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3. MATERIALS AND METHODS
Recent developments in genome analysis of several plant species
have opened up new avenues to decipher the structure and functions
of several unknown genes. Functional annotation of gene sequences
through genomics approach has become inevitable for understanding
the function of genes, identification and characterization of functional
variations and for effective gene manipulation for crop improvement.
The present study was aimed to characterize the function of one of the
calcium dependent protein kinase genes in rice using genetic
transformation approach. The materials and methods followed in the
study are presented in this chapter.
3.1 Materials
3.1.1 The Plant material
For development of transgenic rice plants, two different rice
genotypes were used. Seeds of Taipei 309 (japonica) and BPT 5204
(indica) available at the Directorate of Rice Research (DRR),
Rajendranagar, Hyderabad, India, constituted the plant material used
for transformation.
3.1.2 Source and description of gene constructs
Two gene constructs viz., pB4NU-CK31-Ox (Overexpression) and
pANDA-CK31-Si (Silencing) were kindly provided by Prof. A.K. Tyagi,
University of Delhi South Campus (UDSC), New Delhi. While, the
Agrobacterium strain LBA 4404 was available at DRR Biotechnology
Laboratory, the super virulent Agrobacterium strain EHA105 and
helper plasmid pRK2013 were received from Dr. D. Sudhakar, Tamil
Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India.
3.1.2.1 Overexpression construct
The overexpression construct (CK31-Ox) was made by isolating
a rice cDNA clone of 3373 bp (OsCPK31 gene) from IR64 and then
cloned into the vector pCAMBIA1301. The gene was driven by maize
ubiquitin promoter and nos terminator. Hygromycin and gus genes
were two marker genes (i.e., selection and reporter marker) toward left
and right borders of the T-DNA region. Kanamycin was the bacterial
selection marker gene present in the vector pB4NU. The plasmid was
maintained in the host strain XL-1 Blue MRF.
3.1.2.2 Silencing construct
The PCR-derived 334 bp (5’ coding region and 3’ UTR region)
fragment was inserted into two regions flanked by two recombination
sites (attB1 and attB2) in opposite directions and the gus linker
sequence was flanked by the two inverted repeats. The gene was
driven by maize ubiquitin promoter with first intron and splicing
acceptor site and nos terminator gene. The detailed construction of
pANDA binary vector was described by Miki and Shimamoto (2004).
Hygromycin and kanamycin were two plant selection marker genes in
pANDA vector. The binary vector was maintained in E.Coli strain
DH5α.
3.1.3 Fine chemicals, enzymes and kits
The different chemicals for culture media and ready to use
Mursashige & Skoog medium were purchased from Himedia, Mumbai,
India. All molecular biology fine chemicals/reagents were procured
from Sigma-Aldrich, U.S.A. All Restriction enzymes, Plasmid isolation
kits and Improm-II Reverse Transcription system were purchased from
Promega Corporation, USA. Molecular markers of DNA were
purchased from Fermentas, Lithuania. For Southern blot analysis,
radio label α-32P dCTP, dATP and random primer labeling kit were
procured from JONAKI, BRIT, Hyderabad. Ready To Go DNA Labeling
Beads (dCTP) was purchased from GE Healthcare Ltd. UK.
3.2 Methods
3.2.1 Confirmation of plasmids with restriction digestion and binary vector mobilization
The plasmid was isolated from the host strain E.Coli by plasmid
isolation kit. About 1 µg of plasmid DNA from pB4NU-CK31-Ox was
restricted with 20 units of BamHI for 1 h at 37°C followed by 15 min
denaturation at 65°C. Similarly, DNA from the plasmid pANDA-CK31-
Si was digested with KpnI + SacI enzymes. The restricted DNA
fragments were loaded in a 0.8% of agarose gel (Lonza Inc., USA) pre-
stained with 0.2 µg/ml ethidium bromide and resolved for 3 h at 40
volts in 1X TAE (40 mM Tris-acetate and 2 mM Na2EDTA.2H2O pH
8.0) buffer. The presence of the gene was confirmed by PCR using
gene specific primers (Table 3.4). The DNA fragments specific to
overexpression and silencing gene were resolved through
electrophoresis and documented using Alpha Imager Documentation
System (M/s Alpha Innotech, USA).
3.2.1.1 Introduction of binary vectors into Agrobacterium and its confirmation
The host bacteria E.Coli containing the binary vectors pB4NU-
CK31-Ox, pANDA-CK31-Si, pRK2013 and Agrobacterium strain LBA
4404 and EHA 105 were maintained as glycerol stock. The two binary
vectors were mobilized into Agrobacterium strain LBA 4404 and
EHA105 by triparental mating method with helper plasmid pRK2013
(Lichtenstein and Draper, 1985). Medium composition of LB agar, YEB
and AB minimal medium are listed in Table 3.1, 3.2 & 3.3.
