Reproductive characteristics of Opisthopappus taihangensis (Ling) Shih,an endangered Asteraceae species endemic to China

Opisthopappus taihangensis is an endangered species endemic to China and represents an important genetic resource for chrysanthemum improvement. We describe here its basic reproductive characteristics. The anthers are tetrasporangiate and the anther wall is composed of an epidermis, endothecium, middle layer and tapetum. The middle layer is lost by the microspore tetrad stage, and the tapetum disintegrates at the trinucleate pollen stage. Meiosis in the microspore mother cells is of the simultaneous type, and the tetrad is tetrahedral in shape. Mature pollen grains have three germinal apertures, two sperm nuclei and one vegetative nucleus. The in vitro pollen germination rate is only ∼10%. The ovule is anatropous, dual-integument, tenuinucellatae and the development of the embryo sac follows the Oenothera pattern. The archesporial cell below the nucellus epidermis functions as the megaspore mother cell and forms a linear tetrad. The embryo passes through a globular, heart and torpedo stage before maturing into a cotyledon embryo. The endangerment of O. taihangensis may be associated with low reproductive capacity, as a consequence of poor pollen viability.     

Intergeneric hybridizations between Opisthopappus taihangensis and Chrysanthemum lavandulifolium

For the first time, reciprocal intergeneric hybridizations were produced between Opisthopappus taihangensis and Chrysanthemum lavandulifolium without emasculation. About 20% seed set was similarly obtained from reciprocal hybridization. Only 5 and 6 of the viable plants observed from 45 and 78 seedling survived present some conspicuous intermediate characteristics. Phenotypic evaluation among the progenies of the parents and the putative hybrids was performed carefully since an average of 5.3% seed set was produced in the type of self-pollination using pollen from the same flower and >10% seed set was similarly obtained in the types of self-pollination using pollen from different flowers in a plant and flowers in individual plants from different seeds. One individual of each hybrid shared the inflorescence habit with the pollen plant was confirmed further by the sequence of ncpGS. The two hybrids might be used as bridges of breeding of multi-generic hybrids.     

Comparative anatomy of bisexual and female florets,embryology in Calendula officinalis (Asteraceae), a naturalized horticultural plant

Calendula officinalis is a perennial gynomonoecious herb, often used for medicinal purpose and as ornamentation. The corolla consists of only one petal for ligulate florets, whereas it consists of four to seven petals for disk florets with four to seven stamens accordingly. The bisexual florets are functionally male and their ovaries are either solid or unilocular with no ovule. In contrast, the ovaries of female florets are always unilocular with an anatropous ovule. For bisexual florets, simultaneous cytokinesis in the microsporocyte meiosis leads to tetrahedral, also decussate tetrads. The mature pollen grain is of the three-cell type. The tapetum is mainly of amoeboid type, yet 6.5% antheral tapetum disintegrates in situ, as called glandular tapetum. The young anther wall is composed of epidermis, endothecium, middle layer and tapetum, but the middle layer degenerates at the microspore tetrad stage. The mature anther wall comprises only endothecium, which develops fibrous thickenings. The ovules are unitegmic, tenuinucellatae and the development of the embryo sac follows the monosporic, polygonum type. Before the differentiation of the micropylar cells into egg cell and two synergids, the two polar nuclei fuse into a secondary nucleus and the antipodal cells start degenerating. Two days after blooming, the secondary nucleus has been fertilized by a single sperm nucleus, generating the triploid primary endosperm nucleus.     

The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration

The culture of isolated microspores of kale (Brassica oleracea var. acephala) was studied including the importance of genotype to embryo regeneration, medium composition chiefly the sucrose concentration and the use of colchicine, simultaneously medium renovation. It was initiated using 29 different genotypes as donor plants. Embryos were induced from six of the kale genotypes and these corresponded to the more out-bred genotypes. Embryogenesis was achieved using four different combinations of culture media: (a) microspores initially cultured in NLN medium supplemented with 13% (w/v) sucrose (NLN-13) for 48 h, followed by transfer to fresh NLN-13 medium; (b) microspores cultured for 48 h in NLN-13 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium; (c) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-16 medium; (d) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium. The embryos obtained from four of the genotypes developed into plantlets and these regenerated plants have been successfully transplanted to soil.     

