Table 1.
Composition of various media used for tissue culture and Agrobacterium-mediated genetic transformation studies.
Fig 1.
Isolation, cloning of over-expression vector pCTCN and RNAi vectors pCTSaASN and pCTSbASN for repression in pCAMBIA1300.
(A) Cloning of 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 pUCTSaASN vector. (B) 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. (C) The HindIII EcoRI fragment from the pUC19 intermediate vector (pUCTSaASN) having TOC+CCA1a sense GUS linkerCCA1a antisense +NOS was purified and cloned into pCambia1300 vector to give pCTSaASN named as construct B. (D) 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 named as construct C. (E) The 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 designated as pCTCN for Construct A. (F) PCR amplification and confirmation of 1.35 kb TOC1 band from randomly selected colonies A1, A2, B1, B2, C1 and C2 RNAi constructs from each of the three plates having constructs A, B and C mobilized in Agrobacterium tumefaciens.
Fig 2.
Plasmid constructs employed in the present investigation.
The gfp gene under the control of Ubiquitin promoter (pCUbiGFP). One over expression cassette pCTCN designated as construct 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. Gene cassettes cloned in T-DNA region of binary vector pCAMBIA1300 LB- Left border, RB- Right Border, CCA1- Circadian Clock Associated, TOC1 PRO- TIMING OF CAB EXPRESSION 1 PROMOTER, The CCA1a and CCA1b sense and antisense fragments for making the two RNAi constructs pCTSaASN and pCTSbASN were designed by choosing 400 bp of 5`region of CCA1a gene and 395 bp of 3`region of CCA1b gene, CaMV35S-Cauliflower Mosaic Virus 35 S Promoter, NOS Terminator- Nopaline synthase terminator.
Fig 3.
Callus induction, co-cultivation and Agrobacterium-mediated genetic transformation of Japonica rice variety Taipei 309.
(A) and (B) High frequency plant regeneration from GFP expressing transformed calli. (C, D, E) High expression of GFP in the callus and roots of the transgenic plants; control roots did not show any GFP expression under confocal microscope (F, G) Creamy white to yellow nodular embryogenic calli proliferated well upon subculture, and 3 month old embryogenic calli (H) Selection and proliferation of hygromycin resistant calli on selection medium harboring constructs A, B and C. (I) The regenerated calli transferred on RM-I showing appearance of green spots after 3–4 weeks, produced more than one shoot derived from constructs A, B and C. (J) Regenerated transformed shoots were transferred to RM-II produced multiple shoots and well developed roots derived from constructs A, B and C. (K) 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. (L) 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 4.
Transgenic plants (T0) regenerated from Agrobacterium–mediated genetic transformation of rice calli having 1, 3, 4 & 5 month old using circadian clock gene constructs A, B, & C. (A) Number of transgenic plants regenerated. (B) Percentage of transgenic plants regenerated.
Fig 5.
PCR analysis of T0, T1 and T2 transgenic plants for the presence of the hyg gene.
Arrowhead indicates the 324 bp band corresponding to the hyg gene of the T-DNA. The pCAMBIA1300 served as a Positive Control (+ve) and non-transgenic Taipei 309 served as a Negative Control (–ve). (A) PCR Analysis of T0 Transgenic Lane 1 L—1 kb ladder, lane 2- (–ve control, lane 3 +ve control, lanes 4–8—putative transgenic plants of construct A, lanes 9–13- putative transgenic plants of construct B, lanes 14–18 putative transgenic plants of construct C, lane 19–1 kb ladder. (B) PCR Analysis of T1 Transgenic Plants. Lane 1- L—1 kb ladder, lane 2 –ve control, lanes 3–4 putative transgenic plants of construct A, lanes 5–9- putative transgenic plants of construct B, lanes 10—putative transgenic plants of construct C, lane 11 +ve control. (C) PCR Analysis of T2 Transgenic Plants. Lane 1- L—1 kb ladder, lane 2–9 putative transgenic plants of construct A, lanes 10–16 putative transgenic plants of construct B. (D) PCR Analysis of T2Transgenic Plants. Lane 1- L—1 kb ladder, lanes 2–12 putative transgenic plants of construct B, lanes 13–16 putative transgenic plants of construct C, lane 17 +ve control. The gel images for Fig 5A, 5C and 5D is original. The gel images for Fig 5B have been cropped; full length gel pictures have been shown as S7 Fig.
Fig 6.
Southern blot analysis of T0 transgenic plants derived from wild type, gfp gene containing transgenic plants and twenty five transgenic plants derived from constructs A, B and C. The genomic DNA was digested with HindIII and probed with hyg gene.
Fig 7.
The qRT-PCR expression analysis of CCA1 gene in T0, T1 and T2 transgenic rice plants at different time points; 6:00AM, 12:00 Noon, 6:00 PM and 9:00 PM.
The relative fold change expression of CCA1 gene was monitored as per 2-ΔΔCT method by taking wild type as a calibrator. (A, B, C) CCA1 gene expression in T0 derived transgenic plants of construct A, B and C (D) CCA1 gene expression in T1 derived transgenic plants of construct A, B and C (E) CCA1 gene expression in T2 derived transgenic plants of construct A, B and C.
Fig 8.
Melt curve of 18 S rRNA primers employed as an internal control (GenBank Accession No. AF156675) for qRT-PCR analysis of various T1 transgenic progeny plants of rice.
Fig 9.
Melt curve of CCA1 gene-specific primers employed in qRT-PCR analysis of various T1 transgenic progeny plants of rice.
Fig 10.
Morphological appearance and comparison of seed size of T1 transgenic progeny plants harboring gene constructs A, B and C and that of wild type (WT). Bar size is in mm. (A) morphological appearance of T1 seeds. Bar size depicting seed length for WT-6.75mm, Construct A-5.5mm, Construct B-7.02mm and Construct C-7.43mm. (B) Seed length and width of WT and various lines derived from construct A, B and C are shown graphically.
Fig 11.
Morphological appearance and comparison of seed size of T2 transgenic progeny plants harboring gene constructs A, B and C and that of wild type (WT). Bar size is in mm. (A) morphological appearance of T2 seeds. Bar size depicting seed length for WT is 6.75mm, Construct A- 5.5mm, Construct B-7.02mm and Construct C-7.5mm. (B) Seed length and width of WT and various lines derived from construct A, B and C are shown graphically.
Table 2.
Morphological characterization of T0 transgenic progeny plants.
Table 3.
Morphological characterization of T1 transgenic progeny plants with statistical analysis (student’s t-test).
Table 4.
Morphological characterization of T2 transgenic progeny plants with statistical analysis (student’s t-test).