, India); Milli-Q System:

Gradient A10 (Millipore, USA); PCR (Thermocyclers): Mycycler (Bio-Rad, USA),

Mastercycler gradient (Eppendorf, Germany); Real Time iQ5 Cycler (Bio-Rad, USA),

pH Meter: Eutech pH510 (Eutech, Singapore); Spectrophotometers: Gene Quant Pro

(Amersham Biosciences, UK), SpectraMax Plus (Molecular Devices, USA), T80+ UV–

Vis spectrophotometer (PG Instruments Ltd., UK), Nanodrop: ND1000 (Wilmington

USA); Ultra low freezer (-86ºC): C340 and U410 (New Brunswick Scientific, UK); UV

Crosslinker: UVC 500 Crosslinker (Amersham Biosciences, UK); Vacuum

Concentrator: Eppendorf Concentrator 5301 (Eppendorf, Germany), Water Bath: Julabo

SW23 and F12 Model (Julabo, Germany); Rotary shaker: Wise shake (Wisd Lab

Instruments, Germany) and MT-3 Plant Microtome (NK Systems, Japan).


38

2.2 METHODS

2.2.1 Standardization of regeneration protocol for J. curcas

2.2.1.1 Raising of aseptic cultures and culture conditions

J. curcas seeds were grown in earthen pots in green house conditions and the six

months grown plants were used to obtain node and leaf as in vivo explants. In vitro

aseptic shoot cultures were established by culturing nodal explants collected from 6

months old plants. The nodal explants (1-2 cm) with axillary buds were excised and

surface sterilized with 0.1% mercuric chloride for 15 minutes and rinsed five times with

sterile distilled water under sterile conditions. The sterilized nodal explants were

cultured on MS medium supplemented with 2.22-4.44 μM BAP, 3% (w/v) sucrose and

0.8% (w/v) agar for the formation of shoots from axillary buds. After 4 weeks of

culture, leaves were collected from these axillary shoots and used as in vitro explants.

Young leaves close to apical buds were excised from 6 months old plants, surface

sterilized by 2% sodium hypochlorite for 12 min and used as in vivo explants.

Mature decoated seeds of Jatropha were surface sterilized with 0.1% (w/v)

mercuric chloride or 2% sodium hypochlorite for 15 min and washed 4–6 times with

sterile distilled water. For regeneration and transformation, embryo and cotyledonary

leaf explants were dissected out from endosperm aseptically and carefully. Embryos

were pre-cultured for 5 days and cotyledonary leaves were pre-cultured for 14 days on

optimized solid MS basal media (Murashige and Skoog, 1962) (pH 5.8) supplemented

with 2.22 μM BAP with or without 2.46 μM IBA, 0.8% (w/v) agar and 3% (w/v)

sucrose. All cultures were maintained under controlled laboratory conditions at 25 ±

20C under a 16/8 h light/dark photoperiod with cool white fluorescent lamp of 35μmol

m-2

s-1

light intensity.


39

2.2.1.2 Shoot induction

For shoot induction, different explants viz. embryo axis, hypocotyl, cotyledonary leaf

and mature leaf were cultured on shoot induction medium (SIM) i.e. MS medium

containing 3% (w/v) sucrose, 0.8 % (w/v) agar and different plant growth regulators:

BAP, TDZ and IBA in different combinations. Embryo axis and hypocotyls were put

vertically and leaf explants were put horizontally such that abaxial side of leaf is in

contact with the medium. Observations were recorded after 28 days.

2.2.1.3 Shoot proliferation

For proliferation of newly developed shoot buds from different explants, shoot

regeneration medium (SRM) i.e. MS medium supplemented with 3% (w/v) sucrose,

0.8% (w/v) agar and different concentrations of BAP, IBA, IAA and GA3 was tested.

Explants were subcultured again to the same medium for 28 days for effective

proliferation of shoots. Observations were recorded after 28 days after each subculture.

2.2.1.4 Shoot elongation

Proliferated shoots were elongated by subculturing to shoot elongation medium (SEM)

i.e. MS medium supplemented with 3% (w/v) sucrose, 0.8 % (w/v) agar and BAP for 28

days for further shoot elongation. For required height, shoots were again subcultured to

the same SEM for 28 days. Observations were recorded after 28 days after each

subculture.

