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Alteration of generations in peanut plants was examined by the culturing of seeds. Flowers were induced in more than 50% of the seed cultures in vitro. Benzyl aminopurine stimulated the rate of flower induction most among the factors examined. Development of pegs was stimulated by gibberellic acid given after flowering. Dark culture allowed the development of ovaries in pegs to immature seeds and then to mature seeds; with this, the life cycle of the peanut plant was complete. Mature seeds excised from the original cultures germinated normally in vitro, and the cotyledonary nodes proliferated multiple shoots on shoot-forming medium. Alteration of generations and the in vitro omission of certain steps of the life cycle in plants are discussed.

ソバ植物の種内雑種植物体の試験管内での交雑育種を検討した．圃場での良好な組み合わせであるキタワセソバと福井在来ソバを用い，試験管内で誘導したキタワセソバ( 母方，秋型) の花芽に，ポット栽培福井在来ソバ( 父方，夏型) の花粉を試験管内人工授粉させ，受粉花芽・胚珠を培養して試験管内再生植物の育成を試みた．培養体をココナッツミルクを含む培地で培養することにより，飛躍的に試験管内第2 世代植物の再生率が高まり，連続的な試験管内世代交代が可能となった．得られた再生植物体はザイモグラム，RAPD，数種の形態および農業形質から雑種と判断された．バッククロスを3 回繰り返し，母方形質を理論的に94% 持つキタワセソバ個体を育成した．初回の交雑と3 回のバッククロスを終了するまでに掛かった期間は240 日であった．試験管内での第2 世代植物体形成期間(1 世代期間に相当) は55 ～ 60 日で，試験管内世代交代期間は圃場のそれより速いと考えられた．試験管内再生植物の大量迅速育苗による系統確立は可能と考えられた．

Micropropagation of Tartary buckwheat (Fagopyrum tataricum Gaertn.) plants was examined by the culture of a reproductive organ, seeds in vitro. In initial culture, multiple shoots were induced from a single shoot derived from a 3 week old seedling. Indevidual shoots excised from multiple shoots produced multiple shoots successively at 6 week intervals when cultures were maintained in modified Murashige and Skoog's medium supplemented with 20 μM BAP, 30 g/l sucrose and 1 g/l Gellan Gum in vitro. In some conditions, protrusions were formed and further developed to axillary buds and shoots. One can calculate that about 258 (152,587,890,625) shoots could be propagated from one shoot per year. Plantlets were regenerated by rooting from single shoots in medium containing 1 μM IBA. After acclimatization, almost 100% of regenerated plants were well established in soil. Application of the present micropropagation procedure to multiply inter-specific hybrid plants between common and Tartary buckwheat plant is discussed.

Two monomeric lectins, SSA-b-3 and SSA-b-4, were purified from the bark tissue of Japanese elderberry, Sambucus sieboldiana. SDS-PAGE of the purified lectins showed the presence of single bands of 35 and 33 kDa for SSA-b-3 and SSA-b-4, respectively, irrespective of the presence of reducing agent. MS analysis as well as gel filtration of these lectins indicated that they exist mostly as monomeric lectins. Analysis of the N-terminal amino acid sequences of SSA-b-3 and SSA-b-4 yielded an identical sequence, indicating their close structural relationship. Four cDNA clones with extensive homology were obtained from the bark cDNA library and indicated to encode SSA-b-3 or SSA-b-4 from the comparison with the N-terminal sequences of these lectins. These clones were classified into two groups, three for SSA-b-3 and one for SSA-b-4, based on the predicted isoelectric points. The amino acid sequences of the encoded polypeptides were almost identical with the B-chain of a type 2 ribosome-inactivating protein from the same bark tissue, sieboldin-b, except for the absence of a small peptide containing a cystein residue, which is critical for the heteromeric dimerization with an A-subunit. Carbohydrate binding specificity and biological activity of these lectins are also reported.