3.2.1.1.1 Mobilization of the binary vector into Agrobacterium
tumefaciens by triparental mating
Freshly streaked E.Coli strains harbouring pRK2013 and binary
vector (pB4NU-CK31-Ox) in LB agar medium containing 50 mg/l
kanamycin plates were incubated at 370C in incubator. Agrobacterium
tumefaciens was streaked separately in YEB medium agar plates
containing 10 mg/l rifampicin and incubated at 300C. Another YEB
agar plate without antibiotics was prepared for triparental mating.
One colony each from E.Coli with pRK2013, E.Coli harbouring
the plasmid to be mobilized and Agrobacterium was patched
separately on the YEB plate very close to each other. Using sterile
loop, all the three bacterial strains were mixed very well and the plate
was left at 300C for 12-18 h. After mating, the bacterial colony on the
YEB plate was scrapped and suspended in 1 ml of 0.9% sodium
chloride. A serial dilution was performed by transferring 100 µl of
bacterial suspension into 900 µl NaCl (10-1 dilution). Five dilutions
such as 10-1, 10-2, 10-3, 10-4 and 10-5 were made. From different
dilution, 100 µl of each dilution was added to an AB minimal agar
plate containing rifampicin 10 mg/l and the antibiotic kanamycin 50
mg/l and then spread uniformly using sterile glass rod. The plates
were incubated at 300C for 3-5 days. The same procedure was
followed for the mobilization of binary vector pANDA-CK31-Si into LBA
4404 and EHA 105 Agrobacterium strains as described above.
3.2.1.1.2 Confirmation of binary vector in Agrobacterium
From 10-4 dilution plate, six individual colonies of
transconjugants were selected from AB minimal medium and
confirmed by colony PCR with gene specific primers (Table 3.4) to
produce 606 bp amplicon for CK31-Ox construct. Similarly,
transformed Agrobacterium harbouring pANDA-CK31-Si binary vector
was confirmed by colony PCR of six colonies of recombinant
Agrobacterium from the selection plate. Gene specific primers viz.,
forward primer of Gus linker and reverse primer of coding region of Si
(Table 3.4) were used to obtain approximately 1.3 kb amplicon size for
pANDA CK31-Si vector.
Colony PCR was performed in a 10 µl reaction containing -10X
PCR buffer (10mM Tris, pH 8.4, 50 mM KCl, 1.8 mM MgCl2 and
0.01mg/ml gelatin), 0.1 mM of dNTPs, 200 nM of primers, 1.0 unit of
Taq polymerase (Bangalore Genei, India) and half of the single colony
of either CK31-Ox or CK31-Si was mixed in PCR plate/PCR tube and
kept in Thermal Cycler (Bio-Rad, USA). The PCR cycling conditions
were as followed: Initial denaturation for 10 min at 940C, followed by
35 cycles of denaturation at 940C for 30 sec, annealing at 580C for 30
sec and extension at 720C for 1 min, followed by a final extension at
720C for 7 min. The PCR products were resolved on 1% agarose gels
(Lonza Inc, USA) in 1X TAE buffer in a midi electrophoresis unit (CBS
Scientific, USA) for 3 hours at 50V, pre-stained with ethidium bromide
(0.5 μg/ml) and photographed using gel documentation system.
3.2.1.1.3 Introduction of plasmid into E. coli and its confirmation with restriction digestion
PCR confirmed Agrobacterium colonies with CK31-Ox and
CK31-Si plasmids were streaked separately on YEB agar medium
containing antibiotics (Rif 10 mg/l, kan 50 mg/l) and the plasmids
were isolated by alkaline lysis method using the plasmid isolation kit.
The isolated plasmid DNAs were transformed into E.Coli strain (DH5α)
by heat shock method. The transformed E.coli colonies were streaked
on LB agar plates containing the antibiotic kanamycin 50 mg/l. The
positive colonies were picked up and inoculated in LB liquid medium
with kanamycin 50 mg/l. The cultures were then incubated at 370C
for 12 h with 220 rpm followed by plasmid isolation. After plasmid
isolation, about 1 µg each of CK31-Ox plasmid and CK31-Si plasmid
were separately restricted with restriction enzymes. For example,
CK31-Ox plasmid, was restricted with 20 units each of BamHI, XhoI
and KpnI, while CK31-Si plasmid, was digested either with 20 units of
KpnI or in combination of KpnI and SacI enzymes and then incubated
at 370C for two h. The restricted plasmids were denatured at 65°C for
10 minutes and resolved in 0.8 % agarose gel.
Table 3.1: Composition of LB Agar medium Component g /100 ml Bacto-tryptone 1 Bacto-Yeast Extract 0.5 NaCl 1 Bacto Agar 1.5 pH 7.0
Table 3.2: Composition of YEB Agar medium
Table 3.3: AB minimal medium composition Constituents g / 100 ml
AB Buffer (20X)
K2HPO4 6 NaH2PO4 2 pH 7.0
AB salts (20X)
NH4Cl 2 MgSO4.7H2O 0.6 KCl 0.3 CaCl2.2H2O 0.31 FeSO4.7H2O 0.005
AB Medium
Add 0.5 g of glucose to 90 ml of double distilled water. Add 1.5 g of agar and autoclave. After the medium cools to about 550C, add 5 ml of AB salts and 5 ml of AB buffer and antibiotics 10 mg/l rifampicin and 50 mg/l kanamycin and pour the plates in laminar hood.
