Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet)

Abstract

The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to <5% (<4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20-30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.

Efficient transformation of beet (Beta vulgaris) and production of plants with improved salt-tolerance

“…interested genes into the protoplasts was limited by the difficulty of regenerating plantlets from protoplasts (Hall et al, 1993(Hall et al, , 1996Sevenier et al, 1998). When calluses were used as the material for transformation, transgenic cells could be easily obtained, but the regeneration of plantlets from the transgenic calluses was difficult (Paul et al, 1987;Krens et al, 1988).…”

“…However, the majority of the beet cultivars were recalcitrant and the regeneration of plantlets from calluses was not as expected. Until now only a few genotypes were successfully transformed (Lindsey and Gallois, 1990;Paul et al, 1990;Fry et al, 1991;D'Halluin et al, 1992;Jacq et al, 1993;Hall et al, 1993Hall et al, , 1996Steen and Pedersen, 1993;Konwar, 1994;Sevenier et al, 1998). Fortunately, subsequent research had contributed to the improvement of the genetic transformation protocol in beets (Mannerlof et al, 1996;Joersbo et al, 1998;Kifle et al, 1999;Ingersoll et al, 1996;Ivic et al, 2001;Hisano et al, 2004), and the development of these genetic manipulation methodologies allowed many genes to be introduced into beets, especially in herbicide-resistance and disease-defense, resulted in great progresses in beet genetic breeding (D'Halluin et al, 1992;Mannerlof et al, 1996Mannerlof et al, , 1997Snyder et al, 1999;Dewar and May, 2000;Ivic et al, 2001).…”

Temperature and Abscisic Acid Can Be Used to Regulate Survival, Growth, and Differentiation of Cultured Guard Cell Protoplasts of Tree Tobacco

“…3A). All media used to isolate and culture B. vulgaris GCP contained w-propyl gallate, an inhibitor of lipoxygenase (EC 1.13.11.12; Krens et al, 1990Krens et al, , 1994Hall et al, 1993Hall et al, , 1994. It has been suggested that lipoxygenase plays a role in senescence (Bousquet and Thimann, 1980) and that the enzyme catalyzes synthesis of free radicals that oxidize ACC to ethylene by a nonenzymatic process (Kacperska and Kubacka-Zebalska, 1989).…”

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

“…Papers that specify the method of alginate sterilization mostly report the use of autoclaving. The results vary from species to species: for example, protoplast division frequency of 4-22% in Nicotiana tabacum (Adaoha Mbanaso and Roscoe, 1982), 11-75% in Daucus carota (Grzebelus et al, 2012), 17-92% in Citrus sinensis (Niedz, 1993) and 20-30% in Beta vulgaris (Hall et al, 1993). In reports specifying filter sterilization of alginate, plating efficiency was 1-30% in Arabidopsis thaliana (Dovzhenko et al, 2003), 6-78% in Nicotiana tabacum (Dovzhenko et al, 1998) and 0-35% in Nicotiana sanderae (Draget et al, 1988).…”