Application of treated wastewater for cultivation of roses (Rosa hybrida) in soil-less culture

No information is available today concerning the effect of irrigation with secondary treated sewage water on growth, production or quality of roses or other cut flowers. In the present study we investigated the effect of irrigation with treated sewage water on roses cultivated in two soil-less medium, perlite, an inert mineral medium and Choir (coconut fibers), an organic medium of high ion absorption capacity. During 12 months of exposure to the treated water, the visible appearance of the plants, their growth, the quantity and size of the flowering stems and their postharvest performance were not affected by the irrigation treatments. Contents of macroelements in the leaf tissues were unaffected by the irrigation with the secondary treated sewage water. At the same time, Cl contents increased 47% in perlite and 73% in Choir grown plants reaching levels characteristic of exposure to moderate salinity. Mn, Cu and B contents increased as well under cultivation in both perlite and Choir under irrigation with treated sewage water. On the other hand, contents of Fe, Zn, Mo and Al, were similar in all treatments. In all treatments contents of all the examined micro and macroelements were within the range accepted for proper plant function.     

In vitro propagation of wavy-leaved Indian paintbrush (Castilleja applegatei Fern.)

Castilleja spp. (Indian paintbrush, Orobanchaceae) are desirable ornamental plants with showy floral bracts that are native throughout much of the western U.S. Propagation of these hemiparasites by seed is usually successful only when a host species is present, and asexual propagation through traditional methods has proven to be extremely difficult. In this study we present an effective shoot culture micropropagation system for Castilleja applegatei Fern., the wavy-leaved Indian paintbrush. In vitro shoot tips of three C. applegatei clones were cultured for 28 days on woody plant medium (WPM) supplemented with four concentrations of each of three cytokinins: 6-benzylaminopurine (BA), 6-(y,y-dimethylallylamino)-purine (2iP) and zeatin. Responding explants, shoot number per responding explant and shoot length were determined. In vitro rooting of microcuttings cultured for 42 days on media containing three concentrations of indole-3-butyric acid (IBA) were also evaluated. A high percent response to shoot induction was observed across all treatments, ranging from 94.5 to 100%. C. applegatei responded best to zeatin, which resulted in both the highest mean shoot number and mean shoot length among the cytokinins tested. The best overall shoot multiplication occurred on media with 4.0 μM zeatin, yielding a mean shoot number of 4.11 and mean shoot length of 3.95 cm. The mean highest rooting response (66.7%), root number (13.21) and root length (2.73 cm) were obtained on WPM supplemented with 10 μM IBA. Significant clone × treatment interactions existed for all variables except mean root number, thus the optimum treatments for both shoot multiplication and root induction were clone-dependent. Rooted microcuttings were acclimated ex vitro with an average success rate of 81.2%. This study demonstrates the considerable potential of an in vitro shoot culture system for the asexual propagation of Castilleja spp.     

In vitro proliferation from axillary buds and ex vitro protocol for effective propagation of Syringa × hyacinthiflora ‘Luo Lan Zi’

As a precondition for lilac mass propagation, the optimal shoot-multiplication medium for Syringa × hyacinthiflora ‘Luo Lan Zi’ was ascertained mainly based on clustered microshoot inducement and large leaf area establishment in 6-benzyladenine (BAP) (1.00 mg L⁻¹) and zeatin (Z) (0.10 mg L⁻¹) combination. Medium supplied with lower level of BAP (0.50 mg L⁻¹) and auxin (IAA) (0.25 mg L⁻¹) was not suitable for lilac shoot proliferation, but it could be competent for long-term preservation of the un-rooted shoots so that subsequent proliferation culture could be carried out at anytime. In addition, excess height growth which resulted in low transplanting survival rate was effectively controlled by decrease in node number when paclobutrazol (PBZ) was applied in rooting medium at a concentration of 1.00 mg L⁻¹ after taking into account the effects on shoot height, rooting, persistent leaf area and PBZ carry-over. An important overwintering treatment was to use a plastic chamber covering for plants in the greenhouse prior to field planting to ensure adequate biomass of stem and underground parts not only in the current growing season but also in the subsequent years.     