Component g / 100 ml Yeast Extract 0.1 Beef Extract 0.5 Sucrose 0.5
Magnesium sulphate 0.05 Peptone 0.5 pH 7.0 Agar 1.5
Table 3.4: Primers and its corresponding amplicon length
3.2.2 Tissue culture and genetic transformation of rice
Prior to genetic transformation, standardization of tissue culture
conditions for callus induction and regeneration of Taipei 309 and
BPT 5204 was imperative. The following sections describe the
optimization of tissue culture conditions and genetic transformation
protocols for development of transgenic rice plants.
3.2.2.1 Tissue culture media
Response of to tissue culture conditions always vary to different
genotypes. Hence, it is very difficult to suggest a single universal
tissue culture medium which can be suitable for callus induction and
plant regeneration across the genotypes. Based on earlier efforts made
in the Biotechnology Laboratory of DRR, suitable culture media
combinations could be identified to produce callus and regenerate
plants at optimum levels. The basic MS (Murashige & Skoog, 1962)
medium (purchased from Himedia, India) was used for all the
experiments including callus induction, plant regeneration and
genetic transformation. The MS basal medium comprised MS basic
salts with macro, micronutrients, vitamins, and myo-inositol .
Further, aminoacids such as casein hydrolysate (500 mg/l), L-proline
Gene Forward primer Reverse primer Amplicon size
Ox 5’-tttagccctgccttcatacg -3’ 5’-gagcaggcaatttgagaacc-3’ 606 bp
Si 5'-ttctacaacctgctgcgt -3' 5'-atatggattgagcggctg-3' 334 bp
Gus 5’-catgaagatgcggacttacg -3’ 5’-atccacgccgtattcgg-3’ 636 bp
Ox-Exon 5'-ctggcaccattagctttgagg-3' 5'-ctatgagggtggcgaggaact-3' 150 bp
Si- int 5’-catgtctgtatgaacggcaaac-3’ 5’-actcggacacgcagttcag-3 185 bp
OsActin1 5’-tccatcttggcatctctcag-3’ 5’-gtacccgcatcaggcatctg-3’ 337 bp
(500 mg/l), phytohormones. Either sucrose or maltose was used as
carbon source and added separately to the MS medium. Phytagel was
used as soldifiying agent. To optimize the callus induction with higher
percentage of embryogenic callus and plant regeneration, MS basal
medium with different combinations of carbon sources (sucrose and
maltose), phytohormones (2,4-Dichlorophenoxyacetic acid (2,4-D),
kinetin, 6-Benzylaminopurine (BAP), 1-Naphthaleneacetic acid (NAA))
were used. All tissue culture media used in this study were prepared
in double distilled water followed by pH adjustment with either 1N
NaOH or 1N HCl. The media were added with 3 g/l phytagel (a
solidifying agent) and autoclaved at 1210C (15 psi) for 20 min. Mature
rice seeds of Taipei 309 and BPT 5204 were used as explant for
optimization of callus induction. The seeds were surface sterilized with
70% ethanol for 2 min and 10 min with 0.1% HgCl2, followed by
rinsing for three to four times with sterile distilled water. The seeds
were inoculated in MS medium for callus induction at 26±2ºC in dark
for 18-21 days.
3.2.2.2 Optimization of media for callus induction
To examine the effect of carbon source, growth regulators
during callus induction, the seeds were inoculated in MS basal callus
induction medium (Table 3.5), 2,4-D 2.0 mg/l with two different
carbon sources such as sucrose and maltose (30 g/l) separately. Also,
different combination of growth regulators such as 2,4-D (2 mg/l),
BAP (1.0 mg/l) and kinetin (0.5 mg/l) were tested in three different
callus induction medium (CIM) viz., CIM1 (MS basal media + 2 mg/l
2,4-D), CIM2 (MS basal media + 2 mg/l 2,4-D + 0.5 mg/l BAP) and
CIM3 (MS basal media + 2 mg/l 2,4-D + 0.5 mg/l kinetin). Callus
induction frequency was calculated after 21 days incubation at dark
as follows:
No. of calli induced
Frequency of callus induction (%) = X 100
No. of seeds plated
3.2.2.3 Optimization of plant regeneration medium
Scutellum derived embryogenic calli were selected under
dissection microscope and individually transferred to MS plant
regeneration medium (Table 3.5) with different combinations of
phytohormones such as RM1 (kinetin 2.0 mg/l + NAA 0.5 mg/l), RM2
(BAP 2.0 mg/l + NAA 0.5 mg/l) and RM3 (kinetin 2.0 mg/l + BAP 1.0
mg/l + NAA 0.5 mg/l) solidified with 4 g/l phytagel. The cultures were
incubated at 26±20C under 16 h/8 h continuous light/dark. After 3-4
weeks, the regenerants were transferred to ½ MS basal medium for
rooting, solidified with 4 g/l phytagel. The rooted plants were initially
transferred to Yoshida’s culture solution (Table 3.6) for hardening for
about 10 days and subsequently transferred to earthen pots.