2.2.1.5 Rooting and acclimatization

For rooting, 3-4 cm elongated shoots were transferred to root induction medium (RIM)

containing half-strength MS medium supplemented with 2% (w/v) sucrose, 0.7 (w/v)

agar and IBA for 28-40 days. Alternatively, high concentration of IBA was tested as

pulse treatment and then shoots were transferred to RIM. Additionally, 100 mg/l


40

activated charcoal was also added optionally as additive for adsorption of phenolic

compounds released in the medium, if present. After 28-40 days, plantlets with rooted

shoots were transplanted to autoclaved soil in plastic pots (5x10 cm) covered with

transparent plastic lids and maintained under high humidity for 7–10 days, thereafter

gradually exposed to culture room conditions. Acclimatized plantlets were transferred to

sand: soil: FYM (2:2:1) in earthen pots for establishment in green house conditions.

2.2.2 Determination of salt sensitivity in J. curcas

2.2.2.1 In vitro

Effect of NaCl on seed germination was assessed. J. curcas seeds without seed coat

were sterilized with 2% sodium hypochlorite for 15-20 min and washed 5-6 times with

sterilized distilled water. Embryos were excised from sterilized seeds and cultured on

MS media plates supplemented with 4.44 μM BAP, 2.46 μM

IBA and different NaCl

concentrations (0, 50, 100, 150, 200 and 250 mM). In average, 10 embryo explants were

put in each plate. Experiment was performed in replicate and observations were

recorded after 8 days.

2.2.2.2 In hydroponics

Sterilized seeds without seed coat were put on liquid ½ MS wet cotton in sterilized culture

bottles for 7 days. For hydroponic culture, germinated seedlings were transferred to plastic

pots containing liquid ½ MS basal media (without organic supplements and sugar) for 7

days for acclimatization in hydroponic environment. Pots were put in Plant growth chamber

under controlled conditions i.e. 25oC temperature, 50% RH and 16/8 light/dark cycle. After

7 days, seedlings were treated with liquid ½ MS supplemented with different NaCl

concentrations (0, 50, 100, 150, 200 and 300 mM). Five seedlings were transferred in each


41

pot and all the seedlings taken were healthy and of equal height and weight. Experiment

was performed in replicate and observations were recorded after 14 days.

2.2.3 Determination of lethal dose (LD50) of hygromycin

The lethal dose of hygromycin as selective agent was determined by separately

culturing 14 days pre-cultured cotyledonary leaf explants and 5 days pre-cultured

embryo axes on MS media supplemented with phytohormones viz. 4.44 μM BAP, 2.46

μM IBA and various concentrations of hygromycin (0, 2.5, 5, 7.5, 10 and 15 mg l

-1). In

average, 20 cotyledonary leaf explants and 10 embryo explants were put in each plate.

Experiment was repeated twice and observations were recorded after 30 days.

2.2.4 Genetic transformation of J. curcas

2.2.4.1 Agrobacterium tumefaciens mediated genetic transformation

2.2.4.1.1 Agrobacterium infection and co-cultivation

Transgenic plants of J. curcas were developed by A. tumefaciens mediated genetic

transformation using different explants i.e. in vitro leaf, pre-cultured embryo axes,

cotyledonary leaf and in vivo leaf. Transformation protocol was optimized for

parameters such as duration of infection with Agrobacterium, co-cultivation time,

concentration of acetosyringone and cefotaxime etc.

To overexpress SbNHX1 gene in J. curcas, A. tumefaciens strain EHA105

mobilized with pCAMBIA1301-SbNHX1 construct having CaMV 35S promoter driven

expression of SbNHX1 gene was used. Transgenic plants were developed by using leaf

disc transformation method described by Horsch et al. (1985) and embryo axes

transformation. Single colony of the recombinant EHA105 strain was grown overnight

at 28 °C in 50 ml LB broth containing antibiotics 50 mg/l kanamycin and 25 mg/l

rifampicin. Bacterial culture was collected at late log phase (O.D.600nm = 0.6) (50 ml)


