Isolation, cloning and expression of CCA1 gene in transgenic progeny plants of Japonica rice exhibiting altered morphological traits

Circadian clock genes holds tremendous potential for breeding crops better adapted to environmental fluctuations inherent to climate change. Endogenous TOC1 promoter and CCA1 gene from rice were isolated, cloned and mobilized into pCAMBIA1300 vectors and RNAi constructs A, B and C. Embryogenic calli of varying ages derived from mature seeds of Taipei 309 were employed for Agrobacterium-mediated genetic transformation generating T0, T1 and T2 independent transgenic lines were analyzed for over-expression and repression of CCA1 gene along with various morphological traits. Six hundred and thirty two T0 transgenic plants were generated from rice calli using constructs A, B and C. T0 progeny plants derived from constructs A, B and C did not show any considerable difference in morphological traits. T1and T2 progeny plants derived from construct A exhibited over-expression of CCA1 gene, on the contrary, progeny plants derived from RNAi constructs B and C exhibited repression of CCA1 gene in qRT-PCR analysis at different time points and showed rhythmicity peaking at dawn (6:00 AM) and lowest expression at 12:00 Noon. T1 and T2 progeny plants derived from construct A, namely, A-17 and A-45 exhibited reduced number of tillers/panicles (6–8), reduced thousand seed weight (10.1–16.6g), decreased seed length (4.98 to 6.58mm), decreased seed width (1.1–1.8mm) as compared to wild type plants. T1 and T2 progeny plants of construct B and C showed increased number of tillers/panicles (8–19), better seed yield (4.98–28.9g), increased thousand seed weight (15.6–29.03g), slightly increased seed length (5.7–7.43mm) and slightly increased seed width (1.7–2.98mm) as compared to wild type plants. Chlorophyll content in T1 and T2 progeny plants did not show any significant difference among the three constructs, however, rhythmicity was observed over the period of time in conjunction to CCA1 gene expression. Evidence has been presented which demonstrates that endogenous repression of CCA1 gene resulted in improved morphological traits: increased number of tillers/panicle, thousand seed weight, seed size; whereas, over-expression leads to diminution in morphological traits: decreased number of tillers/panicle, thousand seed weight, seed size as compared to the wild type in T1 and T2 progeny plants. This is first report of successful regulation of endogenous CCA1 gene under control of TOC1 promoter and its effect on improved growth vigor in Japonica rice.

