PEG-mediated, Stable, Nuclear and Chloroplast Transformation of Cyanidioschizon merolae

Zienkiewicz, Maksymilian; Krupnik, Tomasz; Drożak, Anna; Kania, Kinga

doi:10.21769/bioprotoc.3355

Cited by 9 publications

(9 citation statements)

References 18 publications

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“…The agitation of algal cells in the presence of glass beads, polyethylene glycol (PEG), and foreign DNA has become a standard protocol for the delivery of foreign DNA into the model green microalgae C. reinhardtii and other species as Cyanidioschyzon merolae, Chlorella vulgaris, and Dunaliella salina [ 14 , 19 , 20 , 21 ]. This protocol has been adopted for nuclear and chloroplast transformation and can be applied to cell wall deficient cell lines or those with a cell wall when chemo/enzymatic removal is performed before transformation (as with Chlorella strains) [ 13 , 14 , 15 , 22 , 23 , 24 ].…”

Section: Traditional Algal Transformation Techniquesmentioning

confidence: 99%

Gene Delivery Technologies with Applications in Microalgal Genetic Engineering

Gutiérrez

Lauersen

2021

Biology

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Microalgae and cyanobacteria are photosynthetic microbes that can be grown with the simple inputs of water, carbon dioxide, (sun)light, and trace elements. Their engineering holds the promise of tailored bio-molecule production using sustainable, environmentally friendly waste carbon inputs. Although algal engineering examples are beginning to show maturity, severe limitations remain in the transformation of multigene expression cassettes into model species and DNA delivery into non-model hosts. This review highlights common and emerging DNA delivery methods used for other organisms that may find future applications in algal engineering.

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Section: Traditional Algal Transformation Techniquesmentioning

confidence: 99%

Gene Delivery Technologies with Applications in Microalgal Genetic Engineering

Gutiérrez

Lauersen

2021

Biology

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“…The PEG-mediated transformation begins with the protoplast suspension acquisition by performing various enzymatic treatments upon the plant cells. The protoplast suspension is then allowed to undergo a centrifugation process [ 2 , 32 , 33 , 36 , 37 , 41 , 45 , 48 , 64 ]. Further purification step is taken to obtain chloroplast suspension which is free of contaminants and other cellular components.…”

Section: Main Textmentioning

confidence: 99%

“…This was followed by the introduction of 60% PEG solution to the mixture and further incubated for a specific duration. The concentration of PEG solution and the incubation period is depending on the amount of the chloroplast and DNA construct used [ 2 , 32 , 33 , 36 , 37 , 41 , 45 , 48 , 64 ]. After 3 days of introducing PEG to the mixture, the chloroplast is grown in selectable conditions such as with the presence of antibiotics and antifungals, to avoid the growth of bacteria and fungi [ 64 ].…”

Section: Main Textmentioning

confidence: 99%

“…The concentration of PEG solution and the incubation period is depending on the amount of the chloroplast and DNA construct used [ 2 , 32 , 33 , 36 , 37 , 41 , 45 , 48 , 64 ]. After 3 days of introducing PEG to the mixture, the chloroplast is grown in selectable conditions such as with the presence of antibiotics and antifungals, to avoid the growth of bacteria and fungi [ 64 ]. The transformants are monitored by the expression of the selectable marker incorporated in the DNA construct, and the transformation efficiency is also identified.…”

Section: Main Textmentioning

confidence: 99%

“…The transformants are monitored by the expression of the selectable marker incorporated in the DNA construct, and the transformation efficiency is also identified. Further confirmation on the identification of the transformants is performed by using PCR [ 2 , 32 , 33 , 36 , 37 , 41 , 45 , 48 , 64 ]. After the screening of the transformants, the transformed chloroplast is treated with osmotic buffer and sorbitol followed by the culturing of transformant on regeneration medium for recovery process [ 2 , 32 , 33 , 36 , 37 , 41 , 45 , 48 , 64 ].…”

Section: Main Textmentioning

confidence: 99%

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Gene introduction approaches in chloroplast transformation and its applications

Kumar

Ling

2021

Journal of Genetic Engineering and Biotechnology

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Background Chloroplast is a type of plastid that is believed to be originated from ancestral cyanobacteria. Chloroplast besides being a major component for photosynthesis, also takes part in another major plant metabolism, making it one of the major components of plants. Main body Chloroplast transformation is an alternative and better genetic engineering approach compared to the nuclear transformation that has been widely applied in plant genetic engineering. Chloroplast transformation has exhibited various positive effects as compared to nuclear transformation. This is a more preferred technique by researchers. To carry out chloroplast transformation, the vector design must be performed, and a selectable marker needs to be incorporated before the chloroplast could uptake the construct. The common way of introducing a gene into the host, which is the chloroplast, involves the biolistic, PEG-mediated, carbon nanotubes carriers, UV-laser microbeam, and Agrobacterium-mediated transformation approaches. Apart from discussing the processes involved in introducing the gene into the chloroplast, this review also focuses on the various applications brought about by chloroplast transformation, particularly in the field of agriculture and environmental science. Conclusion Chloroplast transformation has shown a lot of advantages and proven to be a better alternative compared to nuclear genome transformation. Further studies must be conducted to uncover new knowledge regarding chloroplast transformation as well as to discover its additional applications in the fields of biotechnology.

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