42

and pelleted by centrifugation for 10 min at 5000 rpm and 4 oC temperature. The

pelleted cells were re-suspended in 50 ml of ½ strength MS containing 2% (w/v)

sucrose and 100 μM acetosyringone. The bacterial suspension was poured in a sterile

petriplate and used for co-cultivation. The pre-cultured in vivo and in vitro leaves were

cut from the edges and cut into 0.5-1 cm2 discs. Pre-cultured leaf discs and slightly

green embryo axes with 4-6 wounds by blade were infected with bacterial suspension

with gentle shaking for about 20 min. Explants were blotted dry on a sterile Whatman

filter paper and co-cultivated in dark on shoot induction medium (SIM) supplemented

with 100 μM acetosyringone for 3 days at 25±1 ºC.

2.2.4.1.2 Selection and regeneration of transformants

After co-cultivation, explants were washed with sterile distilled water containing 500

mg/l cefotaxime, blotted dry on a sterile Whatman filter paper and transferred to SIM

with 500 mg/l cefotaxime. After 14 days, explants were transferred to SIM with

antibiotics (5 mg/l hygromycin and 500 mg/l cefotaxime) for 21 days. For second and

third selection, the explants were subcultured to optimized SIM and/or SRM with 5

mg/l hygromycin and 400 mg/l cefotaxime. After three cycles of selection and

regeneration, putative transformed shoots were transferred for approx. 40-60 days to

SEM + 300 mg/l cefotaxime for further shoot elongation. Transformation efficiency

was calculated as number of GUS or PCR positive explants survived after third round of

selection with respect to total explants used.

For rooting, elongated shoots were transferred to RIM. After 4-6 weeks, plantlets

with rooted shoots were transplanted into autoclaved sand: soil: FYM (2:2:1) mix in

small pots (5x10 cm) covered with transparent plastic lids and maintained under high

humidity for 7–10 days in culture room at 25±1ºC, thereafter gradually exposed to

culture room conditions followed by green house conditions. Established plants were


43

then transferred to 12 inches pots containing a mixture of red soil: sand: FYM (2:2:1).

Overall regeneration efficiency was calculated as total number of transgenic plants

finally regenerated after transformation with respect to total explants used.

2.2.4.1.3 Histochemical GUS assay

Transient gus expression of embryo axis and leaf was assessed according to Jefferson,

(1987) after 24 h of Agrobacterium infection. Leaves of transgenic lines (4- 5 month old

after transformation) were assayed for the constitutive expression of gus gene. GUS

assay was done by using GUS assay kit (Sigma, USA) or manually by incubating the

tissues in freshly prepared GUS assay buffer (1 mg/ml X-Gluc with 0.05 M Na2HPO4,

0.5mM K3Fe(CN)6, 0.5mM K4Fe(CN)6, 10mM EDTA and 0.1% (v/v) Triton X-100) for

12 h at 37oC. Thereafter tissues were destained with 70% alcohol to examine the blue

region.

2.2.4.2 Microprojectile bombardment mediated genetic transformation

2.2.4.2.1 Extraction of recombinant plasmid DNA from E. coli

A single transformed colony of E. coli DH5α or 10 μl of previously frozen cells

resistant to kanamycin were picked and grown overnight in 5 ml LB medium containing

50 mg/l kanamycin. Incubation was at 37 °C with shaking at 200 rpm. The overnight

grown E. coli culture was spinned down for 3 min at 13,000 rpm in a microcentrifuge

tube. Plasmid pCAMBIA1301 or pCAMBIA1301-SbNHX1 construct were isolated by

using plasmid Miniprep kit (Qiagen, Germany) following manufacturer’s protocol.

Plasmid DNA was eluted through column provided with kit and dissolved in 50 μl

sterilized milliQ water. DNA concentration and O.D. (1.80-1.90) were taken by

Nanodrop spectrophotometer and stored at -20 °C for further use.


