Plant Protoplasts: Isolation, Culture and Plant Regeneration

Davey, M. R.; Anthony, Paul; Patel, Deval; Power, J. B.

doi:10.1002/9780470686522.ch9

Cited by 21 publications

(15 citation statements)

References 36 publications

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“…Isolation efficiency examined for seven carrot accessions in the present work was relatively high, reaching from 2 to 5 9 10 6 protoplasts per g of leaf tissue and around 10 6 protoplasts per g of hypocotyl tissue. These values are similar to those reported for other species (Wiszniewska and Pindel 2009;Davey et al 2010). Lower protoplast yield obtained from hypocotyls than from leaves is still sufficiently high, even to perform protoplast fusion.…”

Section: Discussionsupporting

confidence: 88%

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

Grzebelus

Szklarczyk

Barański

2011

Plant Cell Tiss Organ Cult

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An easy and effective regeneration system from leaf-and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 9 10 6 and 10 6 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf-and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds. Keywords Carrot Á Daucus carota Á Plating efficiency Á Protoplast viability Á Somatic embryogenesis Abbreviations CPP Carrot petiole protoplast medium 2,4-D 2,4-Dichlorophenoxyacetic acid ETAF Extra thin alginate film FDA Fluorescein diacetate MES 2-(N-Morpholino)ethanesulfonic acid MS Murashige and Skoog medium (1962) NAA a-Naphthaleneacetic acid PEM Proembryonic mass TAL Thin alginate layer

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Section: Discussionsupporting

confidence: 88%

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

Grzebelus

Szklarczyk

Barański

2011

Plant Cell Tiss Organ Cult

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“…Such recalcitrance of plant in vitro cultures is a widely known phenomenon-its importance was recognized from the very beginning of this research field (Benson 2000). With respect to protoplast cultures recalcitrance was noted for most monocotyledons, perennial woody species and legumes (Papadakis and Roubelakis-Angelakis 2002; Davey et al 2010). This list can be extended to include sugar beet as well, at least with respect to protoplasts of mesophyll origin (Hall et al 1993, MajewskaSawka andMünster 2003).…”

Section: Discussionmentioning

confidence: 99%

Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures

et al. 2012

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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.

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“…The efficiency of protoplast isolation and their quality, expressed by cell viability, are governed by several factors including: genotype; type of source tissue; mechanical procedures on source tissue (slicing or removal of the epidermis); conditioning of source tissue before enzyme maceration (dark or osmotic treatment); conditions of tissue digestion (i.e., composition of enzyme mixture, temperature and duration of enzyme incubation, pH of the enzyme solution, gentle agitation), and the method of protoplast isolation (Frearson et al 1973;Rao and Prakash 1995;Ortin-Parraga and Burgos 2003;Sinha et al 2003;Davey et al 2005c;Kiełkowska and Adamus 2012). When large populations of protoplasts are required, which is the norm for fusions, from 1 g of FW, between 10 5 and 10 7 viable cells should be released (Davey et al 2010). Though the protoplast yield obtained in the present study, varying from 1.4 to 4.5 9 10 6 , was genotypedependent, for all accessions it was even high enough to perform protoplast fusion.…”

Section: Discussionmentioning

confidence: 99%

Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application

Maćkowska

Jarosz

Grzebelus

2014

Plant Cell Tiss Organ Cult

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Protoplasts of four Daucus carota subspecies and three wild Daucus species were isolated from 2-weekold shoot cultures during overnight incubation in an enzyme mixture composed of 1 % (w/v) cellulase Onozuka R-10 and 0.1 % (w/v) pectolyase Y-23. Before the culture, they were embedded in autoclave-or filter-sterilized alginate solution. Modified thin alginate layer (TAL) and extra thin alginate film (ETAF) techniques were applied for protoplast immobilization. A rich mineral-organic medium based on the formulation of Kao and Michayluk supplemented with 0.1 mg l -1 2,4-dichlorophenoxyacetic acid, 0.2 mg l -1 zeatin, and optionally 100 nM phytosulfokine (PSK), a peptidyl plant growth factor, was used for protoplast culture. Plating efficiency was genotype-dependent and in 40-day-old cultures, it varied from 10 % for Daucus pusillus to 73 % for D. carota subsp. sativus. A considerably higher ability in colony formation was observed in the modified TAL culture system using filter-sterilized alginate and in the presence of PSK in the protoplast culture medium. Plant regeneration through somatic embryogenesis stimulated by PSK application occurred for five out of the seven Daucus accessions used in the present study. We believe our data may facilitate the use of wild Daucus in somatic hybridization with cultivated carrot.

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Plant Protoplasts: Isolation, Culture and Plant Regeneration

Cited by 21 publications

References 36 publications

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures

Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application

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