Introduction Circadian Clock Associated1 (CCA1) and Late Elongated Hypocotyl (LHY) are two important transcription factors that are active and expressed at dawn [1,2] and they are partially redundant MYB transcription factors that are members of the larger Reveille (RVE) gene family, which also contains the major clock activators such as RVE8, RVE6, and RVE4 [3][4][5][6][7].
The present day understanding of circadian clock in Arabidopsis thaliana is based on the model proposed [8] and later reviewed [9]. It has been proposed that TOC1 increases LHY/ CCA1 expression allowing LHY/CCA1 expression to reach a peak at dawn beginning the cycle again [8]. In Arabidopsis the two MYB transcription factors, CCA1 and LHY [9] together with the PRR 5, 7, 9 jointly function as a Morning Element Loop, both mRNA and post translational level peaking at dawn. It interlocks with an Evening Element Loop comprising of Timing Of Cab Expression 1 (TOC1) or PRR1, GIGANTEA (GI). The Evening Element Loop also contains Early Flowering 3 and 4 (EFF3 & ELF4) as well as Lux Arrhythmic (LUX). TOC1 expression oscillates peaking during early evening, opposite to CCA1 and LHY. In particular, the transcription factors CCA1 and LHY, which are mostly produced in the morning, are thought to repress the expression of the gene that codes for another transcription factor, TOC1, which is mostly produced in the evening and, in turn, represses expression of the genes CCA1 and LHY. The cycle starts with light induced LHY/CCA1 expression at dawn, this results in accumulation of LHY/CCA1, which further represses TOC1 expression which in turn results in reduced activation of LHY/CCA1. As the cycle progresses there is a decrease in LHY/CCA1 expression allowing increase in TOC1 transcript levels and reach a maximum at dusk, when LHY/CCA1 are at their lowest. Hayama and Coupland [9] proposed that TOC1 act as activator of LHY/CCA1 expression by interaction with a basic helix-loop-helix protein, namely, Phytochrome-Interacting Factor3 (PIF3), that in turn binds to LHY and CCA1 promoters. Full length cDNA of CCA1, PRR1 and ZTL1 genes isolated from Nipponbare rice variety under the control of CaMv35S promoter were over expressed in transgenic plants of Arabidopsis thaliana by Murakami et al. [10]. It was reported that when the rice clock-associated genes were overexpressed in Arabidopsis thaliana, it leads to significant perturbation of the endogenous circadian rhythms indicating that circadian clock genes were highly conserved between the two species. RVE8 activates expression of evening expressed genes such as TOC1, PRR5, PRR9, GI, LUX, and ELF4 in opposition to CCA1 and LHY [7,11]. The CCA1 has been shown to directly modulate TOC1 and other downstream genes in circadian clock, photosynthesis and starch metabolism as well as for increased biomass and growth vigor in Arabidopsis thaliana [12]. Identification of novel genes against downy mildew disease resistance and its regulation by CCA1 gene in Arabidopsis thaliana was reported [13]. The qRT-PCR of ZmCCA1 gene was found to express in abundance in maize stem and leaves in morning hours under long day and short day with a rhythmic pattern; whereas, TOC1 gene was found to express 10-12h after dawn [14]. Over-expression of ZmCCA1 gene in Arabidopsis thaliana resulted in down regulation of GI, Constans (CO) and Flowering locus T (FT) genes leading to longer hypocotyls and delayed flowering.
Later, Huang et al. [15] have shown that TOC1 does not function as an activator but rather functions as a general repressor of oscillator gene expression and repression occurs through TOC1 rhythmic binding to the circadian oscillator gene promoters. CCA1 over-expressing (CCA1-ox) plants have improved ability to grow in ROS stress-inducing conditions due to the fact that CCA1 regulates genes involved in production, response, and transcriptional regulation of ROS in Arabidopsis has been reported [16]. It has been reported that the CCA1 gene products are involved in light-regulated transcriptional activation of gene expression. Increased metabolic vigor in developing embryos when the maternal copy of CCA1 is repressed in inter-specific hybrids of Arabidopsis was reported [17]. Wheels within wheels: the plant circadian system has been reviewed and documented [18]. It has been reported that the CCA1 gene products are involved in light-regulated transcriptional activation of gene expression. CCA1 genes universally control a number of vital plant activities, including development, growth, and reproduction and key agricultural traits [19]. Rice has become a model system for studying gene expression and regulation [20]. So far there has been no report on studies on regulation of endogenous CCA1 gene in transgenic plants of rice. Keeping this in view, gene constructs pCTCN for over-expression vector (A) and RNAi vectors pCTSaASN and pCTSbASN for repression (B and C) have been generated using standard molecular biology protocols in the plasmid vector pCAMBIA 1300 (8.959kb). Transgenic plants of Japonica rice variety Taipei 309 plants using Agrobacterium tumefaciens, strain EHA 105 harboring the above three constructs have been generated by introducing endogenous CCA1 gene under the control of TOC1 promoter for studying CCA1 gene expression in both up-regulated and down-regulated manner in T0, T1 and T2 progeny plants and the morphological characteristics were analyzed.
Although the CCA1 gene sequence has been amplified from rice genome as template, the restriction enzyme recognition sites (employed for cloning) were killed/knocked out without altering the original amino acid sequence CCA1 protein. The 2.172 kb CCA1 gene from rice Os08g0157600 (original) from NCBI was modified in which codon usage has been optimized for plants; and certain restriction enzyme sites have been killed as shown in S1 Fig. Modified CCA1 gene was got synthesized from GenScript USA Inc., Piscataway, NJ, USA as a cDNA clone in pUC57 vector; the CCA1 gene was excised out using SacI and BamHI and it was cloned into pUC19 having TOC1 promoter and NOS gene described above giving PTCN vector (PUC19:TOC1:CCA1:NOS).
Isolation, cloning of CCA1a and CCA1b gene for construction of RNAi vectors. The CCA1a and CCA1b sense and antisense fragments for making the two RNAi constructs pCTSaASN and pCTSbASN were designed by choosing 400 bp of N terminal region of CCA1a gene and 395 bp of C terminal region of CCA1b gene, respectively as shown in S1 Fig. PCR amplification for CCA1a sense BamHI-XHoI (400bp) using gene specific primers Forward 5'-GCT AGG ATC CTC CTC TGG TGA GGA A-3' and Reverse 5'-TAG CCT CGA GCC ATT TGT GCA GTG C-3' and CCA1a antisense SacI-XmaI (400bp) using gene specific primers Forward 5'-GCT AGA GCT CTC CTC TGG TGA GGA A-3' and Reverse 5'-TAG CCC CGG GCC ATT TGT GCA GTG C-3'. PCR amplification for CCA1b sense BamHI-XHoI (395bp) using gene specific primers Forward 5'-GCT AGG ATC CGA GAA AGA TAT AGA C -3' and Reverse 5'-TAG CCT CGA GGT GCT TGC ACT GCT C-3' and CCA1b antisense SacI-XmaI (395bp) using gene specific primers Forward 5'-GCT AGA GCT CGA GAA AGA TAT AGA C-3' and Reverse 5'-TAG CCC CGG GGT GCT TGC ACT GCT C-3'. The amplifications conditions were same as described for TOC1 promoter using AccuPower PCR premix kit (Bioneer) in a final volume of 25 μl.
The amplified products of CCA1a sense and CCA1a antisense genes were digested with BamHI, XhoI and XmaI, SacI, respectively, and cloned into the intermediate RNAi vector psd20 by kicking out the 4CL gene in sense and antisense orientation linked with a GUS linker. Similarly, the amplified products of CCA1b sense and CCA1b antisense genes were digested with BamHI, XhoI and XmaI, SacI, respectively, and cloned into the intermediate RNAi vector psd20 by kicking out the 4CL gene in sense and antisense orientation linked with a GUS linker. The cloning of CCA1a sense and CCA1a antisense genes in intermediate RNAi vector psd20 involved two steps of sequential cloning by first ligating CCA1a sense gene by digesting with BamHI, XhoI, followed by cloning of CCA1a antisense gene using XmaI, SacI restriction enzymes in which CCA1a sense fragment has already been moved in. Similarly, the cloning of CCA1b sense and CCA1b antisense genes in intermediate RNAi vector psd20 involved two steps of sequential cloning by first ligating CCA1a sense gene by digesting with BamHI, XhoI, followed by cloning of CCA1b antisense gene using XmaI, SacI restriction enzymes in which CCA1b sense fragment has already been moved in.
The CCA1a sense:GUS linker:CCA1a antisense fragments was excised out using BamHI SacI and cloned into pUCTOC1NOS vector by ligating into BamHI SacI site to yield pUCT-SaASN vector. Similarly, the CCA1b sense:GUS linker:CCA1b antisense fragments were excised out using BamHI SacI and cloned into pUCTOC1NOS vector by ligating into BamHI SacI site to yield pUCTSbASN vector.
Isolation, cloning of over-expression vector pCTCN and RNAi vectors pCTSaASN and pCTSbASN for repression in pCAMBIA1300. The HindIII EcoRI fragment having TOC +CCA1+NOS gene from this pUC19 intermediate vector PTCN was digested using HindIII and EcoRI, purified using Qiaquick kit and cloned into pCambia1300 vector to give overexpression construct pCTCN (A) using standard molecular biology protocols.
The HindIII EcoRI fragment from the pUC19 intermediate vector (pUCTSaASN) having TOC+CCA1a sense GUS linkerCCA1a antisense +NOS was purified and cloned into pCam-bia1300 vector to give pCTSaASN (B) construct. Similarly, the HindIII EcoRI fragment from this pUC19 intermediate vector (pUCTSbASN) having TOC+CCA1b sense GUS linkerCCA1b antisense +NOS was purified and cloned into pCambia1300 vector to give pCTSbASN (C) construct.
These three constructs A, B and C were then mobilized into chemically competent Agrobacterium tumefaciens strains EHA105 cells. Competent cells were prepared by streaking on LB + Rif 25 mgl -1 agar plates and incubated at 28˚C for obtaining single colonies. Single colony was picked and grown in 5 ml YEP medium + Rif at 28˚C, 200 rpm for 2 days. Five ml of this active culture was added to 50 ml YEP medium + Rif and grown as above (O/N) till the OD600 reaches 0.7. Chill the culture on ice. Centrifuged the cell suspension at 5000 rpm at 4˚C for 5 min in 50ml yellow cap tubes. Discard the supernatant; resuspend the cells in 1.0 ml of ice cold 20mM CaCl 2 . Dispense 0.1ml aliquots into pre-chilled eppendorf tubes. The cells should be frozen in liquid in nitrogen and stored at -80˚C for further use. For transformation added 1 μg of plasmid DNA A, B and C constructs and freeze the cells in liquid nitrogen. Thaw the cells by incubating the tubes at 37˚C for 5 min. Add 300μl of YEP and incubate at 28˚C with gentle shaking at 100-120 rpm to allow the antibiotic resistance gene to express. Spread on YEP + Rif (20 mgl -1 ) + Kan (50 mgl -1 ) agar plates incubate at 28˚C by inverting them for three days for appearance of single colonies.
Single colonies from all the three constructs A, B and C mobilized in Agrobacterium tumefaciens were grown in 5 ml YEP cultures and plasmid DNA was isolated using Qiagen mini prep kit. The DNA was eluted in 100 μl of sterile water and quantified using Nano Drop Spectrophotometer. The presence of cloned gene constructs was confirmed by performing PCR amplification using 2μl of the isolated plasmid DNA of all the three constructs using primers for TOC1 gene promoter.