Flower induction from shoot segments of buckwheat seedlings was examinedin vitro. Cytokinin, (especially kinetin at 0.1μM), short day conditions and a high concentration ofsolidifying agent improved the flower induction from node segments invitro, in up to about 50% of node segments. The use of anaeration membrane on bottle caps and a high content of sucrose in the mediumimproved flower induction in vitro considerably. In theimproved conditions, flowers were induced from 100% cultures and 10bloomed flowers per explant were induced in vitro in 8weeks. Both long and short types of stigmas, and normal set of flowers wereobserved under the microscope. When pollen produced invitro was cultured on an artificial medium, 70% of the pollengrains germinated, indicating normal viability of in vitropollen, and indicating the potential for artificial pollination invitro. All the varieties examined flowered at a similar percentage,suggesting that the process was independent of variety and that flowers couldbeproduced in vitro. Flower induction from buckwheat plantsin vitro and possible cross breeding invitro are also discussed.

Optimization of micropropagation procedure in suntara orange (Citrus reticulata) was examined by a combination of experimentation and cost simulation. Initially, 20 seeds were cultured in modified Murashige and Skoog's medium for 42 days. Excised single shoots from seed cultures were sub-cultured in modified Murashige and Skoog's medium containing 1 μM BAP, 1 μM IBA, 1.5g/l Gellun gum, and 30g/l sucrose every 28 days. Single shoots were then cultured in modified Murashige and Skoog's medium containing 5 μM IBA for 21 days. Regenerated plants were acclimated in a plastic bag containing 125-ml soil for 10 days. Micropropagation of suntara orange was established through plant regeneration without disorganizing the state. Unit production cost of in vitro plant was calculated to be 76.6 yen at 10, 000 level production. This cost is about 46% of the original cost based on the experimentation and about 60% of the first simulation cost. So, the production cost was remarkably reduced by the present procedure.Further improvement on the present production procedure was examined by cost simulation and the results showed that production cost could be reduced to 68 yen.The present result suggests that cost simulation is useful to optimize the production procedure. The usefulness of cost simulation in the improvement of micropropagation procedure and related subjects are also discussed.

The idea, reforestation of Citrus species in mountainous regions is useful both in providing job opportunities to rural people and in environmental conservation. In vitro plants may meet the demand of desired saplings. However micropropagation of Citrus species has not been well established, although plantlet regeneration from disorganized systems has been reported. Micropropagation of suntara orange (Citrus reticulata BLANCO) from Nepal was attempted and improvement of the procedure was estimated by cost simulation.Micropropagation of suntara orange was established by induction of multiple shoots from seeds born from the same tree, successive shoot multiplication, and rooting from excised single shoots. Sixty percent of the seeds germinated in modified Murashige and Skoog's medium containing 1.5g/l Gellun gum, 10μM benzylamino purine and 30g/l sucrose. Multiple shoots were induced from 60% of seedlings and the average number of shoots per seedling was about 3.4. When shoots 10-15 mm in length were cultured in the medium containing 1μMBAP, 1μM IBA, 30 g/l sucrose and 1.5 g/l Gellun gum for 63 days, the average size of the shoots increased 8.9 times. It was calculated that about 1, 800, 000 can be obtained from one single shoot per year. When shoots 10-15mm in length were cultured in the medium containing 5μM IBA for 21 days, 93% of the shoots rooted. The regenerated plants from 5 mm to 20 mm in height were acclimatized for 30 days and 89% of the plants survived on potted soil. It is suggested that instead of meristem culture, virus free plantlets can be propagated by culturing vegetative embryos from citrus seeds, multi-embryonic seeds.Production cost per in vitro plantlet at 10, 000 plantlet production level was calculated to be 165 yen. The improvement of production procedure was attempted by cost simulation and the results showed that production cost would be reduced to 116 yen.The present results indicate that propagation of plant species by plant tissue culture has a great potential in environmental conservation. The usefulness of cost simulation in the improvement of micropropagation procedure and related subjects are also discussed.