44

2.2.4.2.2 Preparation of microcarriers

Microcarriers (0.5 mg gold particles) coated with 1 μg of plasmid DNA and suspended

in 50μl absolute ethanol, were used as a standard for each bombardment. Gold

microparticles were weighed carefully according to number of bombardments and

suspended in 1 ml 70% ethanol (v/v) by vigorous vortexing for 3–5 minutes followed

by soaking for 15 minutes. Microparticles were washed three times with 1 ml sterile

water by spinning for 30 seconds in a microfuge. After third wash, microparticles were

suspended in sterile 50% glycerol and coated with plasmid DNA (pCAMBIA1301 or

pCAMBIA1301-SbNHX1) using CaCl2 (2.5 M) and spermidine (0.1 M) precipitation

method (Klein et al., 1988). After 10 min incubation on ice, the supernatant was

removed and pellet was washed with 70% (v/v) ethanol followed by washing with

absolute ethanol. After washing, the particle DNA pellet was re-suspended in absolute

ethanol for bombardments. Care was taken to ensure uniform particle distribution and

minimize agglomeration.

2.2.4.2.3 Arrangement of explants

Five days pre-cultured 40-50 embryo axis explants were arranged aseptically in 90 mm

petriplate in a circle with diameter of 25 mm on solid MS basal media (pH 5.8)

supplemented with 2.22 μM BAP, 0.8% (w/v) agar and 3% (w/v) sucrose just before the

bombardment. 0.2 M mannitol was added as osmoticum to one media petriplate

(replicate) for each treatment grouped with 1μm microcarrier size to know the effect of

osmoticum.

2.2.4.2.4 Microprojectile bombardment

Bombardments were done with biolistic gene gun (PDS 1000/ He, Bio-Rad) under a

vacuum of 27 inches of Hg, 25 mm distance from rupture disc to macrocarrier and 10


45

mm macrocarrier flight distance for all bombardments. Optimum transformation

conditions i.e. helium pressure, microprojectile travel distance and microcarrier size

were determined using the plasmid pCAMBIA1301, which harbours the gus reporter

gene and the selectable hptII gene, both controlled by the cauliflower mosaic virus

(CaMV) 35S promoter. The variables to be optimized included five rupture disc

pressure (650, 900, 1100, 1350 and 1550 psi), four microprojectile travel distance (3, 6,

9, and 12 cm) and microcarrier size (gold particle size 0.6, 1.0 and 1.6 μm). Non-

bombarded embryo axes and embryo axes bombarded with uncoated microcarriers were

used as controls.

After optimizing the suitable bombardment conditions for getting higher

transformation efficiency, the same optimized parameters were used for transformation

of Jatropha with pCAMBIA1301-SbNHX1 construct (Figure 2.1) for improved salt

tolerance.

2.2.4.2.5 Selection and regeneration of transformants

After bombardment, the explants were kept in dark at 25 ºC for 24 h and then

transferred to optimized shoot induction medium (SIM). After 15 days, explants were

transferred to selection medium (same as above) containing 5 mg/l hygromycin. For

effective selection, the explants were transferred to optimized shoot regeneration

medium (SRM) with increasing concentration (6 and 7 mg/l) of hygromycin. After three

cycles of selection, putative transformed shoots were transferred for approx. 40-60 days

to shoot elongation medium (SEM) for further shoot elongation. Transformation

efficiency was calculated as number of GUS or PCR positive explants survived after

third round of selection with respect to total embryos bombarded.

For rooting, elongated shoots were transferred to root induction medium (RIM).

After 4-6 weeks, plantlets with rooted shoots were transplanted into pots (5x10 cm)


46

covered with transparent plastic lids and maintained under high humidity for 7–10 days,

thereafter gradually exposed to culture room conditions. Established plantlets were

transferred to pots containing a mixture of red soil: sand: FYM (2:2:1) in green house

conditions. Overall regeneration efficiency was calculated as total number of transgenic

plants finally regenerated after transformation with respect to total embryos bombarded.

2.2.4.2.6 Histochemical GUS assay

Transient gus expression was assessed after 24 h of bombardment and randomly 20

transformed embryos per shot per plate were selected (Jefferson, 1987) for optimization

of best suited transformation conditions with pCAMBIA1301 vector. While

transforming pCAMBIA1301-SbNHX1 construct, 3-5 embryo axes were subjected to

GUS assay for primary confirmation of transformation. Whole plantlets and leaves of

transgenic lines (4- 5 month old after bombardment) were assayed for the constitutive

expression of gus gene. GUS assay was done by using GUS assay kit (Sigma, USA) or

manually by incubating the tissues in freshly prepared GUS assay buffer as described

previously. Embryo axis explants with at least one discrete blue spot or region on the

tissue were scored GUS positive for statistical analysis.

