Toshinari Godo

In vitro propagation utilizing suspension cultures of meristematic nodular cell clumps and chromosome stability of Lilium×formolongi hort.

Abstract Suspension cultures composed of meristematic nodular cell clumps of Lilium×formolongi hort. line R13 have been maintained in liquid MS medium containing 1 mg/l picloram for over 4 years. All of the cell clumps checked were diploid and these clumps maintained a high potential for shoot regeneration. The fresh weight of meristematic nodular cell clumps increased 2.5-fold during 14 days of cultivation in a flask or bioreactor. The sucrose concentration in the regeneration medium and the addition of BA affected shoot regeneration from the cell clumps. More than 85% of the clumps produced shoots on solidified regeneration medium (1/2 MS medium containing 5–10 g/l sucrose) after 2 months of culture. A sucrose concentration more than 20 g/l resulted in the reduction of shoot regeneration. Addition of BA to the solidified medium was effective in increasing shoot numbers and stimulating the induction of shoots. The plantlets regenerated from cell clumps were all diploid with 24 chromosomes and grew normally to flowering. It is expected that about 4.5×1010 plantlets per year could be produced from 1 g of meristematic nodular cell clumps through this propagation method.

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

SummarySuspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.

Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus Tricyrtis

SummaryThe liliaceous perennial plants, Tricyrtis spp., are cultivated as ornamental plants in Japan. Natural populations of several Japanese Tricyrtis spp. are severely threatened by indiscriminate collection and habitat destruction. In this study, a plant regeneration system based on somatic embryogenesis has been developed for efficient clonal propagation of T. hirta, T. hirta var. albescens, T. formosana, T. formosana cv. Fujimusume, T. flava ssp. ohsumiensis, and T. macrantha ssp. macranthopsis. Flower tepal explants of these genotypes were cultured on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) alone or in combination with N-(1,2,3-thiadiazol-5-yl)-N′-phenylurea (thidiazuron, TDZ). Calluses induced on media containing 2,4-D produced somatic embryos following their transfer to a plant growth regulator-free medium, indicating that these calluses were embryogenic. A combination of 4.5μM2,4-D and 0.45 μM TDZ was most effective for inducing embryogenic calluses from tepal explants. Among various explant sources, filaments were most suitable for inducing embryogenic calluses on a medium containing 4.5μM 2,4-D and 0.45 μM TDZ. Embryogenic calluses were only obtained from filament explants for T. macrantha ssp. macranthopsis. Embryogenic calluses could be maintained by subculturing monthly onto the same medium, and a 1.5–3.5-fold increase in fresh weight was obtained after 1 mo. of subculture. Depending on the plant genotype, 50–500 somatic embryos per 0.5g fresh weight of embryogenic callus was obtained 1 mo. after transfer to a plant growth regulator-free medium. Most of the embryos developed into plantlets, and they were successfully acclimatized to greenhouse conditions. Regenerated plants showed no alteration in the ploidy level as indicated by chromosome observation and flow cytometric analysis.

Micropropagation of Lysionotus pauciflorus Maxim. (Gesneriaceae)

Numerous shoots were directly regenerated from the leaf explants of Lysionotus pauciflorus on the MS medium containing 0.5-2 microM NAA with or without 1 microM BA. The calli were induced from the leaves on MS medium supplemented with 2 microM 2, 4-D. The calli proliferated about four times in fresh weight in the liquid medium of the same composition as the callus induction medium after 4 weeks of culture on a rotary shaker at 100 rpm. Shoots were induced from these calli on the regeneration medium amended with 32 microM BA or 0.5 microM zeatin. Regenerated shoots rooted easily on (1/2) MS medium without any plant growth regulators. Most of the regenerants from callus were diploid, whereas eight of 66 acclimatized plantlets were tetraploid determined by flow cytometric analysis. Furthermore, flow cytometric analysis of calli also revealed tetraploidy.