Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration

The first efficient cryopreservation procedure for in vitro-grown shoot tips of raspberry (Rubus idaeus L.) has been developed based on encapsulation–vitrification (EnVi) and encapsulation–dehydration (EnDe). EnVi resulted in higher survival (85%) and regrowth (75%) of cryopreserved shoot tips than EnDe (65 and 50%, respectively). In both cryogenic procedures, shoots regenerated from cryopreserved shoot tips without intermediary callus formation. Histological studies showed that a much larger number of meristematic cells survived following EnVi than EnDe. The EnVi procedure was applied to seven raspberry genotypes with an average survival and regrowth of 71 and 68%, respectively. Regenerated plants showed normal morphology. Results here indicate EnVi as a simple and efficient method for long-term preservation of R. idaeus germplasm.     

Properties of an unusual isolate of raspberry ringspot virus from grapevine in Germany and evidence for its possible transmission by Paralongidorus maximus

An isolate of raspberry ringspot nepovirus (RRV-P) commonly found infecting grapevine in localised areas of the German Palatinate, was serologically closely related to, but distinguishable from, the English type strain of this virus (RRV-E) which is transmitted by Longidorus macrosoma. However, unlike RRV-E, RRV-P had a restricted herbaceous host range and produced symptoms reliably in only two hosts, Chenopodium quinoa and Nicotiana occidentalis-accession 37B: these symptoms were a faint systemic vein clearing which, on most occasions in C. quinoa, was transient. In in vitro studies with herbaceous plant sap, RRV-P infectivity was lost after diluting 1/100-1/500, after storage at 20^oC for 1–3 days and at 4^oC for 45 days: for similar studies with RRV-E, the values were 1/125 000, and more than 15 days at 20^oC and 4^oC, respectively. RRV-P was difficult to purify in quantity and in most preparations seemed to sediment as a single component corresponding to ‘bottom’ component of RRV-E. Purified particles of RRV-P, like those of RRV-E, contained a major polypeptide and two RNA species of Mx 54 000, 2.6 × 106 and 1.6 × 106 respectively. There was no evidence from RNA preparations from purified virus particles or, from analysis of dsRNA from infected plants, that RRV-P contained a satellite RNA. The incidence of RRV-P in vineyards was not associated with the presence in soils of Longidorus nematodes, but was associated with the distribution in the Palatinate of Paralongidorus maximus. Furthermore, results from an experiment in Germany in a vineyard planted with healthy grapevines in soil fumigated to destroy nematodes, showed spread of RRV-P into these plants from an adjoining source of infected grapevines and soil infested with P. maximus. In laboratory studies, RRV-P was transmitted by P. maximus at a very low level between grapevines (used as the virus source and test plants) but not to, or between, herbaceous hosts.     

Male lures and male mating competitiveness in a genetic sexing strain of the melon fly (Diptera: Tephritidae)

The Sterile Insect Technique (SIT) has been used to suppress or eradicate populations of the melon fly, Zeugodacus cucurbitae (Coquillett), a pest of various fruits and vegetables throughout Asia, Africa, and South Pacific Islands. The success of the SIT depends largely on the ability of released, sterile males to compete successfully with wild males for copulations with wild females. Unfortunately, both the sterilization process (via gamma irradiation) and artificial selection under mass-rearing conditions generally reduce the biological competence, including the mating competitiveness, of the released males. However, previous data on Z. cucurbitae show that irradiation has minimal adverse effects and prolonged mass-rearing may or may not result in decreased mating ability. The present study first confirms that neither irradiation nor mass-rearing had deleterious effects on male mating ability of males from a pupal-color, genetic sexing strain (termed T1). Then, the influence of male lures (cue-lure and its natural analogue raspberry ketone) on the mating success of sterile T1 males was measured as a potential pre-release procedure to improve the effectiveness of SIT. Feeding on cue-lure enhanced male mating success, but the effect was short-lived (1 d but not 3 d post-feeding). Feeding on raspberry ketone boosted mating success over a longer time, i.e., at least 5 d but not 10 d post-feeding. However, mixing raspberry ketone into the adult diet had no detectable effect on male mating performance. The implications of these results for SIT against Z. cucurbitae are discussed.     

Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts

Raspberry extracts enriched in polyphenols, but devoid of organic acids, sugars and vitamin C, were prepared by sorption to C18 solid phase extraction matrices and tested for their ability to inhibit the proliferation of human cervical cancer (HeLa) cells in vitro. The raspberry extract reduced proliferation in a dose-dependent manner whether this was judged by cell number or measurements of cell viability. However, measurements based on cell viability were more accurate and gave an EC(50) value of 17.5 microg/ml gallic acid equivalents (GAE) at day 4 of culture. Raspberry extracts were fractionated by sorption to Sephadex LH-20 into an unbound fraction, which was obviously enriched in anthocyanins, and a bound fraction. The unbound anthocyanin-enriched fraction was much less effective in reducing proliferation then the original extract and gave an EC(50) value estimated at 67 microg/ml. The LH-20 bound fraction was more effective than the original raspberry extract (EC(50)=13 microg/ml) suggesting that the main anti-proliferative agents were retained in the bound fraction. Analysis of the original extract, the unbound and the LH20 bound fractions by LC-MS confirmed that the unbound fraction was enriched in anthocyanins and the bound fraction primarily contained ellagitannins. The ellagitannin-rich bound fraction had the highest antioxidant capacity as measured by the ferric reducing antioxidant potential (FRAP) assay. The mechanism by which the ellagitannins inhibit proliferation of cancer cells is discussed.     

Tyrosinase inhibitory effect and inhibitory mechanism of tiliroside from raspberry

Tiliroside was found to inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag time of tyrosine oxidation catalyzed by mushroom tyrosinase was obviously lengthened; 0.337?mM of tiliroside resulted in the lag time extension from 46.7?s to 435.1?s. A kinetic analysis shown that tiliroside was a competitive inhibitor for monophenolase and diphenolase with K_i values of 0.052?mM and 0.26?mM, respectively. Furthermore, tiliroside showed 34.5% (p?<?0.05) inhibition of intracellular tyrosinase activity and 54.1% (p?<?0.05) inhibition of melanin production with low cytotoxicity on B16 mouse melanoma cells at 0.168?mM. In contrast, arbutin displayed 9.1% inhibition of cellular tyrosinase activity and 29.5% inhibition of melanin production at the same concentration. These results suggested that tiliroside was a potent tyrosinase inhibitor and might be used as a skin-whitening agent and pigmentation medicine.     

Biological Control of Crown Gall in Grapevine and Raspberry by Two Pseudomonas spp. with a Wide Spectrum of Antagonistic Activity

Pseudomonas aureofaciens B-4117 and P. fluorescens CR330D inhibited the growth of a wide range of plant pathogens, including Agrobacterium tumefaciens , when tested on agar media. In a series of nursery-based trials with natural pathogen inoculum, application of either B-4117 or CR330D significantly reduced the incidence and severity of crown gall caused by A. tumefaciens on grapevine and raspberry. The extent of disease control depended upon the variety tested. Both bacteria reduced disease during seedling root production and grafting. The disease incidence on root cuttings of three grapevine varieties was reduced by 56-80% and the disease severity index (DSI) was decreased by 75-86%. Depending on the scion variety, the number of healthy rooted grafts increased by 2-3.5-fold, while the DSI was reduced by 1.5-3-fold. The results suggest that there is potential in using these antagonists to diminish the influence of latent rootstock infection on graft sensitivity to crown gall. Pretreatment of rooted raspberry seedlings with P. aureofaciens B-4117 prevented the development of crown galls caused by A. tumefaciens strain K24 or by a mixture of A. tumefaciens pathogenic strains previously isolated from raspberry. Both Pseudomonas spp. persisted on the root surfaces of inoculated vine cuttings and in non-sterile soil. The advantages of using the antagonistic bacteria as biocontrol agents of crown gall are discussed.     

