4. RESULTS

During post genomics era, identification and characterization of

genes is considered inevitable to make complete use of genome

sequences and to efficiently manipulate/exploit them for overall

improvement of rice crop. To validate the precise function of the

identified genes, various genetic engineering approaches have become

pivotal. Keeping this in view, an attempt was made to identify

functional aspects of a newly identified rice CDPK gene OsCPK31,

using transgenic approach. The OsCPK31 was used for transformation

of two rice genotypes, Taipei 309 (japonica) and BPT 5204 (indica)

following Agrobacterium-mediated genetic transformation. Further,

elaborate expression studies were carried out to validate gene function

in transgenic plants.

4.1 Plasmid confirmation and vector mobilization into

Agrobacterium strains

4.1.1 Overexpression vector CK31-Ox

The overexpression binary vector (pB4NU-CK31-Ox) was first

confirmed for the presence of OsCPK31 gene by restriction digestion

and PCR analysis. Upon digestion with BamHI, a 3.3 kb fragment was

released (Fig 4.1), while 3.3 kb PCR amplicon was amplified with gene

specific primer pairs for the plasmid CK31-Ox (Fig 4.2). The confirmed

plasmid was mobilized into Agrobacterium strains LBA 4404 and EHA

105 by triparental mating method; six colonies were selected randomly

for colony PCR using promoter (forward) + gene specific (reverse)

primer pairs. About 606 bp amplicon was amplified in all the six

colonies while no amplification was observed in negative control (wild

strain) (Fig 4.3). Plasmid was isolated from the positive single

Agrobacterium colony. By heat shock method the isolated binary

plasmid was transformed into E.coli, then the plasmid was isolated

from E.coli and restriction digestion was made with BamHI, XhoI and

KpnI enzymes. Fragments of 3.3 kb (BamHI), 1.1 kb, 6 kb, vector

backbone (XhoI) and linearized fragment of length 17.4 kb (KpnI)

respectively were released (Fig 4.4). The confirmed vector pB4NU-

CK31-Ox vector was used for rice transformation (Fig 4.5).

4.1.2 Silencing vector CK31-Si Restriction digestion of CK31-Si with double enzymes

combination (KpnI + SacI), released ~1.9 kb fragment containing gene

of interest + gus linker sequence (Fig 4.6). Similarly, the CK31-Si

plasmid confirmed by PCR with gene specific primer pairs showed an

amplified product of 334 bp from the sequence of OsCPK31 gene (Fig

4.7). The confirmed plasmid was mobilized into Agrobacterium strains

and randomly six colonies were selected for colony PCR analysis using

gus (forward) + si (reverse) primer pairs combination. PCR product of

approximately 1.3 kb was amplified in all six colonies except negative

control (Fig 4.8). A positive Agrobacterium colony was selected and

plasmid was isolated.

The isolated plasmid was then transformed into E.Coli and once

again plasmid was isolatedfrom E. coli. The plasmid after restriction

digestion with KpnI + SacI the plasmid released 1.9 kb (gus linker +

gene) and vector backbone, while KpnI digested plasmid released a

single linearized fragment (>20 kb) of CK31-Si vector (Fig 4.9). The

confirmed Agrobacterium harbouring binary vector was used for

transformation (Fig 4.10).

4.2 Optimization of callus induction and regeneration for indica

and japonica genotypes

Success of any transformation experiment depends mostly on

reliable and repeatable protocol for callus induction and regeneration

of the fertile plants from transformed cells. Of the several tissue

culture media combinations available, the MS medium (Murashige

and Skoog, 1962) was a widely adopted medium for generation of

embryogenic calli and plant regeneration from different rice genotypes.

The present experiment was carried out to optimize MS medium with

different composition of carbon sources and phytohormones which

could affect the culture response in the selected two genotypes.

4.2.1 Effect of carbon sources on callus induction Influence of maltose and sucrose (30 g/l each) as a carbon

source in MS basal medium with 2, 4-D (2 mg/l) on callus induction

from mature seeds of BPT 5204 and Taipei 309 (T-309) genotypes was

assessed. Among the two carbon sources, maltose promoted high

frequency of callus induction compared to sucrose (Table 4.1).

Addition of maltose promoted callus induction from 48 to 56 % for

BPT 5204 and 52 to 63 % for Taipei 309 respectively (Fig 4.11).

Embryogenic calli was very prominent in MS medium containing

maltose than sucrose as carbon source.

Table 4.1 Effect of different carbon sources on callus induction for two rice genotypes.

*Mean of six replications

4.2.2 Effect of phytohormones on callus induction

Mature seeds of BPT 5204 and Taipei 309 were cultured on MS

basal medium supplemented with auxins and cytokinins to assess the

effect of phytohormones on callus induction. The genotypes BPT 5204

and Taipei 309 responded differently to growth regulators with respect

to callus induction frequency (Table 4.2, Fig 4.12). Callus induction

medium (CIM) with different combination of 2, 4-D and kinetin or BAP

were tested. Among the three media, CIM3 (2.0 mg/l 2,4-D and 0.5

mg/l kinetin) showed highest callus induction percentage with

embryogenic calli in both genotypes viz., BPT 5204 (77%) and Taipei

309 (82%) respectively than the other two media combinations.

Carbon

source

Mean callus induction %

BPT 5204* Taipei 309*

Sucrose 48.33±1.61 52.00±1.46

Maltose 56.33±1.50 63.33±1.91

CV (%) 7.12 9 LSD (5%) 5.26 7.7

Fig 4.11: Effect of carbon source on callus induction medium.

Fig 4.12: Effect of phytohormones on callus induction medium. A: Seeds of BPT 5204 on CIM3; B: Embryogenic callus from BPT 5204 CIM 1: 2.4-D (2 mg/l); CIM 2: 2,4-D (2 mg/l) + BAP (0.5 mg/l); CIM 3: 2,4-D (2 mg/l) + Kinetin (0.5 mg/l)

A

B

BPT 5204 BPT 5204 BPT 5204

Taipei 309 Taipei 309 Taipei 309

a

b

Fig 4.13: Effect of growth regulators on regeneration of indica and japonica genotypes. (a): Plants regenerated from BPT 5204 and (b): Taipei 309 RM 1: Kinetin (2 mg/l) + NAA (0.5 mg/l); RM 2: BAP (2.0 mg/l ) + NAA (0.5 mg/l) RM 3: Kinetin (2 mg/l) + BAP (1.0 mg/l) + NAA (0.5 mg/l)

Fig 4.14: Frequency of plant regeneration in BPT 5204 and Taipei 309 from three different media.