Morphological and molecular analyses of Rosa damascena × R. bourboniana interspecific hybrids

Rosa damascena Mill is the most important scented rose species cultivated for rose oil production. Rosa bourboniana L. (Edward rose), a related species, is popular on account of its longer blooming period and ease of propagation. With an aim to combine the oil quality of R. damascena and recurrent flowering habit of R. bourboniana, two cultivars (Jwala and Himroz) of R. damascena were crossed with R. bourboniana. The F1 hybrids obtained were evaluated using morphological, random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers. Twenty-two selected RAPD and three SSR primer pairs were utilized for hybrid identification. According to presence or absence of bands RAPD and SSR markers were classified into seven types of markers. The bands specific for the pollen parent and occurring in the hybrids were good markers to confirm the hybridity. The non-parental bands expressing uniquely in hybrids were effective in distinguishing the hybrids from each other. Cluster analysis, based on Jaccard's similarity coefficient using unweighted pair group method based on arithmetic mean (UPGMA), reliably discriminated the hybrids into two main clusters. These results indicate the practical usefulness of RAPD and SSR markers in hybrid identification in scented roses. The approach is advantageous for its rapidity and simplicity, for identification of hybrids at the juvenile stage. One of the studied morphological traits – prickle density, can also complement in the identification of interspecific hybrids between R. damscena (♀) and R. bourboniana (♂).     

In vitro propagation of European aspen (Populus tremula L.) from axillary buds via organogenesis

Axillary buds from 1-year-old twigs of European aspen (Populus tremula L.) were used for propagation of indigenous aspen clones for further phytoremedial studies. The most intense bud development was seen after the break of dormancy, i.e. from the middle of February until mid-March. Lower infection rates were observed from the axillary buds collected at an urban forest border in comparison to a forest location, accompanied by higher percentages of browning. The optimal medium for shoot induction primarily via callus cultures was Murashige and Skoog mineral salts medium (MS) supplemented by 20 μM zeatin riboside and 1 μM indol acetic acid. The shoots were rooted in half-strength MS medium supplemented by the same hormones, after which they were successfully transferred to a commercial soil mixture. The protocol for aspen propagation is proposed.     

Cold storage of oil rose (Rosa damascena Mill.) flowers

Oil rose flowers were stored at 0 °C in four different packaging materials [plastic box + stretch film (PB + SF), Xtend^®, Smartbag^® and polyethylene (PE)] for 60 days. During storage, weight loss, O₂ and CO₂ concentrations in the packages, petal color and sensorial attributes were investigated besides essential oil content and composition. Storage duration and packages had significant (p < 0.01) effects on weight loss. At the end of storage, the lowest weight loss was in PE package (1.696%) whereas the highest weight loss was in Xtend^® (10.081%). The essential oil content was significantly (p < 0.01) affected by storage duration and packages. In addition, the essential oil contents obtained from all packages for a storage period of 10 days and the essential oil contents obtained from unstored (control) petals were included in the same group. At the end of storage, the essential oil contents decreased by 91.3, 57.7, 80.0 and 64.3% in PB + SF, Xtend^®, Smartbag^® and PE packages, respectively as compared to control. In addition, storage duration and package types significantly (p < 0.01) affected petal color, O₂ and CO₂ concentrations in the packages and sensorial scores. The concentration of citronellol, a main component of rose oil, increased in all packages during storage of 10 days in comparison to the control group while it varied in other storage durations and package types. However, nerol and geraniol were lower than the control group during storage while concentrations of nonadecane, heneicosane and eicosane were higher. In conclusion, loss of oil yield and quality, due to various reasons and particularly due to fermentation in oil rose from the harvest of petals to their distillation, can be minimized with storage of petals in all package types for up to 10 days.     

Dormancy and germination in Rosa multibracteata Hemsl. & E. H. Wilson

Most of the achenes produced by Rosa multibracteata Hemsl. & E. H Wilson are dormant on maturity and require pretreatment to stimulate germination. To investigate the mechanism of dormancy and to develop effective methods of improving germination, roles of the pericarp, testa, and embryo of R. multibracteata in regulating dormancy were studied by investigating the effect of different pretreatments on germination. The effects of temperature and water stress were also tested with achenes treated by warm plus cold stratification. In freshly harvested achenes, pericarps are permeable and the embryo fully developed, which eliminates the possibility of physical, morphological, or morphophysiological dormancy. Germination percentage remained low (<5%) despite softening the pericarp or even removing it fully. However, fully removing the testa improved germination significantly (39%), indicating the possible presence of germination inhibitors in the testa. Dry storage, scarification with sulphuric acid (H₂SO₄), and warm stratification proved ineffective by themselves but when combined with cold stratification, improved germination and shortened the cold stratification period needed to break dormancy. Dry storage for 68 weeks followed by cold stratification for 16 or 24 weeks resulted in maximum germination (72–79%) among all the treatments. In achenes scarified with H₂SO₄, germination increased with an increase in the duration of cold stratification. Neither gibberellic acid (GA₃) nor ‘smoke water’ (water through which smoke had been bubbled for 2 h) had any positive effect on germination even on seeds that had been mechanically scarified or stratified. Both high temperature and water stress lowered germination in achenes treated with warm plus cold stratification. Our results suggest that R. multibracteata achenes have an intermediate physiological dormancy, and that dry storage for 68 weeks followed by cold stratification for 16 or 24 weeks is the best method for propagating R. multibracteata from seed.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