Callus induction
Dehusked seeds of rice variety Taipei 309 were surface sterilized with 1:1 Clorox bleach (6.0% sodium hypochlorite) and water in 100 ml total volume for 20 min, washed several times with sterile distilled water. Fifteen to twenty seeds were inoculated on N6 callus induction medium supplemented with 250 mgl -1 Myo-inositol, 1.0gl -1 Casein hydrolysate, 690 mgl -1 Proline, 1.0mgl -1 Thiamine HCl, 30gl -1 Maltose, 3gl -1 Phytagel, pH 5.8 [21,22] autoclaved and upon cooling added 5.0 mgl -1 2,4-D and 0.1 mgl -1 BAP and dispense in 90 mm Petri dishes and incubated in dark at 25±2˚C. The calli was separated from germinating seed after removing the shoot and root axis and sub-cultured on to fresh callus induction medium every two weeks. Detailed composition of various media used for tissue culture, Agrobacterium-mediated genetic transformation studies are given in Table 1.

Agrobacterium-mediated genetic transformation
Embryogenic calli obtained from mature seeds of Taipei 309 were employed for Agrobacterium-mediated genetic transformation of rice calli of varying ages from one month to five months as per protocol reported by Patel et al. [22] with minor modifications. The four constructs, namely, A, B & C mobilized in Agrobacterium tumefaciens, and pCUbiGFP glycerol stocks was used to inoculate in 5 ml of YEP medium supplemented with Rif (20 mgl -1 ) + Kan (50 mgl -1 ) incubate at 28˚C with continuous shaking at 200 rpm overnight. Next day morning transfer all the 5 ml culture in 45 ml Infection media having MS basal media salts supplemented with 1 mgl -1 Thiamine HCl, 250 mgl -1 Myoinositol, 1.0 gl -1 Casein hydrolysate, 690 mgl -1 Proline, 30 gl -1 Glucose, 5.0 mgl -1 2,4-D and 200 μM Acetosyringone, pH 5.2 and grown at 28˚C with continuous shaking at 200 rpm for 2-4 h till the OD 600 reaches 0.5-0.6. Infected the embryogenic calli with the Agrobacterium cells by giving heat shock at 42˚C for 3 min followed by incubation of 12 min at room temperature, excess of suspension drained off and the calli were blotted on to five layers sterile Whatman papers to remove excessive Agrobacterium suspension and co-cultivated for two days on co-cultivation media having 200 μM Acetosyringone at 25˚C in dark.

