Plant Regeneration from Cultured Protoplasts

Xu, Zhihong; Hou, Xue

doi:10.1007/978-94-015-9253-6_2

Cited by 10 publications

(6 citation statements)

References 151 publications

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“…S2). High density of protoplasts or use of feeder cells is generally believed to stimulate cell division, by releasing stimulating substances into the medium (Xu and Xue 1999). However, this needs to be balanced since a high density of viable protoplasts will hamper distinct separation of individually developing calluses.…”

Section: Resultsmentioning

confidence: 99%

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

et al. 2016

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Key message Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. AbstractSite-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2–12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1–10 bp, but also vector DNA inserts of 34–236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-016-2062-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

et al. 2016

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“…Similarly, Schween et al (2003) found that the culture of moss protoplasts (Physcomitrella patens) with high plating density at 3 × 10 5 protoplasts mL -1 sustained viability and promoted the development of cells better than with using a lower density. These results may be due to the fact that high plating density protoplasts are able to release a sufficient amount of the physiologically active compounds, including growth factors and/or amino acids, into the medium to stimulate and sustain mitotic division, normally known as the nurse-effect (Davey et al 2005;Fu et al 2009;Schween et al 2003;Tomar and Dantu 2010;Xu and Xue 1999). In addition, Rákosy-Tican et al (2007) also found that plating efficiency could be improved for all sunflower genotypes and culture protocols (L4 and VKM regeneration protocols) when a low plating density used at 5 × 10 4 protoplasts mL -1 was adjusted to 8 × 10 5 protoplasts mL -1 .…”

Section: Discussionmentioning

confidence: 99%

The effects of cytokinin and plating density on protoplast culture of sunflower

2022

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Sunflower (Helianthus annuus L.) protoplasts were isolated from seven-day-old etiolated hypocotyls of 10 A line and four-week-old fully expanded young leaves of PI 441983 line in vitro seedlings using an enzymatic method. Purified protoplasts were collected by filtration and floatation in sucrose solution. Semi-solid protoplast culture was performed using the L4 regeneration protocol with various culture media and plating densities to achieve the highest efficiencies for protoplast culture of hypocotyl and mesophyll protoplasts of 10 A and PI 441983 lines, respectively. The concentrations in liquid L'4M medium and different plating densities were evaluated in two types of cytokinins, the adenine-type 6-benzyladenine (BA) and the phenylureatype thidiazuron (TDZ). The highest colony formation was achieved in both sunflower lines when 0.5 mgL -1 BA and 0.5 mgL -1 TDZ were applied with a high plating density (3 × 10 5 protoplasts mL -1 ). These conditions led to 38.45% and 39.40% colony formation for hypocotyl protoplasts of the 10 A line and mesophyll protoplasts of the PI 441983 line, respectively. Moreover, many hypocotyl protoplast-derived colonies developed into micro-calli. In addition, superior development of both sunflower protoplasts was observed with all plating densities when BA was used in combination with TDZ. This finding will be applicable to future sunflower hybrid production via somatic hybridization.

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“…Comparative studies have indicated that protoplasts extracted from petals outperform those derived from young leaves [11]. Furthermore, under identical enzymatic conditions, protoplasts isolated from suspension cultures exhibit the highest yield and vigor, followed by sterile seedling leaves, with guaiac tissues yielding the lowest [12]. It is important to note that the optimal choice of isolation materials and enzymatic digestion time can vary among different plant species, necessitating preliminary testing to determine the most suitable materials.…”

Section: Protoplast Isolation 21 Plant Tissues For Protoplast Isolationmentioning

confidence: 99%

Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants

Chen,

et al. 2023

IJMS

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Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts’ usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.

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Plant Regeneration from Cultured Protoplasts

Cited by 10 publications

References 151 publications

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

The effects of cytokinin and plating density on protoplast culture of sunflower

Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants

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