Plant differentiation in terms of calli developing into green
shoots was recorded 3-4 weeks after transferring to regeneration
medium and the regeneration frequency was calculated as follows:
No. of calli with shoots
Regeneration Frequency (%) = X 100
Total no. of calli in regeneration
Table 3.5: Composition of basal media used in the present study
Constituents (Murashige and Skoog, 1962) Modified MS medium (mg /l)
KnO3 1900
NH4NO3 1650
KH2PO4 170
SO4.7H2O 370
CaCl2.2H2O 440
MnSO4.H2O 16.4
ZnSO4.7H20 8.6
H3BO3 6.2
KI 0.8
Na2MoO4.2H2O 0.25
CuSO4.5H2O 0.025
CoCl2.6H2O 0.025
FeSO4.7H2O 27.8
Na2-EDTA 37.3
Nicotinic acid 0.5
Pryidoxine Hcl 0.5
Thiamine Hcl 1.0
Glycine 2.0
Myo-inositol 100
Caesin hydrolysate 500
L-Proline 500
Carbon source Sucrose or maltose
phytohormone -
Phytagel 3.0 g
pH 5.8
Table 3.6: Yoshida’ culture solution composition
3.2.2.4 Optimization of hygromycin concentration for selection of transformants
The antibiotic hygromycin was used as a selection agent for
selection of transformed calli from the large number of non-
transformed ones. Optimum concentration of hygromycin was
decided by preparing kill curves for both varieties using non-
transformed control calli. Hygromycin B, was added to selection
medium at concentrations of 0, 25, 50, 75 and 100 mg/l. One
hundred fresh embryogenic calli (21 days old) of 2-3 mm size from
both genotypes were incubated in the selection medium for three
cycles in three replicates. The optimal concentration of hygromycin
antibiotic was established based on the dead calli % vs
Component Stock solution (g/10L)
Volume (ml) for 4 lt
NH4NO3 914 5 NaH2PO4.2H2O 403 5 K2SO4 714 5 CaCl2 886 5 MgSO4.7H2O 3240 5 MnCl2.4H2O 15
5
(NH4)6Mo7O24.4H2O 0.74 H3BO3 9.34 ZnSO4.7H2O 0.35 CuSO4.5H2O 0.31 FeCl3.6H2O 77 Citric acid (monohydrate) 119 pH 5-6
concentration of hygromycin in mg/l. The mortality rate of callus
from two genotypes was calculated.
3.2.2.5 Statistical Analysis
The tissue culture data were subjected to statistical analysis
with 3-6 replications depending on the experiment. Percentage
values were transformed to using arc sine angular transformation
before performing ANOVA. Significance test for all the factors
examined viz., different basal media with effect of carbon source,
combination of phytohormones and shoot regeneration was
evaluated. Analysis of Variance and Least Significant Difference
(LSD) values for each factor and for their interactions were
calculated at 1% and 5% level of probability, ANOVA was performed
by using the software Statistical Analysis System (SAS) version 9.2
available at Directorate of Rice Research.
3.2.3 Genetic transformation of rice genotypes The selected rice genotypes, Taipei 309 and BPT 5204 were
transformed using Agrobacterium-mediated transformation method
for the deployment of target gene OsCPK31. The schematic protocol
for production of transgenic rice plants using tissue culture method
is shown (Fig. 3.1). The composition of different media used for
transformation is given in table 3.7.
The Agrobacterium tumefaciens strain LBA 4404 and EHA 105
separately harbouring pB4NU-CK31-Ox and pANDA-CK31-Si binary
vector were used for the transformation of Taipei 309 and BPT 5204
genotypes. Neomycin phosphotransferase encoded by npt II gene was
used as bacterial selection marker.
Table 3.7: Media used for transformation of two genotypes
Medium Composition Callus induction medium (MSCIM)
MS basal salt + 2 mg/l 2,4-D + 0.5 mg/l kinetin + 0.5 g L-proline + 100 mg/l myo-inositol + 0.5 g/l casein hydrolysate + 30 gm/l maltose + pH 5.8 + 0.3 gm/l phytagel
Suspension medium (MSSus)
MS basal salt + 2 mg/l 2.4-D + 68.5 gm/l sucrose + 36 gm/l glucose + pH 5.2 + 100uM Acetosyringone (AS)
Co-culitvation medium MSSus + 0.3 % phytagel + 200uM Acetosyringone.
