Hairy roots were induced by inoculation with Agrobacterium rhizogenes (strain 15834) on sterile seedlings of Hyoscyamus niger. The axenic culture of hairy roots, isolated from the seedlings, proliferated 2760 to 3750-fold based on the initial dry weight after five weeks of culture in Gamborg B5 (B5), Murashige & Skoog (MS) and Woody Plant (WP) liquid media. Hyoscyamine as the main tropane alkaloid was isolated in a high yield, together with scopolamine, 6β-hydroxyhyoscyamine and 7β-hydroxyhyoscyamine from the hairy roots of H. niger. Rapid growth of the hairy roots was observed in WP medium, but not in B5 medium. On the other hand, a relatively high content of hyoscyamine was obtained in MS medium, but low in WP medium. B5 medium showed moderate growth and hyoscyamine production. A change in the concentrations of sucrose and potassium nitrate in the B5 medium gave improved results in growth and alkaloid production. The highest content (2.7%, dry weight) of hyoscyamine was observed in MS liquid medium.

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The cells (callus) induced from F₁ variety of celery were cultured by the fed-batch culture of nitrogen. In the culture using a medium containing ammonium and nitrate (Schenk and Hildebrandt medium), the exhaustion of ammonium was the bottleneck of the growth, therefore it could be avoided by the fed-batch culture of ammonium. The fed-batch culture of nitrate reduced the decrease of the specific growth rate caused by the exhaustion of ammonium, and the efficiency of growth in the fed-batch culture of nitrate using a medium containing nitrate as sole nitrogen source was equal to that in the fed-batch culture of ammonium using the Schenk and Hildebrandt medium. Therefore it was suggested that by controling the nitrate concentration in the medium, the efficiency of nitrate could be equal to that of ammonium.

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Culture conditions of watermelon (Citrullus lanatus L.) were investigated for the purpose of establishing an efficient plant regeneration system. Adventitious shoots were induced efficiently (50%) on MS medium containing 10mg/l IAA and 10mg/l BA. Cotyledon explants from young seedlings precultured for 3 days on MS medium containing 10mg/l IAA and 10mg/l BA or precultured for 1 day on MS medium without any phytohormone gave a high frequency of shoot formation (46% and 42%, respectively). The basal region of cotyledon showed higher frequency of shoot formation (55.3%) than the apical region (23.0%). Varietal differences were observed in the efficiency of shoot regeneration. Cultivar ‘Kikodama’ showed the highest frequency of shoot formation (68%).

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Potato (Solanum tuberosum L.) tubers were propagated in a jar fermentor using a 2 step culture method which consisted of the shoot multiplication step (step 1) and the tuber induction and development step (step 2). Shoots which were multiplied in the aerial phase of the jar fermentor were more valuable for the mass propagation of tubers than the shoots cultured under continuously submerged conditions in the liquid medium. The development of tubers was strongly suppressed under the submerged conditions in step 2, whereas the development of tubers was stimulated only at the surface area of the medium, and this suppression might cause significant variation in tuber weight. Tubers contained about 20% (w/w) dry matter, irrespective of size and localization in the jar fermentor.

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The effect of cultural temperatures on Citrus polyembryogenesis through in vitro ovule culture was studied. Ovules of sweet orange [Citrus sinensis (L.) Osbeck] previously hand-pollinated with trifoliate orange [Poncirus trifoliata (L.) Raf.] were collected aseptically at 30 days after pollination, and cultured on Murashige and Tucker (1969) basal medium supplemented with 10mg·liter^-1 adenine and 400mg·liter^-1 malt extract. The ovules were subcultured every 25 days. The growth of ovules was significantly affected by the cultural temperature. The development of ovules was favorable at 25°C and 30°C, but was seriously inhibited at 20°C. Many embryos were subsequently differentiated from the ovules at 25°C and 30°C. When the embryos were transplanted to the basal medium containing 1mg·liter^-1 gibberellic acid (GA₃), they eventually grew into seedlings. Some of them had trifoliate leaves, and the others had monofoliate ones. After 135 days in culture, seedlings with the trifoliate leaves appeared in high frequency at 30°C. Those seedlings were considered to be hybrids. It was, consequently, suggested that Citrus hybrids could be obtained efficiently by in vitro ovule culture under the condition of high ambient temperature.

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The resting period and field performance of potato (Solanum tuberosum L.) tubers derived from a jar fermentor culture were investigated. About 40% (w/w) of the tubers easily lost more than 40% (w/w) of their weight during the first week after being removed from the jar fermentor and stored under room conditions. Sprouting of such easily wilting tubers was clearly delayed whereas other tubers sprouted within 3 months under the same conditions. When the tubers were transplanted and cultivated under field conditions, a yield decrease was observed with the late sprouting tubers. This suggested that the tubers could be selected according to the decrease in their weight after culture.

