Ethylene evolution and sensitivity in cut orchid flowers

The effects of ethylene and the rate of ethylene production were studied in several orchid flowers. Orchid flowers showed a wide range of sensitivity to ethylene; Vanda ‘Miss Joaquim’ was most sensitive, Cattleya, Cymbidium and Paphiopedilum were less sensitive, while Dendrobium and Oncidium were not sensitive. Ethylene production in opened flowers was low, less than 1 nl/flower/h. In Cattleya, a peak in ethylene production was observed at the onset of senescence. Emasculated and/or pollinated flowers of Vanda, Cattleya and Paphiopedilum produced high levels of ethylene; Dendrobium did not react to these treatments in ethylene production.The anionic silver-thiosulfate complex (STS) was partially inhibitory to ethylene production in emasculated Cymbidium flowers, but appeared to stimulate the process in pollinated flowers. It was, however, effective in decreasing anthocyanin synthesis in both emasculated and pollinated Cymbidium flowers. In Cattleya, aminoxyacetic acid (AOA) and STS had no effect on longevity of cut flowers.     

Effects of development,temperature, and calcium hypochlorite treatment on in vitro germinability of Phalaenopsis seeds

There are no standardized procedures for sanitizing orchid seeds for propagation by tissue culture and there is insufficient information about the optimum stage of orchid seed development for best germination. Phalaenopsis amabilis flowers were hand-pollinated and fruits harvested 90, 105, and 120 d after pollination (DAP) for seed developmental analysis. Embryo cell number per seed was counted after staining with 4′-6-diamidino-2-phenylindole and viewing through a confocal microscope. Germination percentage and cell number per embryo increased from 14 to 61% and 41 to 66%, respectively, during fruit development from 90 to 120 DAP. Seeds from mature, browning (∼140 DAP) Phalaenopsis Sogo Lit-Angel and Phalaenopsis spp. breeding line 9450 seed pods failed to germinate until frozen at −196, −80, or −18 °C and thawed or chilled at 4 °C for 10 d. Germinability in 140 DAP seeds was correlated with cracked testa after freezing and thawing. P. amabilis seeds were treated with 0, 5, 10, or 15% calcium hypochlorite (CH) for 5, 10, or 15 min. Ninety six percent of untreated seeds from 90 DAP fruit produced protocorms within 40 d after sowing (DAS). Exposing seeds to 5% CH for 10 or 15 min decreased germination to 85 and 73%, respectively. Exposure to 10 or 15% CH for 5, 10, or 15 min produced seed germination percentages of less than 40%. Protocorms developed root hairs and shoot primordia by 50 DAS and an average of one leaf and root by 85 DAS after treatment with either 0 or 5% CH. Higher concentrations delayed or inhibited protocorm development. Green fruits 120 DAP produced the highest percentage of protocorms, while ∼140 DAP seeds from browning fruit were dormant but cold treatments increased germination.     

Rapid seedlings regeneration from seeds and vegetative propagation with sucker and rhizome of Eremospatha macrocarpa (Mann & Wendl.) Wendl and Laccosperma secundiflorum (P. Beauv.) Kuntze

To develop efficient seedling production methods for Laccosperma secundiflorum and Eremospatha macrocarpa, a study was conducted to examine regeneration using offsets combined with several physical and chemical treatments of seeds. Offsets categorized into small, medium and large diameters, were planted in three conditions: shaded and open nursery, and greenhouse. We tested sucker from E. macrocarpa, and sucker and rhizome from L. secundiflorum. For both species, high viability percentage (ranging from 55% to 100%) were observed for small and medium suckers planted in shaded nursery and greenhouse, against less than 49% for sucker planted in open nursery. The mean seedling emergence times were estimated to 84, 77 and 75 days after planting (DAP) for small, medium and large sucker of L. secundiflorum, respectively under open nursery condition, and 76, 75, 95 DAP for small, medium and large suckers of the same species, respectively in shaded condition. Greenhouse has a significant positive effect on E. macrocarpa seedlings emergence time. For this species, the mean seedling emergence times were estimated to 43 DAP for small sucker and 76, 93 DAP for medium and large suckers. No seedling was obtained from rhizome planted in all the growing conditions tested. Concerning seed dormancy breaking, germination percentages and rates were determined for 13 treatments. The best treatments were pre-soaking unscarified seeds for 4 days in 1.01 g l⁻¹ and 0.10 g l⁻¹ KNO₃, with 79% and 68% of germination, respectively and in 3.46 × 10⁻³ g l⁻¹ GA₃ for 68% of germination. These methods are suggested to improve germination of L. secundiflorum seeds. Successful and recommended methods for E. macrocarpa are pre-soaking scarified seeds in 3.46 × 10⁻³ g l⁻¹ and 3.46 × 10⁻⁴ g l⁻¹ GA₃, 96% and 94% of germination, respectively. Dormancy, probably a combination of mechanical and chemical dormancy, is present in the two species.     