Selection medium
MSCIM + 50 mg/l hygromycin B + 250 mg/l carbenicillin + pH 5.8 + 0.4% phytagel
Regeneration medium (RM1)
MS basal salt + 2 mg/l kinetin + 0.5 gm/l NAA + 100 mg/l myo-inositol + 500 mg/l L-proline + 500 mg/l casein hydrolysate + 30 g/l sucrose + 25 mg/l Hygromycin B pH 5.8 + 0.4% phytagel
Rooting medium
½ MS basal salt + 15 gm/l sucrose + pH 5.8 + 0.4% phytagel
Hardening medium Yoshida’s solution (table 3.6)
Fig. 3.1: A schematic protocol for development of transgenic rice
3.2.3.4 Preparation of Agrobacterium culture The glycerol stock of Agrobacterium culture was freshly streaked
on YEB agar plate containing 1.5% agar agar (Type I) with 10 mg/l
rifampicin and 50 mg/l kanamycin. The culture was incubated
overnight at 28°C. From the streaked fresh plate, a single
Agrobacterium colony was inoculated in 5 ml of AB minimal medium
containing 50 mg/l kanamycin which was incubated at 28°C with
shaking @ 220 rpm for 16 h. From the 5 ml mother culture, add 0.2,
0.4, 0.6, 0.8 and 1 ml of Agrobacterium into 50 ml of fresh AB minimal
medium containing 50 mg/l kanamycin and incubate for 12-16 h at
280C with constant shaking at 220 rpm. The culture density was
adjusted to O.D 0.5 at 600 nm using ND-1000 Spectrophotometer
(NanoDrop Technologies Inc., USA).
3.2.3.5 Bacterial infiltration and co-cultivation
The bacterial culture in YEB liquid medium was centrifuged at
3500 rpm for 20 min at 250C and the pellet was re-suspended in
liquid suspension medium (Table 3.7) used for infiltration, which
contained 100 µM acetosyringone (AS) and it was incubated at room
temperature for 2 h by gentle shaking for induction of the vir genes.
Selected embryogenic calli (18-21 days old) were cut into small pieces
of 2-3 mm size. The pre-incubated Agrobacterium culture was used for
infection of the embryogenic calli. These calli were transferred to the
bacterial infiltration (MSSus) medium in a sterile conical flask which
was incubated for 15 min with occasional shaking. The infected calli
were blotted dry on a Whatman No. 1 filter paper and transferred to
the co-cultivation medium (Table 3.7) supplemented with 200 µM AS.
The plate was layered with Whatman No.1 filter paper to control the
over growth of Agrobacterium. The cultures were co-cultivated in the
dark at 26±2°C for 3 days.
3.2.3.3 Washing of co-cultivated calli
After 72 h of co-cultivation at dark, the calli were washed
manually 3-4 times with sterile distilled water inside the laminar air
flow cabinet followed by shaking 3-4 times on a shaker @ 120 rpm for
5 min each at room temperature, till wash solution became clear. The
calli were again washed 3-4 times with MSCIM liquid medium (Table
3.7) containing 250 mg/l of cefotaxime for LBA 4404 mediated
transformation and 250 mg/l each of cefotaxime and carbenicillin 15
min each. Transformed calli were transferred to a sterile petri dish
layered with a sterile blotting paper to remove excess of medium and
bacteria.
3.2.3.4 Selection of transformed calli
The callus induction medium supplemented with antibiotics 250
mg/l cefotaxime and 50 mg/l hygromycin B for LBA 4404 strain or
400 mg/l of carbenicillin and 50 mg/l hygromycin for EHA105 strain
was used as selection medium. The washed calli were placed on
selection medium to select the transformed hygromycin resistant calli
from the non-transformed calli. After two weeks of incubation in
selection medium, the resistant calli were selected under dissection
microscope and then transferred to fresh selection medium. The
selection was repeated for 3 cycles of 15 days each. During each sub-
culture, the number of dead calli was recorded. At least one plate of
non-transformed calli from same batch was kept on selection medium
with each transformation experiment for trouble shooting the
experiment result.
3.2.3.5 Regeneration of plantlets from transformed calli
Following 3 cycles of selection, resistant proliferating
embryogenic calli were transferred to regeneration medium MSKN
(Table 3.7) supplemented with 25 mg/l of hygromycin and incubated
in dark for 7-9 days, after which the plates were placed under
fluorescent light source in the culture room at 26±2°C for 3-4 weeks.
Calli showing green shoots were transferred carefully to fresh medium
and were incubated for one more cycle, if required. The putative
transgenic plantlets were transferred to rooting medium (Table 3.7) for
15 days and fully rooted plants were transferred to Yoshida’s culture
solution (Table 3.6) for hardening and then transferred to earthen pots
and were grown in transgenic biosafety glass house.
3.2.4 Confirmation of transgene by molecular analysis 3.2.4.1 Isolation of genomic DNA from transgenic and control
non-transformed rice plants
Genomic DNA was isolated from young leaves collected from
putative transgenic plants grown in transgenic biosafety glass house
as per the protocol of Rajendrakumar et al., (2007) for PCR analysis.
Fresh leaves collected from plants at vegetative stage were used to
isolate genomic DNA for Southern blot analysis by following modified
CTAB method (Dellaporta et al., 1983).
