Search Results
Cytokinins were first recognized as a class of phytohormones for their ability to promote cytokinesis in cultured plant cells and have since been shown to be involved in a wide range of physiological processes. Most recently, the availability of phytohorm one-specifying genes from Agrobacterium tumefaciens has allowed for direct in planta manipulation of cytokinin levels. Overexpression of the isopentenyl transferase (ipt) gene by constitutive promoters led to enhanced ability of plant cells to undergo shoot organogenesis but the high endogenous cytokinin levels almost completely suppressed root development. Transient overproduction of cytokinins using promoters regulated by environmental and/or developmental factors did not inhibit regeneration of rooted plants. Transgenic plants in which cytokinin levels can be modulated are being used to characterize the participation of cytokinins in fundamental regulatory mechanisms of morphogenesis, delayed senescence, disease resistance and directed nutrient transport. The potential for using reconstructed cytokinin biosynthesis genes in economically important crops is of tremendous agronomic significance.
Lycopersicon esculentum cv. UC82b cotyledons were co-cultivated with A. tumefaciens carrying vectors with modified isopentenyl transferase (ipt) genes. The ipt gene was placed under the control of the RUBISCO promoter in both the sense and antisense orientation. Over 50 transformants were recovered on kanamycin-containing media. Seeds from RO plants were germinated on selective media and R1 plants transformed with the ipt gene identified by PCR and Southern blot hybridization. Phenotypes of the R1 plants, whether transformed with the ipt gene in the sense or antisense orientation, were comparable to the control plants transformed with an inactive cytokinin gene. Fruit weights from both were similar to those from control plants, however, yields were reduced and ripening delayed. Most fruit had no seeds or very few small seeds. Cytokinin levels are being determined in order to correlate them to the observed phenotypes.
Immature `Redhaven' peach [Prunus persica (L.) Batsch] embryos were infected with a shooty mutant strain of Agrobacterium tumefaciens, tms328::Tn5, which carries an octopine-type Ti plasmid with a functional cytokinin gene and a mutated auxin gene. Shoots were regenerated from embryo-derived callus that was initiated on MS medium lacking phytohormones. Shoots exhibited increased frequency of branching and were more difficult to root than the noninfected. Transcripts of the tms328::Tn5-cytokinin gene were detected using northern analyses of total plant RNA. Polymerase chain reaction of genomic DNA and cDNA resulted in amplification of DNA fragments specific for the cytokinin gene, as determined by restriction enzyme and Southern analyses. The concentrations of the cytokinins zeatin and zeatin riboside in the leaves of regenerated plants were on the average 51-fold higher than in leaves taken from nontransformed plants. None of the shoots or callus tissues were postive for octopine. The expression of the T-DNA encoded cytokinin gene promotes growth of peach cells in the absence of phytohormones, thus serving as a marker for transformation. In addition, this gene appears to promote morphogenesis without an auxin inductive step.
A Lycopersicon esculentum Mill. (tomato) cDNA clone with high similarity to a Nicotiana plumbaginifolia Viv. (tobacco) cytochrome P450 gene was isolated using 5' and 3' rapid amplification of cDNA ends (RACE). The isolated cDNA (GenBank Accession No. AF249329) has an open reading frame of 1494 base pairs (bp) and encodes a protein of 498 amino acids with 75% identity to the N. plumbaginifolia cytochrome P450 (CYP72A2) and 45% to a Catharanthus roseus G. Don (Madagaskar periwinkle) CYP72A1 protein sequence. By Southern-blot analysis, one or two highly homologous genes were detected in the L. esculentum genome. Expression of the cloned P450 gene was regulated by circadian rhythm and enhanced by wounding. Leaf transcripts were detected in the light but not dark. Highest transcript levels were observed 3 hours after mechanical wounding. No increase in expression was seen in response to applications of zeatin as with the N. plumbaginifolia gene. Of the tissues analyzed, shoot tips and young leaves and fruit had the highest detectable transcript levels. Attempts to transform more than 1400 cotyledon explants of L. esculentum with sense or antisense CYP72A2 gene constructs produced no transgenic plants.