Keragaman Jumlah Salinan Transgen Galur T0 Padi Kultivar Nipponbare Berdasarkan Analisis QPCR Dengan Penanda Gen HptII

The development of transgenic crop using Agrobacterium tumefaciens produces different inserted transgenes, whether copy numbers or location in the plant genome. The research was performed to detect chimeric phenomena based on the transgene quantity analysis in tillers of a clump and of some clumps which were derived from the same calli. CsNitr1–L gene and hptII gene as a marker on a binary plasmid pCAMBIA1300 was transformed into the Nipponbare rice genome using A. tumefaciens strain LBA 4404. Molecular analysis was carried out on three tillers of each four clumps of Nipponbare transgenic T0 generation (events number 1, 2, 3, and 4) and four groups of T0 clump derived from one callus. Three T0 clump samples were collected from each of the four groups of T0 clumps. The results of qPCR analysis showed that the transgene copy numbers of tillers which were derived from one T0 clump were the same. qPCR analysis also discovered that not all plants from one callus demonstrated the same transgene copy numbers. This implies that each T0 rice clump which grows from the transformed calli was needed to be split in the acclimatization step so that the uniform T1 seeds would be obtained

Introduksi Konstruk Gen CsNitr1-L dengan Promotor Ubiquitin melalui Agrobacterium Tumefaciens dan Deteksi Molekulernya pada Padi Kultivar Nipponbare

Nitrogen based fertilizers such as urea and NPK are primary needs for rice farmers. To get significant improvement of crop yield, the more quantity of fertilizers are applied. It make negative impact for surrounding environment. Based on that, the efforts should be done to suppress the demand of fertilizers such as by developing Nitrogen Use Efficiency crops. CsNitr1-L is one of gene that related to Nitrogen Use Efficiency trait in plant. The objectives of this research are to develop the construction of CsNitr1-L gene candidate in pCAMBIA1300-Ubi1 promoter and to obtain the transformants of rice cultivar Nipponbare which contain the construction of CsNitr1-L gene candidate. The construction of pCAMBIA1300::Ubi1::CsNitr1-L has successfully assembled and was transformed to immature embryo of rice cultivar Nipponbare using Agrobacterium tumefaciens strain LBA4404. It was obtained 146 lines of T0 Nipponbare. PCR analysis of T0 Nipponbare lines showed that 66 of them was identified as positive T0 lines contained hptII and CsNitr1-L genes. Transformation efficiency obtained was 11,9%. The result of analysis copy number using Southern Hybridization in positive PCR of T0 lines randomly indicated that 4 lines have a single copy of transgene. Based on these results, it can be concluded that CsNitr1-L gene construct was successfully introduced into the genome of the rice plant cultivar Nipponbare and the positive PCR of T0 lines containing the gene of hptII and CsNitr1-L, also a single copy of the transgene was obtained