啤酒花茎段培养及快速繁殖技术研究

以啤酒花的幼嫩茎段作为外植体,研究出理想的一次性成苗培养基：ＭＳ＋６－ＢＡ０．０１ｍｇ／Ｌ＋ＩＡＡ０．１ｍｇ／Ｌ＋琼脂６．５ｇ／Ｌ＋葡萄糖２％。试管苗月增殖率４．２,生根率１００％,成苗率９０％。采用试管苗微扦插的方式进行快速繁殖,经锻炼后直接移入经改造后的土壤中,移栽成活率达９３．８％。     

Eleven new microsatellites for hop (Humulus lupulus L.)

We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite‐enriched libraries for GA_n and GT_n types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.     

啤酒花节间外植体再生体系的建立

在国内首次建立了两个啤酒花主栽品种（麒麟丰禄,青岛大花）的节间外植体再生体系。实验表明：啤酒花无菌苗的节间为适于不定芽再生韵外植体;适宜的再生培养基为Ms基本培养基＋B5维生素＋葡萄糖20g／L,附加IAA0．25mg／L＋TDZ2mg／L（麒麟丰禄）或IAA0．125mg／L＋TDZ1．5mg／L（青岛大花）,以7g／L琼脂固化,pH5．8;不定芽再生频率可达57％（麒麟丰禄）或16％（青岛大花）。啤酒花再生体系的建立,为利用植物基因工程技术培育抗病虫害啤酒花新品系奠定了基础。     

多效唑（MET）对啤酒花试管苗生长和移栽的影响

在培养基中加入0.1-0.2mg/L浓度的多效唑（MET）,啤酒花试管苗植株矮壮,单株生根数量增加、移栽成活率提高13％。0．5－2．0mg/L时,试管苗基部愈伤组织增多,枝叶失绿,生长受到抑制。初步发现多将唑对啤酒花试管苗的效力与葡萄糖用量呈正相关。     

New polymorphic dinucleotide and trinucleotide microsatellite loci for hop Humulus lupulus L

One hundred and thirty-five microsatellite markers were developed for hop Humulus lupulus L. from di- and trinucleotide-enriched libraries. Seventy-eight primers showed amplification in two tested genotypes. Twenty-four loci were further characterized on a population of 34 hop samples and the number of alleles per locus, observed heterozygosity and expected heterozygosity ranged from two to 20 (9.7 on average), from 0.0294 to 0.9412 (0.6234 on average) and from 0.0294 to 0.9170 (0.6720 on average), respectively. These microsatellite markers will be further used for studying population structures and relationships and for identifying important qualitative and quantitative loci of hop.     

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

 A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions. Received: 10 March 1997 / Revision received: 12 November 1997 / Accepted: 22 November 1998     

Isolation and characterization of polymorphic microsatellites for assessment of genetic variation of hops (Humulus lupulus L.)

Hop (Humulus lupulus L.) cultivars are vegetatively propagated and it is difficult to differentiate them during the process of propagation. Fingerprinting with molecular markers based on DNA could be a useful means of identifying different cultivars. Simple sequence repeats, or microsatellite markers, are the most suitable marker for genetic fingerprinting because they are multi‐allelic and co‐dominant. For this purpose, we have developed primers for 10 new polymorphic microsatellite loci that are suitable for genetic fingerprinting in hop.     

Enzymatic Reactions by Five Chalcone Synthase Homologs from Hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

NMR assignment of iso-alpha-acids from isomerised extracts of Humulus lupulus L. cones

Iso-alpha-acids are known to contribute to the characteristic bitter taste of beer. Six iso-alpha-acids were isolated from a carbon dioxide extract of the cones of Humulus lupulus L. by centrifugal partition chromatography followed by separation through beta-cyclodextrin binding. This method overcame the low yield issue of most isolation procedures that results from the low stability of these compounds to light and oxygen. Their full identification was performed using one- and two-dimensional NMR spectrometry, including (1)H- and (13)C-NMR, (1)H-(1)H COSY, HMQC and HMBC and electrospray ionisation mass spectrometry. The results confirmed the structures of the isolated compounds as trans-isocohumulone, cis-isocohumulone, trans-isohumulone, cis-isohumulone, trans-isoadhumulone and cis-isoadhumulone. Epimers can be easily distinguished by observing the chemical shift differences of the H-5, H-1', H-2' and C-5 signals and the different splitting pattern of H-5 and H-2'.     

