Studies of cotyledon protoplast cultures from B napus, B. campestris and B. oleracea. II: Callus formation and plant regeneration

Abstract

Cotyledons from twelve cultivars ofBrassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about I month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2-3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1-22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast cultures derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.

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

“…Differentiation in the in vitro cultures is generally known to be genotype-specific, and Brassica species are no exception (Jourdan and Earle 1989; Loudon et al 1989; Zhao et al 1995; Hansen et al 1999a), as confirmed in this study. Mesophyll- and hypocotyl-derived protoplasts were successfully isolated with satisfactory yields, viability, and division frequencies, especially for the red form of cabbage cv.…”

“…The cytoplasmic male sterility system has been introduced to B. oleracea through asymmetric protoplast fusion (Sigareva and Earle 1997). Positive results, as mentioned above, have been achieved using B. oleracea accessions that were selected for high plant regeneration capacity, but the ability to undergo meiotic divisions and regenerate shoots remained problematic (Jourdan and Earle 1989; Zhao et al 1995; Hansen et al 1999a). Although intensive screening of protoplast culture conditions has been performed, the effects of genotype have still overridden most efforts to improve protocols for plant regeneration from protoplast culture.…”

Peptide Growth Factor Phytosulfokine-α Stimulates Cell Divisions and Enhances Regeneration from B. oleracea var. capitata L. Protoplast Culture

“…Brassica oleracea is a plant species that includes many common foods such as cabbage, broccoli, kale, kohlrabi, and cauliflower. The extensive screening of protoplast culture in B. oleracea showed that the effect of genotype overrides the wide use of protoplast techniques in the breeding and biotechnology of this species (Jourdan et al 1990;Kik and Zaal 1993;Zhao et al 1995;Adamus 2012, 2014); therefore, improvements in the methodology aiming to increase the mitotic activity of cultured protoplasts and plant regeneration are needed.…”

Can protoplast production from in vitro cultured shoots of Tanacetum vary during the season?

“…For example, protoplasts, cells lacking a cell wall, are fragile and sensitive to changes in culture conditions. However, there have been relatively few studies on the effects of light on donor tissues used for protoplast isolation (Zhao et al 1995, Geng-Guang 1996. The effect of light on cell wall structure and chemical composition has been reported however (Parvez et al 1996).…”