Identification of jasmonic acid and its methyl ester as gum-inducing factors in tulips

The purpose of this study was to identify endogenous factors that induce gummosis and to show their role in gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) stems. Using procedures to detect endogenous factors that induce gum in the stem of tulips, jasmonic acid (JA) and methyl jasmonate (JA-Me) were successfully identified using gas–liquid chromatography–mass spectrometry. Total amounts of JA and JA-Me designated as jasmonates in tulip stems were also estimated at about 70–80 ng/g fresh weight, using deuterium-labeled jasmonates as internal standards. The application of JA and JA-Me as lanolin pastes substantially induced gums in tulip stems with ethylene production. The application of ethephon, an ethylene-generating compound, however, induced no gummosis although it slightly affected jasmonate content in tulip stems. These results strongly suggest that JA and JA-Me are endogenous factors that induce gummosis in tulip stems.     

上海地区郁金香引种栽培与品种综合评价

通过建立郁金香(Tulipa gesneriana)品种评价体系,筛选生长适应性好、观赏价值高,适宜应用于园林景观的郁金香品种。对29个郁金香进口品种在上海地区进行引种栽培,观察记录其物候期,并对植株高、冠幅等12个生长特性,以及开花整齐度、花型、花色等32个观赏特性进行了观测。从观测指标中筛选出了包括开花整齐度、花色均匀度、花色等27个评价指标,使用层次分析法与灰色关联度分析法建立了评价体系。综合评价将29个品种分成了4个等级：Ⅰ级包含10个品种, Ⅱ级12个品种,而Ⅲ级和Ⅳ级包含品种数较少,分别只有4和3个品种。Ⅰ、Ⅱ级品种普遍具有较高的观赏价值,同时适应性较强,适合今后推广应用于上海及周边地区园林绿化。这对筛选适于上海及周边地区园林种植的郁金香品种有重要的指导作用,建立的评价体系对评价其他郁金香品种在园林中应用也有积极参考意义。     

Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors

Epicotyl segments and nodus expiants from etiolated seedlings of Pisum sativum were transformed using Agrobacterium tumefaciens strains GV 2260 (p35S GUS INT) and GV 3850 HPT carrying either a neomycin- or hygromycinphosphotransferase-gene as selectable markers. The transgenic character of hygromycin- or kananamycin-resistant tissue was confirmed by detection of nopaline or neomycinphosphotransferase-II- and ß-glucuronidase activity in crude extracts of resistant tissues. Up to 5 % of developing shoots from shoot proliferating nodi were regenerated via organogenesis to kanamycin-resistant plantlets. Transformation frequency in vitro was found to be influenced by expiant source, A. tumefaciens strain, pea genotype and duration of cocultivation. Acetosyringone did not increase the transformation rate.Abbrevations GUS ß-glucuronidase - NAA 1-naphthyl-acetic-acid - BA 6-benzylaminopurine - NPT-II neomycinphosphotransferase-II - HPT hygromycinphosphotransferase     

A rapid and efficient regeneration system for pea (Pisum sativum), suitable for transformation

A method for plant regeneration via organogenesis in pea (Pisum sativum) using nodal thin cell layer segments has been developed.From 10 to 12 days old sterile pea seedlings, nodal expiants were excised from which leaves and axillary buds were removed. Shoot regeneration was consistently obtained from liquid cultures where the expiants were floated on the medium. Shoots could be harvested after two weeks and thereafter up to ten weeks and no important effect of the cultivar (Bodil, Puget, Rondo and Trille) used could be observed as far as shooting capacity was concerned.Rooting frequency of the regenerated shoots was cultivar dependent. Plantlets were obtained within 7 weeks after expiant excision. Agrobacterium tumefaciens carrying a disarmed Tiplasmid and a binary vector containing the ß-glucuronidase reporter gene, were used in cocultivation experiments on pea nodal expiants in order to obtain transgenic shoots.Abbreviations DHZ dihydrozeatin - IBA indole-3-butyric acid - GUS ß-glucuronidase - NPTII neomycin phosphotransferase II     

Role of hormones on flower bud formation in thin-layer expiants of tobacco

Flower bud formation was studied in thin-layer tissue expiants of epidermis plus subepidermal cortex from the inflorescence ramifications ofNicotiana tabacum cv. Samsun. With appropriate hormone concentrations of BA and NAA expiants from flowerv and fruitbearing stalks regenerate flower buds only, while those from the internodes of the inflorescence ramifications produce generative as well as vegetative buds. In both types of expiants the number of buds formed depend mainly on the hormone concentrations but, in addition, the age of stalks and internodes from which expiants are taken also affects bud formation. Both ABA and JA inhibit flower bud formation in expiants of flower stalks. JA was shown to particularly inhibit bud initiation.     

