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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.

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