Table 4.2 Effect of different phytohormone combinations on callus induction for two genotypes.

*mean of six replications

4.2.3 Effect of growth regulators on plant regeneration

Embryogenic calli obtained from callus induction medium

(CIM3) were transferred to three different regeneration media (RM).

Regeneration frequency was determined in terms of number of green

spots and shoots. The growth regulator NAA concentration (0.5 mg/l)

was common in all the three media tested where, RM1 was

supplemented with Kinetin (2 mg/l), RM2 with BAP (2 mg/l) and RM3

with Kinetin (2 mg/l) and BAP (1 mg/l) (Table 4.3) (Fig 4.13). Among

the two genotypes, Taipei 309 showed highest regeneration frequency

of 60 % whereas BPT 5204 recorded 48 % (Fig 4.14). Since RM2

produced multiple shoots, RM1 was selected for regeneration of

transgenic plants from transformed callus.

Media

Mean callus induction %

MS with different Phytohormones BPT 5204* Taipei 309*

CIM1 2,4-D (2 mg/l) 42.66±1.33 46±1.71

CIM2 2,4-D (2 mg/l) + BAP (0.5 mg/l) 56±2.31 59.33±1.91

CIM3 2,4-D (2 mg/l) + Kin (0.5 mg/l) 76.66±1.23 82±1.71

CV (%) 7.25 6.48 LSD (5%) 5.45 5.2

Table 4.3: Effect of different phytohormone combinations on regeneration for two genotypes.

Media

Mean Regeneration %

MS with different Phytohormone BPT 5204* Taipei 309*

RM1 Kin (2 mg/l) + NAA (0.5 mg/l) 48.33±1.92a 60.66±1.31a

RM2 BAP (2 mg/l) + NAA (0.5 mg/l) 43.33±1.92ab 55±4.04ab

RM3 Kin (2 mg/l) + BAP (1 mg/l) +

NAA (0.5 mg/l)

39±1.47b 45.33±0.78c

CV (%) 7.18 8.61

LSD (5%) 7.08 10.47

*mean of three replications; numbers followed by similar letters (DMRT notation) are not significantly different at p=0.05.

4.2.4 Effect of antibiotic hygromycin on non-transformed calli of elite genotypes

Twenty one days old rice embryogenic calli were produced on

callus induction medium and subjected to hygromycin antibiotic

sensitivity/kill curve test. A total of 100 calli were cultured in the

medium containing different concentration of hygromycin viz., 0, 25,

50, 75 and 100 mg/l respectively. After two weeks of incubation, total

number of dead calli % were scored (Table 4.4) and kill curve was

plotted in graph (Fig 4.15). At 50 mg/l concentration hygromycin

killed nearly 90% of calli in both genotypes. As >50 mg/l hygromycin

was lethal to transformed calli, 50 mg/l concentration was considered

as optimum level for selection of transformed calli.

Table 4.4: Determination of optimum concentration of hygromycin for selection of transformed calli.

Hygromycin

(mg/l) Dead calli (%)

Taipei 309* BPT 5204* 0 10 ± 1.15 9 ± 0.67 25 72 ± 1.15 64 ± 1.15 50 90 ± 1.14 89 ± 0.67 75 96 ± 0.0 97 ± 1.76 100 100 ± 0.0 98 ± 1.15

*Avg of three replicas

Fig 4.15: Kill curve for optimum concentration of hygromycin B on non-transformed (control) calli of BPT 5204 and Taipei 309 for se lection of transformed calli.

4.2.5 Genetic transformation

4.2.5.1 Transformation of overexpression gene in Taipei 309 and BPT 5204

The scutellum derived 21 days old embryogenic calli from rice

genotypes Taipei 309 and BPT 5204 were co-cultivated with the

Agrobacterium strain LBA 4404 and EHA 105 harbouring pB4NU-

CK31-Ox for introduction of OsCPK31 gene into the rice genome. From

the different transformation experiments, a total of 1662 Taipei 309

and 3291 BPT 5204 embryogenic calli were used for transformation

with CK31-Ox. After 3 cycles of selection in hygromycin containing

medium, the hygromycin resistant calli were transferred to

regeneration medium RM1 containing 25 mg/l hygromycin. In total

267 and 161 putative transformed plants were obtained for Taipei 309

and BPT 5204 respectively. All the plants were maintained in biosafety

transgenic glass house (Fig 4.16, Fig 4.17 & Table 4.5).

Table 4.5: Development of putative rice transformants with pB4NU-

CK31-Ox construct.

Cultivar Gene Agrobacterium strain

calli used for co-cultivation

Plants regenerated

Taipei 309

CK31-Ox

LBA 4404 945 192 EHA 105 717 75

BPT 5204 CK31-Ox

LBA 4404 1939 118 EHA 105 1352 43

Similarly, embryogenic calli from both genotypes were

transformed with Agrobacterium (LBA 4404/EHA 105) harbouring

pANDA-CK31-Si vector. After 3 rounds of selection in hygromycin,

resistant calli were transferred to regeneration medium (RM1) with 25

mg/l hyg. Out of 1780 calli co-cultivated, 163 putative transgenic

Taipei 309 plants were obtained; similarly 144 putative transgenic

BPT 5204 plants were regenerated from 3027 calli co-cultivated (Fig

4.18 & Table 4.6).

Table 4.6: Putative transgenic plants regenerated from two rice genotypes with CK31-Si binary vector.

Cultivar Gene Agrobacterium strain

calli used for co- cultivation

plants regenerated

Taipei 309 CK31-Si LBA 4404 1179 124 EHA 105 601 39

BPT 5204 CK31-Si LBA 4404 1810 72 EHA 105 1217 72

Fig 4.16: Development of putative transgenic plants of Taipei 309 with CK31-Ox. A: Freshly inoculated seeds after 5 days; B: 21 days old embryogenic callus; C; hygromycin resistant calli; D: plantlets regenerated from RM 1; E: plantlets in rooting medium; G: putative transgenic plants in Yoshida’s solution; H: putative plants transferred into pots at biosafety transgenic glass house.