Selection and regeneration of T0 transformed plants
On third day the calli were transferred on to Selection media (same as callus induction media) supplemented with 200mgl -1 Timentin and 50mgl -1 hygromycin and incubated at 25˚C in dark for two weeks. Three more transfers were made on to fresh selection media after every two weeks by transferring newly induced calli, and discarded the brown or Agrobacterium infected calli. The transformed calli was very slow growing creamy white to yellow in color and very light weight. After eight weeks of selection the calli growing on hygromycin was transferred to Regeneration media, MS media supplemented with 2.0 mgl -1 BAP, 30 gl -1 Maltose, 3.0 gl -1 Phytagel, 200 mgl -1 Timentin and 50 mgl -1 hygromycin and incubated at 25˚C in light for two to three weeks. The calli was transferred to fresh Regeneration media (RM-I) for another two to three weeks for inducing shoots. The regenerated plants were transferred to MS basal media (RM-II) with 200mgl -1 Timentin and 50mgl -1 hygromycin for further proliferation and rooting in Magenta boxes at 25˚C in light. The primary transgenic plants 8-10cm with well developed shoot and root growing on MS basal media supplemented with 200 mgl -1 Timentin and 50 mgl -1 hygromycin in Magenta boxes were carefully removed, washed in running tap water to remove adhering agar were planted in 6 inch pots in 50:50 peat-lite/sand in green house maintained at 26/22˚C with 12h day/night regime and high humidity. Six pots were placed in individual plastic trays half filled with standing water. The plants were initially covered with a glass beaker and the cart was covered with a shade net for acclimatization and for preventing transpiration loss. After 3-4 weeks the transgenic plants were shifted to plant

DNA isolation and PCR analysis of T0, T1 and T2 transgenic progeny plants
The genomic DNA was isolated from young leaf tissues of randomly selected five primary transgenic T0 plants as well as T1 and T2 transgenic progeny plants for over-expression of CCA1 gene constructs A, and repression constructs B & C and wild type (WT) by using modified CTAB method according to [23]. The plasmid DNA was isolated from Agrobacterium strains using a rapid mini-prep method according to [24]. The plasmid DNA amplified with hyg II gene specific primers was used as positive control. PCR was carried out as the first method to confirm the transgenic nature of the regenerated plants and T1 and T2 progeny plants as described [25]. PCR analysis was performed using 100ng of genomic DNA (for plasmid DNA 5ng) in a 25μl reaction mixture with two sets of hyg II gene specific primers, one set The hygromycin sequence in total DNA was amplified using AccuPower PCR premix kit (Bioneer). The PCR cycle comprised of pre-incubation period at 94˚C for 3 min, leading to 35 cycles of denaturation at 94˚C for 1 min, annealing at 60˚C for 1 min and synthesis at 72˚C for 1 min, followed by extension at 72˚C for 5 min. The amplified PCR product (10μl) was subjected to electrophoresis on a 1% agarose gel and visualized under UV Transilluminator.

Southern blot analysis of T0 transgenic progeny plants
For the verification of the stable integration of hyg gene, genomic DNA isolation was performed from leaves of T0 rice plants, digested with Hind III restriction enzyme, subjected to agarose gel electrophoresis and Southern blot analysis was done using DIG High prime DNA Labeling and Detection starter Kit I, Sigma as per protocol reported by 39 with minor modifications. For Southern blot analysis, 10 μg of DNA was digested with Hind III and subjected to electrophoresis on 1.0% agarose and blotted on to a nylon membrane (Immobilion N + , Millipore Corporation, MA, U.S.A.) by capillary blotting. The membrane was UV cross-linked and probed with DIG labeled 700bp hyg gene coding region. Hybridization was carried out at 42˚C. All other procedures were performed according to the manufacturer's instructions.

Raising of T1 and T2 transgenic progeny plants from T0 and T1 seeds
The T0 transgenic rice seeds were harvested after maturity in Phytotron, N C State University, Raleigh, North Carolina, USA. The T0 seeds were imported with the permission of the Department of Biotechnology, Ministry of Science of Technology, Government of India, New Delhi, India through National Bureau of Plant Genetic Resources, New Delhi, India. The T0 seeds derived from three constructs A, B and C and wild type plants (WT) were raised in 6 inches pots in 50:50 peat-lite/sand in Transgenic Green House maintained at 26/22˚C with 12h day/ night regime and high humidity as described earlier to obtain T1 transgenic progeny plants with standard agronomic and management practices in a completely randomized block design with five independent lines of each of the constructs in the Transgenic Green House, Department of Bio & Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India and T1 seeds were harvested. The T0 lines which showed single copy insertion of Hyg gene in Southern analysis were chosen to raise T1 progeny plants. The plants which were performing morphologically better, were selected for raising T1 progeny plants and similar trend was followed in case of T2 progeny plants from T1 seeds. Similarly, T1 seeds derived from three constructs A, B and C and WT plants were raised in 6 inches pots in 50:50 peat-lite/sand in Transgenic Green House maintained at 26/22˚C with 12h day/night regime and high humidity to obtain T2 transgenic progeny plants in a completely randomized block design with five independent lines of each of the three constructs A, B and C in the Transgenic Green House and T2 seeds were harvested.

Collection of morphological data of T0, T1 and T2 transgenic progeny plants and statistical analysis
Morphological data for transgenic progeny plants for over-expression construct A, and repression constructs B & C and wild type (WT) for plant height, numbers of tillers/panicles, yield, thousand seed weight, seed length, seed width and chlorophyll content was collected. The data was subjected to statistical analysis using Student's t-test. All the morphological data of T1 and T2 transgenic progeny is presented as the mean ± SE and P value to compare the obtained parameters from transgenic lines (TG) and wild type plant (WT) and a P value of < 0.05, was considered to be statistically significant.

CCA1 Gene expression analysis of T0, T1 and T2 transgenic progeny plants
Transgenic plants harboring gene constructs for CCA1 gene in over-expression and repression mode were grown in Phytotron/Transgenic Green House for 4 weeks in 12/12-h (day/night) cycles for 24 h CCA1 rhythms analysis and transgenic plants/lines which were found to be PCR positive for hyg gene were chosen from T0, T1 and T2 Transgenic progeny plants. For each genotype, mature leaves from five transgenic plants for each constructs namely, A, B and C and wild type (WT) were collected at 6:00AM, 12:00 Noon, 6:00 PM and 9:00 PM and frozen in liquid nitrogen. Analysis for CCA1gene expression was performed on samples collected at 6:00 AM, 12:00 Noon, 6:00 PM and 9:00 PM using Real time PCR (Applied-Biosystem).