Interspecific crosses in the genus Tulipa L.: identification of pre-fertilization barriers

 Pollen tube growth in the pistil and pollen tube penetration of ovules have both been studied in crosses between cultivars from Tulipa gesneriana L. and 12 tulip species from all eight sections of the genus Tulipa to identify pre-fertilization barriers. Depending on the cross, pollen tubes grew as far as the stigma or the style or continued growing down into the ovary. Pollen tubes penetrated none or only a few percent of the ovules of some crosses, despite the presence of many pollen tubes in the ovary. In other crosses, from which no or only a few hybrids have been obtained after seed maturation on the plant, pollen tube penetration was found in up to 79% of the ovules. Apparently, various kinds of barriers preventing fertilization or normal embryogenesis occur in interspecific tulip crosses. Received: 26 July 1996 / Revision accepted: 31 January 1997     

Gum Formation by Methyl Jasmonate in Tulip Shoots is Stimulated by Ethylene

The promotive effect of methyl jasmonate (JA-Me) on the induction of gum in tulip shoots (Tulipa gesneriana L. cvs. Gudoshnik and Apeldoorn) was studied in the presence of ethylene. Gum formation in the stem and the basal part of the leaves was induced by JA-Me (1% w/w in lanolin) and stimulated strongly by the simultaneous application of 1 or 5 mm 1-aminocyclopropane-1-carboxylic acid (ACC). JA-Me at a concentration of 0.1% did not induce gum, but that together with ACC at a concentration of 1 or 5 mm induced it substantially. Although JA-Me stimulated ethylene production substantially in the stem of intact tulips, ethephon (1% w/w) or ACC (1 or 5 mm) did not induce gum formation in tulip shoots. JA-Me induced gum formation in tulip shoots even in the presence of aminooxyacetic acid or cobalt ions. Moreover, gum formation was also observed in the cut shoot applied with JA-Me as a solution at concentrations of 0.23 mm or more. These results strongly suggest that JA-Me is required for gum formation in tulip shoots, and ethylene probably makes the tissues of shoots sensitive to JA-Me. Received March 23, 1998; accepted June 10, 1998     

郁金香花芽分化过程中鳞茎碳水化合物和蛋白质含量的变化

以郁金香的两个品种‘朱迪斯＇（‘Judith Leyster＇）和‘大笑＇（‘Big Smile＇）为材料,采用石蜡切片法确定其花芽分化进程,并测定花芽分化期鳞茎内碳水化合物和可溶性蛋白质含量的变化。结果表明,郁金香花芽分化过程中,两个品种的碳水化合物含量的变化趋势较为接近。随着鳞茎含水量的下降,可溶性总糖和可溶性蛋白含量均呈先增加后减少的趋势。淀粉含量的变化呈现波动性,而淀粉酶活性则持续增强并在后期达到稳定水平。鳞片中可溶性糖含量和淀粉酶活性同时增加是郁金香花芽形态分化启动的标志。相比于外层鳞片,内层鳞片是为花芽原基生长发育提供营养物质的首要场所。     

In vitro photoinduction of leaf tissue ofStreptocarpus nobilis

Leaf expiants from vegetative plants of the short-day plantStreptocarpus nobilis (C. B. Clarke) developed flower budsin vitro when cultured in 8 h photoperiods. Tn non-inductive photoperiods only vegetative buds were formed.In vitro photoinduction was demonstrated by giving the expiants short-day (SD) cycles and then transferring them to non-inductive photoperiods for expression of flowering. On medium containing 6-benzylaminopurine (BAP) organogenesis was initiated during the photoinductive treatments. Photoinduction of leaf tissue without adventitious bud development was obtained on medium without BAP. The photoinductive state of the leaf tissue was fairly stable, being expressed after 2–3 weeks in non-inductive photoperiods when adventitious buds were formed. The quantitativein vitro flowering response to the endogenous floral stimuli, resulting from photoinduction, could provide the basis of a bioassay for presumptive flower inducing chemicals.     

Genetic transformation ofRhododendron byAgrobacterium tumefaciens

Summary TransgenicRhododendron plants were obtained byAgrobacterium tumefaciens-mediated gene transfer.A. tumefaciens harboring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and (3-glucuronidase (GUS) genes was co-cultivated with stem and leaf segments fromRhododendron tissues culturedin vitro. Adventitious buds were fonned and shoots were regenerated on kanamycin selection medium 3-4 months after inoculation. Integration of the NPTII and the GUS genes was confirmed by polymerase chain reaction (PCR) and by Southern hybridization analyses. Histochemical GUS assay showed that the inserted gene was expressed in all tissues with the cauliflower mosaic virus (CaMV) 35S promoter. This transformation procedure has the potential to expand the range of genetic variation inRhododendron.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene

Summary Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter of cauliflower mosaic virus and an engineered napin (seed storage protein) gene with its own promoter (300 nucleotides 5 to the start of translation). Transformation of B. napus plants was confirmed by detection of NPT II enzyme activity, Southern blot analysis and inheritance of the kanamycin-resistance trait (NPT II gene) in the progeny. Expression of the engineered napin gene in embryos but not in leaves of transgenic plants was observed by Northern analysis. These data demonstrate that morphologically normal, fertile transgenic B. napus plants can be obtained using Agrobacterium as a gene vector and that developmentally regulated expression of reintroduced genes can be achieved.     

