Chromosome observation method at metaphase and pro-metaphase stages in diploid and octoploid strawberries

Chromosome observation is necessary to elucidate the structure, function, organization, and evolution of octoploid strawberry plants’ genes and genomes. However, distinguishing strawberries’ chromosomes from one another using light microscopy is extremely difficult, not only because of their small size and large number, but also because current chromosome observation methods are insufficient. Chromosome preparation and staining using maceration enzymes, acetic acid, and DAPI (4′,6-diamidino-2-phenylindole) were improved for this study to obtain clear images of somatic chromosomes in Fragaria vesca (2n = 14) and Fragaria×ananassa (2n = 56). Collected root tips of octoploid plants were placed in 0.002 M 8-hydroxyquinoline solution for 1 h and stored at 4 °C for 16 h. Subsequently, they were fixed using 3:1 absolute alcohol:glacial acetic acid for 40 min, hydrolyzed in the 1N HCl solution at room temperature for 2 h, macerated using an enzyme solution for 25 min at 42 °C, and stained in 1.5% lacto-propionic orcein solution. On the other hand, in case of DAPI staining, the macerated root tips of octoploid plants were soaked in 60% acetic acid for 5 min before staining. Clear digital images of F. vesca and F.×ananassa were obtained using light and fluorescent microscopy. Their respective 14 and 56 chromosomes were counted. Fluorescent microscopy yielded clear chromosome images at the pro-metaphase in F. vesca and F.×ananassa. This chromosome observation method alleviates the difficulties that have heretofore hindered chromosome analyses of strawberry plants.     

Effect of plant growth temperature on membrane lipids in strawberry (Fragaria × ananassa Duch.)

Changes in membrane lipid composition are important in the acclimation of plants. The influence of four day/night growing temperature combinations (18/12, 25/12, 25/22, and 30/22 °C) on membrane lipids of ‘Earliglow’ and ‘Kent’ strawberry (Fragaria × ananassa Duch.) were studied. The monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) were the major galactolipids in the strawberry leaves. ‘Earliglow’ contained a higher amount of galactolipids in the leaves than ‘Kent’. The major phospholipids in the strawberry leaves, roots, and fruit were phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). PC and PE were the two predominant phospholipids in the strawberry. The leaves and fruit of ‘Earliglow’ contained higher amounts of phospholipids compared to those of ‘Kent’, whereas ‘Kent’ strawberry roots had higher phospholipids. Palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids were major fatty acids in galacto- and phospholipids of the ‘Earliglow’ and ‘Kent’ strawberry. PC is very rich in linolenic acid in leaves compared to the fruit and root tissues. PC had the highest ratio of unsaturated to saturated fatty acids among all phospholipids. There was a significant increase in the content of galactolipids (MGDG, DGDG) and phospholipids (PC, PI, PG and PE) and unsaturation of their fatty acids in the cool day/night growth temperature. Increasing day/night growth temperatures decreased MGDG/DGDG ratios. The shifts in saturation and composition of fatty acids observed with strawberry may be an adaptation response of plants to the temperature changes.     

Improvement of staining method for observation of mitotic chromosomes in octoploid strawberry plants

An important strategy to conduct intentional breeding of octoploid strawberry plants is to recognize the functions of every chromosome. To do so, a methodology must be developed to distinguish chromosomes one by one. We reported the possibility of distinguishing chromosomes using light microscopy when somatic cells of octoploid strawberry plants were stained using ordinary methods with lacto-propionic orcein (LPO). However, karyotype analysis of octoploid strawberry plants required clearer chromosome images. This study obtained clearer chromosome images of octoploid Fragaria × ananassa and F. chiloensis plants. Three staining methods were examined: 60% acetic acid (AA) alone, 1.5% LPO alone, and two-step treatments with 60% AA and 1.5% LPO. Collected root tips of the plants were placed in 2 mM 8-hydroxyquinoline solution for 1 h and were subsequently stored at 4 °C for 15 h. The samples were then fixed in a 3:1 absolute alcohol: glacial acetic acid solution for 40 min, followed by mixture with 1N HCl solutions at room temperature for 2 h and then at 60 °C for 10 min. For separate staining using 60% AA and 1.5% LPO, the root tip was expelled on the glass slide with a drop of each solution for a few minutes to stain the chromosomes. For the two-step staining method, the samples stained with 60% AA were frozen at −80 °C for at least 5 min. The cover slip was removed using a razor blade. Subsequently, the specimens were air-dried and stained with the 1.5% LPO for 3 min. Digital images of chromosomes were obtained using light microscopy. Samples of the two-step staining method produced the clearest chromosome images in both F. × ananassa and F. chiloensis. Furthermore, the greatest color difference between the chromosomes and the cytoplasm was obtained from images of the two-step staining method among the three staining methods. These results demonstrate that the two-step staining method is useful for chromosome counting and karyotype analysis in strawberry plants.     