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.     

Anther wall development,microsporogenesis and microgametogenesis in male fertile and sterile chrysanthemum (Chrysanthemum morifolium Ramat., Asteraceae)

Anther wall development, microsporogenesis and microgametogenesis were compared between a normal male fertile chrysanthemum cultivar ‘NJAU04-29-2’ and the two male sterile selections ‘rm20-11’ (anther indehiscent) and ‘NJAU05-52-2’ (anther aborted). In both of the two male sterile types, the tapetum enlarged abnormally and showed signs of disorganization of walls at the onset of meiosis, the pollen was aborted, the anthers appeared shrunken, and the anther vascular bundle and connective tissue were degenerated by anthesis. In ‘rm20-11’, the two smaller pollen sacs began to degenerate at the microsporogenesis stage, so that only one or two microsporangia developed, while in ‘NJAU05-52-2’, only one or two microsporangia were formed following the archesporial cell stage, and most of the microspore mother cells degenerated during the course of meiosis.     

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.     

Effects of Prohexadione Calcium on growth and gibberellins contents of Chrysanthemum morifolium R. cv Monalisa White

The effect of Prohexadione Calcium (Pro-Ca) and daminozide was observed on the growth characteristics and endogenous gibberellin contents of Chrysanthemum morifolium R. cv Monalisa White. Three concentrations viz. 100, 200 and 400 ppm of Pro-Ca and a single concentration of daminozide (800 ppm) were applied three times with 7 days interval on three weeks old plant under greenhouse condition. Pro-Ca suppressed the plant length up to 30.7% while daminozide inhibited up to 27.12% at optimum concentration. The chlorophyll contents and stem diameter were higher than control, while the fresh weight and flower number insignificantly reduced with such treatments. Gibberellin (GA) analysis showed that Pro-Ca and daminozide application significantly lowered bioactive GA₁ content, although the amount of its immediate precursor GA₂₀ was fractionally higher. Bioactive GA₄ content was slightly higher than the control while significant difference in GA₉ was found between the plants treated with Pro-Ca and daminozide. Current study showed that both early C13 hydroxylation and non-C13 hydroxylation pathways of GA biosynthesis are operational in C. morifolium.     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A::hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach

Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the present study, two open pollinated varieties viz., Arka Gaurav and Arka Mohini were used for transformation. Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301 that carries the genes for β-glucuronidase (uid A) and hygromycin phosphotransferase II (hpt II) was used for transformation. GUS histochemical analysis of T₀ and T₁ plants at various stages of growth followed by molecular analysis using PCR, Southern analysis and RT-PCR allowed selection of transgenics. The method resulted in 17.8% and 11.4% of the T₀ plants in Arka Gaurav and Arka Mohini being selected as chimeric and 35.0% and 29.7%, respectively, were identified as stable transformants in the T₁ generation based on PCR analysis.     

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.     

Identification of seven haplotype-specific SFBs in European pear (Pyrus communis) and their use as molecular markers

The European pear (Pyrus communis) carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. The S-haplotype is conferred by an S-locus, which contains the style-specific expressed S-RNase, and the pollen-specific expressed F-box genes (SFB). Both the S-RNase and the SFB genes are multi-allelic and each is characteristic of one of the S-haplotypes. Therefore, they are ideal markers for molecular S-genotyping. In this work, for the first time, seven haplotype-specific SFBs were isolated from European pears. Particular primers for each of these SFBs were generated, thus providing an additional tool for S-genotyping of European pear cultivars.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

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.     

