Tissue culture methods were investigated for propagation of the epiphytic orchid Neofinetia falcata H. H. Hu. The protocorms were divided into upper and lower portions and cultured. An average of 1.0-1.7 plantlets per explant were derived from upper portions which had survived at frequencies at 10-30%. Plantlets that developed from protocorms were cultured to increase the survival rate. All explants derived from upper portions of the plantlets survived, but explants from lower portions of plantlets did not. Explants derived from darkpreconditioned plantlets showed multiple bud formation when grown on medium containing 0.44 μM N 6 -benzyladenine (BA) and 5.37 μM α-naphthaleneacetic acid (NAA). Leaf explants derived from darkpreconditioned plantlets produced adventitious buds most efficiently when cultured in medium containing 0.44 μM BA and 5.37 μM NAA. These results suggest that the most suitable culture materials for bud formation in N. falcata are the upper portion of plantlets and the leaves of dark-preconditioned plantlets.

We have found that cauliflower mosaic virus (CaMV) 35S promoter-specific transgene silencing is mediated by DNA methylation in gentian (Gentiana triflora ϫ G. scabra). De novo methylation of asymmetric cytosines (CpHpH; where H is A, C, or T) sequence has been detected at the enhancer region (Ϫ148 to Ϫ85) of the 35S promoter in transgenic gentians, and is thought to be responsible for the silencing mechanism. To clarify the concept of de novo methylation, the present study examined the detailed DNA methylation profile of the entire T-DNA sequence (ca. 4 kb) integrated into transgenic gentians. Although highly methylated cytosines at CpG and CpWpG (W is A or T) sequences were broadly distributed, except in the sGFP coding region, highly methylated cytosines at CpHpH and CpCpG sequences were mainly limited to the 35S enhancer region. In addition to the previously identified de novo methylation peak (Ϫ148 to Ϫ85), another peak was discovered at Ϫ298 to Ϫ241. Electrophoretic mobility shift assays showed that gentian nuclear extracts could bind to the corresponding probes (Ϫ149 to Ϫ124 and Ϫ275 to Ϫ250), and that the probes could compete with one another for binding. Thus, a nuclear factor might be involved in the de novo methylation of the two regions. In addition, the present data indicated that the methylation patterns at CpCpG sites could be categorized as CpHpH methylation rather than CpWpG methylation.

The aim of this study was to establish an alternative method to produce clones of tomato plants by modification of the complete decapitation method, which regenerates multiple shoots from the cut surfaces of the main and lateral stems of plants grown in vivo. Shading the stems of tomato plants drastically increased the number of regenerated shoots from 2.4 in controls with unshaded stems to 36.2 in shaded stems. In shaded stems, the concentrations of chlorophyll and phenolic compounds were stable for 3 weeks after cutting, whereas these amounts increased in unshaded stems. Inhibiting the production of phenolic compounds in the shaded stem tissues was associated with an acceleration of shoot formation in vivo.

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