Abstract
Plant biotechnology provides a powerful solution to boost agricultural productivity and nutritional quality. The development process of a transgenic crop includes multiple steps that consist of gene isolation for a target trait, generation of T0 transgenic crops, characterization of the transgene, evaluation of agronomic performance of transgenic crops, selection of elite transgenic lines and assessment of target trait efficacy. Here, we developed elite insect-resistant transgenic rice plants that may satisfy the standards of biosafety assessments. We made a construct with the insecticide cry1Ac gene for a target trait. A total of 310 T0 transgenic lines were generated and underwent extensive analysis. We selected four T3 lines that contain a single-copy transgene inserted into intergenic regions of the rice genome. During this process, we critically analyzed the transgenic lines with five checkpoints that include single copy of transgene, its integration into intergenic region, clean T-DNA arrangement, stability of transgene through generations and substantial equivalence of transgenic plants in agronomic traits other than insect resistance. Consequently, we obtained insect-resistant transgenic rice plants that can be used in practical agriculture.
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Acknowledgments
This work was supported by the Rural Development Administration under the Next-Generation BioGreen 21 Program (Grant No. PJ011135 to J.-K.K) and by the Basic Science Research Program through the National Research Foundation of Korea (Grant No. NRF-2014R1A2A1A11051690 to J.-K.K). We thank the Kyungpook National University for providing a rice paddy field.
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Dong-Keun Lee, Su-Hyun Park and So-Yoon Seong contributed equally to this work as co-first authors.
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11816_2016_410_MOESM1_ESM.pptx
Supplementary Figure S1. Rice RbcS3::TP3:cry1Ac vector. The T-DNA of construct RbcS3:TP3:cry1Ac consists with cry1Ac gene controlled under RbcS3 promoter and an herbicide resistant selection marker, bar gene under the CaMV 35S promoter between left and right T-DNA borders. The plasmid DNA backbone comes from pPZP200 including replication (pVS1 RepA) and stability (pVS1 StaA) region in Agrobacterium. RbcS3, Rubisco small subunit 3; TP3, transit peptide 3; PinII, potato proteinase inhibitor II; NOS, nopaline synthase terminator; SmR, spectinomycin resistant gene. Supplementary Figure S2. Positive controls of TaqMan q-PCR. (A) Vector map for PGD1::AP26 transgenic rice. RB, right border; LB, left border; PGD1, rice PGD1 promoter; AP26, rice AP26 transcription factor; 3’NOS, nopaline synthase terminator from Agrobacterium, Black bar and arrow indicate a probe and a PstI enzyme site for southern blot analysis. (B) Southern blot analysis of three independent single-copy homozygote transgenic lines. 10 µg genomic DNA were digested with PstII restriction enzyme. PstI digested fragments of each genomic DNA were hybridized with 32 P-labeled PGD1 probe. Red arrows indicate single-copy bands. M indicates DNA marker. Supplementary Figure S3. Identification of T-DNA clean borders. For each transgenic rice line, first lane is a PCR product for the RB region, second lane is a PCR product for LB region and final lane is a control PCR product for UBIQUITIN. Either R (right border) or L (left border), or both R, L at the bottom of gel of each line indicates clean border information after sequencing. Supplementary Figure S4. PCR-based retrieval for the whole T-DNA region. PCR primers (arrows) were designed to cover whole T-DNA region between right and left borders (6.2 kb). Seven lines show exact 6.3 kb PCR band. Six lines show unclear or no PCR amplification. Supplementary Figure S5. Genomic DNA Southern blot analysis of the transgenic lines. (A-C) As Figure 4, 5ug genomic DNAs of T2 transgenic rice and nontransgenic (NT) controls were loaded after digestion with indicated two different enzymes. This genomic DNA Southern blot analysis shows information of the copy number in the transgenic rice lines, which are not included in Figure 4. Supplementary Figure S6. Transgenic plants with substantial equivalence in agronomic traits. Representative plants in ripening stage grown in the rice paddy field were transferred to pots for photographing. Supplementary Table S1. Summary of the clean T-DNA borders (PPTX 9369 kb)
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Lee, DK., Park, SH., Seong, SY. et al. Production of insect-resistant transgenic rice plants for use in practical agriculture. Plant Biotechnol Rep 10, 391–401 (2016). https://doi.org/10.1007/s11816-016-0410-y
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DOI: https://doi.org/10.1007/s11816-016-0410-y