Extraction of total RNA and quantitative real time-PCR analysis of T0, T1 and T2 transgenic progeny plants
Total RNA was extracted from young leaves of various lines of transgenic rice plants derived from Agrobacterium-mediated genetically transformed constructs A, B and C and wild type plants using Trizole based method of RNA isolation. First-strand cDNA synthesis was done using Affinity Script Multiple Temperature First-strand cDNA synthesis Kit, and Promega (Go Script Reverse Transcription System Agilent Genomics). Transcript levels of each gene were measured by real-time quantitative qRT-PCR was carried out with Step One Real-time PCR system (Applied Biosystems). Quantitative PCR mixture for expression of plant CCA1 gene was prepared in MicroAmp Fast Optical 96 well reaction plate according to the manufacturer's protocols. The gene-specific primers used for qRT-PCR for CCA1A gene were

Quantification of chlorophyll content in T1 and T2 transgenic progeny plants
Quantification of Chlorophyll from various lines of T1and T2 transgenic progeny plants derived from three constructs A, B and C as per protocol of Hiscox and Israelstam [27] with slight modifications. Briefly, 100 mg of transgenic plant leaf tissue were taken at various time points 6:00AM, 12:00 Noon, 6:00 PM and 9:00 AM the following day, and placed in 12 ml vial containing 7 ml DMSO (Dimethyl Sulphoxide) and incubated at 65°C for 30 min. After incubation, samples were transferred to a 25 ml graduated tube and the volume was made to 10 ml with DMSO 1 ml of above sample was transferred to a glass cuvettes. Spectrophotometer was calibrated against DMSO at 645nm and 663nm (Chlorophyll method). Absorbance was recorded at 645 and 663 nm and chlorophyll content was calculated using equation suggested by Arnon [28] given below:-

Isolation, cloning of TOC1 promoter, NOS terminator and CCA1 gene in PUC 19
In the present investigation endogenous CCA1 gene under the control of TOC1 gene promoter have been isolated, cloned from Japonica rice and mobilized into pCAMBIA1300 vectors and RNAi constructs A, B and C have been employed for Agrobacterium-mediated genetic transformation of embryogenic call derived from rice variety Taipei 309 have been analyzed for altered morphological traits as well as CCA1 gene expression in T0, T1 and T2 transgenic progeny plants. These two essential genes, namely TOC1 and CCA1 genes have been chosen in the present investigation because; TOC1 gene is the master controller of various circadian clock genes; simultaneously, CCA1 gene products are known for controlling a large number of important plant's attributes: growth, development, flowering, reproduction and many other important agricultural traits. The TOC1 is a Transcription Factor which is under circadian control and involved in regulation of its own feed-back-loop and part of the five Pseudo-Response-Regulators (PRR) in plants. TOC1 is known to contribute to plant fitness in carbon fixation and biomass production. That's why TOC1 promoter was chosen over CCA1 gene own promoter. The 1.35 kb TOC1 gene promoter has been PCR amplified using primers using genomic DNA obtained from two weeks old germinated seeds of rice variety Taipei [14] reported cloning and expression of CCA1 gene in maize (ZmCCA1) and revealed that the ZmCCA1 transcript was highly homologous with AtCCA1 from Arabidopsis and OsCCA1 from rice [12].

Isolation, cloning of CCA1a and CCA1b gene for construction of RNAi vectors
The RNAi is a homology dependent gene silencing technology through RNA cleavage or DNA methylation. The sense and antisense fragment against the target gene, say, CCA1 forms a hairpin stem loop dsRNA structure which acts as a substrate for the Dicer. Since the entire fragment of CCA1 gene (2.172kb) of rice as shown in S1 Fig. is quite large and has not been used for construction of RNAi vectors in sense and antisense orientation for complete inhibition, but instead 5'and 3'ends of CCA1 gene have been chosen in order to assess any variation in efficiency of gene silencing. Therefore, in the present investigation sense and antisense fragments have been selected as; one 400bp fragment designated as CCA1a derived from 5'region of CCA1 gene and second 395bp fragment designated as CCA1b derived from 3'region of CCA1 gene were amplified as described below. It is known that the length and the sequence of the stem region comprising the sense and antisense fragments directly control the efficiency of gene silencing in RNAi technology. Wesley et al. [29] reported that RNAi constructs having sense and antisense fragments of complete cDNA of FLC1 gene (600bp) or the two third part of 3'region (400bp) of FLC1 gene showed early flowering in transgenic Arabidopsis. On the other hand, Heilersig et al. [30] reported that RNAi constructs having sense and antisense fragments from 5'region (488bp) of GBSS1 rice starch synthase gene is more efficient in gene silencing than the middle or 3'region of the target gene in transgenic potato. Therefore, two fragments were derived from PCR amplification for The CCA1a sense:GUS linker:CCA1a antisense fragments was excised out using BamHI SacI and cloned into pUCTOC1NOS vector by ligating into BamHI SacI site to yield pUCT-SaASN vector. All the eight clones (1-8) gave 1.6 kb fragment having CCA1a sense fragment + GUS linker + CCA1a antisense fragment along with remaining pUCTOC1NOS fragment as shown in Fig 1A. Similarly, the CCA1b sense:GUS linker:CCA1b antisense fragments were excised out using BamHI SacI and cloned into pUCTOC1NOS vector by ligating into BamHI SacI site to yield pUCTSbASN vector. All the eight clones (1-8) except clone1, gave 1.6 kb fragment having CCA1b sense fragment + GUS linker + CCA1b antisense fragment along with remaining pUCTOC1NOS fragment as shown in Fig 1B.

