Induction of Continuous Tepal Differentiation from in Vitro Regenerated Flower Buds of Hyacinthus orientalis

Continuous differentiation of tepals was successively induced from regenerated flower buds in Hyacinthus orientalis L. cv. White Pearl by controlling the exogenous hormones and explant ages. In 250 days of subculture, each flower bud differentiated an average of more than 70 tepals, with a maximum of over 140 tepals. Studies on the morphogenesis and characteristics of growth and development of the flower buds indicate that the first whorled organ of the flower bud was perianth which consisted of perianth tube and tepals grown at the top of the perianth tube, which is the same as the flower bud of the wild type in H. orentalis. The second and third whorls of the flower bud, which should be stamen and pistil in the wild type, but remained as the tepals in the regenerated flower bud. Growth of the regenerated flower bud was faster in the first several months of culture, then slowed down gradually with time. After 150 days in culture the flower bud growth and organ differentiation became very slow. Other than the tepal differentiation the regenerated flower buds also differentiated at random positions some small flower buds that also differentiated the tepals only. Histological observation revealed that the origin of the regenerated flower buds was jointly participated by some cells in the epidermal and subepidermal layers at the inner surface of the perianth explant, and the inner small flower buds were originated from the meristem which was formed by the transformation of the parenchyma at the base of the very young tepal. The authors also compared and discussed the similarities and differences of the phenotypes between the regenerated flower bud in Hyacinthus and agamous flower in Arabidopsis, from which, they have hypothesized on the role of the hormones in the promotion and termination of the gene expressions by an order of development in plant.     

In vitro plantlet formation from juice vesicle callus of satsuma (Citrus unshiu Marc.)

Callus was induced from juice vesicles of satsuma mandarin on Murashige & Skoog medium supplemented with -naphthaleneacetic acid (NAA), kinetin (K) and gibberellin (GA). Adventitious embryoids arose from the callus tissue on the medium containing 1 mgl^–1 NAA alone. The embryoids grew into embryos which resulted in a plantlet on medium containing 1 mgl^–1 GA.Abbreviations GA gibberellin - K kinetin - NAA -naphthaleneacetic acid     

Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase

Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3beta-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1H NMR and CD spectroscopies as (-)-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non-specific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.     

The Formation and Distribution of Ice within Forsythia Flower Buds

Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0°C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between −10 and −25°C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze-fixed at −5°C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil.     

Regulation by Abscisic Acid of In Vitro Flower Formation in Torenia Stem Segments

In Torenia stem segments cultured on a defined medium withoutphytohormones, in vitro flower formation was influenced by thephysiological states of the explants. Endogenous contents ofABA, but not those of IAA, were closely correlated with thephysiological states of the explants. Application of ABA (100ng/ml) to the culture medium stimulated flower formation inthe originally vegetative explants which otherwise had littleflower-forming capacity. Thus, endogenous ABA seems to be oneof the factors controlling the flower-forming capacity of Toreniastem segments. The highest rate of flower formation in the stemsegments was obtained when endogenous contents of ABA (whichresulted from both endogenously present and externally appliedABA) in the stem tissues was between 16 and 20 ng/g fresh weight. ¹ Present address: Bioscience Research Center, Mitsui PetrochemicalIndustries Ltd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received November 22, 1984; Accepted March 1, 1985)     

Enhancement of Tuberization of Axillary Shoot Buds of Potato (Solarium tuberosum L.) Cultivars Cultured In Vitro

Factors controlling growth and tuberization of axillary budsin shoots of plantlets of potato (Solarium tuberosum L.) culturedin vitro were investigated. Correlative inhibition restrainedgrowth and tuberization of the axillary buds. Exposure of intactplantlets for various periods (4 to 48 h) to low (2 or 12C)or high (30 C) temperatures as comparedto 18C, did not alleviatecorrelative inhibition. Removal of the apical part of the shoot,the roots or both was generally ineffective Elevating sucroseconcentration from 30 to 80 g dm^–3 promoted tuberizationon axillary buds, and the cytokinin 6-(-dimethylallylamino)purine (2iP), alleviated correlative inhibition and enhancedtuberization in intact plantlets. In the whole plantlet mostof the tubers were formed on the basal nodes, however, oncecorrelative inhibition was eliminated by the dissection of theshoot to single node sections, tubers were formed on every axillarybud. The single most effective factor inducing tuberizationin single node sections was the growth retardant ancymidol,an inhibitor of giberellin biosynthesis. Key words: Potato, Solanum tuberosum L., in vitro tuberization, correlative inhibition     

Histological Study of Adventttious bud Formation on Lactuca Sativa Cotyledons Cultured In Vitro