Parental chromosome differentiation in citrangequat [Fortunella sp. × (Citrus sinensis × Poncirus trifoliata)] using CMA banding patterns

The efficiency of chromomycin A₃ (CMA) staining was examined for parental chromosome differentiation in citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and citrangequat (Fortunella sp. × citrange). All of the accessions analyzed had the same chromosome number of 2n = 18. CMA staining revealed six characteristic banding patterns on the basis of the number and position of CMA positive bands (CMA⁺) as follows; A: two terminal and one proximal band, B: one terminal and one proximal band, C: two terminal bands, D: one terminal band, E: no band, and F: one proximal band. Chromosome CMA banding patterns of the accessions were 1A + 1B + 2C + 13D + 1F in Fortunella margarita, 2B + 2C + 7D + 7E in ‘Fukuhara’ orange, 2B + 10D + 6E in Poncirus trifoliata, 1B + 1C + 10D + 6E in citrange and 1A + 1C + 11D + 4E + 1F in citrangequat. The results of this study confirmed the intergeneric and tri-generic hybridity of citrange and citrangequat, respectively.     

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.     

Agrobacterium × plant factors influencing transformation of ‘Joseph's coat’ (Amaranthus tricolor L.)

A protocol is developed for Agrobacterium-mediated genetic transformation of Amaranthus tricolor via explant co-cultivation with Agrobacterium rhizogenes. Bacteria-plant specific factors which influenced transformation were optimized. Of the two Agrobacterium strains employed, LBA9402 was more infectious compared to A4. Bacterial suspensions grown overnight with 100 μM acetosyringone and experiencing O.D.₆₆₀ = 0.6 followed by dilution to a density of 10⁹ cells ml⁻¹ were the most effective. Explants from garden-grown plants were more responsive than those from in vitro cultures; stem internodes being better than leaves. Immersion of the pre-pricked explants in bacterial suspension resulted in a markedly higher transformation frequency compared to the direct injection method. The infection of internode explants with the LBA9402 strain followed by co-cultivation on growth regulator-free MS medium (MS0) for 5 days resulted in emergence of hairy roots up to a maximum frequency of 97.22%. Roots were individually cultured in MS0, but fortified with bactericidal antibiotic (500 μg ml⁻¹ cefotaxime). Rhizoclones showing prolific growth were renewed through successive subcultures in MS0. Opine gene expression was revealed by positive agropine and mannopine synthesis in all selected transformed rhizoclones. Shoot regeneration from root clones, capable of auxin-independent growth and opine proficiency, was stimulated in MS augmented with 2.0 mg l⁻¹ zeatin. pRi TL–DNA rolB and pRi TR–DNA man2 ORF were detected in leaf tissues of regenerated plants from selected hairy root clones through PCR amplification. The implication of such findings is discussed on the possibility of conferring protection to crop amaranths against biotic stress challenges, particularly due to insects, viruses or fungal pathogens.     

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.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

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.     

Changes in morphological phenotypes and essential oil components in lavandin (Lavandula × intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes

Hairy roots were induced from leaf-derived calli of lavandin (Lavandula × intermedia Emeric ex Loisel.) by infection with wild-type strains of Agrobacterium rhizogenes, A-5 (MAFF 02-10265) and A-13 (MAFF 02-10266). A-5-inoculated calli formed hairy roots more efficiently than A-13 ones. The transgenic shoots could be obtained from hairy root segments mediated by each Agrobacterium strain. However, different plant growth regulators were required for efficient adventitious shoot formation in each strain. In A-5, the most efficient adventitious shoot formation rate of 23.8% was observed in a medium with 4.4 × 10⁻⁶ M of 6-benzylaminopurine. On the other hand, a significantly higher rate of 13.2% was detected in a medium with 4.0 × 10⁻⁷ M of N-(2-chloro-4-pyridyl)-N′-phenylurea in A-13. Most of the regenerated plants showed dwarfism with closed internodes and extensive lateral branching, which were typical characteristics of ‘hairy root syndrome’. On the other hand, only nine of the 45 regenerated plants formed flower buds in early June, a delay of about one month compared with nontransgenic regenerated plants. The floral stalks and spikes of these plants were very short, resulting in a compacted form. Many regenerants showed a significantly lower productivity of essential oil than nontransgenic regenerants. Moreover, the relative percentage of the linalyl-cation-derived compounds, linalool and linalyl acetate, decreased in most of the regenerated plants. Compact plants with the ability of flower bud formation are assumed to be valuable not only for lavandin breeding, but also for clarifying the interaction between rol genes expression and essential oil production.     

