Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex

Abe, Jun; Hirano, Naoko; Komiya, Ayumi; Kanda, Naho; Fujiwara, Anri; Hori, Sachie; Tsuchikane, Yuki; Sekimoto, Hiroyuki

doi:10.21769/bioprotoc.1813

Cited by 6 publications

(6 citation statements)

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“…Second, the efficiency of transformation is not very high; the average transformation efficiency following selection in 0.3 µg ml −1 phleomycin was 5.5 × 10 −6 cells or 0.03 transformants µg –1 DNA (Abe et al ., 2011). Therefore, many cells must be prepared in order to establish a sufficient number of transformants (Abe et al ., 2016), which requires a large amount of incubator space. We have tried to establish other transformation methods, such as electroporation using protoplasts.…”

Section: Introductionmentioning

confidence: 99%

Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum‐strigosum‐littorale complex by square‐pulse electroporation

et al. 2021

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The zygnematophycean algae occupy an important phylogenetic position as the closest living relatives of land plants. Reverse genetics is quite useful for dissecting the functions of genes. However, this strategy requires genetic transformation, and there are only a few reports of successful transformation in zygnematophycean algae.Here, we established a simple and highly efficient transformation technique for the unicellular zygnematophycean alga Closterium peracerosum-strigosum-littorale complex using a square electric pulse-generating electroporator without the need for cell wall removal.Using this method, the transformation efficiency increased > 100-fold compared with our previous study using particle bombardment. We also succeeded in performing CRISPR/Cas9based gene knockout using this new method.Our method requires only small amounts of labor, time and incubator space. Moreover, our technique could also be utilized to transform other charophycean algae with available genome information by optimizing the electric pulse conditions.

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Section: Introductionmentioning

confidence: 99%

Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum‐strigosum‐littorale complex by square‐pulse electroporation

et al. 2021

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“…Recently, several genetic and genomic tools have been developed for Closterium . Reverse genetic analysis has been performed by knockdown (Hirano et al ., ) or CRISPR/Cas9‐based knockouts (Kanda et al ., ) using a stable nuclear transformation system with particle bombardment (Abe et al ., , ).…”

Section: Introductionmentioning

confidence: 99%

The genus Closterium, a new model organism to study sexual reproduction in streptophytes

Tsuchikane

Sekimoto

2018

New Phytologist

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Contents Summary99I.Introduction99II.Life cycle of Closterium100III.Sexual reproductive processes in the heterothallic Closterium peracerosum‐strigosum‐littorale complex101IV.Homothallism in the C. psl. complex102V.Sexual reproduction and inheritance of mating types in Closterium ehrenbergii102VI.mt‐determining gene of the C. psl. complex103VII.Future perspectives103Acknowledgements103References103 Summary Closterium occupies a key phylogenetic position as an ancestor of land plants and is the best‐characterized Charophycean alga in terms of the process of sexual reproduction. Zygospores form as a result of sexual reproduction between genetically determined mating type plus (mt+) and mating type minus (mt−) cells in heterothallic strains, or between clonal cells in homothallic strains. Here we review knowledge on the intercellular communication and mating type determination for successful sexual reproduction in Closterium. Using genomic information and transgenic techniques, the genus could be a model organism to study the mechanisms and evolution of sexual reproduction in streptophytes.

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“…Success in achieving this goal will require optimization of the electroporation as well as testing alternative approaches of transgene delivery. This includes alternative procedures for direct uptake of DNA by protoplasts [19, 25], particle bombardment [57, 58] as well as Agrobacterium -mediated conjugative transformation [59]. Also, it will be important to determine whether stable transformation occurs via homologous recombination (like in Physcomitrella , [60, 61]) or random integration (like in most land plants, [62]).…”

Section: Discussionmentioning

confidence: 99%

Regeneration and transient gene expression in protoplasts of Draparnaldia (chlorophytes), an emerging model for comparative analyses with basal streptophytes

Caisová

Jobe

2019

Plant Methods

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Background Green plants comprise two lineages: (1) the streptophytes that colonised land and (2) the chlorophytes that have adaptations to land but remained mostly aquatic. To better understand what made streptophytes so successful, we are currently establishing the chlorophyte alga Draparnaldia sp. (Chaetophorales, Chlorophyceae) as a model for comparative analyses between these two lineages. However, establishing Draparnaldia as a valuable model requires that it can be transformed. Thus, our goal is to develop a transformation protocol for this alga. Results We have established the first transformation protocol for Draparnaldia . This protocol is based on protoplast transformation by electroporation. It includes instructions on protoplast isolation, regeneration and transient transfection. It also provides a list of the effective selective agents for future Draparnaldia transformations. Conclusions Our protocol opens a way for Draparnaldia functional genomics analyses. Moreover, it also provides an important base for establishment of stable transformation. Electronic supplementary material The online version of this article (10.1186/s13007-019-0460-6) contains supplementary material, which is available to authorized users.

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Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex

Cited by 6 publications

References 5 publications

Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum‐strigosum‐littorale complex by square‐pulse electroporation

Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum‐strigosum‐littorale complex by square‐pulse electroporation

The genus Closterium, a new model organism to study sexual reproduction in streptophytes

Regeneration and transient gene expression in protoplasts of Draparnaldia (chlorophytes), an emerging model for comparative analyses with basal streptophytes

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