Fig 4.17: Development of putative transgenic plants from CK31-Ox in BPT 5204 genotype. A: Seed inoculation after 10 days; B: seed inoculation after 21 days; C: Embryogenic callus; D: hygromycin resistant calli; E: plantlets regenerated from RM 1; F: plantlets in rooting medium; G: putative transgenic plants in Yoshida’s solution; H: putative plants transferred into pots at biosafety transgenic glass house.

A

C

E

G

B

D

F

H Fig 4.18: Development of putative transgenic plants from CK31-Si in Taipei 309 genotype. A: Seed inoculation after 7 days; B: callus after 18 days; C-D: hygromycin resistant calli E: plantlets regenerated from RM1; F: plantlets in rooting medium; G: putative transgenic plants in Yoshida’s solution; H: putative plants transferred into pots at biosafety transgenic glass house.

4.3 Molecular characterization of transgenic plants 4.3.1 PCR analysis of T0 overexpression plants

Genomic DNA was isolated from putative transgenic rice plants

as well as from the non-transformed control plants. PCR analysis of

all putative over-expressed transgenic rice plants amplified a 606 bp

fragment when amplified with the primer pair containing promoter +

coding sequence of CK31-Ox. Twenty four plants of Taipei 309

obtained via transformation of LBA 4404 and (Fig 4.19) were found to

be positive for PCR. In case of BPT 5204, seven plants from LBA 4404

and thirteen plants from EHA 105 were observed to be PCR positive

(Fig 4.20).

4.3.2 Southern blot analysis of overexpression plants

The genomic DNA was isolated from the PCR positive transgenic

plants obtained from both constructs and genotypes were subjected to

Southern blot analysis for further confirmation of T-DNA integration

into plant genome. The genomic DNA were digested with BamHI and

probed with full length Ox coding sequence. The hybridized blots

confirmed the presence of 3.3 kb (full length) band in 9 plants of

Taipei 309 and 8 plants of BPT 5204 (Fig. 4.21 & Fig. 4.22). Non-

transformed control plants did not show any band. Hereafter, the

Southern confirmed overexpression transgenic plants obtained from

Taipei 309 is designated as TP followed by line number while plants

obtained from BPT 5204 as BP followed by the line number.

M C B 1 2 3 4 5 6 7 8 9 10 11 121314 15 16 17 18 19 20 21 22 23 24 25 26 P

Fig 4.19: PCR analysis of Taipei 309 putative over expression transgenic plants. M: 1 kb ladder; C: non-transformed control; B: blank; 1-26: putative transgenic plants; P: positive control

606 bp

Fig 4.20: PCR analysis of putative overexpression transgenic plants obtained from BPT 5204. M: 1 kb ladder; C: non-transformed control; B: blank; 1-10/1-13: putative transgenic plants obtained from LBA 4404 (A) and EHA 105 (B); P: positive control

4.3.3 Copy number detection in overexpression primary transformants

Genomic DNA of three Taipei 309 (TP5, TP6 and TP13) and seven BPT

5204 (BP1, BP2, BP5, BP7, BP8, BP9 and B12) overexpression

primary transformants were analyzed for copy number detection. The

genomic DNA was digested with KpnI (unique enzyme site) and probed

with the hpt coding sequence to analyze left border junction fragment.

In nine plants a distinct hybridizable band of >2.3 kb was observed in

which seven single copy and two double copy T-DNA integrations in

both Taipei 309 and BPT 5204 were identified (Fig. 4.23). BP1 plant

DNA was degraded hence no band was observed.

4.3.4 Molecular analysis of T0 Silencing transgenic plants

Similarly putative silencing Taipei 309 transformants were

analyzed by PCR with gene specific primer pairs which showed a 334

bp amplicon representing CK31-Si in four plants obtained from LBA

4404 and seven from EHA 105 strain respectively (Fig 4.24). None of

the BPT 5204 plants were confirmed PCR positive.

For Southern confirmation, silencing plants were digested with

KpnI and SacI to release ~ 1.9 kb fragment from the T-DNA region. For

hybridization, PCR amplified product of 334 bp of OsCPK31 gene was

used as probe. A hybridizable band of ~1.9 kb was found in four

plants from LBA 4404 and five plants from EHA 105 in the genome of

Taipei 309 (Fig. 4.25). None of the BPT 5204 silencing plants were

found positive in Southern hybridization. Southern confirmed

silencing plants obtained from Taipei 309 is designated as TS followed

by line number hereafter.

Fig 4.25: Southern blot analysis of Taipei 309 silencing plants. C: control Taipei 309; 1,97,98 and 100 silencing plants obtained from LBA 4404. 2,5,11,15,17 silencing plants obtained from EHA105; P: positive control

Fig 4.24: PCR analysis of putative silencing transgenic plants obtained from Taipei 309. a: plants developed with LBA 4404 (pANDA-CK31-Si) b: plants developed with EHA 105 (pANDA-CK31-Si) control (c); Blank (B); empty lane (E); positive control (P)

a

b

4.3.5 Gus histochemical analysis of overexpression plants

The Southern confirmed overexpressed plants from Taipei 309

and BPT 5204 were tested for gus gene expression with various

tissues viz., leaf, flower, seed and endosperm. The gus gene was

confirmed by the presence of blue foci in all the positive plant tissues

(Fig. 4.26) while no such expression was found in non-transformed

plant tissues. Thus, the T-DNA of overexpression plants was

integrated in the genome without any rearrangement.

Fig 4.26: Gus expression analysis of different tissues of overexpression. T: Transgenic (T0 plants); C: control non-transformed tissue

LEAF

C T

4.3.6 Inheritance pattern of transgenes in T1 and T2 generations

4.3.6.1 Inheritance of overexpression transgenic lines

Out of 17 confirmed primary transgenic plants (T0) with

overexpression gene only four plants of Taipei 309 (TP4, TP5, TP6 and

TP13) and one from BPT 5204 (BP2) were fertile while the other

remaining plants were sterile. All the five fertile plants were advanced

to further generations. Selfed seeds from Southern confirmed T0

transformants were germinated normally on dry soil bed and T1

generation plants were raised. All the T1 seedlings were analyzed by

PCR with gene specific primer pairs. A PCR product of 606 bp was

amplified in nine progenies from four independent Taipei 309

overexpression lines and 9 PCR positive plants from BPT 5204

overexpression line (Table 4.7, Fig. 4.27 & Fig. 4.28).

