Lupulus Var Research Articles

Host-plant phenology mediates facultative ant mutualism in the rare butterfly, Celastrina humulus (Lepidoptera: Lycaenidae).

Host-plant phenology can directly and indirectly mediate the abundance of insect herbivores. Our objective was to determine how host-plant flowering phenology shapes a facultative ant-lycaenid mutualism. The focus of our research was the hops azure, Celastrina humulus Scott & D. Wright, a rare species whose larvae feed on the pollen-bearing inflorescences of Humulus lupulus var. neomexicanus. We used several approaches to evaluate the role of host-plant flowering phenology in this system. First, we monitored larvae over three study years (2020-2022) to parse the role of host-plant flowering phenology and other factors in shaping the likelihood of ant tending. Second, we tested larval performance at various phenological stages of the host plant. We also quantified variation in soluble proteins and secondary metabolites among inflorescences at varying phenological stages. Lastly, we treated artificial sugar-protein baits with extracts from different phenological stages of the host plant; this allowed us to assess how chemical variation among stages could impact ant recruitment. Monitoring results revealed that the likelihood of ant tending was lowest for larvae on host plants with early-stage inflorescences. These floral stages had the greatest concentrations of both soluble proteins and α-acids (humulone and cohumulone), and in the feeding trial, early-stage flowers enabled greater weight gain for larvae. However, extracts from early-stage flowers reduced ant recruitment to sugar-protein baits. Altogether, these results suggest that early-stage inflorescences enhance larval growth while also reducing the recruitment of mutualist ants. This shows an indirect mechanism whereby changing host-plant phenology can mediate herbivore populations through interactions with ants.

Registration of seven powdery mildew‐resistant wild hop germplasm lines

AbstractCultivation of common hop (Humulus lupulus L.) has increased dramatically during the past decade, with the primary growth occurring in the Pacific Northwest of the United States. A major limitation to hop production is hop powdery mildew [caused by Podosphaera macularis (Wallr.) Braun & Takam]. To date, a comprehensive evaluation of wild hop germplasm resources for resistance to hop powdery mildew has yet to be conducted. Herein, we report the first extensive powdery mildew resistance screening of 244 wild H. lupulus populations (6,732 individuals) collected from North America and Eurasia. We used an elimination screening approach by sequentially inoculating four P. macularis isolates containing virulence on all previously described R genes. We detected the presence of powdery mildew resistance to isolates representing extant pathogenic diversity in seven germplasms (UMN‐PMR‐002 Male [Reg. no. GP‐38, PI 699936], UMN‐PMR‐005 [Reg. no. GP‐39, PI 699937], UMN‐PMR‐006 Female [GP‐40, PI 699938], UMN‐PMR‐007 Male [Reg. no. GP‐41, PI 699939], UMN‐PMR‐008 [Reg. no. GP‐042, PI 699940], UMN‐PMR‐010 Male [Reg. no. GP‐43, OU 699941], UMN‐PMR‐013 Female [Reg. no. GP‐44, PI 699942]). Based upon genotypic profiling, the seven germplasms were identified in H. lupulus var. lupulus originating from Eurasia. These accessions could be used to introgress broad‐spectrum powdery mildew resistance in hop breeding programs worldwide.

Cytosine Methylation in Genomic DNA and Characterization of DNA Methylases and Demethylases and Their Expression Profiles in Viroid-Infected Hop Plants (Humulus lupulus Var. 'Celeia').

