Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences
| Autor: | Elisabeth A. Mudd, Anil Day, Panagiotis Madesis, Elena Martin Avila |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Genetics Homoplasmy fungi food and beverages Biology 01 natural sciences Marker gene 03 medical and health sciences 030104 developmental biology Chloroplast DNA Recombinase Direct repeat Plastid Homologous recombination 010606 plant biology & botany Transplastomic plant |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071614716 |
| DOI: | 10.1007/978-1-0716-1472-3_4 |
| Popis: | Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops. |
| Databáze: | OpenAIRE |
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