Table 4.7: PCR analysis of Taipei 309 and BPT 5204 overexpression

transgenic T1 progenies.

Plant # seed sowed seed germinated PCR +ve PCR-ve

TP4 5 2 1 1 TP5 10 3 3 - TP6 11 3 2 1 TP13 15 3 3 - BP2 22 15 9 6

Further, Southern blot analysis of PCR positive T1 progenies

showed 6 Taipei 309 (Fig. 4.29) and 9 BPT 5204 (Fig. 4.30) progenies

were found positive for OsCPK31 gene (Table 4.8). All the Southern

positive plants were healthy at biosafety glass house and set seeds

normally.

Table 4.8: Molecular analysis of T1 progenies of overexpression plants.

4.3.6.2 Generation advancement of T1 plants of overexpression transgenic lines

From the T1 progenies, 6 Taipei 309 and 4 BPT 5204 transgenic

lines were selected for T2 generation advancement. Segregation

analysis of T2 progenies was checked by seeds germinated on ½ MS

basal medium supplemented with 50 mg/l hygromycin antibiotic and

also gus gene expression assay. Thirty seeds (T2) from each selected

Southern positive T1 progenies were inoculated in ½ MS basal

medium with 50 mg/l hygromycin for 2 weeks. All the hygromycin

resistant plants were checked for gus gene expression to check any

deviation in segregation pattern. From the T2 segregating progenies,

two lines of Taipei 309 (TP5-6 and TP6-10) and one line of BPT 5204

(BP2-10) were found to be homozygous for hygromycin resistant gene

(Fig. 4.31 & Fig. 4.33A) and gus gene (Fig. 4.32 & Fig. 4.33B) whereas

the other two Taipei 309 overexpression lines and three BPT 5204

lines segregated in 15:1 (Two copy) and 3:1 (single copy) (Table 4.9).

T1 Plant # PCR Southern T1 Plant # PCR Southern TP4-2 + + BP2-1 + + TP4-5 - - BP2-5 + + TP5-4 + + BP2-7 + + TP5-6 + + BP2-9 + + TP6-4 + + BP2-10 + + TP6-10 + + BP2-11 + + TP13-10 + + BP2-12 + + TP13-12 + - BP2-13 + + BP2-15 + +

The ratio 15:1 in TP4-2 and TP13-10 indicated that these two lines

might have two copy insertions at two different loci of rice

chromosomes. The hygromycin resistant overexpression plants were

further confirmed by PCR (Fig 4.34 & Fig 4.35) and Southern analysis

(Table 4.10, Fig 4.36 & Fig. 4.37). Further, randomly selected lines

from T2 progenies were advanced to T3 generation. About 30 seeds per

line were sown in dry soil bed for 2-3 weeks at biosafety glass house.

The germinated plants were transferred to pots and leaf material was

collected for PCR analysis from one month old plants. Properly grown

plants were tested for transgene presence by PCR analysis. Among

four Taipei 309 T2 lines selected, two homozygous lines (TP5-6-21 and

TP6-10-10) were found to be positive for PCR and no segregation was

observed (Fig. 4.38 A & B). But selected lines viz., TP4-2-21 and TP13-

10-10 segregated in 15:1 ratio (Fig. 4.38 C & D). Homozygous line

BP2-10-10 was found PCR positive and no segregation was found

(Table 4.11 and Fig. 4.39). Phenotype and expression analysis was

studied at T3 generation.

Table 4.9 : Progeny analysis of T2 overexpression transgenic lines. Plant # seed

inoculated

HygR HygS Gus +ve

Ratio Chi value

P-value

TP4-2 30 27 3 27 15:1 0.536 0.464

TP5-4 30 22 8 22 3:1 0.186 0.666

TP5-6 30 30 0 30 1:0 - -

TP6-4 30 21 9 21 3:1 0.745 0.388

TP6-10 30 30 0 30 1:0 - -

TP13-10 30 28 2 28 15:1 0 1

BP2-5 25 16 9 16 3:1 1.974 0.160

BP2-10 25 25 0 25 1:0 0 -

BP2-13 25 22 3 22 3:1 1.974 0.160

BP2-16 25 19 6 19 3:1 0 1.0 R: Resistant; S: sensitive

Table 4.10: Molecular analysis of T2 progenies of overexpression lines.

- Not analyzed.

T2 plant # No. of plants analyzed Molecular result

PCR Southern PCR+ve Southern+ve

TP5-6 25 17 25 17

TP6-10 26 13 24 13

TP4-2 22 - 22 -

TP13-10 24 - 24 -

BP2-10 25 17 25 17

A

Fig 4.31: Germination of T2 seeds with hygromycin (50 mg/l) medium. A. Seeds of the T2 line TP5-6; B: TP6-10

B

Fig 4.32: GUS assay in selected T2 transgenic lines of TP5-6 (A) and TP6-10 (B).

A B

A B

Fig 4.33: Phenotype analysis of T2 seeds/leaves of BP2-10. A: T2 seeds inoculated in ½ MS basal medium with hygromycin (50 mg/l) B: Hygromycin resistant leaves were checked for gus gene expression

Fig 4.34: PCR analysis of transgenic T2 progenies of selected lines of TP5-6 (A); TP6-10 (B); TP4-2 (C); TP13-10 (D).

606 bp

606 bp B

A

C

D

606 bp

606 bp

Fig 4.35: PCR analysis of T2 progenies of BP2-10.

M B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 C P

606 bp

Fig 4.36: Southern blot analysis of T2 progenies of TP5-6 (A) and TP6-10 (B).

M 1 4 6 8 10 11 13 17 1 9 20 21 22 23 C P

3.3 kb

M 1 3 5 6 7 9 10 12 13 15 16 18 19 21 22 23 25 C P

3.3 kb

A

B

Fig 4.37: Southern blot analysis of T2 progenies of BP2-10.