§ Two gms of young leaf from putatively transformed plants were
taken and cut into small pieces. The leaf samples were ground in a
autoclaved pestle and mortar with liquid nitrogen and 10 ml of
CTAB buffer (2% w/v CTAB, 100 mM Tris Hcl, pH 8.0, 20 mM
EDTA, 1.4 M NaCl) was added to it. Transfer the powder into 50 ml
Polypropylene tube.
§ Incubate the mixture at 650C for 1 h followed by equal volume of
Phenol:chloroform: isoamyl alcohol (25:24:1) was added to the tube
and the contents were mixed well by inversion and centrifuged at
13000 rpm for 15 minutes at 250C.
§ The aqueous phase was collected in a fresh tube without
disturbing the intermediate layer and ~5µl of RNase (10 mg/ml)
was added followed by incubation at 37oC for 30 minutes.
§ About equal volume of chloroform:isoamyl alcohol was added to the
contents in tube and mixed well by inversion. The mixture was
centrifuged at 13,000 rpm for 10 minutes and the supernatant was
collected in a tube.
§ To the clear supernatant, an equal volume of chilled isopropyl
alcohol was added. The contents were mixed gently by inverting
and centrifuged at 13,000 rpm for 10 minutes.
§ The supernatant was discarded and the pellet was washed with
200 µl of 70% ethanol. The pellet was air dried at room
temperature for about 1-2 hours and dissolved in 100 µl TE pH 8.0
(10 mM Tris-HCl and 1 mM EDTA).
§ Two µl of DNA in TE was loaded along with reference λ-DNA in a
0.8% agarose gel, electrophoresed for about an hour and the gel
was documented by using Alpha Imager UV gel documentation
system (M/s Alpha Innotech Corporation, USA). The DNA
concentrations of test samples were determined by comparing
intensity of the band with that of reference DNA.
3.2.4.2 PCR analysis
PCR analysis was carried out using the DNA isolated from the
putative transgenic plants and non-transformed controls. Genomic
DNA concentration was quantified by NanoDrop® ND-1000
spectrophotometer and diluted to 50 ng. The DNA from the non-
transformed plants was used as negative control and the plasmid DNA
was used as positive control. Primers for PCR analysis of
overexpression plants and silencing plants were listed in Table 3.4.
PCR reaction mixture (10 µl) was prepared with 50 ng of genomic
DNA, 1X Assay Buffer (containing 1.5 mM MgCl2), 125 µM of dNTPs, 2
µM of each (forward and reverse) primer (Table 3.4) and 1 unit of Taq
DNA polymerase (Bangalore Genei, India) and amplified on a Thermal
cycler (Bio-Rad, USA). The PCR profile condition was 950C for 5 min +
35 cycles of (950C for 30 sec + 580C for 30 sec + 720C for 1 min) +
720C for 7 min. The amplicons were electrophoresed in a 1 % agarose
gel prestained with ethidium bromide in 1X TAE (40 mM Tris-acetate
and 2 mM Na2EDTA.2H2O pH ~8.5) buffer as per Sambrook and
Russell (2001). The electrophoresed products were visualized under
UV light and documented using Alpha lmager Documentation System
(M/s Alpha Innotech, USA).
3.2.4.3 Southern blot analysis
Genomic DNA from the transformed and non-transformed
control plants was used for Southern blot analysis. To check the
integrity of genomic DNA 2 µl of genomic DNA was electrophoresed on
0.8% TAE gel and was observed under UV-trans illuminator and
concentration was measured in NanoDrop® ND-1000
spectrophotometer. The genomic DNA (10 µg) of overexpression
putative transgenic plants was digested with 20 units of BamHI along
with non-transformed control. The reaction set up was as follows: 10
µg DNA, 20 units of enzyme, 1X buffer, 1X BSA in a total volume of 50
µl and kept digestion at 370C water bath for 12-16 h. Before stopping
the enzyme activity, 3-5 µl of restricted samples were pre-run in 0.8%
TAE gel for complete or incomplete digestion. In case of incomplete
digestion, additional 20 units of enzyme to the sample was added and
incubated for 1-2 h at 370C. The enzyme activity was denatured at
650C for 15 min and the digested DNA was loaded on 0.8% agarose gel
and electrophoresed over night at 16 volts and the gel was
documented. For left border junction fragment analysis of
overexpression plant, genomic DNA was digested with KpnI enzyme of
20 units per reaction.
Similarly, for Southern blot analysis of plants with silencing
gene, the genomic DNA was restriction digested with two enzymes
combination viz., KpnI + SacI to release 1.9 kb fragment from the
inserted T-DNA. About 10 µg of genomic DNA was digested with 20
units of each enzyme in presence of 1X buffer and 1X BSA in a total
volume of 50 µl and incubated overnight at 37°C. The agarose gel was
electrophoresed at 16V for 16-18h.