Effects of plant growth promoting bacteria (PGPB) on yield,growth and nutrient contents of organically grown strawberry

The effects of plant growth promoting bacteria (PGPB) on the fruit yield, growth and nutrient element content of strawberry cv. Fern were investigated under organic growing conditions between 2006 and 2008. The experimental plot was a completely randomized design with 3 replicates. Three PGPB strains (Pseudomonas BA-8, Bacillus OSU-142 and Bacillus M-3) were used alone or in combination as bio-fertilizer agent in the experiment. Data through 3 years showed that the use of PGPB significantly increased fruit yield, plant growth and leaf P and Zn contents. Root inoculation of M3 and floral and foliar spraying of OSU-142 and BA-8 bacteria stimulated plant growth resulting in significant yield increases. M3 + BA-8, BA-8 + OSU-142, M3, M3 + OSU-142 and BA-8 applications increased cumulative yield by 33.2%, 18.4%, 18.2%, 15.3% and 10.5%, respectively. Number of fruits per plant significantly increased by the applications of M3 + BA-8 (91.73) and M3 (81.58) compared with the control (68.66). In addition, P and Zn contents of strawberry leaves with bacterial inoculation significantly increased under organic growing conditions. Available P contents in soil were increased from 0.35 kg P₂O₅/da at the beginning of the study to 2.00, 1.97 and 1.82 kg P₂O₅/da by M3 + OSU-142, M3 + BA-8 and M3 + BA-8 + OSU-142 applications, respectively. Overall, the results of this study suggest that root inoculation of Bacillus M3 alone or in combination with spraying Bacillus OSU-142 or Pseudomonas BA-8 have the potential to increase the yield, growth and nutrition content of strawberry plant under organic growing conditions.     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

Effect of phosphite fertilization on growth,yield and fruit composition of strawberries

Traditionally, phosphates (Pi, salts of phosphoric acid, H₃PO₄) have been used for plant fertilization, and phosphites (Phi, salts of phosphorous acid, H₃PO₃) have been used as fungicides. Nowadays several Phi fertilizers are available in the EU market despite the fact that in research trials Phi has often had a negative influence on plant growth. The objective of this study was to elucidate the effect of a Phi fertilizer on plant growth, yield and fruit composition of strawberries (Fragaria × ananassa Duch.). Experiments were carried out with ‘Polka’ frigo plants in South Estonia in 2005 and 2006. The number of leaves per plant, total and marketable yields, fruit size, fruit ascorbic acid content (AAC), soluble solids content (SSC), titratable acidity (TA), anthocyanins (ACY) and total antioxidant activity (TAA) were recorded.     

Inoculation of growing media with the rhizobacterium Variovorax paradoxus 5C-2 reduces unwanted stress responses in hardy ornamental species

Inoculation of growing media with plant growth promoting rhizobacteria (PGPR) has a number of potential benefits for the production of ornamental plants. Certain rhizobacteria synthesise the enzyme ACC deaminase, which cleaves ACC, the precursor of the plant hormone ethylene. Bacterial metabolism is now known to lead to a reduction in [ACC] in the plant transpiration stream and bacteria are hypothesised to act on ACC exuded from roots. This in turn reduces the ethylene generated in plants growing in growing media inoculated with these bacteria. Here we tested if applications of the ACC deaminase containing rhizobacteria Variovorax paradoxus⁵C–²could be of benefit to ornamental growers by reducing ethylene generation in stressed plants. Ethylene is produced at levels that are inhibitory to growth and development under a number of abiotic stresses. The propagation and production of hardy ornamentals is deleteriously affected by abiotic stresses that involve ethylene signalling, including drought and physical wounding. Inoculation of growing media with V. paradoxus 5C-2 lowered ethylene emission from mature leaves of Cytisus × praecox experiencing drought stress. In addition, bacterial inoculation of the growing media resulted in significantly reduced abscission of the mature leaves under drought treatment. Beneficial effects of inoculation where also found in the wounding response of Fargesia murielae following divisional propagation and late season senescence in Aquilegia × hybrida in response to drought stress. Together these results demonstrate that V. paradoxus⁵C–²has real potential for use on ornamental nurseries in situations where plant stresses are unavoidable.     

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 (♂).     