3.3 kb

M B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C P

M B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 C P

M B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C P

M B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C P

Fig 4.38: PCR analysis of T3 progenies of T-309 overexpression lines of TP5-6-21 (A); TP6-10-10 (B); TP4-2-19 (C); TP13-10-10 (D).

606 bp

606 bp

606 bp

606 bp

Fig 4.39: PCR analysis of T3 progenies of BP2-10-10 overexpression lines.

606 bp

B

A

C

D

Table 4.11: Molecular analysis of T3 progenies of overexpression lines.

T3 plant # No. of plants analyzed

Molecular result PCR +ve PCR -ve

TP5-6-21 26 26 0 TP6-10-10 25 25 0 TP4-2-19 26 24 2 TP13-10-10 26 23 3 BP2-10-10 25 25 0

4.3.6.3 Inheritance of transgenic silencing T1 lines

Selfed T1 seeds of nine Southern positive Taipei 309 silencing

lines were collected (Table 4.12). Three independent lines viz., TS1,

TS2 and TS11 were selected for generation advancement, inheritance

study and molecular confirmation of transgene. T1 seeds of the line

TS1 were sowed normally on dry bed soil at biosafety glass house

whereas TS2 and TS11 seeds were kept on ½ MS basal medium with

50 mg/l hygromycin. The germinated seedlings were transferred to

pots and leaf material was collected for transgene confirmation. A

multiplex PCR was used for transgene presence in all the three

silencing progenies. Progenies of TS1 showed 12 positive and two

negative for PCR and Southern blot (Fig. 4.40 a & b). Similarly all the

hygromycin resistant plants were found PCR and Southern positive for

the lines TS2 (Fig. 4.41 a & b) and TS11 (Fig. 4.42 a & b) ). In TS11,

only 13 plants were used for southern blot while the other two plants

were died after transplanting into the pots (Table 4.13).

Table 4.12: T1 seeds collected from silencing primary transformants.

Plant # Total T1 seeds

TS1 22 TS97 27 TS98 29 TS100 21 TS2 33 TS 5 39 TS11 45 TS15 28 TS17 26 TC** 350

** Avg of 4 control plants Table 4.13: Molecular analysis of Silencing lines at T1 generation. Plant #

seed sowed /inoculated

seed germinated

PCR +ve

PCR -ve

plants analyzed

S+ve S-ve

TS1 22 14 12 2 14 12 2

TS2 25 17 hygr 17 - 17 17 0

TS11 20 15 hygr 15 - 13 13 0

Hyg: hygromycin; r: resistant; S: Southern

4.3.6.4 Generation advancement of T1 plants of silencing plants

To study the inheritance pattern of transgene in three

independent silencing transgenic plants, T2 seeds were used. Five

different transgenic T1 lines from TS1, TS2 and TS11 were selected

and observed the inheritance pattern for hygromycin gene. About 30

seeds were inoculated in ½ MS basal medium contained 50 mg/l

hygromycin antibiotic and maintained in tissue culture room at

28±2ºC for two weeks. All the lines were segregated as mendelian

monogenic ratio 3:1 (Table 4.14). The line TS1-2 was found to be

homozygous for hygromycin gene while other four lines were

hemizygous. For further studies, transgenic T2 plants viz., TS1-2, TS2-

13 and TS11-11 were selected. The hygromycin resistant plants from

the selected three T2 plants were transferred into pots and molecular

characterization was done. The hygromycin resistant transgenic T2

plants of TS1-2, TS2-13 and TS11-11 were confirmed by PCR and

Southern analysis (Table 4.15, Fig. 4.43, Fig. 4.44 & Fig. 4.45).

From the Southern confirmed T2 silencing plants, three lines

i.e., TS1-2-24, TS2-13-17 and TS11-11-10 were advanced to T3

generation. About 30 seeds were sowed for germination. Healthy and

fully grown plants were transferred into pots and maintained in

biosafety transgenic glass house. Genomic DNA was isolated from

these plants of silencing progenies and analyzed by PCR (Fig. 4.46).

Except the line TS1-2-24, the other two T3 lines were segregating and

hemizygous condition even at T3 generation (Table 4.16, Fig. 4.46B &

C). The PCR positive T3 progenies were used for phenotype and

expression studies.

Table 4.14: Segregation analysis of T2 progenies of silencing lines.

Plant # Seed inoculated

Hygr Hygs Ratio Chi value

P-value

TS1-2 30 30 0 1:0 - - TS1-3 30 23 7 3:1 0 1 TS1-4 30 20 10 3:1 0.195 0.658 TS1-9 30 22 8 3:1 0.186 0.666 TS1-11 30 22 8 3:1 0.186 0.666 TS2-1 25 20 5 3:1 0.219 0.639 TS2-4 25 22 3 3:1 1.974 0.16 TS2-9 25 18 7 3:1 0.219 0.639 TS2-12 25 21 4 3:1 0.877 0.349 TS2-13 25 19 6 3:1 0 1 TS11-1 25 22 3 3:1 1.974 0.16 TS11-3 25 21 4 3:1 0.877 0.349 TS11-7 25 18 7 3:1 0.219 0.639 TS11-8 25 20 5 3:1 0.219 0.639 TS11-11 25 19 6 3:1 0 1

Table 4.15: Molecular analysis of T2 progenies of silencing lines.

Table 4.16: PCR analysis of T3 progenies of silencing lines.

Plant # Seed sowed

Seedling germinated

plant analyzed PCR +ve PCR-ve

TS1-2-24 30 23 23 23 0 TS2-13-17 30 24 24 16 8 TS11-11-10 30 23 23 17 6

T2 plant # No. of plants analyzed Molecular result

PCR Southern PCR+ve Southern+ve

TS1-2 24 17 24 17 TS2-13 19 19 19 19

TS11-11 19 19 19 19

1.9 kb

1.9 kb

1.9 kb

a

b

a

b

a

b

Fig 4.42: Molecular confirmation of T1 progenies of silencing line TS11. a: multiplex PCR analysis; b: Southern blot analysis

Fig 4.41:Molecular confirmation of T1 progenies of silencing line TS2. a: multiplex PCR analysis; b: Southern blot analysis

Fig 4.40: Molecular confirmation of T1 progenies of silencing line TS1. a: multiplex PCR analysis; b: Southern blot analysis