Abiotic and biotic stresses can lead to changes in host DNA methylation, which in plants is also mediated by an RNA-directed DNA methylation mechanism. Infections with viroids have been shown to affect DNA methylation dynamics in different plant hosts. The aim of our research was to determine the content of 5-methylcytosine (5-mC) in genomic DNA at the whole genome level of hop plants (Humulus lupulus Var. ‘Celeia’) infected with different viroids and their combinations and to analyse the expression of the selected genes to improve our understanding of DNA methylation dynamics in plant-viroid systems. The adapted HPLC-UV method used proved to be suitable for this purpose, and thus we were able to estimate for the first time that the cytosine methylation level in viroid-free hop plants was 26.7%. Interestingly, the observed 5-mC level was the lowest in hop plants infected simultaneously with CBCVd, HLVd and HSVd (23.7%), whereas the highest level was observed in plants infected with HLVd (31.4%). In addition, we identified three DNA methylases and one DNA demethylase gene in the hop’s draft genome. The RT-qPCR revealed upregulation of all newly identified genes in hop plants infected with all three viroids, while no altered expression was observed in any of the other hop plants tested, except for CBCVd-infected hop plants, in which one DNA methylase was also upregulated.

Phytochemical Characterization of Native New Mexico Hops

Neomexicanus hops (Humulus lupulus var. neomexicanus) are receiving increased attention within the craft beer and nutraceutical industries. Characterization of bittering acids and essential oils in two neomexicanus varieties revealed wide ranges of bittering acid compositions and distinct essential oil profiles compared with ‘Cascade’ common hops (H. lupulus). Total phenolic content (TPC), expressed as gallic acid equivalent (GAE), in neomexicanus hops ranged from 50 to 100 mg·g−1 GAE, consistently higher than published literature values for hop TPC (2 to 50 mg·g−1 GAE). Results indicate that, compared with ‘Cascade’, neomexicanus hops have unique phytochemical characteristics, which may lead to new applications in brewing and nutraceutical fields.

Germplasm sources of protective glandular leaf trichomes of hop (Humulus lupulus)

We report here that small but numerous foliar glands are present near the base of the abaxial leaf blades of hop (Humulus lupulus L.) at about 2.6 times the density found in the more distal regions, and this concentration of glands appear to protect the basal region from damage by insects. Distinctive wild taxa of H. lupulus are native to distinctive regions of the north temperate world. The much higher presence of protective glands in the three native North American Humulus taxonomic varieties in comparison with the varieties native to Europe and Asia suggests specialized leaf feeders in North America have been responsible for the evolution of greater gland density. Commercial domesticated hop cultivars trace most of their germplasm to the European hop (H. lupulus var. lupulus), which possesses a much lower concentration of leaf glands than the native hops of all other regions of the world, and therefore is likely relatively susceptible to damage. We suggest that breeding for increased density of protective foliage glands may be a non-chemical way of alleviating foliar feeding damage in cultivated hops.

First report of Dahlia latent viroid and Potato spindle tuber viroid mixed‐infection in commercial ornamental dahlia in Japan