Flower yield performance and stability of various Rosa damascena Mill. landraces under different ecological conditions

The flower yield stability of Damask rose as an important medicinal and aromatic plant at different environments has not been well documented. In order to evaluate flower yield and stability, 35 landraces of Damask rose were studied at 8 locations in Iran during 2007–2008. Analysis of variance revealed significant differences (p ≤ 0.01) among landraces (G), environments (E), locations (L) and for landrace × environment (GE) and landrace × location (GL) interactions. Both GE and GL interactions were mainly crossover, a large portion of which was accounted for by non-linear (unpredictable) component. The landraces of IS9, YZ2, WA1, IS7 and IS1 with 3120.63, 2941.63, 2894.62, 2769.15 and 2716.92 kg/ha respectively produced the highest flower yield among studied landraces. Kerman with average flower yield of 3635.46 kg/ha produced the highest yield among studied locations. According to the results, most of landraces that originated from temperate, warm temperate and arid regions produced higher flower yield than those from cool, cool temperate, semi-arid and humid regions. The landraces of YZ2, IS5, IS8, IS4, KZ1, AR1_, IS3 and BA1 were stable and YZ2, IS5, IS8, IS4, KZ1, AR1 IS6, IS3, BA1, IS10 and YZ1 were adaptable landraces for flower yield according to Eberhart and Russell (1966) model. The presence of some high flower yield and stable landraces such as YZ2 and IS5 suggests that a genotype can demonstrate high flower yield and stability for yield simultaneously. Thus, simultaneous selection for flower yield and stability using nonparametric methods could be possible. In addition, taking into consideration flower yield and stability potential, the landraces of YZ2, IS5, IS8, IS4 and KZ1 as general stable, adaptable, and high flower yield are recommended. Furthermore, the landraces of IS9 and WA1 as high flower yield and specific adaptable landraces can be recommended for temperate and arid areas and the landraces of IS7 and IS1 for semitemperate and cool areas.     

Encapsulation of in vitro-derived explants of olive (Olea europaea L. cv. Moraiolo): II. Effects of storage on capsule and derived shoots performance

The experiments described in this paper were designed to examine the effect of storage at different temperatures for various periods on regrowth and proliferation ability of encapsulated olive microcuttings (capsules). The proliferation and rooting ability of the shoots derived from the capsules were compared with those maintained in normal proliferation by 45 days period of subculturing. A simple method of encapsulated microcuttings storage in plastic cuvettes was used. Interesting feature was the ability of the capsules to retain their viability and regrowth after storage at room temperature as well as at 4 °C for 15 and 30 days and both axillary buds on the nodes of almost all capsules sprouted successfully. Improvement in proliferation and rooting abilities were found in subsequent culturing of the shoots developed from the capsules. The study indicates bright prospects of commercial application of alginate nutrient capsule technology in various situations like supply of certified plant material from both official or private organizations to the plant nurseries and/or exchange of germplasm at international level without refrigerated containers just in plastic cuvettes.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Embryo germination and proliferation in vitro of Juglans regia L.

This paper presents the optimal culture conditions for the in vitro embryo germination and proliferation of Juglans regia L. rootstock cv. Peralta, selected by the IMIDA fruticulture team. J. regia L. rootstock cv. Peralta is characterised by its resistance to salinity, lime and by its vigour. The first experiment determined the best culture medium for in vitro embryo germination. The Murashige and Skoog medium (MS; Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol. Plant. 15, 473–497), the medium developed by our team for walnut (NGE), the Lloyd and McCown medium (WPM; Lloyd, G., McCown, B., 1981. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by the use of shoot tip culture. Proc. Plant Prop. Soc. 30, 421–427) and that of Driver and Kuniyuki (DKW; Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19 (4), 507–509), were compared, all without growth regulators. The best germination percentage was obtained in WPM (81%) with significant differences between the different media. In the second experiment, the optimal benzylaminopurine and indole butyric acid concentrations were determined for the proliferation stage of the explants obtained in the first experiment. The proliferation rates obtained varied from 0 in the medium without cytokinins to 6 in the medium with 0.5 mg l⁻¹ BAP. The cluster proliferation quality and other parameters studied indicate that the optimal treatment was 0.5 mg l⁻¹ BAP.     