Humulus lupulus L. cv. Cascade grown in Northern Italy: morphological and phytochemical characterization

Abstract

Several aroma hops (Humulus lupulus L.) were recently introduced in Northern Italy as a small-scale production of excellence. In this preliminary study, the American cultivar Cascade was investigated in a combined morphological and phytochemical survey. Morphological investigation on trichome structure, density and distribution was performed by scanning electron microscopy and light microscopy. Essential oil composition, α/β-acid and polyphenol profiles over 3 years were determined by GC-MS and HPLC analyses. Two types of non-glandular (simple and cystolithic) and glandular (peltate and bulbous) trichomes were observed on leaves and female inflorescences. The peltate trichomes resulted as the main sites of terpene production and accumulation. The essential oil profiles showed myrcene, β-caryophyllene, (E)-β-farnesene and humulene epoxide II as the dominant compounds over the three collection times, although with different relative abundances. The presence of two exclusive compounds, γ-muurolene and trans-γ-cadinene, characterized the investigated cv. Cascade, potentially enhancing herbal, woody and spicy aroma traits of this cultivation in Northern Italy. The bitter acid composition showed quantitative values consistent with literature data only for the second and third monitoring year. Qualitative differences in polyphenol content were also recorded, for the presence of quercetin-3-O-malonylglucoside and kaempferol-3-O-rutinoside, which may characterize this peculiar Italian cultivation.     

Preparation of green colorimetric pH sensor using Humulus lupulus L. (common hop) biomolecular extract for sweat monitoring

Novel smart cotton diagnostic assay was developed toward onsite sensing of sweat pH variations for possible medical applications such as drug test and healthcare purposes. Humulus lupulus L. extract was obtained according to previously reported procedure. As reported by high-performance liquid chromatography (HPLC), the extract demonstrated the presence of hop acids, prenylchalcones, and prenylflavanones, which is responsible for the colorimetric changes. The extract was applied to cellulose fibers employing potassium aluminum sulfate as mordant. This was observed by the formation of mordant/xanthohumol nanoparticles onto cotton surface. The absorption spectra and CIE (Commission Internationale de l'Eclairage) Lab screening of the prepared cotton assay showed colorimetric changes in association with hypsochromic shift from 600 nm to 433 nm upon exposure to sweat simulant fluid (pH < 7). The biochromic activity of the xanthohumol-finished cotton depends mainly on the halochromic performance of the xanthohumol chromophore to show a colorimetric switch from yellow to white owing to intramolecular charge transfer in the xanthohumol molecule. No substantial defects were detected in gas-permeability and stiffness of the treated fabrics. Satisfactory fastness was approved for the xanthohumol-dyed diagnostic cotton assay.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

Modulation of breast cancer cell survival by aromatase inhibiting hop (Humulus lupulus L.) flavonoids

Hop flavonoids are being regarded as attractive molecules to prevent or treat certain forms of cancer. Studies have focused mainly on xanthohumol, the most abundant prenylated chalcone existing in hops extract. However, during the production of beer, or after its ingestion, xanthohumol originates different metabolites, among which isoxanthohumol and 8-prenylnaringenin. The aim of this work was to study the effect of the prenylflavonoids xanthohumol, isoxanthohumol and 8-prenylnaringenin on the breast cancer Sk-Br-3 cell line proliferation, apoptosis and activity of the enzyme aromatase (estrogen synthase). Aromatase activity was determined by a tritiated water assay, cell proliferation was assessed by [³H]thymidine incorporation, sulforhodamine B protein measurement and Ki-67 immunostaining and apoptosis was determined by TUNEL. Our results show that all tested prenylflavonoids were able to inhibit aromatase activity and thus, estrogen formation. Additionally, breast cancer cell line proliferation was decreased and apoptosis induced by all three compounds. The presence of 17β-estradiol in treatment medium was able to revert the effect of the prenylflavonoids on cellular proliferation. These observations strengthen the idea that hop flavonoids may have anti-breast cancer effects and shed new light on a possible mechanism of action by which these effects occur, namely through their ability to decrease estrogen synthesis.     

Anti-inflammatory and anti-tumor-promoting effects of 5-deprenyllupulonol C and other compounds from Hop (Humulus lupulus L.)

A new phloroglucinol derivative, 5‐deprenyllupulonol C ( 1 ), along with four other phloroglucinol derivatives, 2 – 5 , five chalcones, 6 – 10 , four flavanones, 11 – 14 , two flavonol glycosides, 15 and 16 , and five triterpenoids, 17 – 21 , were isolated from the female inflorescence pellet extracts of hop (Humulus lupulus L.). Upon evaluation of these compounds against the Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Raji cells, twelve compounds, i.e., 1 – 4, 11 – 14, 17 – 19 , and 21 , showed potent inhibitory effects on EBV‐EA induction, with IC₅₀ values in the range of 215–393 mol ratio/32 pmol TPA. In addition, eleven compounds, i.e., 1 – 4, 6, 11, 12, 14, 17, 18 , and 20 , were found to inhibit TPA‐induced inflammation (1 μg/ear) in mice, with ID₅₀ values in the range of 0.13–1.06 μmol per ear. Further, lupulone C ( 2 ) and 6‐prenylnaringenin ( 14 ) exhibited inhibitory effects on skin‐tumor promotion in an in vivo two‐stage mouse‐skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter.     

