Optimised expression cassettes of hpt marker gene for biolistic transformation of Miscanthus sacchariflorus

Sobańska, Karolina; Cerazy-Waliszewska, Joanna; Kowalska, Monika; Rakoczy, Magdalena; Podkowiński, Jan; Ślusarkiewicz‐Jarzina, A.; Ponitka, A.; Jeżowski, S.; Pniewski, Tomasz

doi:10.1016/j.biombioe.2019.105255

Cited by 4 publications

(3 citation statements)

References 26 publications

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“…Two species of Miscanthus have been successfully transformed using Agrobacterium tumefaciens or particle bombardment: Msa [18][19][20], and Msi [21][22][23][24][25]. Trieu [26] gave specific examples for transformation of Mxg which is confirmed in this report.…”

Section: Introductionsupporting

confidence: 67%

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Transformation and gene editing in the bioenergy grass Miscanthus

Trieu

Belaffif

Hirannaiah

et al. 2022

Biotechnol Biofuels

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Background Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. Results We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. Conclusions We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop. Graphical Abstract

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Section: Introductionsupporting

confidence: 67%

“…Plant growth regulators and non-standard additives included with amounts given per liter; further details provided in text. Msa and Msi media designations followed by -1; M x g media followed by -2 Msa M. sacchariflorus , Msi M. sinensis , M x g M. x giganteus 1 Reference [ 61 ] 2 Sugar alcohols based on [ 19 ] 3 Reference [ 26 ]…”

Section: Resultsmentioning

confidence: 99%

Transformation and gene editing in the bioenergy grass Miscanthus

Trieu

Belaffif

Hirannaiah

et al. 2022

Biotechnol Biofuels

View full text Add to dashboard Cite

show abstract

“…Since Miscanthus plants are self-incompatible, this precluded the use of seeds for clonal regeneration of the mother plant, and explant choices for Mxg were limited due to its sterility. Two species of Miscanthus have been successfully transformed using Agrobacterium tumefaciens or particle bombardment: Msa [18, 19,20], and Msi [21,22,23,24,25]. Trieu [26] gave speci c examples for transformation of Mxg which is con rmed in this report.…”

Section: Introductionmentioning

confidence: 61%

Transformation and gene editing in the bioenergy grass Miscanthus

Trieu

Belaffif

Hirannaiah

et al. 2022

Preprint

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Background: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites.Results: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1-26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors.Conclusions: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

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Developing a long-term and powerful in vitro culture and Agrobacterium-mediated transformation system for Miscanthus sinensis (Poaceae)

Zhou

et al. 2021

Industrial Crops and Products

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Optimised expression cassettes of hpt marker gene for biolistic transformation of Miscanthus sacchariflorus

Cited by 4 publications

References 26 publications

Transformation and gene editing in the bioenergy grass Miscanthus

Transformation and gene editing in the bioenergy grass Miscanthus

Transformation and gene editing in the bioenergy grass Miscanthus

Developing a long-term and powerful in vitro culture and Agrobacterium-mediated transformation system for Miscanthus sinensis (Poaceae)

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