Dahlia (Asteraceae) has been cultivated since 1841 in Japan. Two viroids belonging to the family Pospiviroidae, Potato spindle tuber viroid (PSTVd) and Dahlia latent viroid (DLVd), were identified separately from dahlia plants in Japan and in the Netherlands, respectively (Tsushima et al., 2011; Verhoeven et al., 3). Because DLVd was characterised as a novel species, the findings drew attention towards the study of DLVd infection in dahlia and its potential risk in domestic dahlia cultivation. A total of four dahlia samples infected with PSTVd-dahlia (Tsushima et al., 2) were collected, and total RNA was extracted from approximately 200 mg of leaf tissue using TRIzol® reagent (Life Technologies, Carlsbad, USA) according to manufacturer's instructions. RNA was separated in 2-dimensional polyacrylamide gel (2D-PAGE) (Schumacher et al., 1) and two specific bands appeared, one corresponding to PSTVd infection and the other being unknown (Fig. 1). The unknown circular RNA samples were gel-excised and first tested with PSTVd-specific primers using a two-step reverse-transcription-polymerase chain reaction (RT-PCR) (Tsushima et al., 2), with, however, negative results. Therefore, a DLVd-specific primer set, DLVd-P1 (5'-GGGGCTCCTCAGAGAGTCTC-3') and DLVd-P2 (5'-GGGGCAACTCCGAGAGTGCTG-3') (Verhoeven et al., 3), was then used in two-step RT-PCR for the unknown viroid-like circular RNAs. The amplified DNA bands of the expected size (~340 bp) from these positive samples were purified, ligated to pGEM®-T easy vector (Promega, Madison, USA) and sequenced. Identical 342 bp sequences were obtained from all four dahlia isolates (GenBank Accession No. LC036322), corresponding with DLVd reported from the Netherlands (JX263426). Furthermore, a total of 78 dahlia leaf samples were collected from fields around Japan, irrespective of disease symptoms, and verified by two-step RT-PCR using the same DLVd-specific and pospiviroid-specific primers (Tsushima et al., 2). Of these samples, DLVd infection was detected in 38, suggesting that DLVd is widespread in Japan. New PSTVd infection was not found. To evaluate the pathogenicity of DLVd and PSTVd, these viroids were co-inoculated into wild hop (Humulus lupulus var. cordifolius) and dahlia seedlings. DLVd alone was inoculated into members of the Asteraceae family, Chrysanthemum pacificum, Tithonia rotundifolia, Helianthus annuus and Glebionis coronaria. Inoculated dahlia seedlings were positive for both DLVd and PSTVd, but wild hop and the remaining members of Asteraceae were negative. Dahlia seedlings that were positive for DLVd and PSTVd showed mild leaf curling in the early stage of growth (Fig. 2). To the best of our knowledge, this is the first report of DLVd infecting commercial dahlia in Japan and also of the mixed-infection of DLVd and PSTVd in commercial dahlia plants. A possible negative synergistic interaction of DLVd and PSTVd during dahlia cultivation must be considered. This work was supported in part by Japan Society for the Promotion of Science KAKENHI grant no. 24380026. TT received Research Fellowship of the Japan Society for the Promotion of Science.

The Draft Genome of Hop (Humulus lupulus), an Essence for Brewing.

The female flower of hop (Humulus lupulus var. lupulus) is an essential ingredient that gives characteristic aroma, bitterness and durability/stability to beer. However, the molecular genetic basis for identifying DNA markers in hop for breeding and to study its domestication has been poorly established. Here, we provide draft genomes for two hop cultivars [cv. Saazer (SZ) and cv. Shinshu Wase (SW)] and a Japanese wild hop [H. lupulus var. cordifolius; also known as Karahanasou (KR)]. Sequencing and de novo assembly of genomic DNA from heterozygous SW plants generated scaffolds with a total size of 2.05 Gb, corresponding to approximately 80% of the estimated genome size of hop (2.57 Gb). The scaffolds contained 41,228 putative protein-encoding genes. The genome sequences for SZ and KR were constructed by aligning their short sequence reads to the SW reference genome and then replacing the nucleotides at single nucleotide polymorphism (SNP) sites. De novo RNA sequencing (RNA-Seq) analysis of SW revealed the developmental regulation of genes involved in specialized metabolic processes that impact taste and flavor in beer. Application of a novel bioinformatics tool, phylogenetic comparative RNA-Seq (PCP-Seq), which is based on read depth of genomic DNAs and RNAs, enabled the identification of genes related to the biosynthesis of aromas and flavors that are enriched in SW compared to KR. Our results not only suggest the significance of historical human selection process for enhancing aroma and bitterness biosyntheses in hop cultivars, but also serve as crucial information for breeding varieties with high quality and yield.