Transformation of Stylosanthes spp. using Agrobacterium tumefaciens

Tumours were incited on leaf sections of Stylosanthes humilis, S. hamata, S. guianensis and S. scalra following infection by Agrobacterium tumefaciens. The suitability of 2 binary vectors (pGA472, BIN6) for gene transfer in S. humilis was tested and kanamycin-resistant tumour tissue was obtained from infected leaf pieces. The presence and expression of the neomycin phosphotransferase (NPT II) gene in the plant cells was demonstrated by hybridization of the coding region of the NPT II gene of the transposon Tn5 to DNA and RNA of kanamycin resistant tumours and by detection of significant NPT II activity in tissue extracts. Tumours also produced teratomatous shoots expressing the NPT II gene, but these could not be rooted.     

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.     

Effect of silymarin on plant tissue cultures

The effect of silymarin complex on various types of expiants differing in their nutrition requirements was investigated. The growth of tumorous periwinkle (Catharanthus roseus [L.] G. Don) callus tissue was still identical with the control tissue at the silymarin concentration of 35 mg in 1000 ml of the nutrient medium. However, this silymarin concentration totally inhibited the growth of habituated periwinkle callus tissue; in the presence of 10 mg of silymarin, the growth of this tissue was similar to that of the corresponding tissue grown without silymarin. The growth of tobacco callus tissue (D-strain) requiring for its growth kinetin was reduced by 46.2% at the concentration 10 mg of silymarin in 1000 ml of nutrient medium, but its dry weight was increased by 21% in comparison with the control. Silymarin was most effective on the growth of callus derived from tobacco (Nicotiana glauca Grah:) stem pieces; callogenesis was observed in control tissue in 89.5% cases while in the presence of silymarin (10 mg) only in 48.6%. The primary callus growth was strongly inhibited, too (by 89.9%). The organogenesis onset was never observed on tobacco stem pieces cultured on a nutrient medium with kinetin and IAA in the presence of silymarin. When all types of expiants were transferred from the medium with silymarin on control medium, normal growth appeared very soon and the differences between the experimental and control expiants were smoothed out during two months. These results indicate that the observed changes might be due to the blocking of membrane system permeability leading to an insufficient supply of cells with nutrients and growth substances.     

Control of Ethylene Induced Permeability of Plant Cells by a Substituted Benzothiadiazole

The permeability of bulb tissues of onion (Allium cepa L.), amaryllis (Hippeastrum hybrids), tulip (Tulipa × hybrida) and daffodil (Narcissus tazetta L.) treated with Ethephon and 5-methyl-7-chloro-4-ethoxycarbonylmethoxy-2,1,3-benzothiadiazole (TH6241), to water and solutions of 1,3-dimethylurea was determined by the plasmometric methods. Ethylene treatments promoted, while those with TH6241 generally decreased the cell membrane permeability; TH6241 negated the effects of ethylene on cell permeability.     

Morphological changes in transgenic poplar induced by expression of the rice homeobox gene OSH1

Genetically transformed lombardy poplar (Po-pulus nigra L. var. italica Koehne) plants were regenerated after co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector which included the rice gene for a homeodomain protein (OSH1) and a gene for neomycin phosphotransferase. The expression of the OSH1 gene under control of the cauliflower mosaic virus 35S promoter induced morphological abnormalities in the leaves and stems of the newly generated transgenic poplar plants. This result suggests that OSH1 can function as a regulator of morphogenesis in transgenic poplar, as it does in transgenic rice, Arabidopsis, and tobacco plants. Received: 16 October 1998 / Revision received: 27 November 1998 / Accepted: 12 December 1998     

Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein γTIP

Many bulbous plants need a low-temperature treatment for flowering. Cold, for example, affects the elongation of the stalk, thereby influencing the quality of the cut flower. How the elongation of the stalk is promoted by cold and which physiological and biochemical mechanisms are involved have remained obscure. As invertase has been shown to be involved in the cold-induced elongation of the flower stalks of tulips (Lambrechts et al., 1994, Plant Physiol 104: 515–520), we further characterized this enzyme by cloning the cDNA and analysing its expression in various tissues of the tulip (Tulipa gesneriana L. cv. Apeldoorn) stalk. In addition, the role of sucrose synthase was investigated. Since turgor pressure is an important force driving cell elongation, the role of a water-channel protein (γTIP) was studied in relation to these two enzymes. The mRNA level of the invertase found was substantially up-regulated as a result of cold treatment. Analysis of the amino acid sequence of this invertase revealed the presence of a vacuolar targeting signal. Two different forms of sucrose synthase were found, the expression of one of them appeared to be restricted to the vascular tissue while the other form was present in the surrounding tissue. Both sucrose synthases were present in the stalk during the entire period of bulb storage and after planting, but their activities declined during stalk elongation. The expression of the γTIP gene was restricted mainly to the vascular tissue and its expression profile was identical to that of invertase. Simultaneous expression of invertase and γTIP possibly leads to an increase in osmotic potential and vacuolar water uptake, thus providing a driving force for stretching the stalk cells. Received: 12 January 1999 / Accepted: 24 March 1999