2.2.5 Molecular analysis

2.2.5.1 DNA extraction from plant material

DNA was isolated from leaf using modified CTAB method (Doyle and Doyle, 1987).

T0 plant leaves were collected to extract DNA for molecular confirmation of true

transgenic lines. Plant material was crushed in liquid nitrogen in mortar & pestle or by

using small pestles to homogenize tissue in a 1.5 ml microcentrifuge tube. According to

the brief protocol described here, putative transformed tissue (50-100 mg) was

homogenized in 1 ml CTAB buffer [2 % CTAB, 2 % PVP (polyvinylpyrrolidone), 2 M


47

NaCl, 50 mM EDTA (pH 8.0), 100 mM Tris-HCl (pH 8.0) with 2 % mercaptoethanol

(freshly added)]. Microcentrifuge tubes were incubated in a water bath for 30 min at 65

ºC. Samples were centrifuged at 12,000 rpm for 10 min at room temperature. Equal

volume of chloroform: isoamyl alcohol (24:1) was added and mixed well by inversion.

Samples were again centrifuged at 12,000 rpm for 10 min at room temp. The upper

aqueous phase was transferred to a new tube and DNA was precipitated with equal

volume of isopropanol by inverting the tubes several times and incubating the tubes at -

20oC for 1-2 hours or overnight. Tubes were then centrifuged at 12,000 rpm for 12 min.

Pellet was dissolved in milliQ water and RNase treatment was given for 1-1.5 hours at

37oC. Then sample was extracted with equal volume of phenol: chloroform: isoamyl

alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) consecutively and precipitated

with double volume of ice cold isopropanol and 3M sodium acetate at -20oC for 2-3

hours or overnight. Samples were centrifuged at 12,000 rpm for 12 min at 4oC. Pellet

was washed with 70 % ethanol, air dried to remove all the traces of ethanol and

dissolved in 50 μl sterilized milliQ water. Extracted DNA was quantified and stored at -

20oC for further use. The isolated DNA was also checked by agarose gel electrophoresis

using 0.8 % agarose.

2.2.5.2 DNA quantification

DNA quantification is an important and necessary step prior to most DNA analysis

methods. The concentration of DNA was measured using the Nanodrop

spectrophotometer. For reading, 1 μl sterilized milliQ water was loaded for calibration

and then as reference. Then DNA samples were loaded one by one and absorbance and

concentration was measured at 260 and 280 nm wavelength. A260/A280 ratio of 1.8 is

indication of highly purified DNA (Sambrook and Russell, 2001).


48

2.2.5.3 PCR based confirmation of transgenic plants

The integration of transgene in different lines was confirmed by PCR analysis using

gene specific primers. The primers used for PCR confirmation were SbNHX1 gene

specific [(SbNHX1TF: 5´-GCG GTA CCA TGT GGT CAC AGT TGA GCT C-3´ and

SbNHX1TR: 5´-TCG TCT AGA CTA TGT TCT GTC TAG CAA ATT G-3´); (RT-

NHX1F 5’-ATG GTG TTT GGG TTG CTG A-3’ and RT-NHX1R 5’-CTG CTT CGT

CTT GGT TGT CC-3’)], gus (reporter gene) specific [(gusF: 5´-GAT CGC GAA AAC

TGT GGA AT-3´ and gusR: 5´-TGA GCG TCG CAG AAC ATT AC-3´); (gusRTF2:

5’-CGA CTG GGC AGA TGA ACA T-3’ and gusRTR2: 5’-CTG TAA GTG CGC

TTG CTG AG-3’)] and hptII (hygromycin selection marker gene) specific (hptIIF: 5´-

TTC TTT GCC CTC GGA CGA GTG-3´ and hptIIR: 5´-ACA GCG TCT CCG ACC

TGA TG-3´). The PCR amplification reaction was performed using the following

thermal profile: one cycle of initial denaturation at 94°C for 10 min, 35 cycles of 94°C

for 1 min (denaturation), 55°C for 1 min (annealing), 72°C for 2 min (extension) for

SbNHX1, 35 cycles of 94°C for 0.45 min (denaturation), 60°C for 1 min (annealing),

72°C for 1.5 min (extension) for hptII & gus, 35 cycles of 94°C for 0.30 min

(denaturation), 60°C for 0.30 min (annealing), 72°C for 0.45 min (extension) for RT-

NHX & gusRT and one cycle of 72°C for 4-8 min (final extension).