Seed-coat anatomy and proanthocyanidins contribute to the dormancy of Rubus seed

Rubus seed has a deep double dormancy that restricts germination due to seed coat structure and chemical composition. Improved germination of diverse Rubus species required for breeding improved blackberry and raspberry cultivars is partly dependent on the seed coat structure. This study evaluated the seed coat structure of three species with thin (R. hoffmeisterianus Kunth & C. D. Bouché), medium (R. occidentalis L.) and thick (R. caesius L.) seed coats. The three species exhibited distinctive seed-coat cell composition. The very thin testa (0.086 mm) of R. hoffmeisterianus had little exotesta (surface) reticulation; with the meso- and endotesta composed of sclereids of homogenous shape and size. R. occidentalis had a thick testa (0.175 mm) and a highly reticulate exotesta; the meso- and endotesta were composed of several diverse types of sclereids. R. caesius had the thickest seed coat (0.185 mm) but only moderate exotesta reticulation; the meso- and endotesta were composed of large, irregular, loosely arranged sclereids. R. occidentalis, a medium size seed, was the most heavily lignified with seed-coat thickness similar to R. caesius, the largest seed. Proanthocyanidins (PAs) from dry seed of six Rubus species were extracted and quantified by high performance liquid chromatography. R. hoffmeisterianus, a thin only slightly hard seed, had half the PA (0.45 μg/seed) of R. occidentalis with a thick, extremely-hard seed coat and diverse sclereids (1.07 μg/seed). PA content and sclereid composition both appear contribute to seed coat hardness and resulting seed dormancy. The effectiveness of sulfuric acid for Rubus seed scarification is likely due to degradation of PAs in the testa.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Effects of the antiauxin PCIB on microspore embryogenesis and plant regeneration in Brassica rapa

An efficient protocol to improve microspore embryogenesis and plant regeneration in Brassica rapa was established. The antiauxin p-chlorophenoxyisobutyric acid (PCIB) was used to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. All the 4 tested genotypes responded positively to PCIB. The optimum concentration of PCIB application was found to be 40 μM in NLN-13 medium, which resulted in a 3.4- to 6.2-fold increase in the number of embryos (8.27–19.2 embryos per bud) and a 9.6-fold increase (21.33%) in the plant regeneration frequency in comparison with the controls. Heat-shock treatment by incubation at 35 °C for 1 day was more efficient in inducing embryogenesis in the 2 tested genotypes. The embryos, produced in NLN medium supplemented with 40 μM PCIB and transferred at the 21-day-old followed by a treatment at 4 °C for 5 days, reached the highest direct plant regeneration rate of 58.00%.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Production protocol development for greenhouse cut Linaria, Lupinus, and Papaver flowers

Linaria maroccana Hook. f. Ann., ‘Lace Violet’, Lupinus hartwegii ssp. cruikshankii Lindl. ‘Sunrise’ and Papaver nudicaule L. ‘Meadow Pastels’ seeds were directly sown into 105 cell plug trays and received either ambient light or supplemental high intensity discharge (HID) lighting. For each species, a 2 × 3 × 3 factorial was used with two light intensities during propagation, three transplant stages, and three night temperatures. Seedlings were transplanted at the appearance of 2–3, 5–6, or 8–9 true leaves. Transplanted Linaria and Papaver seedlings were placed at 5/11, 10/16, or 15/21 ± 1 °C night/day temperatures and Lupinus seedlings were placed at 15/24, 18/25, or 20/26 ± 2 °C night/day temperatures. For this study, the optimum production temperature for Linaria was 10/16 °C as the cut stems produced at 15/21 °C were unmarketable and production time was excessively long at 5/11 °C. At 10/16 °C, Linaria seedlings should be transplanted at the 2–3 leaf stage to maximize stem number, stem length and profitability. For Lupinus the optimum temperature was 15/24 °C due to long stems and high profitability per plant. Lupinus seedlings should be transplanted at the 2–3 leaf stage when grown at 15/24 °C to obtain the longest and thickest stems; however, $/m² week was higher for plants transplanted at the 8–9 leaf stage due to less time in finishing production space. For Papaver, the 15/21 °C temperature was optimal as that temperature produced the longest stems in the shortest duration, resulting in the highest $/m² week. At 15/21 °C Papaver plants should be transplanted at the 2–3 leaf stage. Supplemental HID lighting had no effect on any of the species.