猕猴桃属种间嫁接亲和性试验研究及抗根结线虫砧木的初步筛选

以中华猕猴桃（Actinidia chinensis)武植2号（Wuzhi-2),通山5号（Tongshan-5)品种和美味猕猴桃（A.deliciosa)金魁（Jinkui)品种为接穗,采用美味猕猴桃等9种类型猕猴桃一年生实生苗为砧木进行嫁接,研究其短期嫁接亲和性,并对这9种砧木进行抗根结线虫（Meloidogyne)筛选试验。结果表明,种间的嫁接亲和性及抗根线虫性 均有显著差异：以美味猕猴桃和中华猕猴桃优良品种实生苗为砧木,与3个品种均有高的亲和性,成活率高达100％,且抗性在9种砧木中最强;其它物种作砧木均有不足,武植2号／山猴猴桃,通山5号／M16,通山5号／山梨猕猴桃3个嫁接组合的亲和性均较好,成活率在80％以上,但山梨猕猴桃较易感病且与金魁的亲和力较低,M16抗性虽较好,但与金魁,武植2号的亲和力也较低,漓江猕猴桃是抗性较强的物种,但它与3种接穗品种的亲和力都较小,其它5种砧木,对根结线虫的抗性均不强,因此结合成活率及抗根结线虫两因素,在供试材料中,美味猴猕桃和中华猕猴桃接适应性广且抗性强,是较好的砧木或选育砧木的亲本材料。     

Differential expression and cellular localization of ERKs during organogenic nodule formation from internodes of Humulus lupulus var. Nugget

The expression and subcellular localization of extracellular signal-regulated kinase 1 or 2 (ERK1/2) homologues (HLERK1/2) during the process of organogenic nodule formation in Humulus lupulus var. Nugget was studied using antibodies specific for ERK1 and ERK2, and for phosphorylated mitogen-activated protein kinases (MAPKs). The increase in HLERK levels, detected by Western blotting 12 hours after wounding suggests their involvement in response to the wounding treatment applied for morphogenesis induction. In dividing cambial cells, occurring in between 4 and 7 days after morphogenesis induction, as well as in dividing prenodular cells (15 days after induction) HLERK1 and/or 2 were localized in the nucleus. However, as soon as nodular cells start proliferating to form shoot meristems, HLERK1 and 2 were detected in the cytoplasm and not in the nucleus. The data reported account for a differential expression and activation of HLERK1 and HLERK2 throughout the process of nodule formation and plantlet regeneration. HLERK1 appears to be expressed in the stages of nodule formation and plantlet regeneration, playing a possible role in controlling cell proliferation and differentiation. HLERK2 may be induced as a response to reactive oxygen species (ROS) generated by wounding of internodes as its expression is reduced in liquid medium with less oxygen availability compared to solid medium. However, addition of a ROS inhibitor to the liquid medium does not result in a further decrease in the HLERK2 level.     

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.     

Root-Knot Nematodes and the Process of Ageing in Plants

Infection of plants by root-knot nematodes is often accompanied by physiological changes characteristic of ageing. Ultra-low tissue luminescence of infected plants indicated oxidation of cell-membrane lipids. Cells with membranes subjected to oxidation lose some of their capacity for water retention. Treating tomato and radish with lidocaine hydrochloride, an inhibitor of lipid oxidation, retarded above-ground symptoms of root-knot nematode infection and of ageing.     

Structure and inheritance of ribosomal DNA variants in cultivated and wild hop, Humulus lupulus L.

Genetic variation was assessed among cultivated and wild hop, Humulus lupulus, by restriction fragment length polymorphisms (RFLPs) of the ribosomal RNA genes (rDNA). Two rDNA length variants of 10.3 and 9.3 kbp represented by three phenotypes designated A, B and C were detected with XhoI. Restriction-site mapping showed that hop rDNA is structurally similar to those of most higher plants. A high level of homogeneity existed in rDNA repeat lengths among the diverse hop genotypes. Generally, phenotype A was predominant in wild and cultivated European and Asian genotypes; phenotype B in North American cultivars; while phenotype C was present only in native North American hop, providing a potential molecular marker for the identification of this germ plasm. The rDNA data provided genetic evidence for the separation of native and cultivated American genotypes and supports the hypothesis that North American hop cultivars are of hybrid origin from European and native American genotypes. The segregation of rDNA phenotypes in four F₁ families suggests that a single locus with two co-dominant alleles controls genetic variability for rDNA variants in hop.     

Comparison of Sequences from the Ribosomal DNA Intergenic Region of Meloidogyne mayaguensis and Other Major Tropical Root-Knot Nematodes

The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications.