Arabis mosaic and Prunus necrotic ringspot viruses in hop (Humulus lupulus L.)

Purified virus preparations made from nettlehead-diseased hop plants, or from Chenopodium quinoa, to which the virus was transmitted by inoculation of sap, contained polyhedral virus particles of 30 mμ diameter which were identified serologically as arabis mosaic virus (AMV). There were serological differences between AMV isolates from hop and from strawberry, and also differences in host range and in symptoms caused in C. quinoa and C. amaranticolor. AMV was always associated with nettlehead disease. The nematode Xiphinema diversicaudatum occurred in small numbers in most hop gardens, but was numerous where nettlehead disease was spreading rapidly. Preparations from nettlehead-affected hops also contained a second virus, serologically related to Prunus necrotic ringspot virus (NRSV), in mild and virulent forms which infected the same range of test plants but showed some serological differences. Mild isolates did not protect C. quinoa plants against infection by virulent isolates. Hop seedlings inoculated with virulent isolates of NRSV developed symptoms indistinguishable from those of split leaf blotch disease. Latent infection with NRSV was prevalent in symptomless hop plants. Nettlehead disease is apparently associated with dual infection of AMV and virulent isolates of NRSV. An unnamed virus with rod-shaped particles 650 mμ long was common in hop and was transmitted by inoculation of sap to herbaceous plants. Cucumber mosaic virus was obtained from a single plant of Humulus scandens Merr.     

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.     

High throughput isolation of microsatellites in hop (Humulus lupulus L.)

Procedures to generate usable microsatellite marker sequences should be optimized for cost-effectiveness in each species. For hop, we have used a cocktail of several restriction enzymes to digest the genomic DNA. This is followed by capture of microsatellite-containing sequences with long microsatellite probes attached to a membrane. The enrichment level for GA and GT libraries was 37% and 35%, respectively, and 100% of the clones contained microsatellite sequences. Libraries can be generated from genomic DNA in approximately 10 d.     

Development of a shoot multiplication system for hop (Humulus lupulus L.)

Summary Nodal explants from hop were exposed to plant growth regulators to determine suitable media for initiation from axillary buds and subsequent micropropagation. Efficient culture establishment (96.6% of explants) was achieved on Murashige and Skoog (MS) medium (modified to contain 1 mg l⁻¹ thiamine hydrochloride) supplemented with 0.57 μM indoleacetic acid (IAA) and 2.22 μM 6-benzylaminopurine (BA). Subsequent transfer of explants to treatments containing an auxin ([1-naphthaleneacetic acid], NAA or IAA) and BA, 6-[γ,γ-dimethylallylamino]purine (2iP), kinetin (KIN) or thidiazuron (N-phenyl-N′-1,2,3-thidiazol-5-ylurea [TDZ]) resulted in significantly different amounts of multiplication. Optimal TDZ-supplemented media elicited a greater than threefold increase in the number of shoots and nodes generated per explant compared to optimal media containing BA, 2iP and KIN. Shoots were successfully rooted using half-strength MS supplemented with 5.71 μM IAA and 4.9 μM indolebutyric acid (IBA), and regenerated plants were successfully transferred to soi. An overall micropropagation schedule, which can be implemented into hop commercialization programs, includes: (i) establishment in MS with 0.57 μM IAA and 2.22 μM BA; (ii) multiplication in MS with 0.57 μM IAA and 2.27 μM TDZ; (iii) elongation in MS; and (iv) rooting in half-strength MS with 5.71 μM IAA and 4.9 μM IBA.     

Development of novel EST-derived resistance gene markers in hop (Humulus lupulus L.)

Although sources of resistance to major pathogens exist in cultivated hop germplasm, little effort has been invested to date in developing resistance-linked markers. The aim of this study was to design and evaluate resistance gene analogs (RGAs) potentially useful for marker-assisted selection towards novel resistant hop cultivars. A set of 34 putative hop RGAs was retrieved by screening publicly available hop expressed sequence tags (ESTs) for conserved sequence motifs of common resistance protein domains. Seventeen of these were identified as putative RGAs by BLAST analyses. Exon/intron boundary prediction enabled re-sequencing of 24 EST-RGAs, allowing the acquisition of approximately 5 kbp of novel intronic sequence and 8 kbp of re-sequenced exons. Fifteen EST-RGAs exhibited polymorphisms and were added to a framework linkage map of hop. In addition to providing EST-derived markers potentially useful for resistant hop cultivar development, this study provides valuable insights into the utility of targeting hop introns for marker development.     

