The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a significant step toward the commercial application of this technology.
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http://dx.doi.org/10.1007/978-1-62703-995-6_22 | DOI Listing |
STAR Protoc
January 2025
National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Electronic address:
The plastid-encoded RNA polymerase (PEP) plays an essential role in the transcription of the chloroplast genome. Here, we present a strategy to purify the transcriptionally active protein complex from transplastomic tobacco (Nicotiana tabacum) lines in which one of the PEP core subunits is fused to an epitope tag. We describe experimental procedures for designing transformation constructs for PEP purification, selection, and analysis of transplastomic tobacco plants.
View Article and Find Full Text PDFMicrob Biotechnol
December 2024
State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China.
Chlamydomonas reinhardtii, a model green alga for expressing foreign proteins, faces challenges in multigene expression and enhancing protein expression level in the chloroplast. To address these challenges, we compared heterologous promoters, terminators and intercistronic expression elements (IEEs). We transformed Chlamydomonas chloroplast with a biolistic approach to introduce vectors containing the NanoLuc expression unit regulated by Chlamydomonas or tobacco promoters and terminators.
View Article and Find Full Text PDFACS Synth Biol
December 2024
Center for Agricultural Synthetic Biology (CASB), University of Tennessee, 2640 Morgan Circle Dr., Knoxville, Tennessee 37996, United States.
Plastids represent promising targets in plant genetic engineering for many biotech applications, ranging from their use as bioreactors for the overproduction of valuable molecules to the installation of transgenes for improving plant traits. For over 30 years, routine methods of plastid transformation have relied on homologous recombination integrating vectors. However, nonintegrating episomal plasmids have recently received more attention as an innovative tool for the plastid genetic engineering of plant cells.
View Article and Find Full Text PDFPlant Cell Rep
November 2024
Center for Agricultural Synthetic Biology (CASB), University of Tennessee, 2640 Morgan Circle Dr., Knoxville, TN, 37996, USA.
This study describes an optimized plastid genetic engineering platform to produce full marker-free transplastomic plants with transgene integrated at homoplasmy in one step in tissue culture. Plastid engineering is attractive for both biotechnology and crop improvement due to natural bio-confinement from maternal inheritance, the absence of transgene positional effects and silencing, the ability to express transgenes in operons, and unparalleled production of heterologous proteins. While plastid engineering has had numerous successes in the production of high-value compounds, no transplastomic plants have been approved for use in agriculture.
View Article and Find Full Text PDFPlant Mol Biol
November 2024
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, India.
Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. In this study, two contrasting cultivars differencing in their in vitro regeneration efficiency were identified. Tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency through multiomics analysis.
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