Abstract:Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. 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 efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell wal… Show more
“…Although there are some known protocols for protoplastto-plant systems in a number of species (Davey et al 2005;Godel-Jędrychowska et al 2019), especially cultivated species, the efficiency to regenerate complete plant or even callus tissue is still low or occasional. In comparison to agricultural crops, there is a limited number of reports on protoplast cultures applied to medicinal plants (Pan et al 2003;Thomas 2009;Aoyagi 2011;Eeckhaut et al 2013) and only two of them refer to N. sativa (Jha and Roy 1979) and N. damascena (Binding et al 1981).…”
Section: Figmentioning
confidence: 99%
“…about 3×10 5 cells per g of callus fresh weight, with the average cell viability reaching 60%. These values have been considered sufficient for most protoplast-based applications (Davey et al 2010;Maćkowska et al 2014;Godel-Jędrychowska et al 2019). Callus tissue is a much more convenient protoplast source than the more common suspension cultures, which are an excellent source of protoplasts due to their high embryogenic ability, however, the establishment and maintenance of suspension culture are laborious and time consuming (Grzebelus et al 2012a).…”
In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus.
The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time.
“…Although there are some known protocols for protoplastto-plant systems in a number of species (Davey et al 2005;Godel-Jędrychowska et al 2019), especially cultivated species, the efficiency to regenerate complete plant or even callus tissue is still low or occasional. In comparison to agricultural crops, there is a limited number of reports on protoplast cultures applied to medicinal plants (Pan et al 2003;Thomas 2009;Aoyagi 2011;Eeckhaut et al 2013) and only two of them refer to N. sativa (Jha and Roy 1979) and N. damascena (Binding et al 1981).…”
Section: Figmentioning
confidence: 99%
“…about 3×10 5 cells per g of callus fresh weight, with the average cell viability reaching 60%. These values have been considered sufficient for most protoplast-based applications (Davey et al 2010;Maćkowska et al 2014;Godel-Jędrychowska et al 2019). Callus tissue is a much more convenient protoplast source than the more common suspension cultures, which are an excellent source of protoplasts due to their high embryogenic ability, however, the establishment and maintenance of suspension culture are laborious and time consuming (Grzebelus et al 2012a).…”
In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus.
The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time.
“…The obtained results of cell viability and mitotic activity show that the chosen electric conditions had no adverse effect on carrot protoplast cultures. The available reports describing carrot cell behaviour in protoplast cultures present a similar level of cell viability before culture and a similar cell growth kinetics with respect to entering into the first mitotic division as well as formation of cell aggregates 6 , 68 , 81 , 82 . Figure 6 A 20-day-old hybrid cells aggregate after DH-GFP and DH-RFP protoplast fusion.…”
Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.
“…Molecular signalling exerted through peptide hormones plays a key part in regulating plant growth and reproduction [1][2][3]. These phytohormones are also responsible for an environmental response crucial to plant survival and adaptation to different conditions [2,4].…”
Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor’s island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel “druggable” binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.
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