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.     

High-throughput genotyping of hop (Humulus lupulus L.) utilising diversity arrays technology (DArT)

Implementation of molecular methods in hop (Humulus lupulus L.) breeding is dependent on the availability of sizeable numbers of polymorphic markers and a comprehensive understanding of genetic variation. However, use of molecular marker technology is limited due to expense, time inefficiency, laborious methodology and dependence on DNA sequence information. Diversity arrays technology (DArT) is a high-throughput cost-effective method for the discovery of large numbers of quality polymorphic markers without reliance on DNA sequence information. This study is the first to utilise DArT for hop genotyping, identifying 730 polymorphic markers from 92 hop accessions. The marker quality was high and similar to the quality of DArT markers previously generated for other species; although percentage polymorphism and polymorphism information content (PIC) were lower than in previous studies deploying other marker systems in hop. Genetic relationships in hop illustrated by DArT in this study coincide with knowledge generated using alternate methods. Several statistical analyses separated the hop accessions into genetically differentiated North American and European groupings, with hybrids between the two groups clearly distinguishable. Levels of genetic diversity were similar in the North American and European groups, but higher in the hybrid group. The markers produced from this time and cost-efficient genotyping tool will be a valuable resource for numerous applications in hop breeding and genetics studies, such as mapping, marker-assisted selection, genetic identity testing, guidance in the maintenance of genetic diversity and the directed breeding of superior cultivars.     

Inhibition ofStreptococcus mutans and Other Oral streptococci by hop (Humulus lupulus L.) constituents

We report the inhibition of the causative agents of dental caries, Streptococcus mutans and other oral streptococci, by the antimicrobially active ingredients of the hop plant (Humulus lupulus L.). The hop constituents studied were purified beta acid, xanthohumol, isoalpha acid and tetra iso-alpha acid. Cruder hop extracts were also investigated. The antimicrobial activity of these hop constituents was tested against four strainsof Streptococcus mutans as well as one strain each ofStreptococcus sanguis andStreptococcus salivarius and compared to antimicrobial essential oils used in mouthwashes in two independent assay systems. We found that all tested hop constituents inhibited the Streptococci. The minimum inhibitory concentration at pH 7.5 ranged from 2 to 50 μg/ml depending on the microorganism and hop phytochemical tested. Contrary to a previous report, there was no activity enhancement by ascorbic acid over and above the enhancement due to pH lowering. Thére was no resistance development to beta acid after 10 passages in a subinhibitory concentration of this acid. Antimicrobial activity of hop constituents was found to be greater than other plant products such as thymol, nerol, cinnamon oil, oil of clove, menthol and eucalyptol. The possibilities of using hop constituents in mouthwashes are discussed.     

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     

Modern biotechnologies in estimation of genetic diversity of Ukrainian varieties of hop (Humulus lupulus L.)

Genetic variety estimation of hop gene pool using DNA-typing of highly polymorphic microsatellite loci and optimization of introduction to the culture of in vitro conditions is the important stage of national varieties resources forming, basis of modern nursery and protect mean of varieties property, and also it is necessary for development of molecular methods of selection of planting-stocks free from pathogens.     

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.     

In vitro tetraploid induction and generation of tetraploids from mixoploids in hop (Humulus lupulus L.)

This paper describes an efficient colchicine-mediated technique for the in vitro induction of hop tetraploids and its confirmation by flow cytometry. A window of conditions generated a high percentage (>20%) of tetraploid induction, with the highest induction (25.6%) achieved with 0.05% colchicine for 48 h. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploid and tetraploid hops were compared, and it was determined that stomatal length and width are suitable parameters for identifying putative hop tetraploids. As well as generating tetraploids, this technique generates mixoploid hops. Calli, derived from mixoploid leaves, were induced to form shoot buds and shoots. Individual shoots were classed as diploid, mixoploid or tetraploid after screening by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but fails to generate tetraploids.     

Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus lupulus L.) cones.

Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide synthase, was purified to homogeneity from 4.2 mg protein extract obtained from hop (Humulus lupulus L.) cone glandular hairs. The enzyme uses isovaleryl-CoA or isobutyryl-CoA and three molecules of malonyl-CoA to form phlorisovalerophenone or phlorisobutyrophenone, intermediates in the biosynthesis of the hop bitter acids (alpha- and beta-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. Km values of 4 microm for isovaleryl-CoA, 10 microm for isobutyryl-CoA and 33 microm for malonyl-CoA, were found. The amino-acid sequences of two peptides, obtained by digestion of VPS, showed that the enzyme is highly homologous to plant chalcone synthases. The functional and structural relationship between VPS and other aromatic polyketide synthases is discussed.     

Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.)

A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop (Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38-51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N6 amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity.     

Efficient and stable transformation of hop (Humulus lupulus L.) var. Eroica by particle bombardment

To the best of our knowledge, this is the first accurate and reliable protocol for hop (Humulus lupulus L.) genetic transformation using particle bombardment. Based on the highly productive regeneration system previously developed by us for hop var. Eroica, two efficient transformation protocols were established using petioles and green organogenic nodular clusters (GONCs) bombarded with gusA reporter and hpt selectable genes. A total of 36 hygromycin B-resistant (hyg^r) plants obtained upon continuous selection were successfully transferred to the greenhouse, and a first generation group of transplanted plants was followed after spending a complete vegetative cycle. PCR analysis showed the presence of one of both transgenes in 25 plants, corresponding to an integration frequency of 69.4% and an overall transformation efficiency of 7.5%. Although all final transformants were GUS negative, the integration frequency of gusA gene was higher than that of hpt gene. Petiole-derived transgenic plants showed a higher co-integration rate of 76.9%. Real-time PCR analysis confirmed co-integration in 86% of the plants tested and its stability until the first generation, and identified positive plants amongst those previously assessed as hpt ⁺ only by conventional PCR. Our results suggest that the integration frequencies presented here, as well as those of others, may have been underestimated, and that PCR results should be taken with precaution not only for false positives, but also for false negatives. The protocols here described could be very useful for future introduction of metabolic or resistance traits in hop cultivars even if slight modifications for other genotypes are needed.     

Mechanical transmission of Apple mosaic virus in Australian hop (Humulus lupulus) gardens

The transmission of Apple mosaic virus (ApMV; hop, H and intermediate, I serotypes) in Australian hop cultivars was assessed in glasshouse and field trials. Under field conditions, the rate of ApMV transmission was halved when contact between neighboring plants was prevented by early season applications of paraquat to restrict basal shoot growth. However, in a separate field trial the presence of root grafts between hop plants, which may contribute to virus transmission, was also suggested. In glasshouse trials, ApMV was transmitted successfully to hop by the mechanical inoculation of infective sap, simulated pruning, foliar contact, and root grafting, but not by root contact. The rate of mechanical transmission of ApMV to the hop cultivar ‘Victoria’ was greater than to other hop cultivars commonly grown in Australia. However, success of mechanical transmission of ApMV also appeared to be influenced by the cultivar from which inoculum was obtained. ApMV was detected throughout the year in all tissues, in chronically infected field grown plants of cultivar ‘Victoria’, suggesting a uniform virus distribution. The reliability of ApMV detection by serology did not decline in ‘Victoria’ plants later in the growing season as occurred in other cultivars.     

Cloning and analysis of valerophenone synthase gene expressed specifically in lupulin gland of hop (Humulus lupulus L.)

Resin and essential oil derived from hop (Humulus lupulus L.) cones are very important compounds for beer brewing, and they specifically accumulate in the lupulin gland of hop cones. In order to identify the genes responsible for the biosynthetic pathway of these compounds and use the identified genes for hop breeding using Marker Assisted Selection and transformation techniques, genes expressed specifically in the lupulin gland were cloned and sequenced. One of them was suggested to be similar to the chalcone synthase gene from the DNA sequence. The translation product of the gene had the activity of valerophenone synthase, which catalyzes a part of the synthesis reaction of alpha-acid and beta-acid. Northern analysis showed that the valerophenone synthase gene seemed to be expressed specifically in the lupulin gland.