Isolation, cloning of over-expression vector pCTCN and RNAi vectors pCTSaASN and pCTSbASN for repression in pCAMBIA1300
The HindIII EcoRI fragment having TOC+CCA1+NOS gene from this pUC19 intermediate vector PTCN was digested using HindIII and EcoRI, purified using Qiaquick kit and cloned into pCambia1300 vector to give over expression construct pCTCN (A) using standard molecular biology protocols.
The HindIII EcoRI fragment from the pUC19 intermediate vector (pUCTSaASN) having TOC+CCA1a sense GUS linkerCCA1a antisense +NOS was purified and cloned into pCam-bia1300 vector to give pCTSaASN (B) construct. Out of seven clones, only clone 3 gave 3.5 kb CCA1a sense fragment + GUS linker + CCA1a antisense fragment after EcoRI HindIII digestion as shown in Fig 1C. Similarly, the HindIII EcoRI fragment from this pUC19 intermediate vector (pUCTSbASN) having TOC+CCA1b sense GUS linkerCCA1b antisense +NOS was purified and cloned into pCambia1300 vector to give pCTSbASN (C) construct. Out of three clones, clone 2 and 3 gave 3.5 kb CCA1b sense fragment + GUS linker + CCA1b antisense fragment after EcoRI HindIII digestion as shown in Fig 1D. These three constructs A, B and C were then mobilized into chemically competent Agrobacterium tumefaciens strains EHA105 cells and single colonies were obtained on LB + 25 mgl -1 Rif + 50 mgl -1 Kan as shown in Fig 1E  for pCTCN (A), similar results were obtained for the other two RNAi constructs, namely, pCTSaASN (B) and pCTSbASN (C).
Two colonies A1, A2, B1, B2, C1 and C2 were randomly picked from each of the three plates having constructs A, B and C mobilized in Agrobacterium tumefaciens and plasmid DNA was isolated using Qiagen mini prep kit. The presence of cloned gene constructs was confirmed by performing PCR amplification using 2μl of the isolated plasmid DNA of all the three constructs using primers for TOC1 gene promoter. All the constructs showed the appearance of 1.35 kb TOC1 band amplification as shown in Fig 1F. In addition, the purified plasmid DNA of all three constructs were sent for sequencing of TOC1, CCA1 gene and were found to be correct and in the right orientation. Thus, one over expression cassette pCTCN (A) having TOC1:CCA1:NOS was generated and two additional RNAi constructs, namely, pCTSaASN (B) and pCTSbASN (C) for repression of circadian clock genes have been made by cloning sense and antisense part of the CCA1 gene (a & b) linked with GUS linker in pCAMBIA1300 under the control of TOC1 promoter as shown in Fig 2. Similar results of cloning and construction of RNAi vectors have been reported for FLC1 gene in Arabidopsis [29], GBNSS1 gene in potato [30], and CCA1 gene in Arabidopsis [12].

Callus induction, co-cultivation and agrobacterium-mediated genetic transformation
Agrobacterium-mediated genetic transformation of Japonica rice variety Taipei 309 using scutellum derived calli was optimized having gfp gene under the control of ubiquitin promoter (pCUbiGFP) which resulted in high regeneration frequency on hyg containing medium as shown in Fig 3A and 3B and also high level of gfp expression in roots of transformed plants, whereas, control roots did not show any GFP expression under confocal microscope as shown in Fig 3C and 3D. Calli, regenerating shoot buds and roots growing on hyg exhibited high level of gfp expression whereas, control roots did not show any gfp expression as shown in Fig 3E. Germination of seeds occurred 72h after transferring on callus induction medium and the creamy white to yellow nodular embryogenic calli proliferated well upon subculture as shown in Fig 3F and 3 month old embryogenic calli (Fig 3G). Scutellum derived calli of 1, 3, 4 and 5 months old were co-cultivated with Agrobacterium harboring gene constructs A, B and C. The hyg resistant calli were recovered on selection medium. The hygromycin restricted the growth of non-transformed calli, which turned brown and the transformed calli showed active cell proliferation derived from constructs A, B and C as shown in Fig 3H. The hyg resistant calli were transferred on to RM-I. The regenerated calli transferred on RM-I showed green spots after 3-4 weeks, produced more than one shoot derived from constructs A, B and C as shown in Fig 3I. The shoot-regenerating calli were transferred to RM-II and the remaining calli were kept on fresh RM-I for another 2 weeks to check further regeneration potential. The plantlets on RM-II medium have produced multiple shoots, derived from constructs A, B and C as shown in (Fig 3J) and initiated root formation. The well developed putative transgenic plants derived from constructs A, B and C were transferred to plastic pots and maintained in Phytotron for acclimatization, hardening and further proliferation (Fig 3K). The transgenic plants of GFP, A, B, C construct and wild type were maintained in Phytotron for panicle formation, seed setting and grown till maturity (Fig 3L). It was observed that the responses of 1 month old callus were better for transformation and plant regeneration than 3, 4 and 5 month old callus as well as inclusion of BAP in callus induction medium. Similar results were reported for Japonica rice variety Taipei 309 and perennial rye grass [22]. Similarly, high frequency plant regeneration from embryogenic calli of Bermuda grass by inclusion of BAP in callus induction medium was reported [31].
Maximum  (Fig 4). Therefore, plant regeneration frequency decreases with increase in the age of calli. Similar results were reported in which the response of 2-3 month old rice callus was better for transformation and plant regeneration than 4-6 month-old callus [22]. A total of 90 transgenic plants comprising 30   inch pots in peat-lite/sand. In the present study MS based infection medium with 200 μM Acetosyringone has been successfully used for 15 min of Agro-infection inclusive of 3 min of heat shock at 42˚C. MS based Agro-infection medium has been successfully reported earlier [22,[32][33][34][35]. It is widely believed that transformation normally takes place within actively dividing cells and MS medium is better for plant cell growth compared to YEP medium. Similarly, Han et al. [35] also reported improved transformation efficiency in Artemisia annua L. while using MS as an infection medium rather than the LB medium.  (Fig 6). Thus, stable integration of T-DNA in the T0 transgenic plants derived from construct A, B and C was confirmed as shown in Fig 6. Similarly; molecular analysis were carried out by most of the workers to check the integration pattern and copy number by Southern blot analysis [36][37][38], reported one to two copy number of the transgene in T0 plants and one and three copy number integration of transgene in T1 independent transformants using Southern blot analysis.