Genome size in Humulus lupulus L. and H. japonicus Siebold and Zucc. (Cannabaceae)

We analysed chromosome lengths, karyotype structure, and nuclear DNA content (flow cytometry) in diploid (2n=20) and triploid (2n=30) European <em>H. lupulus</em> var. <em>lupulus</em>, American <em>H. lupulus</em> var. <em>neomexicanus</em> (2n=20) and Japanese ornamental hop, <em>H. japonicus</em> (F/2n=16; M/2n=17). Diploid female representatives of <em>H. lupulus</em> var. <em>lupulus</em> and <em>H. l.</em> var. <em>neomexicanus</em> differed in total length of the basal chromosome set (23.16 µm and 25.99 µm, respectively) and nuclear 2C DNA amount (5.598 pg and 6.064 pg) but showed similar karyotype structure. No deviation from the additivity, both in chromosome length and 2C DNA amount was evidenced in triploid monoecious <em>H. lupulus</em> (2n=30, XXY). <em>H. japonicus</em> showed different karyotype structure, smaller basal chromosome set (F/18.04 µm, M/20.66 µm) and lower nuclear DNA amount (F/3.208 pg and M/3.522 pg). There are first evaluations of nuclear genome size in diploid, not commercial representative of European <em>H. lupulus</em> var. <em>lupulus</em> and American <em>H. lupulus</em> var. <em>neomexicanus</em> and first attempt to determine the absolute male and female genome size in two Humulus species.

USE OF DIVERSITY ARRAY TECHNOLOGY (DART) FOR GENOTYPING OF HUMULUS LUPULUS L.

Diversity Arrays Technology (DArT - www.diversityarrays.com) is a micro-array based DNA marker technique for genome wide discovery and genotyping of genetic variation. DArT potentially allows simultaneous scoring of thousands of restriction site polymorphisms between genotypes and does not require DNA sequence information or site specific oligonucleotides. An international consortium (Australia, USA, UK, Slovenia) screened 86 (cultivated, wild; female, male) accessions of hop from Europe, North America, Asia and Australia, including examples of Humulus lupulus var. lupuloides, H. lupulus var. pubescens and H. lupulus var. neomexicanus, using DArT. The accessions included key current and historical, high and low alpha-acid, aroma, triploid, dwarf, and powdery and downy mildew resistant and susceptible cultivars. DArT identified 730 polymorphic markers, with reproducibility approaching 100%, and a high call rate. Preliminary studies of the data indicate that a large proportion of the markers reflect differences between wild and cultivated material. Distinction can be made between and within material of European, North American and Asian origin, with related accessions, and accessions from a single cultivar known by several synonyms clustering together. Initial investigation suggests that the reproducibility of DArT markers, the level of genetic variation detected with this system in hop, and the resolution of known genetic affinities are positive signs that DArT will provide the platform for future exploration of marker-trait associations to develop marker assisted selection technologies in hop. Based on this study, the DArT platform provides a reproducible, accurate, high throughput genotyping tool for hop. In the future this work will be developed to increase the number of markers available for hop using DArT, and to generate linkage maps and identify quantitative trait loci for key traits in a number of families.

POWDERY MILDEW RESISTANT HOP GERMPLASM RELEASE: 'KAZAK 2000'

A USDA-sponsored plant collecting expedition in collaboration with the Vavilov Institute of Plant Industry (VIR), St. Petersburg, Russian Federation, and the Aral Sea Experiment Station for Plant Genetic Resources, Chelkar Town, Kazakhstan, was conducted in the fall of 2000. Hop (Humulus lupulus var. lupulus) cones were collected from wild plants growing on trees in a moist area about 50 km NE of Emba, Kazakhstan. A portion of the seeds were brought to the US and donated to the US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon. Of 74 seedlings that were germinated from Kazakhstani seedlots, four seedlings from PI 635262 demonstrated resistance to powdery mildew, caused by Podosphaera macularis (Braun and Takamatsu). One of these resistant seedlings, 'Kazak 2000', exhibited a hypersensitive response after challenge by powdery mildew, especially when incubated at temperatures greater than 29°C post-infection. In greenhouse assays with an Oregon field population of P. macularis, macroscopic signs of powdery mildew were not observed following repeated inoculations. In laboratory assays at 18°C, 'Kazak 2000' did not develop powdery mildew when challenged with a P. macularis isolate capable of overcoming resistance genes R b , R 3 , and R 5 . Infection of 55% of detached leaves was observed when challenged with characterized isolates capable of overcoming R b , R 3 , R 4 , R 5 , R 6 or R b , R 1 , R 2 , R 3 , R 5 , and R 6 , respectively. In experimental field plots in Oregon from 2003 to 2007, only one powdery mildew colony was observed despite inoculation and exposure to natural inoculum sources each year. Infection of cones was not observed. 'Kazak 2000' provides a novel source of powdery mildew resistance that is effective at high temperatures against multiple races of the pathogen. Limited quantities of cuttings of 'Kazak 2000' are available for research or breeding. Requests should be made to the Hop Curator, USDA-ARS, National Clonal Germplasm Repository, Corvallis, OR 97333.