The 25 μl PCR reaction mixture contained 1 μl genomic DNA (~100 ng), 2.5 μl

10X Taq buffer containing 2 mM MgCl2, 2.5 μl dNTP (2.5 mM concentration for each

of the 4 different deoxyribonucleotides), 0.5 μl sense Primer (20 μM), 0.5 μl antisense

Primer (20 μM), 0.25 μl Taq polymerase enzyme (5U/μl) and 17.75 μl sterilized milliQ

water. The amplified product was assayed by electrophoresis in 1-1.5 % agarose gels in

1X TBE (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 8.0).


49

2.2.5.4 Determination of copy number in transgenics

2.2.5.4.1 Southern hybridization

This technique is widely being used to identify the copy number of integrated gene in

the genome based on hybridization of gene-specific probe to the genomic DNA.

Genomic DNA (20 μg) was isolated from fresh leaves of transformants and wild type

plants and then digested with EcoRI at 37 °C for 10-12 h. Alternatively, FastDigest

EcoRI was used for rapid digestion (Fermentas, USA).

Restriction Digestion Reaction Mix:

Restriction endonuclease (10 U/µg DNA) X µl

10 X restriction endonuclease buffer 5 µl

Sterile distilled water X µl

Total Volume 50 µl

Digested gDNA was separated by electrophoresis in a 0.8% agarose gel and

transferred onto a Hybond N+ membrane (Amersham Pharmacia, UK) by capillary

method using alkaline transfer buffer (0.4 N NaOH with 1 M NaCl). The membrane

was neutralized with Neutralization buffer (0.5 M Tris-Cl of pH 7.2 with 1 M NaCl),

air-dried and DNA was fixed to the membrane by UV cross-linking using 56 mJ cm-2

energy for 1 min in a UVC 500 cross-linker (Amersham Biosciences, UK).

The membrane was placed in hybridization tube and pre-hybridized using 20 ml

DIG EasyHyb buffer solution (Roche, Germany) at 68 °C for 30 min in a rolling tube

hybridization oven. Then, membrane was hybridized with PCR-generated probe for

hptII gene labeled with DIG-11-dUTP, amplified from plasmid pCAMBIA1301 using

0.1 mM DIG-11-dUTP, 1.9 mM dTTP and Taq DNA polymerase, following

manufacturer user guide (Roche, Germany). Purified pCAMBIA1301 and PCR


50

amplified hptII gene served as a positive controls while DNA from non-transformed

plant as a negative control. Hybridization was carried out at 68 ºC overnight in probe-

DIG EasyHyb buffer solution. The membrane was then washed 2-3 times at room

temperature for 5 min in 2x SSC, 0.1% SDS, and twice for 15 min in 0.2x SSC, 0.1%

SDS at 68 ºC. The membrane was then rinsed with washing buffer provided with kit for

5 min. Then, the membrane was incubated in 100 ml of blocking solution (provided

with kit) for 30 min, followed by incubation in 50 ml of Antibody solution (50 ml

blocking solution + 5 µl Antibody). It was washed twice with washing buffer for 15

min. It was then equilibrated in 100 ml of detection buffer for 5 min.

The membrane was then placed on development folder and approximately 1 ml of

CDP star chemiluminescent solution from DIG Northern Starter kit was applied on the

membrane. Immediately the membrane was covered with second sheet of the folder to

spread the substrate evenly. The membrane was incubated for 5-10 min in dark and

excess CDP star solution was squeezed out. The folder having the membrane was

placed in phosphoimager cassette along with X- ray film and exposed for 15-30 min.

