Molecular characterization and n use efficiency of LeAlaAT ‘Mekongga’ transgenic rice lines

Genetic engineering is one of the strategies for developing nitrogen (N)-use-efficient rice (Oryza sativa) varieties. One gene that plays an indirect role in N metabolism is alanine aminotransferase (AlaAT). It can efficiently increase N content and crop yield. In a previous study, the tomato AlaAT gene (LeAlaAT) was successfully isolated and introduced into ‘Mekongga’ rice. The present research was conducted during 2018 and 2019 at the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor, Indonesia. The objectives of the present study were to perform the molecular characterization of LeAlaAT ‘Mekongga’ rice lines on the basis of the hpt marker gene, the direct PCR of the LeAlaAT fragment, and the phenotypic evaluation of the selected LeAlaAT T1 ‘Mekongga’ rice lines in response to different N fertilizer rates (0 kg ha−1 [control] and 60, 90, and 120 kg ha−1). This research involved three activities, namely (1) Southern blot analysis, (2) direct PCR, and (3) N use efficiency (NUE) test of ‘Mekongga’ transgenic lines. Southern blot analysis revealed that in T0 transgenic lines, the copy number of the hpt marker gene varied from 1 to 3. Direct PCR confirmed the presence of the AlaAT fragment in the T1 generation of five ‘Mekongga’ transgenic lines. The five transgenic lines showed high panicle number, biomass weight, shoot dry weight, and total grain weight under 120 kg ha−1 nitrogen. The high agronomical NUE of transgenic lines under 120 kg ha−1 N implied that the transgenic rice lines have the potential for efficient N use at a certain minimum level of N (120 kg ha−1 of nitrogen) and should be further evaluated at high N levels.

GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans

Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans

Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

IDENTIFICATION OF CDNA FRAGMENT TIGHTLY LINKED TO NV AND TM-2 LOCI IN TOMATO

Tm-2 is a resistance gene in tomato to Tomato Mosaic Virus (ToMV), located in heterochromatic region of chromosome nine. Since map based cloning difficult to perform for identify the gene on that region, we apply differential display approach by using two near-isogenic tomato lines (NILs), one without Tm-2 and the other with Tm-2 to identify cDNAs of the transcripts from the region surrounding the Tm-2 locus. Among the 150 combinations of three anchor primers and fifty arbitrary primers, 10 combinations generated cDNA polymorphic bands. Out of them, only one combination of CA6, exhibited polymorphic band under southern blot analysis, subsequently a genetic experiment showed that the CA6 locus tightly linked to the Tm-2 locus. The CA6 fragment also hybridized to genomic DNA fragments from a tomato line carrying Tm-2a, a line of L. peruvianum from which Tm-2a originated, and a tomato line carrying another Tm-2-like gene. A northern hybridization blotting result suggested that the gene corresponding to CA6 fragment was constitutively transcribed.