AFLP Discrimination of Native North American and Cultivated Hop

Hop (Humulus lupulus L.) is cultivated for the female flowers, or “cones,” which traditionally have been used as a bittering and flavoring agent in beer. Hop breeding historically relied on relatively simple selection techniques within established breeding lines. Supplementing current breeding material with new genetic sources would enhance a hop breeder's ability to select for new traits. The objective of this research was to assess the genetic relationship of H. lupulus var. lupuloides and H. lupulus var. pubescens accessions with hop germplasm currently utilized by hop breeding programs. A total of 60 hop accessions representing both cultivated (n = 21) and native (n = 39) Humulus were evaluated using amplified fragment length polymorphism (AFLP) molecular markers. The four primer combinations used generated 296 scorable fragments of which 176 (59.5%) were polymorphic. Principal components analysis and hierarchal cluster analysis showed the native American accessions clustering separately from the cultivated germplasm. Within each of the two main groups, two smaller subgroups were evident with H. lupulus var. lupuloides and H. lupulus var. pubescens segregated into unique clusters. This research provides the first molecular genetic evidence of H. lupulus var. pubescens and H. lupulus var. lupuloides being two separate botanical varieties. Results from this research suggest incorporating the native American hop accessions studied in this experiment would enhance the genetic diversity within hop breeding programs.

Genic SSRs for European and North American hop (Humulus lupulus L.)

Eight genic SSR loci were evaluated for genetic diversity assessment and genotype identification in Humulus lupulus L. from Europe and North America. Genetic diversity, as measured by three diversity indices, was significantly lower in European cultivars than in North American wild accessions. Neighbor Joining cluster analysis separated the hop genotypes into European and North American groups. These eight SSRs were useful in uniquely identifying each accession with the exception of two sets of European landraces and a pair of Japanese cultivars, ‘Shinshuwase’ and ‘Kirin II’. An accession from Manitoba grouped with the European (EU) cluster reflecting the group’s genetic similarity to older Manitoba germplasm used to develop ‘Brewer's Gold’ and the gene pool arising from this cultivar. Cultivars grouped closely with one of their immediate parents. ‘Perle’ grouped with its parent ‘Northern Brewer and ‘Willamette’ grouped with its parent ‘Fuggle H’. Wild American accessions were divided into two subgroups: a North Central group containing mostly H. lupulus var. lupuloides and a Southwestern group containing H. lupulus var. neomexicanus accessions. These eight SSRs will be valuable for genotype identification in European and wild American germplasm and may potentially prove useful for marker-assisted selection in hop. PCR products from four previously reported primer pairs that amplify the same intronic SSR regions as do the genic SSRs in this study were compared in eight common cultivars. Different primer pairs generated robust markers at the chs2 and chi loci. However, only the HLC-004B and HLC-006 primer pairs amplified successfully at the chs3 and chs4 loci.