Production of intraspecific hybrids between wild-type and petaloid-sepal cultivars in Habenaria radiata

Orchids are commercially important plants with flowers that are unique and very specialized in shape and color. The flowers consist mostly of sepals, lateral petals, lip (labellum) and column, and are zygomorphic and resupinate. Whereas most orchid species have petaloid tepals in the first and second whorls, Habenaria radiata has a flower with greenish sepals and white lateral petals and lip. ‘Hishou’, one of the cultivars of H. radiata, is a floral homeotic mutant and has a petaloid median sepal and lip-like lateral sepals in the first whorl. Additionally, this cultivar often has non-resupinate flowers whereas wild-type H. radiata flowers are resupinate. In the present study, we investigated the genetic inheritance of these characters in the ‘Hishou’ cultivar by crossing it with wild-type plants. Some intraspecific hybrids, which were confirmed by PCR-RFLP analysis, had flowers with a petaloid median sepal and lip-like lateral sepals in the first whorl, indicating that these were dominant characters. Since the remainder of the intraspecific hybrids had wild-type flowers, these characters must be heterozygous in ‘Hishou’ plants. Although ‘Hishou’ plants had non-resupinate flowers, intraspecific hybrid flowers were resupinate, even though they had the petaloid median sepal and lip-like lateral sepals. This result indicates that non-resupination must be a recessive character. Since sepal-petalization and triple lip characters of ‘Hishou’ inherited dominantly, these characters can be utilized for the breeding of Habenaria species by intra- and interspecific crosses.     

Senescence of Dendrobium `Pompadour' flowers following pollination

Summary

Flowers of Dendrobium `Pompadour' developed premature petal and sepal senescence following pollination. Pollination induced an ethylene climacteric accompanied by a small respiratory climacteric, epinasty and increased flower or inflorescence fresh weight and water uptake. Pollination did not alter petal and sepal anthocyanin content and ion leakage, and lip, petal and sepal fresh weight and dry weight. The fresh weight and dry weight of stigmas (columns) together with pedicels increased significantly after pollination. Ovary growth of pollinated orchid flowers with petals and sepals intact was greater than that of pollinated orchid flowers without petals and sepals, while their water uptake was not significantly different.     

Reproductive organography of Bougainvillea spectabilis Willd

Bougainvillea spectabilis Willd. is of prime importance for horticulture, as well as potentially for pharmaceutical industries, agriculture and environmental industries. However, its floral development is not yet well understood. A detailed study on floral structure and floral organography in the species was first completed using microscopy of paraffin microtome sections of buds. The results were indicated as follows: first, the three trumped flowers in the cymose inflorescence develop asynchronously. Secondly, Varieties with multi-whorl bracts do not develop any sexual organs, i.e., perianth, pistil and stamens. Thirdly, the wall of the two-loculus anther consists of two kinds of cells: the inner wall, consisting of thick-cytoplasmed cells and the outer wall, consisting of fibrous cells. Fourthly, the pollen grains, with three germination colpi, vary substantially in the form and size in summer under the highest day temperature of 40 °C. Fifthly, the pistil is characterized with betalain-acumulating stylar brush. Followed the developmental course, only one basal ovule is developed in the superior ovary. Finally, organs of one flower develop consecutively from the outer to the inner, i.e., from bracts, to calyx, stamen, and carpel while the three flowers bloomed one by one in one cymose inflorescence. It almost takes 1 week from first bud to the third flower blooming. Our research showed a series of special characteristics of reproduction organography of B. spectabilis which can be useful for understanding its reproduction biology and its sterility.     

High frequency early flowering from in vitro seedlings of Dendrobium nobile

Dendrobium nobile Lindl. is a popular temperate Chinese orchid commonly marketed as a traditional medicinal plant. Seedlings of Dendrobium nobile Lindl. produced floral buds (33.3–34.8%) precociously on a defined basal medium (1/2 MS) containing paclobutrazol (PP₃₃₃) at 0.5 mg L⁻¹ or thidiazuron (TDZ) at 0.1 mg L⁻¹ within 4 months of culturing. The frequency of floral buds formation can be further increased to 95.6% by growing seedlings in a PN (PP₃₃₃ 0.3 mg L⁻¹ + NAA 0.5 mg L⁻¹)-containing medium followed by transfer onto 1/2 MS medium with PP₃₃₃ and TDZ (PP₃₃₃ + TDZ). However, flower developed was deformed under 25 °C but it developed fully when grown in a lower temperature regime (23 °C/18 °C, light/dark) for 45 days. Under optimal condition, in vitro flowering was observed about 6 months after seed sowing.     

Ovules that failed to form seeds in zinnia (Zinnia violacea Cav.)