The X- ray photofilm (Eastman Kodak, USA) was then developed using developer and

fixer solutions to visualize the signals.

2.2.5.4.2 Determination of copy number by real time PCR

Genomic DNA concentration determined by NanoDrop Spectrophotometer was diluted

to 1, 10 and 100 ng/μl concentration. Real time quantitative PCR (RTqPCR) condition

was optimised for gus gene primers (gusRTF2: 5’-CGA CTG GGC AGA TGA

ACA T-3’, gusRTR2: 5’-CTG TAA GTG CGC TTG CTG AG-3’) and JcKASIII gene

(NCBI accession no. DQ987701.1) primers (JcKAS1F: 5’-GCA CTT GGC TGC AAA

ACA AAT-3’, JcKAS1R: 5’-CGT CCA GTC AAC ATA TCG AG-3’). JcKASIII was

used as an internal control because this gene has single copy in J. curcas genome (Li et


51

al., 2008a). The PCR reactions were carried out using 0.25 μM primers for both the

genes in 20 μl reaction using QuantiFast SYBR Green PCR reaction kit (Qiagen, USA).

RTqPCR assay mix

Sterilized distilled water (kit) 8.5 μl

2X Qiagen Sybermix 10.0 μl

Forward primer (20 μM) 0.25 μl

Reverse primer (20 μM) 0.25 μl

Template DNA 1.0 μl

Total volume 20 μl

The following PCR conditions were maintained for RTqPCR:

Cycle 1: (1 cycle)

Step 1: 95.0 °C 5 min

Cycle 2: (40 cycles)

Step 1: 94.0 °C 30 sec

Step 2: 60.0 °C 30 sec

Step 3: 72.0 °C 45 sec

Cycle 3: (1 cycle)

Step 1: 95.0 °C 1 min

Cycle 4: (1 cycle)

Step 1: 60.0 °C 1 min

Melt curve analysis (increase set point temperature after 2nd

cycle by 0.5 °C)

Cycle 5: (71 cycles)

Step 1: 60.0 °C-95.0 °C 30 sec

At the end of the PCR cycles, the products were put through a melt curve analysis. The

amplified product was run on a 1.5% agarose gel to confirm expected size. The

experiments were repeated twice independently with three replicates each time.

Reactions were run in Real-Time iQ5 Cycler (Bio-Rad, USA), and standard curves were

plotted using threshold cycle (CT) values to determine reaction efficiencies. The

efficiency values were put in the following formula (Equation 1) (Shepherd et al., 2009)

to determine the copy number ratio of gus to JcKASIII:


52

Ratio = …………………. (1)

Copy number was determined by the following ratio range:

Ratio Copy number

< 0.5 Zero

0.5-1.5 single

1.5-2.5 double

2.5-3.5 triple

> 3.5 multiple

2.2.6 Statistical analysis

For regeneration, all experiments were performed for three times and uniform number

of explants was used. For optimization of best suited protocol for genetic transformation

via microprojectile bombardment, at least two plates with three replicates were

bombarded for each treatment. Frequency of GUS activity was calculated as number of

embryos showing gus expression to the total number of explants stained after

bombardment and is expressed as percentage. Data on the regeneration, transformation

and transgenic analysis was subjected to analysis of variance (ANOVA) for analysis to

determine differences (Sokal and Rohlf, 1995) and were expressed as mean ± SE. A

Tukey HSD multiple comparison of mean test was used when significant differences

were found and p<0.01 or p<0.05 was considered as significant.

1+ Effieiency (C

T gene of interest)

gene of interest

1+ Effieiency (C

T control)

control


53

2.2.7 Physiological analysis

2.2.7.1 Leaf disc assay

Leaf disc assay was performed for analysis of transgenic plants for its salt tolerance

according to procedures described by Fan et al. (1997). Healthy leaves from similar age

WT and transgenic plants (T0 generation) were detached. Leaf discs of 5 mm diameter

were punched out and floated in 5 ml sterilized distilled water with different

concentrations of sodium chloride (0, 50, 100 and 200 mM) for 8 days. The leaf discs

were kept under 16 hours white light (35μmol m-2

s-1

)/ 8 hours dark at 25±2 oC. The

effects of this treatment on leaf discs were assessed by observing phenotypic changes

and quantification of chlorophyll content spectrophotometrically after extraction in 80%

acetone. The experiment was repeated twice.