Expression of Allene Oxide Cyclase and Accumulation of Jasmonates during Organogenic Nodule Formation from Hop (Humulus lupulus var. Nugget) Internodes

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its stereoisomeric precursor, cis-(+)-12-oxophytodienoic acid (OPDA), which is catalyzed by allene oxide cyclase (AOC, EC 5.3.99.6). A cDNA of AOC was isolated from Humulus lupulus var. Nugget. The ORF of 765 bp encodes a 255 amino acid protein, which carries a putative chloroplast targeting sequence. The recombinant protein without its putative chloroplast target sequence showed significant AOC activity. Previously we demonstrated that wounding induces organogenic nodule formation in hop. Here we show that the AOC transcript level increases in response to wounding of internodes, peaking between 2 and 4 h after wounding. In addition, Western blot analysis showed elevated levels of AOC peaking 24 h after internode inoculation. The AOC increase was accompanied by increased JA levels 24 h after wounding, whereas OPDA had already reached its highest level after 12 h. AOC is mostly present in the vascular bundles of inoculated internodes. During prenodule and nodule formation, AOC levels were still high. JA and OPDA levels decreased down to 10 and 118 pmol (g FW)(-1), respectively, during nodule formation, but increased during plantlet regeneration. Double immunolocalization analysis of AOC and Rubisco in connection with lugol staining showed that AOC is present in amyloplasts of prenodular cells and in the chloroplasts of vacuolated nodular cells, whereas meristematic cells accumulated little AOC. These data suggest a role of AOC and jasmonates in organogenic nodule formation and plantlet regeneration from these nodules.

(397) Atypical Disease Response to Hop Powdery Mildew

Open-pollinated hop seeds (Humulus lupulus var. lupuloides E. Small, H. l. var. pubescensE. Small, H.l. var. neomexicanusNelson and Cockerell, and H.l. var. lupulusL.) were collected from the United States, Canada, and Kazakhstan and screened for resistance to hop powdery mildew (Podosphaeramacularis Braun & Takamatus). A total of 2108 seedlings were repeatedly inoculated with high levels of P. macularis. Under greenhouse conditions, most seedlings exhibited a compatible disease response typical of susceptible hosts with sporulating colonies covering both leaf surfaces. Three genotypes from hop seed native to Emba, Kazakhstan, remained uninfected in greenhouse assays. Further experimentation of those genotypes in growth chambers at 18 °C revealed that they demonstrated an atypical disease response. Six days after inoculation, necrotic lesions on the adaxial leaf surfaces were visually apparent. Microscopic examination showed areas of collapsed epidermal cells, collapsed hyphae, and golden-brown discolorations extending out from the center of the infection. These symptoms contrasted those of a hypersensitive response in which fungal growth is restricted by the collapse of epidermal cells in advance of the fungus. The infection frequency of Kazakhstani genotypes was intermediate compared to H.l. cultivars Symphony (susceptible) and Nugget (resistant) after exposure to three preinoculation temperature regimes. These genotypes could represent a new source of polygenic resistance to hop powdery mildew, and could broaden hop germplasm available to global breeding programs.

Biosynthetic experiments with tall plants under field conditions. 18O 2 incorporation into humulone from Humulus lupulus

Five segments of a large hop plant ( Humulus lupulus var. Hallertauer Magnum) carrying several cones were enclosed in sealed glass vessels that were gassed with 18O 2. After 14 days, the segments were harvested and humulone and cohumulone were analysed by NMR spectroscopy and mass spectroscopy. The oxygen atoms in position 6 of humulone and cohumulone showed 9% 18O enrichment, respectively. It follows that the C-6 hydroxy groups were introduced by oxygenase catalysis.

Expression of lipoxygenase during organogenic nodule formation from hop internodes.