The previously described cost simulation model was used to improve micropropagation of Brassica campestris cv. Kukitachina in vitro. The simulation was based on data obtained from different practical experiments including the use of simple medium and food containers, rooting from excised single shoots and acclimatization at a time, and so on.There was no significant difference between the effect of Otsuka hydroponic culture nutrient or Hyponex with additives and the modified Murashige and Skoog's medium on successive shoot multiplication. Also the reagent grade sucrose could be replaced with table sugar, granular sugar or reagent grade glucose without adverse effect on the successive shoot multiplication. The above results suggested that cheap carbon sources could be successfully used in micropropagation. Also in comparison with culture bottles, food containers were equally effective for successive shoot multiplication. The shoots multiplied three times every 20 days. Rooting from excised shoots and acclimatization of in vitro plants were possible in nursery boxes and seventy per cent of regenerants were successfully planted out.The production cost of in vitro plants at 10, 000 level production, using the above modified procedure was calculated to be 61. 0 yen per plant. This represents a 39% cost reduction when compared with the previously described laboratory procedure. Improvement of micropropagation by simulation of production cost was discussed.

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acalpha2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S(197), A(233) and Q(234), in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acalpha2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acalpha2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution.

The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacterial, is usually undetected in higher plants and animals. The enzyme also catalyzes conversion of non-toxic 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU), a toxic compound for plant growth. The gene encoding cytosine deaminase (codA) from Escherichia coli was fused to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101. The resulting plasmid pLABR102 contained two marker genes for plants: a positive marker gene, bialaphos resistance (bar) gene driven by the promoter from nopaline synthase gene (Pnos) and a negative one, P35S-codA. The binary vector pLABR102 was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation. In transgenic progenies (T3) of the second (T2) generation heterozygous for a single T-DNA insertion, a 3:1 segregation ratio was observed on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected). From T2 plants homozygous for the T-DNA insert, on the other hand, no segregation was detected: all the T3 seedlings were resistant to bialaphos and sensitive to 5-FC. PCR and Northern analyses showed that the 5-FC sensitivity in transgenic descendants was caused by the integration and expression of the chimeric codA gene in the Arabidopsis genome. The results indicated that cytosine deaminase from E. coli is functional and useful for negative selection in Arabidopsis, and that sensitivity to 5-FC as well as the positive bialaphos resistance are dominant traits in Arabidopsis.

The capacity of various carbohydrates to support growth of Japanese morning-glory callus in the dark over a 14-day period was examined. Sucrose was the most effective compound, but glucose, fructose, trehalose, maltose, cellobiose, raffinose and soluble starch supported significant growth. The callus remained alive in the presence of inulin, mannitol, inositol, methyl-α-glucoside and glycerol; while cells grown on galactose, mannose, sorbose, xylose, arabinose, melibiose, lactose, dextran, carboxy-methyl cellulose, sorbitol, galactitol, ethylene glycol were necrotic. Examination of the effects of these various carbon sources on the cell wall and cytoplasmic activities of acid invertase, trehalase, maltase, cellobiase, melibiase and lactase could not be correlated with the growth-promoting activity of their substrates. Extracellular hydrolysis of sucrose, trehalose, maltose, cellobiose, lactose, raffinose, and inulin occurred as a consequence of the presence of cell wall hydrolases in the morning-glory callus, and hydrolytic products could be detected in the medium.

Gibberellin A5 (GA5), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, 3H] GA5 (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [3H] GA5. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [3H] GA5 was converted to [3H] GA1 (2%), [3H] GA3 (4%), [3H] GA6 (2%), [3H] GA22 (1%) and their glucosyl conjugates, and also to [3H] GA8 glucoside, and [3H] GA5 glucosyl conjugates. The major conjugate-like substances were [3H] GA1 and [3H] GA3 glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.