An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Abstract

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 106 ml−1) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 106 ml−1). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.

Progress in plant protoplast research

“…Rotation of the alginate/protoplast suspension during application and before polymerization minimizes layer thickness (Grzebelus et al 2012a). Also the embedding agent type affects the final outcome, possibly by interacting with genotype, osmolarity, temperature, culture system or aeration (Prange et al 2010a;Kielkowska and Adamus 2012). This is in accordance with earlier postulations on the positive effect of embedding by membrane stabilization through lipid peroxidase inhibition, the prevention of leakage of cell wall precursors or other metabolites, and lower ethylene levels (Bajaj 1989).…”

“…In Cichorium, a universal regeneration system could be accomplished by agarose bead culture (Deryckere et al 2012). For other crops, as well beads, discs, layers, thin layers or extra thin films are used (Pati et al 2005;Rakosy-Tican et al 2007;Prange et al 2010a;Grzebelus et al 2012a;Kielkowska and Adamus 2012). A major advantage of embedding systems is the easy refreshment of the cultures.…”

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

“…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).…”

“…Protoplasts embedded in alginate matrix can be dispensed as beads (Damm and Willmitzer 1988), blocks (Pan et al 2003), or layers with different thickness such as thin alginate layers (TAL, Damm et al 1989) and extra thin alginate films (ETAF, Pati et al 2005), and then cultured in liquid media. Alginate has been successfully used as a gelling agent in protoplast cultures of many species; among others, in Lotus covniculatus and Nicotiana tabacum (Pati et al 2005), Citrus sinensis (Niedz 2006), Helianthus annuus (Rákosy-Tican et al 2007), D. carota (Grzebelus et al 2012a), Beta vulgaris (Grzebelus et al 2012b), and Brassica oleracea (Kiełkowska and Adamus 2012).…”

Embedding in Filter-Sterilized Alginate Enhances Brassica Oleracea L. Protoplast Culture

“…B. napus protoplasts embedded in alginate layers showed 3-5% division frequency (Dovzhenko, 2001). There is only one report of its use for B. oleracea: embedding in alginate layers stimulated division in protoplasts but the process was strongly genotype-dependant (Kiełkowska and Adamus, 2012).…”