Fig 4.43: Phenotype and molecular confirmation of T2 progenies of silencing line TS1-2. a: T2 seeds inoculated in ½ MS basal medium with hygromycin (50 mg/l) b: PCR confirmation of hygromycin resistant plants with Gus gene forward and Si- reverse primers; c: Southern blot confirmation

TS1-2 a

b

c

1.3 kb

1.9 kb

TS2-13

Fig 4.44: Phenotype and molecular confirmation of T2 progenies of silencing line TS2-13. a: T2 seeds inoculated in ½ MS basal medium with hygromycin (50 mg/l) b: PCR analysis. Primer combination gus forward + Si- reverse primer pairs c: Southern blot confirmation

a

b

c

1.3 kb

1.9 kb

TS11-11

Fig 4.45: Phenotype and molecular confirmation of T2 progenies of silencing line TS11-11. a: T2 seeds inoculated in ½ MS basal medium with hygromycin (50 mg/l) b: PCR analysis with gus gene forward and Si- reverse primers c: Southern blot confirmation

a

b

c

1.3 kb

1.9 kb

Fig 4.46: PCR analysis of T3 progenies of T-309 Silencing lines TS1-2-24 (A); TS2-13-7 (B); TS11-11-10 (C).

B

A

C

4.4 Phenotypic expression study of OsCPK31 in transgenic plants

Molecular confirmed T3 progenies of overexpression and

silencing plants were allowed to maturity. Phenotypic expression of

the T3 progeny plants of OsCPK31 transgenic plants were studied.

4.4.1 Expression of OsCPK31 in transgenic and control plants 4.4.1.1 Endogenous gene OsCPK31 expression on different tissues

of Taipei 309 and BPT 5204 Comparison of microarray and Q-PCR data of OsCPK31 gene

with the vegetative (three stages) and reproductive stages (six stages of

panicle and five stages of seed development) clearly indicated that the

expression of OsCPK31 gene in wild type ‘IR64’ rice started at S3 stage

(5-10 DAP) of seed development and further proceeded to S4 (11-20

DAP) and S5 (21-29 DAP). Thus this gene expressed only in the seed

and it did not up-regulated in other vegetative stage or stress

conditions (Fig. 4.47, unpublished data kindly provided by Prof. A.K.

Tyagi, UDSC, New Delhi). Based on the above information, gene

expression and phenotypic study was undertaken to elucidate the

functions of the OsCPK31 gene in transgenic plants.

To determine the endogenous OsCPK31 gene expression, total

RNA was isolated from different tissues such as leaf, root, stem,

panicle and seed (flowers collected from 0-29 DAP) of BPT 5204 and

Taipei 309 control plants. After reverse transcription, the cDNA was

analyzed with gene specific primer pairs. RT-PCR result of different

tissues of both genotypes showed, a 334 bp fragment amplified only in

seed and no amplification was observed in other tissues tested (Fig.

4.48).

02

468

10

1214

LEAF

ROOT

SEEDLING P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5

COLDSALT

DESCCATION

VEGETATIVE PANICLE SEED STRESS

Nor

mal

ised

Mea

n Si

gnal

Inte

nsity

Microarray

1.2

0

0.2

0.4

0.6

0.8

1

1.2

LEAF

ROOT

SEEDLING

P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5COLD

SALT

DESCCATION

VEGETATIVE PANICLE SEED STRESS

Rel

ativ

e T

rans

crip

t lev

el Q-PCR

Fig 4.47: Expression profiling of OsCPK31 gene during reproductive and abiotic stress conditions Microarray (A); Real-time PCR (B). Pictures provided by Prof. A.K. Tyagi, UDSC, New Delhi

A

B

4.4.1.2 Northern dot blot of seed collected at different stages of seed development in BPT 5204 (wild type)

Northern Dot blot hybridization was performed with seedlings of

non-transformed control plants of BPT 5204. The seed developmental

stage was categorized into S1-S5 starting from pollination to maturity.

Dot blot assay clearly indicated that the endogenous gene (OsCPK31)

started its expression gradually from S3 to S5 stage, but no

expression was detected at S1 or S2 stage (Fig. 4.49). Thus, the

endogenous gene OsCPK31 was preferentially expressed only in the

endosperm.

4.4.1.3 RT-PCR analysis of overexpression leaf and seed

Leaf tissues of control (Taipei 309) and overexpression

transgenic plants of BP2, TP5, TP6, TP4 and TP13 were selected at T3

generation for analyzing transgene expression with gene specific

primer pairs. Transgene was expressed in all the overexpression

leaves whereas no expression was found in control leaf (Fig. 4.50).

Similarly, seeds of different developmental stage of overexpression

plants and control plants (S1 to S5) were subjected to RT-PCR

analysis. Three overexpression plants of T3 generation viz., BP2 (single

copy), TP6 (single copy) and TP4 (two copies) were considered for RT-

PCR analysis for transgene expression. In the control tissues, the

endogenous gene was expressed in S4 and S5 stage whereas no

expression was detected in S1-S3 stage. The transgene (150 bp)

fragment was ubiquitously overexpressed in all the three selected

overexpression lines (Fig. 4.51). OsActin1 gene was used as an

internal control to check equal amount of cDNA from the same

samples analyzed for RT-PCR.

4.4.1.4 RT-PCR analysis of silencing leaf and seed To examine the transgene expression of OsCPK31 in silencing

lines, RT-PCR analysis was carried out with leaf tissues of control and

three silencing lines viz., TS1, TS2 and TS11 at T3 generation. The

result showed that the transgene expressed in leaves of all the three

transgenic silencing lines whereas no expression was found in leaves

of control plants (Fig. 4.52). To check the OsCPK31 gene suppression

or silencing at the seed developmental stage, leaf samples from S4 and

S5 stage was taken for RT-PCR analysis since S1 to S3 stage the

endogenous gene expression was undetected in the control tissues. In

all the three silencing lines, on an average of 50% low expression of

the endogenous gene in both stages was observed as compared to

control (Fig. 4.53).