2.2.7.2 Chlorophyll estimation

Leaf discs of WT and two transgenic lines L2, L8 were treated with different

concentrations of NaCl as described earlier and used for chlorophyll estimation. 7-8

treated leaf discs (4-9 mm2 area) were homogenized thoroughly in 80% acetone and

centrifuged at 3,000g for 2–3 min in dark. The O.D. of supernatant was taken at 645 and

665 nm and chlorophyll was calculated per gram fresh weight of tissue by the following

formula (Equation 2) (Arnon, 1949).

Chlorophyll content =

(mg/ g tissue)

………………………… (2)

[(20.2 X O.D. of 645 nm) + (8.02 X O.D. of 665 nm)] X Volume

1000 X g tissue


54

2.2.8 Other molecular techniques used in this study

2.2.8.1 Preparation of competent Agrobacterium cells

Competent cells of A. tumefaciens were prepared according to method of Vincze and

Bowra, (2006). A. tumifaciens strain EHA105 cells were streaked on LB agar plates

containing 25 mg/l rifampicin antibiotic and incubated at 28 °C for 18-24 hours. A

single colony of A. tumefaciens grown on plate was inoculated in 50 ml of LB medium

containing 25 mg/l rifampicin and incubated at 28 °C for 16-20 hours with shaking (240

rpm) until O.D.600 = 0.6 was reached. The cells were chilled on ice for 15 min and spun

down by centrifugation at 4,500 rpm for 10 min at 4 °C. The culture medium was

discarded and pellet was dissolved in 2 ml of ice-chilled 20 mM CaCl2 solution

containing 10 % (v/v) glycerol. 100 μl aliquots of the suspension were dispensed into

pre-chilled microfuge tubes, frozen immediately in liquid nitrogen and stored at -80 °C.

2.2.8.2 Transformation of A. tumefaciens EHA105 by freeze-thaw method

The pCAMBIA1301-SbNHX1 construct was mobilized into A. tumefaciens strain

EHA105 by a freeze-thaw method (Holsters et al., 1978). A. tumefaciens was thawed

carefully, 5μl (0.2-1.0 μg) of construct (pCAMBIA1301-SbNHX1) DNA was mixed

gently with the competent cells and incubated in liquid N2 for 5 min. The cells were

then exposed to 37 °C for 5 min and then placed on ice for 2 min. After that, 1 ml of LB

medium was added and incubated at 28 °C for 2-4 h with vigorous shaking at 240 rpm.

Cells were centrifuged at 5000 rpm for 3 min at 4 °C, 900 µl of supernatant was

discarded and cells were suspended in the remaining medium. Finally, transformed

Agrobacterium cells were spread on LB-Rif-Km agar medium plates and incubated at

28 °C for 20-24 hours. Presence of colonies was checked and the plates were stored at 4

°C for further use. Five independent transformed colonies of strain were randomly

selected and checked for the transformation by PCR.


55

LB-Rif-Km agar medium: LB agar medium plates supplemented with 50 mg/l

kanamycin and 25 mg/l rifampicin.

2.2.8.3 Growth of recombinant A. tumefaciens and preparation of glycerol stocks

Single colonies of A. tumefaciens were examined for the presence of plasmid construct

by colony PCR. Positive colonies were inoculated in 10 ml of LB-Rif-Km medium and

incubated at 28 °C for 16-20 h with vigorous shaking at 240 rpm. The culture was

transferred to oakridge tubes and Agrobacterium cells were pelleted by centrifugation at

4000 rpm for 10 min at 15 °C. The cells were resuspended in a 1:1 volume of LB-Rif-

Km medium and glycerol stock media. 100 μl aliquots of the suspension were dispensed

into pre-chilled microfuge tubes, frozen immediately in liquid nitrogen and stored at -80

°C for further experiments.

Glycerol stock media: Glycerol 50 % (v/v), MgSO4 100 mM, Tris 25 mM and pH 7.4.

chapter 2 materials and methods - shodhganga :...

Documents