3.2.4.3.1 DNA blotting to membrane
The gel removed from gel tray was placed in a glass tray and
treated for 15 min in depurination solution containing 0.25N HCl. The
gel was rinsed with sterile double distilled water for twice and soaked
with denaturation solution containing 0.4N NaOH for 20 min with
constant and gentle agitation on a rotary platform at 30 rpm. The
treated gel was ready to transfer by vacuum blot apparatus (Hoefer
Scientific, USA). A window was cut using polythene sheets to exactly
fit the gel area of transfer (not the complete gel size). One sheet of
Whatman No. 3 filter paper and Hybond N+ blotting membrane (GE
healthcare Ltd., UK) was cut according to the gel area of transfer. The
nylon membrane was soaked in sterile distilled water and placed over
the Whatman sheet. The window was replaced and the safety clamps
were fixed. The gel was carefully placed over the window where the gel
carrying DNA was to be transferred. A vacuum pump was connected
through a liquid trap and operated at 35 kPa. The alkali transfer
solution (0.4N NaOH) was poured over the gel to cover the gel
completely and the transfer was carried out for 20 min. The vacuum
was then released and the membrane was washed briefly with 2X
SSC. The 2x SSC was prepared from the stock solution 20X SSC
(175.3 g NaCl + 88.23 g Tri sodium citrate in 1000 ml distilled water).
DNA was cross linked to the membrane in a UV cross linker at 1200
µJ (Hoefer® Inc., USA) and the membrane was dried at 650C for 2 h
and was stored at 4°C in a air tight polythene bag.
3.2.4.3.2 Pre – hybridization and Hybridization
The membrane was transferred either to a hybridization bottle
or a plastic trough with 50 ml of pre hybridization buffer containing
0.5 M sodium phosphate buffer pH 7.2, 1mM EDTA pH 7.0, 7% w/v
SDS, 100 µg/ml denatured salmon sperm DNA and 100 µg/ml Bovine
albumin serum. The bottle/trough was kept in the hybridization oven
at 650C for 6 h. After 6 h of pre-incubation, 25 ml of freshly prepared
hybridization solution was added to the bottle/trough.
3.2.4.3.3 Radio labeling the Probe DNA
The CK31-Ox, hpt or CK31-si coding sequences were used for
preparation of radio labeled probe. Radio labeling was done using the
ready to go DNA labeling beads (GE healthcare Ltd., UK). The final
reaction volume of 50 µl buffer contained, dATP, dGTP, dTTP, klenow
fragment (12 units). The probe DNA (50-100 ng) was denatured by
keeping in boiling water for 10 min and quick chilled on ice. The probe
DNA was added to the labeling beads and the volume was made up to
45 µl. To this mixture, 5 µl (50 µCi) of α-32P dCTP was added and
mixed thoroughly. The reaction mixture was left undisturbed at 370C
for 45 min. The reaction was stopped by keeping the tube in boiling
water for 5 min. Radio labeled probe was added to the hybridization
buffer and the hybridization bottle/trough was placed back in the
hybridization chamber. After 16-20h of hybridization, the membrane
was washed in 50 ml of wash solution-I containing 2X SSC and 0.1 %
SDS at 650C. After 20 min wash, solution-I was replaced with wash
solution-II containing 1X SSC and 0.1% SDS at 650C for 20 min.
Finally, the membrane was washed at 650C for 20 min with wash
solution-III containing 0.1X SSC and 0.1% SDS. At each stage
radioactivity was measured by Geiger counter. Later, the membrane
was blotted on a Whatman no. 3 filter paper and covered with saran
wrap. The membrane was then placed on X-Ray cassette and exposed
to X-Ray film for 24 to 48 h at -700C.
3.2.5 Gus histochemcial studies on transgenic plants
Histochemical assay of gusA gene expression in overexpression
plants was performed according to the method of Jefferson et al.
(1987), Matured leaves of Southern confirmed transgenic plants were
placed in Phosphate buffer containing 1% Triton X-100 and incubated
for 1 h in a 370C incubator. The explants were then transferred to X-
Gluc (5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide; Biosynth AG,
Staad, Switzerland) staining solution containing 50 mM Sodium
Phosphate buffer (pH 7.0), 1 mM EDTA (pH 7.0), 1 mM Potassium
ferricyanide, 1 mM Potassium ferrocyanide, 1mM X-Gluc. The
explants were incubated at 370C in an incubator for 16-24 h.
Chlorophyll clearing was done with acetone: methanol (1:3) solutions.
3.2.6 Inheritance studies
Seeds harvested from the primary transgenic plants (T0) were used for
raising T1 generation plants. All the confimed transgenic events were
advanced to subsequent generations (T1, T2, T3 etc.) by selfing. Seeds
from selected T2 transgenic lines of CK31-Ox and CK31-Si and non-
transformed plants were surface sterilized with 70% ethanol and 0.1%
HgCl2 solution. The sterilized seeds were inoculated in hormone free ½
MS basal medium with 50 mg/l hygromycin. The plates were
incubated under the light for 15-20 days. Number and percentage of
germinating seedlings resistance/sensitive to hygromycin seedlings
were scored.