Fig 4.48: Tissue specific expression of endogenous OsCPK31 gene in wild-type plants. Taipei 309 (A); BPT 5204 (B). A 334 bp fragment was amplified in seed (0-29) DAP but not in other tissues. OsActin1 - internal control

Fig 4.49: Northern dot blot analysis of endogenous OsCPK31 gene at different stages of seed in wild-typeBPT 5204 plants. Upper panel: Autoradiogram of Northern dot blot. The gene OsCPK31 expressed from S3 to S5 stage. No expression in leaf. Lower panel: Formaldehyde-agarose gel electrophoresis

A

B

Fig 4.50: RT-PCR analysis of transgene OsCPK31 in leaves of overexpression transgenic plants. BP2- BPT 5204 over expression, TP5,6,4 and 13:Taipei 309 over expression; B: Blank; P: positive control; OsActin1 - internal control

Fig 4.51: RT-PCR analysis of transgene OsCPK31 in different stages of seed in overexpression plants. C: control BPT 5204; BP2- BPT 5204 over expression, TP4 and TP5:Taipei 309 over expression; B: Blank; P: positive control; OsActin1 - internal control

Fig 4.53: Transcript accumulation of endogenous OsCPK31 gene in seed of silencing transgenic plants. Suppression of endogenous OsCPK31 gene in silencing lines (TS1, TS2 and TS11) than control tissues. OsActin1- internal control

(185 bp)

Fig 4.52: RT-PCR analysis of OsCPK31 transgene expression in silencing plants. Transgene expressed in the selected silencing lines TS1, TS2 and TS11. B: Blank; P: Positive control. OsActin1: internal control

4.4.2 Phenotype observation of overexpression and silencing

transgenic plants

4.4.2.1 Pollen viability test

During reproductive stage of rice plants, before anthesis,

matured anthers were collected from overexpression and non-

transformed control plants. To check pollen viability, a few drops of

2% I2-KI solution was added. The fertile pollens were stained as dark

blue while sterile pollens did not get staining and appeared empty.

Five overexpression T0 transformants (four Taipei 309 and one BPT

5204) were tested. Number of fertile pollen grains was less compared

to its control non-transformed in both genotypes whereas no fertile

pollen was observed in other Southern confirmed overexpression

transformants (data not shown). Pollen test was conducted in the

subsequent generation also (T1, T2 and T3). No difference in the

number of fertile pollen grains was observed. Pollen test of

representative T0 and T3 overexpression plants is presented in Fig 4.54

and Fig 4.55. Similarly, pollen fertility was tested on silencing plants

at different generations viz., T0, T1, T2 and T3 plants. The number of

viable pollen grains was almost equal with its non-transformed control

(Fig. 4.56).

Fig 4.54: Pollen test of over expression T-309 transgenic plants at T0 and T3 generation. At T0, the number of pollen grains were less compared to control whereas in the T3 generation, number of pollen grains were equal with its non-transformed control.

Fig 4.55: Pollen test of over expression BP2 transgenic plants at T0 and T3 generation. At T0, the number of pollen grains were less compared to control whereas in the T3 generation, number of pollen grains were equal with its non-transformed control.

Fig 4.56: Pollen test of Silencing transgenic plants at T0 and T3 generation. At T0, the number of pollen grains were less compared to control whereas in the T3 generation, number of pollen grains were equal with its non-transformed control.

4.4.2.2 Microscopy study of seed development after pollination in

overexpression plants

Based on the Northern Dot-blot analysis of endogenous

OsCPK31 gene in control plants, the grain development changes in

overexpression plants were examined from S3 to S5 stage. Microscopic

observation of spikelets were collected at regular intervals viz., 5, 11,

15, 20, 22 and 25 days after pollination (DAP). It was observed that in

overexpression plants milky stage was started early at 5 DAP in both

genotypes whereas in control the spikelet was in milky stage at 10

DAP. It was also observed that in transgenic overexpression plants

(TP5, TP13 and BP2) spikelets reached hard dough stage at 15 DAP

while the spikelets in control plants were in milky stage and embryos

could be excised at the same stage (Fig. 4.57 and Fig. 4.58). The entire

developmental process (0-29 DAP) was appeared to be fast in

overexpression plants. Hence the seeds matured within 22 days in the

transgenic line, TP5 (Fig. 4.59) and within 20 days in the line , BP2

(Fig. 4.60), but in control plants the entire process took 29-30 DAP.

However, no such differences were observed in all the silencing lines

which also took 29 -30 days to maturity.

Fig 4.57: Comparison of seed development at 15 DAP of Taipei 309 overexpression spikelets. The overexpression seed attained dough stage but in control the seed was in milky stage. Black arrow represent an embryo was excised from the control seed. Number indicated: image magnified

Control BP2 over expression

Fig 4.58: Comparison of seed development at 15 DAP of BPT 5204 overexpression spikelets. A-B: Control spikelet collected at 15 DAP. Red arrow: excised embryo from the corresponding spikelet. C-D: BPT 5204 overexpression spikelet collected at 15 DAP. The overexpression seed was in dough stage. Black arrow: embryo E: Transverse cut of same spikelet. Number indicated image magnified

Fig 4.59: Microscopic observation of seed development stages (S3 to S5) of control (NT) and overexpression plants (TP5). At 5 days after pollination (DAP), the endosperm was in milky stage in transgenic spikelet but in control the milky stage at 11 DAP. Red arrow: Embryo excised from control at 11 DAP. The spikelet of transgenic plant was in early dough stage at 11 DAP. At 22 DAP, the control endosperm was in hard dough stage while transgenic spikelet was matured. Black arrow represent rice seed after remove the palea and lemma. But in control, seed matured at 28 DAP.

Fig 4.60: Microscopic observation of seed development stages (S3 to S5) of control (NT) and overexpression plants (BP2). At 5 days after pollination (DAP), the endosperm was in milky stage in transgenic spikelet but in control the milky stage at 11 DAP. The spikelet of transgenic plant was in early dough stage at 11 DAP. At 20DAP, the transgenic spikelet was completely matured but the control endosperm was in soft dough stage But in control, seed matured at 29 DAP.

4.4.2.3 Floral characteristics of transgenic overexpression and silencing plants

To study the floral characteristics, five independent

overexpression plants were forwarded to T3 generation and their

significance was statistically analyzed by student t-test (Table 4.17).