Abstract

Cyto-histological changes accompanying the formation of adventitious buds in excised cotyledons of Lactuca sativa were studied during the first 12 days after planting in vitro. Prospective proliferating cells can first be recognized, already on the first day after planting, by a marked increase in nuclear and nucleolar volumes, followed on the second day by a burst of cell divisions involving particularly mesophyll cells. Then lignified elements develop together with meristematic center, forming a callus-like tissue in the inner part of the cotyledons. At the third day of culture, the epidermal cells start to divide with a periclinal wall followed by an anticlinal division. In the following days of culture the epidermal cells, which divide mainly with periclinal walls, form layers of cells below the surface, gradually filling up the intercellular spaces. From the 8^th day on, the buds protude above the surface and develops into shoots. These results are discussed in relation to DNA content of nuclei of Lactuca sativa cotyledons and to the time course of cell division and tracheary element formation. The very regular sequence of changes associated with the initiation and development of the bud makes the in vitro culture of Lactuca cotyledons an appropriate System for histochemical and biochemical studies.     

Effects of Wounding on Adventitious Bud Formation in Torenia Stem Segments Cultured In Vitro

In in vitro cultured stem segments of Torenia fournieri Lind.,the formation of adventitious buds can be induced when the culturemedium contains cytokinin. When long stem segments (2.0 cm ormore) were cultured with cytokinin, a large number of buds wereformed in the marginal regions, namely, within the limits of0.5 cm from the cut ends of explants, while only a few budswere initiated in the middle part of the explants. If a slightinjury was made transversely with a scalpel in the central partof an explant, a significant increase in the number of budswas noted within the limits of 0.5 cm from the wound site. Whena wounding treatment was given lengthwise to an explant, a largenumber of adventitious buds were formed over the entire surfaceof the explant compared to the control. Excision itself of explantsfrom mother plants and the additional wounding given to theexplants seemed to trigger the induction of adventitious buddifferentiation in Torenia stem segments. These wounding treatmentsdid not affect the uptake into explants and/or the distributionpattern of radioactive benzyladenine applied to the culturemedium. Key words: Torenia fournieri, Adventitious bud formation, Cytokinin, Wounding     

Characterization of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin)

Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.     

In Vitro Culture of Hermaphrodite Floral Buds of Cucumis melo L.: Microsporogenesis and Ovary Formation

A method has been described by which floral buds of hermaphroditemelons isolated at various stages from archesporium to preleptotenein the pollen mother cells (PMC) can be grown through to pollen-grainformation in culture. Floral buds cultured for this period ina medium with thymidine-methyl-³H (³H-T) showed incorporationof label into the nuclei of tapetal cells and PMCs. The labelincorporated into the PMC was probably due to pre-meiotic DNAsyn-thesis. In other tissues of the floral buds, ³H-T incorporationproceeded through meiosis. Metabolic and environmental factorsinvolved in normal differentiation of the microsporogenous tissueof floral buds cultured in vitro are discussed.     

Functional expression and mutational analysis of flavonol synthase from Citrus unshiu.

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C. Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.     

Wheat Germ Agglutinin-Gold as A Novel Marker for Mesectoderm Formation in Mouse Embryos Cultured In Vitro

The routes of movement of mesectoderm cells in mammalian embryos have not yet been investigated experimentally due to technical problems. However, the recent development of in vitro culture methods have made an experimental approach to this problem in mouse and rat embryos possible. We have used combined lectin and colloidalgold (WGA-Au) probe as a nontraumatic, easily detectable mesectaderm marker. The probe is introduced into the amniotic cavity by microinjection. All of the cells lining the cavity, including the mesectoderm precursors, phagocytose the colloidal gold, which is then stored in membrane bound vesicles. The probe remains inside the target mesectoderm cells after their migration into the mesoderm compartment. Vesicles containing gold are detectable in both ultrathin and semithin sections. The applicability of WGA-HRP as a probe was also assessed because of the many properties it shares with WGA-Au, but it proved to be unsatisfactory for this purpose because it is transfed between cells and also to the extracellular spaces.     

Isolation of microprotoplasts from a partially synchronized suspension culture of Citrus unshiu

The effect of hydroxyurea (HU) and amiprophos-methyl (APM) on the synchronization of suspension cultures of Satsuma mandarin (Citrus unshiu) and micronucleation of the suspension cells sequentially treated with both, HU and APM, were investigated. When suspension cultures in early-log phase were treated with 4 or 10mM HU for 24h, the number of cells in the S-phase and the mitotic index (MI) increased significantly. Exposure of the early-log phase suspension culture to 32microM APM led to a marked increase in the MI 12 and 24h after treatment, while higher as well as lower concentrations (16, 24 and 48microM) had no effect. Suspension cultures subjected to sequential treatment, e.g. pretreatment with 10mM HU for 24h followed by treatment with 32microM APM for 24h, also showed a considerably increased MI. Furthermore, 61.5% of the protoplasts isolated from the sequentially treated suspension cells were micronucleated, whereas only 3.6% of the control protoplasts, isolated from untreated cells, showed micronucleation. Ultra-centrifugation of the micronucleated protoplasts generated microprotoplasts of different sizes, most of them below 5microM in diameter, with 1 or few chromosomes. The potential application of microprotoplasts in citrus genetic improvement is discussed.     