3.2.7 Expression studies of OsCPK31 gene in transgenic rice plants
Expression of rice endogenous gene ‘OsCPK31’ in wild plant
(non-transformed control plant) of Taipei 309 and BPT 5204 were
studied with different tissues viz., leaf, root, stem, panicle and seed (0-
29 Days after pollination (DAP). The different stage of flower
development after pollination was designated as S1 (0-2 days), S2 (3-4
days), S3 (5-10 days), S4 (11-20 days) and S5 (21-29 days)
respectively. Similarly expression study of OsCPK31 gene in leaves
and flowers (S1 to S5 stage) from transgenic over expression plants
and transgenic silencing plants (S4 and S5 stages only) were also
carried out.
3.2.7.1 RNA isolation
Total RNA from leaf was isolated by Trizol® (In Vitrogen)
according to manufacturer’s instructions while RNA isolation protocol
from starchy grain was followed according to Singh et al (2003). About
100 mg of each tissue were used for isolation and further the RNA
from all tissues was treated with DNase I (Qiagen) and purified the
total RNA by RNeasy minelute kit (Qiagen). The integrity of RNA was
checked in 1.2% formaldehyde-agarose gel (Sambrook and Russel
2001). RNA was quantified by Nanodrop® ND-1000
spectrophotometer. RNA samples with 260/280 ratio between 1.9 and
2.0 and 260/230 ratio on or above 2.0 were used for rt-PCR analysis.
3.2.7.2 First strand cDNA synthesis
Three µg of total RNA was taken for first strand cDNA synthesis
using oligo d(T) primers (ImProm-II Reverse Transcription System,
Promega Corp., USA) according to manufacturer’s instructions.
Initially the RNA was denatured at 65°C for 5 min and quick chilled
on ice for 5 min followed by a short spin to condensate and maintain
the original volume. The cDNA was synthesized in 50 µl reaction
volume containing 4-6 µl RNA, 0.5 µg Oligo d(T) primers, 3 mM MgCl2,
0.4 mM each dNTPs, 0.8 units of rRNasin® (RNase inhibitor) and 1
unit of Reverse Transcriptase in 1X ImProm-II reaction buffer and
incubated at 25ºC for 5 min to allow the primer to anneal in a thermal
cycler followed by 42ºC for 1 hour for reverse transcription. The
reverse transcriptase was denatured at 70°C for 15 min before
proceeding to PCR amplification.
3.2.7.3 Northern Dot blot hybridization and reverse transcription PCR
About 20 µg of total RNA from leaf and flowers of non-
transformed BPT 5204 were taken for Northern Dot blot. Each sample
was mixed with denaturants such as 10XMOPS : 5µl, Formaldehyde :
5µl, Formamide : 15µl respectively. Samples were denatured at 65ºC
for 15 min and immediately placed on ice and then 20µl of ice cold
20XSSC was added. A nylon membrane was placed in 10XSSC
solution for 10 min and dried on a Whatman 3mm filter paper a for
few min. The samples were placed on the membrane one by one and
then the membrane was UV-cross linked. The membrane was
transferred to hybridization bottle and poured pre-hybridization
solution and incubated for 16 h at 42ºC in hybridization oven. The
constituents of pre-hyb and hyb solutions were the same as used for
Southern blot analysis. After 16 h pre-hybridization 15 ml fresh
hybridization solution with 10% Dextran sulphate was replaced. To
the hyb-solution, 50 µl of denatured probe solution was added and
hybridized for 24 h at same temperature. Instead of dCTP, 50 µCi of α-
p32 dATP was used. Preparation of probe DNA, washing and auto
radiogram procedure was same as followed for Southern hybridization.
The single strand cDNA was quantified in NanoDrop®
spectrophotometer and analyzed with gene specific primer of OsCPK31
gene using standard PCR conditions as mentioned above. OsActin1
primer was used as internal control for normalizing the equal amount
of cDNA used.
3.2.8 Phenotypic studies of transgenic plants
3.2.8.1 Pollen viability test
After panicle initiation, the mature anthers of transformed and
non-transformed control plants were used for pollen viability test. The
anthers were dehisced from florets and macerated with a few drops of
2% I2-KI solution to observe the fertile and sterile pollens under a
compound microscope.
3.2.8.2 Microscopic observation of rice seed development
The rice spikelets emerged from panicle were tagged and
collected from first day after pollination (DAP) to seed maturity i.e., 0-
30 days at regular interval of 5 days. Developmental stages from
flowering to seed were observed under the stereomicroscope in
transgenic plants with OsCPK31 (overexpression) and non-
transformed control plants.
3.2.8.3 Floral characteritics of transformed and non-transformed plants
During reproductive stage of both transformed and non-
transformed control plants, the floral characters such as number of
panicles per plant, number of filled grain/panicle, total
spikelet/panicle, sterility percentage, days to grain filling and average
maturity days were recorded at T3 generation. All the recorded data
were validated using student t-test for statistical difference for each
phenotype characters between control and transgenic plants.
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