Fig 1.
Schematic diagram of the T-DNA region of binary vectors for marker elimination and an DNA excised product.
Restriction sites within the MCS were unique digestion sites in the vector. The structure of pCMF was the same to as of pHWMF except that the modified FLP gene was replaced with the native FLP gene. pCMF-TC was derived from inserting the NtTC gene, which is a tocopherol cyclase ortholog isolated from tobacco, into multiple cloning sites of pCMF. After gene excision, the CaMV 35S promoter was inserted adjacent to the NtTC coding region. P35SF, HPTR, and TCR primers were designed to detect the DNA excision. The PCR product amplified with P35SF/TCR would be 1.5 kb if DNA excision occurred and 5.7 kb otherwise. P35S, CaMV 35S gene promoter; hpt, hygromycin phosphotransferase gene; T35S, 35S CaMV gene terminator; Ppod, stress-inducible peroxidase gene promoter; FLP; recombinase gene from Saccharomyces cerevisiae; Tnos: Agrobacterium nopaline synthase gene terminator; MCS, multiple cloning site; LB, left border; RB, right border; NtTC, tocopherol cyclase gene isolated from tobacco; FRT, FLP recognition site.
Table 1.
Differences in codon usage between Oryza sativa, native FLP, and S. cerevisiae and codons used for mFLP gene synthesis.
Fig 2.
PCR and sequencing analysis for identification of transgenes and gene excision in transgenic T0 plants.
(a) PCR results with the primer pair P35S/TCR using the genomic DNAs prepared from fresh leaves of transgenic plants after regeneration. (b) Sequence analysis to confirm gene excision from the genome of transgenic T0 plants. The PCR fragments amplified using the primers P35S and TCR were sequenced. (c) PCR results with the primer sets P35SF/HTPR and P35SF/TCR using genomic DNAs prepared from the mature leaves of five randomly selected transgenic T0 lines. M, molecular marker. Line numbers are indicated at the top of each lane.
Fig 3.
Southern blot analysis of T1 progenies of the transgenic lines.
Genomic DNA was digested with EcoRV, which created one cut in pCMF-TC, and hybridized with the DIG-labeled NtTC (a) or hpt (b) probe. M, DIG-labeled molecular marker; P, positive control; lanes 1–5, progenies of the TC2 line; lanes 6–8, progenies of the TC3 line.
Fig 4.
Identification of the T-DNA in transgenic rice plants to select marker-free transgenic rice lines.
(a) PCR analysis of T1 transgenic rice plants for marker excision. (b) RT-PCR analysis of NtTC and hpt from leaves of T1 transgenic rice plants. The rice tubulin (tub) gene was used for normalization.
Fig 5.
Germination test of T1 progenies derived from the transgenic lines.
Seeds of the transgenic primary plants and nontransgenic wild-type plants were sown on germination medium containing hygromycin or without antibiotics.
Fig 6.
Tocopherol contents from seeds of marker-free transgenic rice plants and wild-type rice plants.
Black and gray bars correspond to transgenic TC3-1 plants and wild-type rice plants. Values are means ± SDs (n = 5), and student’s t tests were used to compare values obtained from transgenic lines to those of the wild-type plants. ** p < 0.01.
Fig 7.
Schematic showing the production of selectable marker-free transgenic rice.