Isolation and characterization of new limonoid glycosides from Citrus unshiu peels.

Three limonoid glycosides were isolated from Citrus unshiu peels, and their structures were determined based on MS and NMR spectroscopic data as nomilinic acid 17-O-beta-D-glucopyranoside (1), methyl nomilinate 17-O-beta-D-glucopyranoside (2), and obacunone 17-O-beta-D-glucopyranoside (3). In particular, the location of the sugar moiety was clearly determined by the B/E constant linked scan FABMS method. No limonoid glycosides obtained here were found to have antitumor activity in NCI-H292 and EL-4 cell lines.     

枣树离体叶片不定芽再生体系建立的研究

建立了木枣无菌试管苗快繁体系,以无菌苗叶片为外植体,对影响离体叶片不定芽直接再生的因素进行了研究.试验结果表明,TDZ比BA能更有效地诱导叶片不定芽的再生;褐化是抑制不定芽再生频率提高的关键因子,培养基中添加PVP、V c及改变生长素的种类和浓度均不能促进不定芽再生;添加A gNO3能够减轻褐化并可以大幅度提高再生频率,同时培养初期经过3周避光培养更有利于提高再生效率.因此,以附加2.0 m g/L TDZ和0.2 m g/L IBA的M S培养基,并添加5.0 m g/L A gNO3,可以高效诱导木枣离体叶片不定芽再生,再生频率最高达98.3%.不定芽在附加0.2 m g/L IBA和0.5 m g/L GA3的M S培养基上进行继代伸长培养,当不定芽长至3 cm时,转接至附加0.4 m g/L IBA的1/2 M S培养基上可以良好地诱导生根.     

Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus aurantium

The distribution of the flavanones naringin and neohesperidin has been analyzed during the development of the leaves, flower buds, and fruits of Citrus aurantium. These flavonoids are at maximum concentration in the organs studied during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the naringin and neohesperidin concentration in leaves, flower buds, and fruits is due to a dilution of the flavonoids caused by cell growth, because total content per organ continues to increase. The levels of neohesperidin are always greater than those of naringin, although the ratio between the relative concentrations is different in the three organs studied. Leaves have the highest ratios, varying between 8.83 and 5.18, followed by flowers (3.15-1.85), and fruits (2.23-1.02). These observations suggest different relationships between the respective enzymic activities in their biosynthetic pathway.     

Microbial Formation of Nitrosamines In Vitro

Mortierella parvispora and an unidentified bacterium converted trimethylamine to dimethylamine, and the bacterium (but not the fungus) formed dimethylnitrosamine in the presence of nitrite. Dimethylnitrosamine also appeared in cell suspensions of Escherichia coli and Streptococcus epidermidis and in hyphal mats of Aspergillus oryzae incubated with dimethylamine and nitrate. Suspensions of a number of microorganisms produced N-nitrosodiphenylamine from diphenylamine and nitrite at pH 7.5, and soluble enzymes catalyzing the N-nitrosation of diphenylamine were obtained from two of these organisms. In the presence of these enzymes, several dialkylamines were converted to the corresponding N-nitroso compounds.     

Development of Oilseed Rape Buds, Flowers, and Pods In Vitro

A technique for growing buds, flowers, and pods of oilseed rape(Brassica napus L. cv. Haplona) on stem explants in vitro hasbeen developed. Open flowers and young pods underwent normaldevelopment on a basal medium of minerals, vitamins, and sucrosebut the development of buds was less successful. Young buds(3 mm long) did not develop and only limited development ofthe older buds (5 mm long) took place. Some 3 mm-long buds wereinduced to develop to open flowers by adding naphthyl aceticacid or gibberellic acid. Pod and seed set in open flowers werenot affected by adding plant growth substances to the medium,but pod elongation and pod dry weight were promoted by gibberellicacid, 105 M, and benzyl amino purine, 10⁷ M, respectively. Reducingthe supply of sucrose or minerals to open flowers reduced seedset, pod elongation and pod weight but did not affect pod set.The physiological significance of the results is discussed. Key words: In vitro cultures, oilseed rape, pod development, flower development     

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana

Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.