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Hypocotyl and cotyledon explants excised from two in vitro-grown sesame species, Sesamum indicum and Sesamum orientale were inoculated for callus induction on MS media supplemented with different concentrations and combinations of α-naphthaleneacetic acid (NAA), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and indole-3-acetic acid (IAA), and 6-benzylaminopurine (BAP). A combination of 1-2mg·l^-1 NAA and 0.2-0.6mg·l^-1 BAP was efficient for the formation of calli from hypocotyl and cotyledon explants. After three to four weeks of inoculation, embryo-like structures were formed in hypocotyl-derived calli on the medium supplemented with only 1mg·l^-12, 4-D. Addition of casein hydrolysate (1-2g·l^-1) to the regeneration media containing 0.1mg·l^-1 NAA plus 1-4mg·l^-1 BAP effectively increased the rate of adventitious shoot formation from the hypocotyl- (22-42%) and cotyledon derived calli (16-34%) in S. orientale. However, in S. indicum, only hypocotyl-derived calli showed an increase (14-44%) in adventitious shoot formation rate. Generally, high concentrations of BAP (3-4mg·l^-1) in combination with casein hydrolysate increased the formation of multiple shoots while low concentrations of BAP (1-2mg·l^-1) induced the formation of a single shoot. Even in case of low BAP, addition of a high concentration of casein hydrolysate tended to increase the multiple shoot formation. Regenerated shoots formed roots on 1/2 MS medium supplemented with 0.5mg·l^-1 NAA.

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The isolation, culture, and plant regeneration of protoplasts derived from callus of Phalaenopsis was established. Callus was obtained by the culture of a lateral bud on a young flower stalk. Protoplasts were isolated enzymatically from callus which was precultured on P basal medium (without coconut water (CW) and sucrose) for 30 days. The first cell division was observed after 7-10 days of culture in the basal medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) or CW. After 60 days of culture, the protoplasts cultured in the medium supplemented with 0.05-0.1 mg/l 2, 4-D and 10% (v/v) CW formed numerous colonies by the addition of fresh medium. The colonies developed into small calli and turned green under continuous light condition, and small calli produced PLBs in the same liquid medium. The PLBs derived from protoplasts regenerated shoots readily on P basal regeneration medium (10% (v/v) CW, 0.3% (w/v) Gelrite). The shoots developed into whole plants after transfer onto Hyponex medium (3g/l Hyponex (N:P:K, 6.5:6:19), 2.0% (w/v) sucrose, 0.05% (w/v) activated charcoal, 0.3% (w/v) Gelrite).

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Expression of the β-glucuronidase (GUS) gene in shoot primordia of Haplopappus gracilis was obtained by particle bombardment. The efficiency of gene delivery was tested by accelerating the pressure from 115 to 200kg/cm², the number of bombardments from one to three, the amount of gold particles from 0.1 to 0.4mg per projectile, and the amount of plasmid DNA from 0.2 to 0.8μg/mg gold particles. Double bombardments of 200kg/cm² with projectiles having 0.2mg of gold particles coated with 8μg DNA/mg of gold gave 884±103 blue spots per petri dish (5.2cm diameter).

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Complete plants were regenerated from callus cultures of Taraxacum officinale. Shoot cultures were first established on half strength Murashige-Skoog's medium containing 0.1ppm N⁶-benzyladenine with or without 0.1ppm α-naphthaleneacetic acid. These shoots regenerated roots on phytohormone-free medium, and then, upon transfer to soil in a pot, grew into complete plants with normal flowers and seeds. Triterpenoid constituents of the dedifferentiated callus cells, wild and regenerated shoot and root organs, and also of latex, were analyzed. Triterpene acids (oleanolic and ursolic acids) were found predominantly in callus cells. The composition of triterpen-3-ols was characteristically different between the organs: α-and β-amyrins were found in all the tissues, whereas taraxasterol and lupeol were detected in differentiated organs.

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In vitro mass propagation techniques of takanebiranji (Silene keiskei var. akaisialpina) were studied. Apical and axillary buds isolated from aseptically germinated seedlings were used as culture materials. They were cultured in MS (Murashige and Skoog) medium supplemented with 0.5mg/l of BAP for primary culture. Apical and axillary buds were isolated from the shoots elongated in primary culture. They were subcultured in the same MS medium as the primary culture. Apical and axillary buds obtained in primary culture and subculture were transplanted to the MS rooting medium and successful plantlet regeneration was attained. Regenerated plantlets were transplanted to pots filled with vermiculite for acclimation. Theoretically 4.5×10⁷ acclimated plantlets will be obtained in a year by 6 subcultures of apical and axillary buds from a single seed.

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[in Japanese]

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Meristem tissues were excised from axillary buds of rose plants (Rosa hybrida cv. Carl Red) and cultured on media containing various concentrations of BA in order to clarify the condition for micro-propagation. The meristem tissues showed rapid and effective shoot formation when cultured with 1.0μg/ml BA. After 1 month of incubation, small leaflets of shoots were harvested and cultured on MS medium cotaining either IAA and BA or NAA and BA for callus induction. Adventitious buds were frequently induced in callus tissues cultured in the presence of 0.25μg/ml NAA and 0.005μg/ml BA, and effectively differentiated into shoots when transferred to MS medim containing 1.0μg/ml BA. Abundant roots were formed when shoots were cultured on 1/4×strength regeneration medium.

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