Root Specific Expression of CaCKX1 Gene Alters Root Architecture in Chickpea

Activity of root apical meristem (RAM) regulates developmental plasticity of root. A balance between auxin and cytokinin determines the rate of cell division in RAM. Ratio of auxin to cytokinin is high in the cell division zones at RAM and lateral root initials. Auxin functions by polar gradient while cytokinins are mostly synthesized locally. Therefore, to promote root elongation, it is easier to increase ratio of Auxin:Cytokinin by degrading cytokinins. CYTOKININOXIDASE1 (CKX1) plays major role in regulating cytokinin levels in plant tissues by degradation. We have developed the transgenic chickpea and tomato plants expressing chickpea CKX1 gene driven by chickpea WRKY6 promoter to reduce endogenous cytokinin content specifically in root. An efficient genetic transformation system was developed by using single cotyledon & half embryo of chickpea var. Pusa-362 and healthy seeds of Pusa Ruby as explants. These explants were transformed with Agrobacterium tumifaciens strain GV3101 harbouring P_WRKY6:GUS or P_WRKY6:CaCKX1 plasmid containing NPTII gene for kanamycin selection. A total of 10 and 30 T₀ transgenic plants of tomato and chickpea were generated, respectively. These plants were further advanced up to T₃ generation. PCR and Southern hybridization analysis and histochemical GUS assay of these transgenic plants have confirmed the integration of trans-gene. GUS-staining of plant transformed with P_WRKY6:GUS showed function of this promoter was restricted in roots only. Phenotyping of these plants had shown enhanced root biomass and lateral root formation as compared to the wild type without any shoot/yield penalty. Transgenic plants performed better under low irrigation conditions in controlled environments. Our work established an efficient and rapid Agrobacterium mediated transformation system for chickpea and tomato. Our observations are consistent with the hypothesis that cytokinins are negative regulators of root growth and lateral root formation. This work will definitely accelerate the development of genetically engineered crop plants with desired traits.