The structure of ovules that failed to form seeds after compatible pollination in double-flowered zinnias (Zinnia violacea Cav.) was investigated by observing histological sections, and comparing the normal and abnormal development of the seeds. In an embryo sac of zinnia at anthesis, a large nucleus of fused polar nuclei was clearly recognized. Success in double fertilization was determined by the disappearance of this nucleus. In normally developing seeds, the cellular endosperm became vague at 5–6 days after pollination (DAP) and the developing embryos occupied the entire portion of the seeds up to 10 DAP. A number of ovules failed in fertilization for the lack of an embryo sac. In most of the aborted seeds, the embryo sacs degenerated, whereas the aborted embryos were still alive in a small number of aborted seeds. A small percentage of the aborted seeds exhibited an aborted embryo and an unfertilized fused nucleus of two polar nuclei or ‘single fertilization’. The three major problems suggested for the failure in seed formation in pollinated florets of zinnia included failure in fertilization, ovules lacking embryo sacs, and abortion of developing seeds.     

Scent profiling of Cymbidium ensifolium by electronic nose

The performance of electronic nose (E-nose) for Chinese Cymbidium scent profiling has been evaluated. Changes in scent profiles of two Cymbidium ensifolium cultivars have been monitored at different flowering stages (initial flowering, full flowering, and terminal flowering) and different times combined with two gas collecting devices. Samples were collected by static headspace (SHS) method. How E-nose can be used for pattern recognition and for studying the releasing of flower scent were proposed. Data obtained were subjected to principal component analysis (PCA) and discriminant function analysis (DFA). PCA was performed on the initially instrumental data to explore the structure of each data set and such result showed that the sensory data contained information related to the cultivar and to time spots. DFA was performed to improve the results, leading to clear separations between the sample groups. Gas collecting device did not seriously affect the result of PCA and DFA. Relative aroma intensity (RAI) was proposed as an alternative concept to compare scent intensity between samples on different time points. These results demonstrate the potential application of the E-nose to evaluate the scent profile of flower.     

The importance of male red mason bee (Osmia rufa L.) and male bufftailed bumblebee (Bombus terrestris L.) pollination in blackcurrant (Ribes nigrum L.)

Summary

Male wild bees visit flowers to feed and, despite their lack of specialised organs for carrying pollen, simultaneously pollinate them. We analysed the pollinating efficiency of the males of two bee species for blackcurrant (Ribes nigrum L.): red mason bee (Osmia rufa L.) and bufftailed bumblebee (Bombus terrestris terrestris L.). Both bee species are commercial pollinators and many males are produced during their rearing. Gauze sleeves were used to isolate the inflorescences and to keep the males inside (one per sleeve). In each experimental group, there were six shoots with four neighbouring inflorescences (n = 24). The sleeves were also used to prevent other bees from reaching the flowers, and to allow self-pollination. Non-isolated flowers were pollinated by free-range bees. The effect of male bee pollination was similar to that of free-range bee pollination. The percentages of flowers that developed into fruit were as follows: male bufftailed bumblebee pollination, 79.5%; free-range bee pollination, 71.1%; male red mason bee pollination, 65.1%; and self-pollination, 46.4%; while the mean numbers of fruit per raceme were 5.0, 4.4, 3.6, and 2.4, respectively. The number of non-pollinated flowers per raceme (ranging from 1.3 – 2.8) exhibited an inverse relationship to these numbers of fruit. Our observations showed that male wild bees can pollinate blackcurrant flowers successfully, and could potentially be useful in other crops.     

Mechanism of seedlessness in Iranian seedless barberry (Berberis vulgaris L. var. asperma)

Species of barberry (Berberis vulgaris L. var. asperma) is cultivated in arid and semi arid areas of Iran (South Khorasan province). It is widely used as a food additive. Fruits of this species are seedless, while wild type barberries produce seeds in the same area. In this study, we investigated the mechanism of seedlessness in seedless barberry by pollen viability test, field pollination experiments and microscopic observation of pollen tube growth in pistil and ovule development. For comparison, we also examined ovule development in wild type barberry (B. crataegina DC). In seedless barberry pollen germination was about 54%. Seedless barberry produced 20% seeded fruits when pollinated with pollen of wild type barberry. There was a sharp decrease in fruit set in emasculated unpollinated flowers of seedless barberry. In seedless barberry, a large number of pollen grains (about 370) were observed on stigma of each flower at 12 h after balloon stage (ABS). Most of them germinated and penetrated intracellular area of stigma surface, but no pollen tube reached ovary. In seedless barberry, many ovules did not have any embryo sac or had a very small incomplete embryo sac. In addition, unfused polar nuclei were clearly recognized in some cases at 14 days after full bloom (AFB). However, in wild type, double fertilization was accompanied by disappearance of polar nuclei. In seeded barberry, the cellularized endosperm became apparent at seven days AFB. At 21 days AFB, all ovules of seedless barberry were degenerated, while at the same time in wild type, one or two ovules of each flower were normal and were developing into complete seeds. Results showed that self-incompatibility has a main role in seedlessness of seedless barberry. However, the high frequency of abnormal ovules and single fertilization can be considered as two other reasons of seedlessness. Due to our results, fruits of seedless barberry were set by stimulative parthenocarpy.     