Quantitative real time expression analysis of CCA1 gene and morphological data of T0, T1 and T2 transgenic progeny plants
Quantitative real time PCR of various transgenic T0 lines, T1 lines and T2 progeny lines was carried out for ascertaining the CCA1 gene expression for constructs A, B and C. The 2 -ΔΔCT method was used to calculate relative changes in expression of CCA1 gene. Amplicon abundance was monitored in real-time by measuring SYBR Green fluorescence. The level of CCA1 gene transcript in T0, T1 and T2 plants was normalized with reference to 18S rRNA taken as an internal control as shown in Fig 7. The specificity of primers employed in the present investigation for 18 S rRNA as an internal control and the gene of interest CCA1 in T1 transgenic progeny plants of rice in qRT-PCR analysis as Melt curve is exhibited in Figs 8 and 9, respectively, similar results were obtained in T2 transgenic progeny plants. The expression profile of the T0, T1 and T2 transgenic lines was made in comparison to the wild type (WT) non transformed plant, which was taken as the calibrator. The expression profile of T0 transgenic lines showed that all the transgenic lines derived from construct A, B and C showed highest CCA1 gene expression at 12:00 Noon and lowest level of CCA1 gene expression at 6:00 PM as shown in Fig 7A to 7C. All the T0 transgenic plants derived from RNAi constructs B and C, namely, Similarly, the CCA1 gene expression profile of T1 transgenic lines showed that the transgenic lines derived from construct A, A-17, A-45 showed high level of CCA1gene expression at 6:00 AM and lowest level of CCA1 gene expression at 12:00 Noon, which again increased by 6 folds and 1 fold at 6:00 PM followed by decrease at 9:00 PM in a rhythmic manner as shown in Fig 7D. Similarly, T1and T2 transgenic plants derived from constructs B and C, namely, B-17, B-23, B-28, B-34, B-45, and C-19 showed lowest CCA1 gene expression at 12:00 Noon and highest level of CCA1 gene expression at 6:00 AM except B-17 which again show high expression of CCA1 gene expression at 6:00 PM as shown in Fig 7D and Fig 7E. Similar results of robust expression and association of ZmCCA1 with circadian clock in maize was reported [14] and CCA1 gene expression peaking at dawn, which began to decrease rapidly in Arabidopsis thaliana was reported [12].
The results of the present study are in conformation with the findings of Ni et al. [12] that repression of CCA1 gene promotes growth, biomass, yield, sugar and starch content and chlorophyll content while the over-expression of CCA1 would result in decreased growth vigor in diploids of Arabidopsis. The CCA1 gene expression of T1and T2 progeny lines in the present investigation is highest at dawn (6:00 AM), lowest at 12:00 Noon, again increases until dusk at 6:00 PM and then starts decreasing at 9:00 PM in a rhythmic pattern. At the same time construct A derived transgenic T1 and T2 plants exhibited several fold higher expression of CCA1 gene at dusk, whereas, construct B and C derived transgenic T1and T2 plants exhibited several folds lower expression of CCA1 gene at 12:00 Noon. Likewise, Ni et al. [12] observed that the CCA1 gene expression peaked at dawn (Zeitgeber time 0, ZT0), decreased 6 h after dawn (ZT6), and continued declining until dusk (ZT15). They reported that at ZT6 (noon), CCA1 and LHY were repressed, 2-fold, whereas TOC1 was up-regulated, 2-fold in the F1 hybrids relative to the parents. They also reported that the CCA1 gene expression TOC1::CCA1(RNAi) transgenic Arabidopsis plants, CCA1 messenger RNA and protein amounts were down-regulated 2-10-fold and 1.4-3-fold, respectively, and CCA1 gene repression results in increased chlorophyll synthesis, starch metabolism and growth vigour and this data is in agreement to the data obtained in the present investigation. Similarly, Wang et al. [14] characterized CCA1 gene of maize (ZmCCA1), and reported rhythmic expression of ZmCCA1 in leaves and stem apex meristems under long day and short day conditions, and its peak gene expression Similarly, quantitative-PCR techniques are used extensively to assay and characterize clock gene expression, most notably CCA1 and LHY, directly over a sampling time-course in circadian studies in conjunction with high-throughput techniques [1,2,14,39]. Wang and Tobin quantified CCA1 RNA by competitive RT-PCR in transgenic Arabidopsis plants and showed that constitutive expression of CCA1 results in longer hypocotyls and substantially delayed flowering [2]. Similarly, Lee et al. [40] reported a rhythmic expression of MYB96-a transcription factor that is connected with the clock oscillator that peaked around dusk using qRT-PCR analysis in Arabidopsis.
Morphological characteristics of T1 transgenic progeny plants at different developmental stages from germination of seeds to flowering and seed setting are shown in S4   Fig. T1 and T2 transgenic progeny plants were further analyzed for their morphological traits and growth characteristics such as plant height, numbers of tillers/panicle, yield, thousand seed weight, seed length, seed width and chlorophyll content at different point of time and subjected to statistical analysis using Student's t-test.
Characterization of T0 transgenic lines of construct A, B and C did not show any considerable difference in any of the morphological traits over the wild type and in student's t-test was also not found statistically significant as shown in Table 2. While, T1 transgenic progeny plants derived from constructs B and C showed increased number of tillers/panicles, improved plant condition, better seed yield, increased thousand seed weight, slightly increased seed size as compared to wild type plants, at the same time T1and T2 transgenic progeny plants derived from constructs A exhibited reduced number of tillers/panicles, reduced yield, reduced  Tables 3 and 4 and Figs 10 and 11. The T1transgenic progeny plants derived from constructs A, namely, A-17 and A-45 exhibited reduced number of tillers/panicles (6-7), reduced thousand seed weight (16.6-15.5g), decreased seed length (5.5-5.4mm) and decreased seed width (1.3-1.75mm) as compared to wild type plants as shown in Table 3, S1 Table and Fig Table 4, S2 Table and Fig 11. On the contrary, T1 transgenic progeny plants derived from constructs B and C showed increased number of tillers/panicles (8)(9)(10)(11)(12)(13)(14)(15)(16), improved plant condition (3)(4), better seed yield (3.95-23.01g), increased thousand seed weight (16.8-28.5g), slightly increased seed length (6.75-7.43 mm), and increased seed width (1.8-2.18 mm) as compared to wild type plants as shown in Table 3, S1 Table and Fig 10. Similarly, T2 transgenic progeny plants derived from constructs B and C showed increased number of tillers/panicles (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), improved plant condition (3)(4), better seed yield (6.5-28.9g), increased thousand seed weight (16.9-29.03g), slightly increased seed length (6.7-7.5 mm) and increased seed width (1.75-2.98 mm) as compared to wild type plants as shown in Table 4, S2 Table and Fig 11.The P value in all the above cases was found to be less than 0.05 so the differences in morphological characters were found to be statistically significant as shown in Tables 3 and 4 in majority of the transgenic lines derived from B and C constructs of T1 and T2 progeny plants.
Increase of seed size in wild type Japonica rice variety Nipponbare has been reported by Liu et al. [41], GW5 protein is expressed in various rice organs and acts in the brassinosteroid signaling pathway regulating grain width and weight. Similarly, in wild type tropical Japonica rice, GLW7 encoding the plant-specific transcription factor OsSPL13 positively regulates cell size in the grain hull, resulting in enhanced rice grain length and yield has been reported [42]. Recently, over-expression of OsEXPA10 gene in rice enhancing growth, which lead to increased susceptibility to BPH (Brown Plant Hopper) being one of the nastiest insect pests of rice and blast attack has been reported by Tan et al. [43]. Repression OsEXPA10 gene reduced morphological characters but improved resistance to BPH and blast attack. The results of present investigation for over-expression of CCA1 gene in T1 and T2 transgenic progeny plants resulting in detrimental effect on morphological attributes, whereas, repression of CCA1 gene by RNAi constructs in T1and T2 transgenic plants resulted in improved morphological characteristics are in conformity of data presented [12,14] and for increased chlorophyll synthesis, starch metabolism and growth vigour [40] by RNAi constructs harboring CCA1 gene in transgenic Arabidopsis thaliana plants. Recently, mathematical models and experimental data in Arabidopsis thaliana showed that Myb transcription factors CCA1/LHY proteins serve as a repressor and RVE8 acts as an activator of gene expression even though both of them bind to the same cis-element [44]. They have shown that multiple feedback loops of the plant clock genes guarantee rhythmicity under unfavorable environmental cues. A variable response of gene expression between scion and rootstock upon homo grafting of Arabidopsis thaliana in various organs was reported from my laboratory [45]. The flower buds of scion showed overrepresentation of the transcription factor genes, such as Homeobox, MYB and NAC. Differential transcription of genes related to gibberellic acid, ethylene and other stimuli was observed between scion and rootstock.
Our results of gene silencing by RNAi vectors are in conformity to the data reported for FLC1 gene in Arabidopsis [29], GBNSS1 gene in potato [30], and CCA1 gene in Arabidopsis [12]. The chlorophyll content measured in T1 (S3 Table) and T2 (S4 Table) progeny plants showed rhythmicity with passage of time in the CCA1 gene expression. It was at peak at 12:00 Noon when CCA1 gene expression is lowest and again decreases until dusk (6:00 PM) and then start increasing afterwards as shown in S6 Fig. A similar correlation between CCA1 gene expression and chlorophyll content has been reported [12] in Arabidopsis. However, no significant difference was observed in the level of chlorophyll content with an average of 0.04mgg -1 in T1 (S3 Table) and T2 (S4 Table)