Morphological and oil content variations amongst Damask rose (Rosa damascena Mill.) landraces from different regions of Iran

Forty landraces of Damask rose were collected from 28 provinces (13 origin sites) in Iran and evaluated to determine the diversity among them. The investigated characters were: flower weight, flower diameter, peduncle length, number of petals, number of stamens and oil content. Pearson's coefficients showed that number of petals was positively correlated with flower weight (r = 0.642) and number of stamens (r = 0.630), while its correlation with peduncle length was negative (r = −0.53). A negative correlation was obtained between oil content and number of stamens (r = −0.60) which is useful for indirect selection of landraces with low number of stamens and therefore high amount of oil content. Cluster analysis based on five botanical characters and oil content separated the origin site 12, which included Guilan, Mazandaran and Golestan provinces with a specific climate condition of temperate humid, from other origin sites. This distinctiveness was already confirmed by the bi-plot of the principal components. The distribution of Damask rose landraces provided useful information for the future collection and for the genetic resource management in these origin sites.     

Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): Induction,conversion and synthetic seeds

Pineapple guava (Acca sellowiana) syn. Feijoa sellowiana, a Brazilian indigenous Myrtaceae is under domestication in South Brazil. Previous works showed that this species is responsive to somatic embryogenesis and recalcitrant to conventional methods of clonal propagation. In the present work it was evaluated the role of components of culture medium in the induction and development of somatic embryos. The technology of synthetic seeds was also evaluated. Zygotic embryos were inoculated in LPm medium supplemented with 8 mM glutamic acid and 8 mM l-glutamine, 2,4-dichlophenoxiacetic acid (20 μM) and myo-inositol. For conversion of somatic embryos and synthetic seeds it was tested the effect of 6-benzylaminopurine and gibberellic acid combined or not with activated charcoal. The highest values for embryogenetic induction (100%) and number of somatic embryos/explant (113) were observed in the LPm medium supplemented with Glu (8 mM), and 2,4-D. The culture medium supplemented with BA (0.5 μM) and GA₃ (1 μM) and activated charcoal (1.5 g L⁻¹) enhanced the conversion of somatic embryos to plantlets. Pre-germinated somatic embryos encapsulated in sodium alginate with BA (0.5 μM) and GA₃ (1 μM) developed radicles. The use of synthetic seed was a requisite for the survival of plantlets.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Clonal propagation of mulberry (Morus indica L. cultivar M-5) through in vitro culture of nodal explants

A high frequency of sprouting (80.0%) and shoot differentiation was observed in the primary cultures of nodal explants of Morus indica L. cultivar M-5 on MS medium supplemented with 2,4-D (0.3 mg/l). In vitro proliferated shoots were multiplied rapidly by culture of shoot tips on MS medium with BAP (0.5 and 1.0 mg/l) which produced the greatest multiple shoot formation. Multiplication was also achieved by culture of shoot tips on MS medium with BAP (4.0 mg/l) and GA₃ (0.05 mg/l) which facilitated the elongation of shoots followed by sprouting of axillary buds of in vitro grown shoots. A high frequency of rooting (86.7%) with development of healthy roots was observed from shoots cultured on medium with 2,4-D (1.0 mg/l). Plants with well developed roots were transferred to soil with a survival frequency of 80%.     

Pome fruit viruses at the Canadian Clonal Genebank and molecular characterization of Apple chlorotic leaf spot virus isolates

A survey for apple and pear viruses was carried out at the Canadian Clonal Genebank (CCG), Harrow, Ontario, Canada, during the fall/winter of 2007 and spring of 2008. Leaves and/or dormant cuttings were randomly collected from 438 to 122 accessions of apple and pear, respectively. Samples were tested by Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) for the presence of Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). Infection rates for apples were ACLSV (48.1%), ASGV (10%), ASPV (6.6%) and ApMV (7.1%), and for pears ACLSV (42.6%). ACLSV was detected and characterization by multiplex RT-PCR with primers targeting a fragment of 677 bp corresponding to the partial coat protein (CP), movement protein (MP) and untranslated (3′UTR) region in 22 accessions of apple and pear. Multiplex RT-PCR showed a higher sensitivity over the ELISA test. The nucleotide and amino acid deduced partial CP identities ranged from 82.6–100% to 91–100%, respectively, while partial MP identities was 62.5–100% at aa level based on the amplified fragment appropriate for partial MP using a frame shift, among 22 ACLSV isolates. Phylogenetic analyses based on the partial CP region clustered CCG ACLSV isolates in two different groups, while those based on the partial MP region embraced CCG ACLSV isolates in two sub-clusters within the same group. This is the first report on the detection of ACLSV, ASPV, ASGV and ApMV at CCG, and the molecular characterization of ACLSV isolates in apple and pear plants from worldwide countries to deduce possible heterogeneity and evolution.     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.