Two carotenoid biosynthetic genes, phytoene synthase (Psy) and carotene desaturase (CrtI) linked via synthetic 2A sequence under control of CaMV 35S promoter (two T0 plants 5 and 6) or β- conglycinin promoter (three T0 plants 7, 13 and 16) were transformed into soybean variety Kwangan. After agronomic and phenotypic selection at early generations, T5 progeny of PAC soybean were analyzed by Southern blot to confirm T-DNA copy numbers. A total of 27 homologous lines derived from one of three T0 plants (line 7 under the control of β- conglycinin promoter) with one copy T-DNA insertion, were separated and planted into greenhouse. Flanking sequence analysis was carried out on one of homologous line 6-2-3 and results indicated the T-DNA was intergenic inserted into chromosome 14 from 10,873,131 to 10,872,998 base of soybean chromosome. T-DNA insertion structure, flanking sequence and inserted gene expressions need to be analyzed in the further study.
Resveratrol rice Iksan526 was developed by overexpession of T-DNA (RB::P-Ubi::RS::T-NOS::P-35S::PAT::T-35S::LB) in rice variety Dongjin. To confirm one locus insertion of T-DNAs, Mendelian genetic analysis was carried out on selection marker bar gene and objective RS gene separately by using a F2 population derived from a cross of Dongjin/Iksan526 (T6). A total of 450 four-leaf-old plants from F2 population were treated by 0.3% basta, and a phenotypic separation ratio of 3:1 (321 survival: 129 dead, p>0.90) complied with Mendelian inheritance indicating one locus insertion of bar gene. Genotypic separation was analyzed by using PCR with specific primers for 300 plants, which were selected from 321 survival plants after phenotypic separation. Results revealed a ratio 1:2 of homologous to heterozygous (92:208, p>0.90), which further confirmed one locus insertion of RS gene. In addition, comparison on agronomic traits and resveratrol contents between transgenic rice and the donor variety were launched to evaluate the phenotypic performance over multi-generations (years).