Study of lipoxygenase expression (LOX; EC 1.13.11.12) during organogenic nodule formation in hop (Humulus lupulus var. Nugget) showed that LOXs are developmentally regulated throughout the process, suggesting their involvement in the response of internodes to wounding, nodule formation, and plantlet regeneration from these nodules. LOX activity and lipid peroxides exhibited a huge increase during the first week of culture, which may indicate a role for LOX and LOX products in response to wounding in hop, as reported for other systems. Western blotting analysis showed a de novo synthesis of LOX isoenzymes in response to wounding and the detection of three different isoenzymes. Confocal analysis of LOX immunofluorescence revealed the presence of the enzyme in cortical cells of induced internodes and in prenodular cells, mostly appearing as cytoplasmic spots. Some of them were identified as lipid bodies by cytochemical and double immunofluorescence assays, suggesting the involvement of a lipid body LOX during nodule formation. Immunogold labeling detected LOX in peroxisomes, lipid bodies, and plastids of nodular cells. Quantification of the labeling density provided statistical significance for the localization of LOX (three different isoenzymes) in the three compartments, which suggested a possible involvement of LOX in metabolic functions of these organelles during organogenic nodule formation and plantlet regeneration.

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.

Comparative chemical attributes of native North American hop, Humulus lupulus var. lupuloides E. Small

The genetic diversity of 159 representative genotypes of native hop ( Humulus lupulus var. lupuloides E. Small, Cannabaceae) from 34 selected populations was assessed by relative magnitudes and ranges of alpha acids (AA), beta acids (BA), and the cohumulone (CoH) component of alpha acids, with reference to temporal changes between 1989–1990 and 2001, and to the same attributes in American and European hop cultivars, principally H. lupulus var. lupulus L. Chemical profiles of these genotypes were generated by high pressure liquid chromatography (HPLC) of methanol extracts from their processed samples (cones). The alpha ratio (AR, alpha acids / alpha+beta acids) measured the degree to which alpha acids predominated in cone extracts. Synchronous ranges of AR and CoH were also selected for graphic portrayals of native hop genotypic diversity. Cones sampled and analyzed from eight populations that were accessible in both 1989 and 2001 were distinct in chemical attributes, indicating a succession of genotypes, and suggesting temporal cycling of H. lupulus var. lupuloides germplasm. The principal distinctions between the two sub-species were a markedly higher proportion of CoH (38–88% vs. 19–41%) in alpha acids of H. l. var. lupuloides, and generally higher concentrations of AA in cultivars of both American and European commercial hop cultivars, predominantly H. lupulus var. lupulus. All of the 159 native hop genotypes also contained detectable levels of xanthohumol and xanthogalenol, prenylflavonoids recently reported to have mammalian anti-cancer activity. Some native genotypes had previously exhibited natural repellence of insect and mite pests; thus H. lupulus var. lupuloides germplasm offers a diverse resource of underutilized and yet undefined biochemicals.

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget.

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.

An electron probe X-ray microanalysis study during organogenesis from internode-derived nodules of Humulus lupulus var. Nugget

Elemental changes during the induction of organogenesis from internode-derived nodules of Humulus lupulus var. Nugget were studied by electron probe X-ray microanalysis (EPMA) of specimens submitted to physical fixation procedures. X-ray spectra were collected from cambial and cortical cells. Four days after explants inoculation an increase of K and Ca was detected in cells of both regions. Four to twelve days after explants inoculation an increase of Cu, Zn, Fe, S and Mn was therein detected. Values of Cu, Zn, Fe, K and S were lower in control explants than in induced explants 12 days after induction. Although S presented fluctuations it increased throughout the induction period. X-ray spectra collected from organogenic nodules revealed higher levels of Ca, K, Fe, P and S on peripheral regions where regeneration was occurring. Ca was mobilized in several directions, from inner regions of nodules towards their periphery at the onset of plantlet regeneration. Levels of Mg and Na were low or absent. Control explants neither formed nodules nor regenerated plantlets. The results suggest that EPMA can be used to study relative elemental changes during plant morphogenesis induction and enables the early establishment of organogenic regions in nodules.