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.