GENETIC MANIPULATION OF CYTOKININ LEVELS IN NEW PLANT TYPE RICE

The changes in chlorophyll content, rate of senescence and cytokinin (CK) levels in the top three leaves of field-grown New Plant Type (NPT) rice were determined during the grain filling period. The chlorophyll content in the leaves decreased from flowering to maturity. Fast-senescing and slow-senescing NPT rice lines were identified. The presence of 22 different CKs in the leaves of fastsenescing and slow-senescing NPT lines was reported for the first time. The 22 CKs were placed into four functionally different groups of CKs: active CKs (CK bases and ribosides), storage CKs (CK O-glucosides), inactive CKs (CK-7-Nglucosides and CK-9-N-glucosides), cis-zeatin and its derivative CKs. The results showed that it is not the actual content of CKs but the changes in dynamics of CKs which are decisive for fast senescence. The grain filling percentage was positively and significantly correlated with the rates of senescence in the top three leaves. The grain yield was significantly correlated with the rate of senescence only in the flag leaf which indicates the importance of the flag leaf in supplying the photo assimilates to the grains. The isopentenyl transferase (ipt) gene, from Agrobacterium-tumeJaciens, is involved in the first rate-limiting step in cytokinin biosynthesis. Using biolistic transformation, the ipt gene under the control of a senescence-specific SAG 12 promoter (SAG 12-ipt) was introduced into NPT rice to delay leaf senescence. The ipt gene was successfully integrated into the genome and expressed in the leaves of transgenic plants. The grain filling percentage was lower in the transgenic SAG12-ipt To plants compared to the control plants. The levels of Z, Z9R, iP and iP9R in the leaves of plant No. T2068 and the levels of Z9R, DHZ9R and iP in the leaves of plant No. T1193 were higher than in the leaves of control plants. PCR analysis showed that the ipt gene was inherited in the TI progeny of the transgenic To plants. The ipt gene was inherited in a 3:1 segregation ratio in the TI progeny of transgenic plant No. Tl193 which indicates that the ipt gene was inherited as a single Mendelian locus. The grain filling percentage was lower in the ipt-positive T I plants of plant No. T1l93 compared to the ipt-negative T I plants of plant No. Tl193 and the untransformed control plants. Four T I progeny of plant No. Tl193 and their T 2 progeny showed delayed leaf senescence, shorter plant height, two to four week delay in flowering and lower grain filling compared to the control plants which might be due to overexpression of the cytokinins. The levels of Z9R and iP in the leaves of Tl193-24-9 plant and the levels of Z9R, DHZ9R, iP and iP9R in the leaves of T1193-27-8 plant were higher than in the leaves of the control plant. Since the Arabidopsis thaliana SA G 12 promoter did not seem to work well in the mono cot background of rice, efforts were made to isolate the homologue of the SAG12 promoter in NPT rice. The expression of Arabidopsis SAG12 and senescence-related genes was examined in various parts of NPT rice. A phylogenetic tree showed that the SAG12 gene had homology with several cysteine proteases in cereals, such as, maize, barley and rice and clustered closest to two rice BAC clones, namely, OSM146118 and OSM146316. However, the OSMl46118 rice BAC clone was not senescence-specific. The OSMl46118 BAC clone was expressed in equal intensity in the non-senescing, senescing and senesced leaves, hence, it is not senescence-specific. After RT -PCR analysis of the OSM146316 rice BAC clone, the transcripts found in non-senescing, senescing and senesced leaves were cloned and sequenced. The clones obtained in the senescing leaves showed homology with Prunus armeniaca's ethyleneforming enzyme. Tblastn result of the ethylene-forming enzyme showed that it had homology with OSM13394 rice BAC clone. However, this rice BAC clone was expressed in equal intensity in all parts of the riceplant, hence, it is not senescence-specific. The expression of senescence-related genes, namely, SAG101, ORE9, red chlorophyll catabolite, YLS3 and chlorophyllase, in NPT rice was determined. These senescence-related genes had some homology with several rice BAC clones, such as OSM14989, OSM1359, OSM151086, OSM1366 and OSM1282. RT-PCR analysis showed that these rice BAC clones were expressed in all or most parts of the rice plant, hence, none of them were found to be senescence-specific. In further studies, differential display or screening the cDNA library may be used to isolate the rice homologue of the SAG12 promoter.