Toshiki Nakashima

Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium

Plant regeneration from isolated protoplasts of 8 cultivars of lisianthus, Eustoma grandiflorum (Griseb.) Schinners, has been established by using activated charcoal. Protoplasts were isolated from lisianthus leaves grown in vitro and started to divide within 3–4 days of culture, but successful colony formation was only achieved by adding gellan gum blocks containing 1% (w/v) activated charcoal immediately after culture. Colonies consisting of as many as 50–100 cells formed after 30 days of culture and were transferred to fresh medium for callus proliferation and shoot regeneration, respectively. These shoots rooted on MS medium containing 0.5 mg l−1 indolebutyric acid(IBA) and the plantlets were finally transplanted to pots. Morphological characteristics, growth habit and pollen fertility of protoplast-derived plants of one cultivar were not different from those of seed-grown plants as control.

Normalization of asparagus somatic embryogenesis using a maltose-containing medium

Summary Embryogenic calli derived from internal buds were cultured and maintained on Murashige and Skoog (MS) medium containing 2 mg/L 2,4-D, 3 % sucrose and 0.2 % gellan gum. Normal somatic embryos were induced from the embryogenic calli by pretreating with ½MS liquid medium for 7 days, sieving through 600 μm stainless steel mesh and then partially desiccating on plant growth regulator-free MS medium containing a high concentration of gellan gum (1.0% w/v) for 1 week. Furthermore, use of maltose as a sugar and/or osmoticum in the embryo induction medium promoted development of normal somatic embryos. Using these methods, non-vitrified bipolor embryos were induced 30 days after transfer (approximately 1,500 embryos per 0.1 mL packed cell volume). The somatic embryos induced on maltose-containing medium exhibited a much higher germination rate (more than 80 %) with cold treatment (14 days at 4 °C) than that induced on sucrose-containing medium.

Somaclonal and chromosomal effects of genotype, ploidy and culture duration in Asparagus officinalis L.

Chromosome and morphological variations of embryogenic calli-derived plants of gynogenic haploid, diploid, triploid and tetraploid asparagus (Asparagus officinalis L.) were investigated. Artificial tetraploids were produced using colchicine treatment of seeds of diploid cultivar, ‘Poultom’. ‘Haidel’ (2X) was crossed with the artificial tetraploids, from which one gynogenic haploid, one diploid, 6 triploid, 3 mixoploids were obtained. Embryogenic calli were first obtained from crown buds, subsequently induced to form somatic embryos, and after 30 days, induced to germinate. Chromosome variation in embryogenic calli-derived plants increased with increasing duration of subculture, particularly when low ploidy levels of plants such as haploid and diploid were used as explants. Approximately 80% of haploid-derived plants showed morphological variations such as dwarfness and abnormal morphological characteristics, although no differences were observed in cladodes and stem characteristics between other polyploid-derived plants and their parents. The data presented here would supply important fundamental information for commercial mass-propagation using somatic embryogenesis.

Production of interspecific somatic hybrid plants between Asparagus officinalis and A. macowanii through electrofusion

Interspecific somatic hybridization was performed between embryogenic callus protoplasts of Asparagus officinalis cv. Mary Washington and callus protoplasts of A. macowanii by electrofusion. By utilizing iodoacetamide (IOA) inactivation treatment for A. officinalis protoplasts and the difference of cell division capacity between the two species, one callus line, which had an ability to produce somatic embryos, was selected as a putative hybrid. Normal germination of the somatic embryos was induced only when they were exposed to a period of cold treatment (14 days at 4°C). However, the regenerated plants showed intermediate but abnormal morphological characters in vitro as well as after transfer to soil. The hybrid nature of the plants derived from this callus was confirmed by isozyme, nuclear rDNA and random amplified polymorphic DNA (RAPD) analyses and counting the chromosome number.

Polyacetylenes in hairy root cultures of Campanula medium L.

Summary Campanula medium L. hairy roots induced by infection with Agrobacterium rhizogenes A 13 grew well in three hormone-free liquid media, Murashige-Skoog (MS), Gamborg B5 (B5) and Woody Plant (WP). In these media, the hairy roots produced a high amount of the polyacetylene monoglucoside, lobetyolin (maximum content: 3.74 % in MS medium, 2.69 % in B5 medium and 3.13 % in WP medium, dry weight) whose levels were over 130 times higher than that of the intact plant.

Polyphenol production in hairy root cultures of Fragaria x ananassa

Abstract Hairy roots of Fragaria × ananassa cv. Reikou, induced with Agrobacterium rhizogenes ATCC 15834, grew well in hormone-free Murashige-Skoog (MS), root culture and Gamborg B5 liquid media. Particularly, in MS medium, hairy roots whowed maximum growth (539 mg per flask, dry wt at week 8) producing high contents of polyphenols (especially (+)-catechin (0.59% dry wt at week 8) and procyanidin B-3 (0.80% dry wt at week 7). Polyphenol contents in the intact plant (leaf blade, petiole, calyx, receptacle and root) were also investigated.