Crocin, picrocrocin, and safranal as the major secondary metabolites are important for the high quality of spice saffron. Crocin and picrocrocin were detected in stigma-like structures proliferated in vitro. Safranal appeared after heat treatment. The contents and relative ratio of these substances in the stigma-like structures were similar to those in intact young stigmas, indicating that these stigma-like structures showed not only morphological but also biochemical similarity to the intact stigmas. Saffron, dried stigmas of Crocus sativus L., is widely used as a spice and a drug. It contains three major secondary metabolites which are important for the high quality of saffron, a yellow pigment "crocin",1~6) a bitter-tasting substance "picrocrocin",2' 7' 8) and a good perfume "safranal'\2'8'9) Many stigma-like structures can be proliferated in vitro from the excised young stigmas and ovaries of saffron of Linsmaier-Skoog10) and Nitschn) agar media supplemented with auxin and cytokinin in combinations, and these structures, colorless at first, turned yellow and then deep orange during maturation.12) In this paper, we report the analysis of the secondary metabolites in these stigma-like structures. Our purpose of this research is to produce the spice, saffron, economically. MATERIALS AND METHODS Plant materials. Bulbs of Crocus sativus L. were purchased from Sakata-no-Tane Co., Yokohama. The intact stigmas were picked up from the flower buds and used for the control analysis (Tables II and III). The stigma-like structures were formed on young half ovaries, which were aseptically excised from young flower buds and cultured on agar media supplemented with auxin (NAAand IBA) and cytokinin (BA and kinetin) in combination (Tables I and II). Cultivation was done at 20°C in the dark. Reagents. Crocin isolated from gardenian fruits was the product of Taito Co., Tokyo. Safranal, chemically synthesized, was the gift of Mr. Kiyonori Suzuki of Sota Koryo Co., Chiba. /?-Glucosidase isolated from almond seeds was purchased from Sigma Co. Quantitative analyses of saffron secondary metabolites. Amountsof crocin, picrocrocin, safranal, and HTCCwere measured from their peak areas at 443nm, 250.5nm, 308 nm and 250.5 nm, respectively, using a HPLCcolumn, Hiber LiChrosorb RP-18 (particle size, 5 /mi; column size, 4 x 250mm) (Cica-Merck). Crocin and picrocrocin con

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Some bacteria belonging to the genera Arthrobacter, Brevibacterium and Corynebac-.terium, so-called glutamic acid-producing bacteria, are highly important as they are used for the industrial fermentation production of various amino acids. The genetic breeding of such amino acid-producing strains has so far been carried out by conventional mutation and selection methods. After a phenol treatment of the supernatant, DNAwas recovered as ethanol precipitation. These DNA samples were analysed by agarose gel electrophoresis with a Tris-acetate buffer system as described by Sharp et al.7) As a result of the screening, two low-molecular-weight plasmids were found. One was found in Brevibacterium lactofermentum ATCC13869 Recentlyand named pAM330.The other was found in Corynebacterium glutamicum ATCC1 3058 and named pHM1519. They were purified.by the CsCl-EtBr equilibrium density gradient centrifugation method, and were confirmed to exist as closed circular molecules. The molecular weights of pAM330 and pHM1519 were determined as 3.0 Md and 1.8 Md, respectively, from electrophoretic mobility in a 0.8% agarose gel. Restriction enzyme analysis was carried out on those plasmids, and the results are shown in Fig. 1. The contents of pAM330 and pHM1519 were roughly analysed by the agarose gel electrophoresis method8'9* as follows: Cells were cultured in 4ml of the CM2Gmediumto the exponential growth phase, and 600 jig/ml of ampicillin was added to the culture medium to ensure the lysozyme digestion. The cells were harvested at 2.5 hr after the addition of ampicillin, and were lysed with lysozyme (lOmg/ml, 37°C 2hr) and SDS (4%). The lysis was completed by heating at 65°C for lOmin, followed by phenol extraction and ethanol precipitation. The DNAsamples were dissolved in TEN buffer and treated with RNase (20/ig/ml, 37°C fir 20min) and pronase (100/^g/ml, 37°C for 20min) sequentially.After phenol extraction and ethanol precipitation, the recovered DNAwas dissolved in TENbuffer, and electrophoresed in a 0.8% agarose gel. The photographs of the gel were taken under irradiation by ultra-violet light, and the negative films of them were analysed by a densitometer (Shimadzu CS-910). As a result, the amounts ofpAM330 and pHM1519 were approximately 1.4% and 9.1% of the total DNA, respectively.To estimate the