Hybrids sugary × sugary enhancer of sweet corn: A valuable option for cool environments

Sugary (Se1Se1) sweet corn hybrids do not have a high quality for human consumption according to the preferences of today's consumers, although they have a good stand establishment in cool environments. On the contrary, sugary enhancer (se1se1) hybrids have a high table quality, but a poor stand establishment in areas with a cool growing season. Sugary × sugary enhancer (Se1se1) sweet corn genotypes could be a valuable alternative for cool environments, but no information is available about the performance of sugary × sugary enhancer hybrids under such conditions. The objective of this paper is to evaluate the potential of sugary × sugary enhancer sweet corn genotypes in cool environments in comparison with sugary and sugary enhancer genotypes. Eight sugary lines and eight sugary enhancer lines were used as the parental lines of 15 sugary, 12 sugary enhancer, and 57 sugary × sugary enhancer hybrids that were evaluated in three different cool environments. The results of this work show that sweet corn cultivars with the Se1se1 genotype are an appropriate alternative for cool areas because they have better plant and ear types and a better eating quality than the Se1Se1 genotypes, and better emergence, early vigor, and shorter flowering time than se1se1 genotypes.     

Stoloniferous shoot protoplast,an efficient cell system in potato for somatic cell genetic manipulations

The present study reports that protoplasts isolated from stoloniferous shoots (SS) of potato represent an efficient system for somatic cell genetic manipulations. SS were established from single-node cuttings on MS medium supplemented with either 0.1 or 0.2 M sucrose (Suc), and protoplasts were isolated and cultured within the alginate strip, following an improved method. SS induced by 0.1 M Suc yielded 8–22 × 10⁵ protoplasts g⁻¹ fresh mass, with a high morphogenic competence. However, 0.2 M Suc-induced SS yielded protoplasts that contained large amounts of starch grains, resulting in their high degree of fragility, delayed cell division and poor morphogenic competence. For symmetric somatic hybridization (electrofusion) between Solanum tuberosum Gp. Tuberosum androgenic (di)haploid (2n = 2x = 24) ‘C-13’ and diploid (2n = 2x = 24) wild species S. pinnatisectum, protoplasts isolated from 0.1 M Suc-induced SS were also found to be most responsive. Out of several putative somatic hybrids, there were two tetraploids and five diploids, with 48 and 24 chromosomes, respectively at all the three shoot layers (L₁–L₃). This precluded the occurrence of mixoploidy vis-à-vis chimaerism in regenerants, as common in somatic fusion involving mesophyll protoplasts of S. pinnatisectum. Nuclear microsatellite analyses based on the two single-locus nSSR loci (STM0037 and STM2030) confirmed that one of the tetraploids was a true nuclear hybrid (heterokaryon), while the other a homokaryon of the Tuberosum parent ‘C-13’. The use of 0.2 M Suc-induced SS protoplasts for fundamental studies on tissue- and/or cell type-specific transient gene expression underlying tuberization has been discussed.     

Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture

Anther culture was successfully developed in Anthurium andreanum Linden ex André cv. ‘Tropical’, but resulted in variation in morphology and growth response of regenerants. Ploidy levels varied in different morphological variants. Finding a convenient, rapid, reliable, practical and indirect method to screen and determine anthurium ploidy level is important not only for anthurium, but for ornamentals in general. Regenerants derived from anther culture showing three different ploidy levels (haploids, diploids, triploids) were used in this study. Five indirect methods were used to assess chromosome number: chloroplast number in stomatal guard cells (M1), stomatal length and width ratio (M2), stomatal density (M3), ratio of length and width of leaves (M4), and microspore number per anther (M5). These were compared to chromosome counting as the direct and control method (M6). Through simple regression correlation analysis, when compared to M6, M1 was the most convenient and reliable indirect method to determine the ploidy level. This method was highly and significantly positively correlated to anthurium ploidy level (r = 0.945; p < 0.01). This method could also be applied much faster than the conventional chromosome method.     

Taxonomic revision of Dendrobium moniliforme complex (Orchidaceae)

Taxonomic revision of Dendrobium moniliforme complex is presented. D. moniliforme complex is characterized by the even slim stems, bracts with brownish zone, semi-spherical anther cap and the hairy disc of lip. Dendrobium tosaense, Dendrobium officinale and Dendrobium guangxiense were excluded by having membranous bracts lacking brownish zone, anther cap conical and bifid. Two species are recognized in this complex, i.e., D. moniliforme and Dendrobium wilsonii. D. wilsonii differs from D. moniliforme by having elliptic leaves about 1.3–2 cm wide, dorsal sepal 3.0–4.0 cm long, 0.6–0.9 cm wide, petals elliptic to oblong, 3.0–4.0 cm long, 1.0–1.5 cm wide, lip elliptic to ovate–lanceolate, 2.6–3 cm long, 1.2–1.5 cm wide.