An efficient method for plant regeneration from suspension cultures of onion was studied. For callus induction, sterilized seeds were inoculated on solid MS medium supplemented with 50μM 4-FPA, 1μM 2iP, 1g/l casein hydrolysate and 0.1M sucrose. For suspension culture, callus was mechanically disaggregated and transferred into liquid MS medium with 10mM MES added, and placed on a rotary shaker. Suspension cultures were routinely subcultured. After pre-culture in liquid medium which increased sucrose concentration to 0.2M, two types of cell clumps developed, a deep yellow, small type, and a light yellow, large type. The large type showed higher regenerative ability than the small type. Frequent regeneration of plantlets occurred on solid MS medium supplemented with 1-5μM 2iP. Regenerated plantlets developed to whole plants.

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Transgenic phalaenopsis plants have been obtained by particle bombardment with a pneumatic particle gun device. All promoters of cauliflower mosaic virus (CaMV) 35S, maize ubiquitin and rice actin genes could be expressed in phalaenopsis using transient assay of β-glucuronidase (gus) gene as a reporter. Bialaphos resistance gene (bar) which confers tolerance to herbicide bialaphos, was used as a selectable marker. Protocormlike bodies (PLBs) of phalaenopsis derived from the leaf segment culture were bombarded by gold particles coated with pMSP38 and pWI-GUS DNA containing the bar gene and the gus gene respectively driven by 35S promoter. Newly-formed PLBs were selected on the medium including bialaphos. Finally seven transgenic plants resistant to bialaphos were obtained, and one of them contained and expressed gus gene. PCR analysis confirmed their transgenic nature and western analysis and histochemical GUS assay showed the expression of bar and gus gene respectively.

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The effects of sampling timing of runners on plantlet growth were examined in shoot apex culture of strawberry ‘Belle rouge’.
Those plantlets sampled from June 23 to Aug. 4 differed in development from those plantlets sampled from Aug. 18 to Sept. 21. The plantlets sampled at the late term had fewer developed leaves and greater plant heights than those sampled at the early term.
Auxin activities of apical meristems of stock plants were examined using Avena bioassay. The auxin activity was highest on the first date of sampling and then declined. These results suggested that shoot apex excised at the early term would be desirable for in vitro culture.

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Protoplasts were isolated from cell suspension cultures induced from a seed of Alnus firma Sieb. in 1/2MS liquid medium containing 1μM 2, 4-D and 0.1μM BAP. The optimal enzyme combination for protoplast isolation was 1% Cellulase Onozuka RS and 0.5% Pectolyase Y-23. Protoplast yield was of 1×10⁷/g fresh weight, and the protoplasts showed high viability of more than 90%. Colony formation from the protoplasts was induced efficiently in 1/2 MS liquid medium containing 0.6M mannitol, 0.09M sucrose and factorial combinations of 0.1-1μM 2, 4-D and 0.1-10μM BAP or 4-PU at the plating density of 5×10⁴/ml. Continuous callus proliferation was achieved by transferring colonies to fresh liquid medium containing 1μM 2, 4-D and 0.1μM BAP without mannitol.

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Maximal productivity of arbutin per cell at various culture stages was examined. Catharanthus roseus cells from early log phase to stationary phase were collected on nylon mesh, then aliquots of cells were suspended in fresh medium or the original medium from which the cells had been removed, for comparison, and hydroquinone was added at 2mM every day until the cells died, in order to obtain the maximal yield. Biotransforming ability based on dry weight of cells in fresh medium dramatically increased with advancing cell stage. In the original medium, however, the biotransforming ability decreased in cells older than day 5. When sucrose (1%) was added to 6-day cells in the original medium, arbutin yield drastically increased. These results suggest that older cells possess higher ability to produce arbutin, but can not manifest this in batch culture because of depletion of essential component (s) in the medium; one of these components was sucrose.
UDPG (uridine 5′-diphosphate glucose) was strongly induced in 6-day cells in fresh medium after the addition of hydroquinone even though it was barely detected before. UDPG had originally been supplied on the 3rd day. In those young cells, the hydroquinone supplied UDPG molecules were presumed to be used competitively both for the glucosylation of hydroquinone and for synthesizing cell wall polysaccharide. The enzyme activity to glucosylate hydroquinone was sufficient in cells on the 3rd, 6th and 8th day. Cell volume of 8-day cells was 1.4-fold as much as that of 3-day cells with the increament of vacuole volume; this might be related to arbutin accumulation.
In conclusion, older cells have a greater ability to produce arbutin by biotransformation. The larger amount of UDPG which is available for the glucosylation of hydroquinone, as well as the larger vacuole, contribute to its ability at least partly.

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Smashing of callus before regeneration in rice (Oriza sativa L.) cell culture was studied for the purpose of increasing plant regeneration efficiency. Using a blender was efficient as the method for smashing callus. The culture in hormone free medium for a week after smashing was efficient for decreasing the browning of callus and increasing the number of regenerated plantlets. In the culture using bioreactor with aeration of enriched carbon dioxide (5%), 9700 plantlets were obtained. The number of plantlets regenerated from the callus which size were 1000-1700 and 1700-2360μm were much more than that regenerated from another size of callus. From this result, more efficient regeneration would be obtained, if a method of smashing the callus uniformly into those sizes could be developed.

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Cryopreservation approach for the germplasm conservation of three useful tropical forest tree species (Cedrela odorata L., Guazuma crinita Mart., and Jacaranda mimosaefolia D. Don.) were carried out using shoot-tip or roottip explants from in vitro-grown plantlets by four cryopreservation methods ((1) simple freezing, (2) rapid freezing, (3) slow pre-freezing, and (4) dehydration method). The effects of cold hardening treatments (5, 10, 15, and 20/10°C) and pre-culture (dehydration treatments) were also studied.
The best results were achieved when the shoot-tips were cooled by slow pre-freezing before immersion in liquid nitrogen (LN). Survival and plant recovery rates of 50 and 20%; and, 50 and 15%; were obtained in cryopreserved shoot-tips of C. odorata and G. crinita, respectively. Although the effects of cold hardening and/or pre-culture treatments are not clear, apparently they were not effective in enhancing the survival or plant recovery rate after immersion in LN because these species may be intolerant of low temperature treatments of long duration and/or to drastic dehydration treatments.
The cryopreservation of shoot-tips from tissue-cultured plants can be considered as a feasible alternative for the long-term storage of C. odorata, G. crinita, and J. mimosaefolia germplasm.

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Root explants of Eustoma grandiflorum were bombarded with plasmid pBI221 harboring the uidA gene encoding β-glucuronidase (GUS) driven by a cauliflower mosaic virus 35S promoter, and the effects of pre- and post-bombardment culture in various starvation media on the number of blue spots of GUS-expressing cells were studied. It was found that pre- and post-bombardment culture in nitrogen-depleted media increased the expression frequency of the transgene, by 4- to 8-times that of the control, and that 3-h pre-culture in the nitrogen-depleted medium gave the highest expression frequency.

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Calli were induced from tuber explants of Colocasia esculenta Schott cv. Dodare on a basal MS medium supplemented with BA, NAA, 30g/l sucrose and 8g/l agar for 130 days at 26°C in the dark. Protocorm-like calli were obtained by culturing whitish calli on a phytohormone-free MS medium for 30 days at 26°C with a 16-hour photoperiod, and plants were regenerated from them. An in vitro clonal propagation method was achieved by culturing regenerated young plants derived from whitish callus on a phytohormone-free MS medium at 26°C with a 16-hour photoperiod.

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