Phenolic derivatives in raspberry (Rubus L.) germplasm collection in Bulgaria

Leaves of 22 accessions of the Bulgarian raspberry (Rubus L.) germplasm collection – five Bulgarian and seven foreign cultivars, eight elite clones and two wild species, Rubus occidentalis L. and Rubus odoratus L., were analysed for phenolic constituents. The quantitative determination of caffeic (1), p-coumaric (2) and ferulic (3) acids, hyperoside (4), tiliroside (5), and isoquercitrin (6) was performed by RP-HPLC using linear gradient elution and UV detection at 254 and 310 nm. The detection limits ranged from 0.23 μg/ml (4) to 0.55 μg/ml (2). Caffeic acid was the dominant phenolic acid in the majority of the samples being present in amounts between 0.05 ± 0.01 mg/g dry weight in R. occidentalis and 1.43 ± 0.06 mg/g in the cultivars. The highest content of flavonols was found in the Bulgarian raspberry elite clones: 1.70 ± 0.002 mg/g (6), 0.60 ± 0.004 mg/g (5) and 0.97 ± 0.01 mg/g (4). Data were analyzed by hierarchical clustering (HC) and principal component analysis (PCA). The HC and PCA scoring plot showed that the samples could be classified into three clusters. Cluster C1 comprised cultivars characterized by high content of phenolic acids (1–3). Seven cultivars and the wild species R. occidentalis formed the cluster C2 presenting low content of phenolic compounds. Cluster C3 grouped the Bulgarian raspberry elite clones and R. odoratus sharing the highest content of flavonols (5 and 6). The Bulgarian elite clone E23617 displayed the highest content of the studied phenolic derivatives. The PCA loading plot showed that 1 can be used to distinguish between different raspberry varieties.     

Identification,characterisation and mapping of simple sequence repeat (SSR) markers from raspberry root and bud ESTs

Raspberry breeding is a long, slow process in this highly heterozygous out-breeder. Selections for complex traits like fruit quality are broad-based and few simple methodologies and resources are available for glasshouse and field screening for key pest and disease resistances. Additionally, the timescale for selection of favourable agronomic traits requires data from different seasons and environmental locations before any breeder selection can proceed to finished cultivar. Genetic linkage mapping offers the possibility of a more knowledge-based approach to breeding through linking favourable traits to markers and candidate genes on genetic linkage maps. To further increase the usefulness of existing maps, a set of 25 polymorphic SSRs derived from expressed sequences (EST-SSRs) have been developed in red raspberry (Rubus idaeus). Two different types of expressed sequences were targeted. One type was derived from a root cDNA library as a first step in assessing sequences which may be involved in root vigour and root rot disease resistance and the second type were ESTs from a gene discovery project examining bud dormancy release and seasonality. The SSRs detect between 2 and 4 alleles per locus and were assigned to linkage groups on the existing ‘Glen Moy’ × ‘Latham’ map following genotyping of 188 progeny and examined for association with previously mapped QTL. The loci were also tested on a diverse range of Rubus species to determine transferability and usefulness for germplasm diversity studies and the introgression of favourable alleles.     

秦岭黄果悬钩子野生种质资源与引种试验研究

对黄果悬钩子在秦岭山区的分布、习性、果实经济性状、营养成分、产量构成以及引种栽培条件下的物候期、根系特征、发枝习性,繁殖特点等内容的研究结果表明,黄果悬钧子植株矮小,具有较耐旱的根系组成特点;果大,质优、风味好,营养丰富;适应性较强,容易繁殖,是一种优良的栽培树萄果树引种驯化,矮化育种野生种质资源。