28 research outputs found
Characterization of a Genomic Region under Selection in Cultivated Carrot (Daucus carota subsp. sativus) Reveals a Candidate Domestication Gene
Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae)
Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus Is Affected by Phytosulfokine (PSK)
Phytosulfokine- (PSK), a peptidyl plant growth factor, has been recognized as a promising
intercellular signaling molecule involved in cellular proliferation and dedi erentiation. It was shown
that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading
to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in
e ciency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical
components of the cell walls during the protoplast regeneration process. So far, especially for the carrot,
a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein
(AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells.
Even less is known about the correlation between wall regeneration and the presence of PSK during
the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during
protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19,
LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate
a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development,
and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures
A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum
The CRISPR/Cas9 system enables precise genome editing and is a useful tool for
functional genomic studies. Here we report a detailed protocol for targeted genome
editing in the model grass Brachypodium distachyon and its allotetraploid relative
B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA
efficiency, Agrobacterium-mediated transformation and the selection and analysis of
regenerated plants. In B. distachyon, we targeted the gene encoding phytoene
desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway.
The albino phenotype of mutants obtained confirmed the effectiveness of the protocol
for functional gene analysis. Additionally, we targeted two genes related to cell wall
maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin
methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent
kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were
targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions
or deletions, resulting in frameshift mutations that, may lead to premature stop codons.
Because of the close phylogenetic relationship between Brachypodium species and key
temperate cereals and forage grasses, this protocol should be easily adapted to target
genes underpinning agronomically important traits
A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum
The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits
Response of carrot protoplasts and protoplast-derived aggregates to selection using a fungal culture filtrate of Alternaria radicina
Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures
The aim of this work was to improve plating efficiency of sugar beet mesophyll protoplast cultures. Preliminary experiments showed that cultures of good quality, viable protoplasts were obtained in rich media based on the Kao and Michayluk formulation and with the calcium alginate as an embedding matrix. Nevertheless, in these cultures cell divisions were either not observed or very seldom confirming earlier reported recalcitrance of sugar beet protoplasts. The recalcitrant status of these cultures was reversed upon application of exogenous phytosulfokine (PSK)—a peptidyl plant growth factor. The highest effectiveness of PSK was observed at 100 nM concentration. Plating efficiencies obtained in the presence of PSK reached approximately 20% of the total cultured cells. The stimulatory effect of phytosulfokine was observed for all tested breeding stocks of sugar beet. Our data indicate that PSK is a powerful agent able to overcome recalcitrance of plant protoplast cultures