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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
Materials and Methods
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.
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
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).
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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)
Materials and Methods
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
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
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)
Materials and Methods
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
Materials and Methods
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).
Materials and Methods
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).
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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:
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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
Materials and Methods
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
Materials and Methods
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.
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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.