Conclusions
Therefore, following conclusions can be drawn from the present investigation. The overexpression of CCA1 gene under the control of TOC1 promoter follows circadian rhythm in  thousand seed weight and increased seed size over the wild type control which is statistically significant. Additionally, RNAi Construct-C having CCA1 gene derived from the 3'terminal region of CCA1 gene was found to have better morphological attributes than Construct B which was derived from 5'terminal region of CCA1 gene. Similar results of gene silencing by RNAi vectors have been reported for FLC1 gene in Arabidopsis [29], GBNSS1 gene in potato [30], and CCA1 gene in Arabidopsis [12]. Circadian clock genes including CCA1 are potent tools to change single or multiple traits simultaneously and their potential application by exploiting clock-regulated activities is just beginning to be understood. Circadian clock genes hold the potential for crops breeding better adapted to environments and the fluctuations inherent to climate change. It is envisaged that data presented herein regarding endogenous CCA1 gene expression studies in transgenic Japonica rice variety Taipei 309 lines and its correlation with growth vigor will significantly initiate the rice breeding programs both qualitatively and quantitatively and further help in better understanding the role of CCA1 gene in plant growth, biomass and metabolism in future. This is the first report of successful genetic transformation of an important Japonica rice variety Taipei 309, by employing Agrobacterium tumefaciens strain EHA 105 harboring   Table. Comparison of average chlorophyll content (mgg-1) in T1 transgenic progeny plants at different time points; 6:00AM, 12:00 Noon, 6:00 PM and 9:00 AM the following day.