There was no significant difference in panicle number and total

spikelets/panicle in all the transgenic overexpression lines as

compared to its respective control plants. However, the trait filled

grain/panicle was significantly reduced in all T3 progenies of

overexpression lines. Sterility percentage was observed to be moderate

in both the genotypes. Apart from the yield parameters, days to grain

filling and average maturity days of the plants were also observed. As

mentioned in the materials and methods, panicles were tagged after

they emerged from the leaf sheath and studied the difference in grain

filling days. Majority of the overexpression panicles were matured

within 19-21 days compared to the control (29-30 days). There was a

decrease of 9 – 11 days in duration of grain filling in overexpression

plants. Because of the decrease in grain filling days in overexpression

plants, the overall crop duration was also decreased i.e. 124 to 126

days in Taipei overexpression lines and 135 days in BPT

overexpression line. But the Taipei 309 control plants matured in 135

days and the control BPT 5204 plants matured in 147 days. These

two parameters were highly significant in overexpression plants than

its control (Fig 4.61 & 4.62).

Fig 4.61: Floral parameters of OsCPK31 overexpression transgenic plants of Taipei 309 at T3 generation. All the data were recorded for individual T3 progenies and calculated mean values. The mean values were tested for significant by student t-test. Bar represents mean ± SE. NT- non-transformed control; P value = 5% (*), 1% (**) levels; non-significant (NS)

Fig 4.62: Floral parameters of OsCPK31 overexpression transgenic plants of BPT 5204 at T3 generation. All the data were recorded for individual T3 progenies and calculated mean values. The mean values were tested for significant by student t-test. Bar represents mean ± SE. NT- non-transformed control; Significant effect of P value = 5% (*), 1% (**), non-significant (NS)

Table 4.17: Floral parameters of overexpression transgenic plants at T3 generation.

Plant # No. of panicles

Filled grain/ panicle

Total spikelet/ panicle

Sterility %

Days to grain filling

Avg maturity days

NT (T-309) 7±0.8 87±8.7 100±9.2 14±2.0 30±0.1 135±1.1

TP5-6-21 6±1.1 73±7.9 108±7.8 29±4.4 21±0.3 126±0.7

TP6-10-10 5±0.6 61±6.3 92±7.2 33±4.4 21±0.3 125±0.5

TP4-2-19 5±0.8 64±5.5 101±6.3 38±1.9 21±0.3 124±0.7

TP13-10-10 5±0.6 69±3.3 103±4.8 32±1.8 21±0.3 125±0.6 NT (BPT 5204) 5±0.7 157±12.8 234±12.2 33±3.9 29±0.3 147±0.5

BP2-10-10 6±1.1 92±10.7 180±10.7 50±4.9 19±0.2 135±1.0 Each value represents the mean value ± SE (n= 26,25,24,25,25) for overexpression and its non-transformed control plants (NT)

Similarly, phenotype observations were made for T3 transgenic lines

with silencing gene. Five of the six yield traits of silencing lines were

found to be significantly reduced than its corresponding control.

Panicle number, filled grain/panicle and total spikelets/panicle were

significantly reduced (Fig. 4.63 & Fig. 4.64). Over all, the sterility %

was observed to be very high in silencing lines (52 – 64%) than its

control (14%) which correlates the suppression of endogenous

OsCPK31 gene in selected silencing lines which in turn impaired the

grain filling process contributing to reduction in yield. However, days

to grain filling and average maturity days did not show any significant

difference as compared to control plants (Table 4.18).

Fig 4.63: Panicles after harvested from control (NT) and transgenic silencing lines of Taipei 309 at T3 generation. Lower panel: seeds collected from individual plants of control, TS1, TS2 and TS11

C Fig 4.64: Floral parameters of s ilencing transgenic plants of Taipei 309 at T3 generation. All the data were recorded for individual T3 progenies and calculated mean values. The mean values were tested for significant by student t-test. Bar represents mean ± SE. Significant effect of P value = 5% (*), 1% (**), non significant (NS)

Table 4.18: Floral parameters of T3 transgenic silencing plants Plant No. of

panicles Filled grain/ Panicle

Total spikelet/ panicle

Sterility %

Days to grain filling

Avg maturity days

NT (T-309) 7±0.8 87±8.7 100±9.2 14±2.0 30±0.1 135±1.1

TS1 3±0.4 20±1.3 41±1.9 52±2.3 29±0.1 135±0.5

TS2 4±0.4 11±1.1 29±2.1 64±2.3 29±0.2 136±0.4

TS11 2±0.2 11±2.1 29±3.0 63±5.1 29±0.2 136±0.8

Each value represents the mean value ± SE (n= 23, 16, 17) for pANDA-CK31-Si and its non-transformed control plants (NT) 4.4.2.4 Phenotype comparison of overexpression and silencing

plants of Taipei 309 Trait comparison of overexpression and silencing plants showed

that there was reduction in filled grain/panicle in silencing plants

whereas moderate reduction in overexpression plants. No change in

total spikelets/panicle was observed in overexpression plants whereas

drastic reduction in total spikelets/panicle was observed in silencing

lines. Sterility % was moderate level in overexpression lines but it was

observed to be very high in silencing lines. Besides, overexpression

plants were decreased in days to grain filling and avg. maturity days

but no such differences was observed in silencing plants rather it

matured like control plants (Table 4.19).

Table 4.19: Trait comparison of overexpression and silencing transgenic plants obtained in Taipei 309.

Traits

Over expression Silencing Control

TP5 TP6 TP4 TP13 TS1 TS2 TS11 NT

No. of panicles 6±1.1 4±0.6 5±0.8 4±0.6 3±0.4 4±0.4 2±0.2 7±0.8 Filled grain/ panicle 83±16.9 61±6.3 64±5.5 69±3.3 20±1.3 11±1.1 11±2.1 87±8.7 Total spikelets/ panicle 108±16.8 92±7.2 101±6.3 103±4.8 41±1.9 29±2.1 29±3.0 100±9.2

Sterility % 29±4.4 33±4.4 38±1.9 32±1.8 52±2.3 64±2.3 63±5.1 14±2.0

Days to grain filling 21±0.3 21±0.3 21±0.3 21±0.3 29±0.1 29±0.2 29±0.2 30±0.1

Avg maturity days 126±0.7 125±0.5 124±0.7 125±0.6 135±0.5 136±0.4 136±0.8 135±1.1