title: Down-Regulation of Cytokinin Oxidase 2 Expression Increases Tiller Number and Improves Rice Yield.
abstract: BACKGROUND
  Cytokinins are plant-specific hormones that affect plant growth and development. The endogenous level of cytokinins in plant cells is regulated in part by irreversible degradation via cytokinin oxidase/dehydrogenase (CKX). Among the 11 rice CKXs, CKX2 has been implicated in regulation of rice grain yield.
  
  
  RESULTS
  To specifically down-regulate OsCKX2 expression, we have chosen two conserved glycosylation regions of OsCKX2 for designing artificial short hairpin RNA interference genes (shRNA-CX3 and -CX5, representing the 5' and 3' glycosylation region sequences, respectively) for transformation by the Agrobacterium-mediated method. For each construct, 5 independent transgenic lines were obtained for detailed analysis. Southern blot analysis confirmed the integration of the shRNA genes into the rice genome, and quantitative real time RT-PCR and northern blot analyses showed reduced OsCKX2 expression in the young stem of transgenic rice at varying degrees. However, the expression of other rice CKX genes, such as CKX1 and CKX3, in these transgenic lines was not altered. Transgenic rice plants grown in the greenhouse were greener and more vigorous with delayed senescence, compared to the wild type. In field experiments, both CX3 and CX5 transgenic rice plants produced more tillers (27-81 %) and grains (24-67 %) per plant and had a heavier 1000 grain weight (5-15 %) than the wild type. The increases in grain yield were highly correlated with increased tiller numbers. Consistently, insertional activation of OsCKX2 led to increased expression of CKX2 and reduced tiller number and growth in a gene-dosage dependant manner.
  
  
  CONCLUSIONS
  Taken together, these results demonstrate that specific suppression of OsCKX2 expression through shRNA-mediated gene silencing leads to enhanced growth and productivity in rice by increasing tiller number and grain weight.
authors.affiliation: Biodiversity Research Center and Genome Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Su-Ying
authors.initials: SY
authors.lastname: Yeh
authors.affiliation: Department of Bioagricultural Science, National Chiayi University, Chiayi, 600, Taiwan.
authors.forename: Hau-Wen
authors.initials: HW
authors.lastname: Chen
authors.affiliation: Biodiversity Research Center and Genome Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Chun-Yeung
authors.initials: CY
authors.lastname: Ng
authors.affiliation: Biodiversity Research Center and Genome Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Chu-Yin
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Division of Biotechnology, Taiwan Agricultural Research Institute, Taichung, 413, Taiwan.
authors.forename: Tung-Hai
authors.initials: TH
authors.lastname: Tseng
authors.affiliation: Biodiversity Research Center and Genome Research Center, Academia Sinica, Taipei, 115, Taiwan.|Department of Ecology and Evolution, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Wen-Hsiung
authors.initials: WH
authors.lastname: Li
authors.affiliation: Department of Bioagricultural Science, National Chiayi University, Chiayi, 600, Taiwan. mku@mail.ncyu.edu.tw.|School of Biological Sciences, Washington State University, Pullman, WA, 99164, USA. mku@mail.ncyu.edu.tw.
authors.forename: Maurice S B
authors.initials: MS
authors.lastname: Ku
keywords: Cytokinin oxidase/dehydrogenase; Growth; Productivity; Rice; shRNA