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.     

Identification of Petunia hybrida cultivars that diurnally emit floral fragrances

In order to identify genetic resources for breeding fragrant petunias for use as bedding plants, volatile compounds released by day from the flowers of 40 commercial Petunia hybrida cultivars were analyzed using a solid-phase micro-extraction technique coupled with GC–MS. The three cultivars with solid deep-blue flowers that accumulate malvidin in corollas with high tissue pH were found to emit abundant iso-eugenol as the principal floral fragrance. Several other cultivars that emitted considerable amounts of methylbenzoate and/or benzylbenzoate from the flower were also identified. Association between the floral fragrance and the other floral traits such as floral anthocyanin composition and corolla-tissue pH was discussed.     

Reduced pollen tube growth and the presence of callose in the pistil of the male floral stage of the avocado

Pollen tube growth was studied in the pistil of the male floral stage of the avocado. Self-pollination of the male stage occurred but pollen tubes did not reach the ovary. When female and male stages were hand pollinated, fewer pollen tubes were present in the pistil of the male than in the female stage. A tube reached the ovary in only 1 of 110 male stage pistils, as compared with all female stage pistils observed. Flowers pollinated in the female stage were retained on the plant for longer than flowers pollinated in the male stage.Aniline blue-positive and resorcin blue-positive material (callose) was not present in the pistil of flowers in the female stage but occurred in 46% of the aniline blue-stained and 30% of the resorcin blue-stained pistils in the male floral stage. By 42 h after first opening, callose was present in every pistil observed, in association with the cell walls of the stigmatic papillae, the transmitting tissue, the vascular tissue or the cortex and epidermis or a combination of these tissues. Callose occurred earlier in pollinated than in unpollinated pistils, particularly in the transmitting tissue.It is suggested that reduced pollen tube growth in the pistil of the male floral stage of the avocado is associated with the presence of callose.     

Regulation of flower development in Dendrobium crumenatum by changes in carbohydrate contents,water status and cell wall metabolism

The involvement of carbohydrates, water potential, cell wall components and cell wall-based enzymes in regulating flower development in Dendrobium crumenatum was investigated. Plants were subjected to cold treatment to release floral buds from dormancy, and the various parameters were investigated from young floral bud stage till flower senescence. Development of floral buds was accompanied by progressive decrease in concentrations of fructans and starch. Upon full flower opening, concentration of soluble sugars was maximum, accompanied by a more negative water potential. High pectin methylesterase activity was observed during early bud development and decreased thereafter. Significant increase in activities of β-galactosidase, β-mannosidase and β-xylosidase was also observed during floral bud development. The cell walls of sepals and petals were modified extensively during floral bud and flower development, as observed by changes in the amounts of celluloses, hemicelluloses and total pectin. Pectin solubilisation was also observed to commence during early floral bud development. These results indicated that carbohydrate hydrolysis, osmotic changes and cell wall dissolution that began early in young floral buds, all regulated flower development in this sympodial orchid. Possible applications of the findings in the horticultural industry are discussed.     

Genetic diversity and phylogenetic relationships among and within species of oriental cymbidiums based on RAPD analysis

The classifications of oriental cymbidiums that are native to Korea, China, Taiwan and Japan were examined by molecular analysis. A total of 21 cymbidiums, 15 species including three Cymbidium gyokuchin, 4 Cymbidium kanran and 2 Cymbidium goeringii cultivars, were analyzed by using randomly amplified polymorphic DNA (RAPD) to determine the interspecific and intraspecific relationships. Twenty-two primers were used in the RAPD analysis to distinguish, by comparing differences in DNA banding patterns, all species and cultivars. Similarity values ranged from 0.501 for Cymbidium aloifoilum and C. kanran to 0.935 for Cymbidium ensifolium and Cymbidium marginatum with analysis of total bands score. Phylogenetic tree analysis of the RAPD results from the 21 cymbidiums identified specific groupings. The cymbidiums could be divided into two clusters based upon ecological traits. One trait was temperate zone preference, with each cymbidium preferring either an Asian or subtropical temperate zone. The group that comprised the subtropical cymbidiums was C. aloifoilum, Cymbidium insigne and Cymbidium lowianum. Additionally, we found that Cymbidium lancifolium and Cymbidium aspidistrifolium could be separated based on different flowering physiology and unique leaf form. The groups identified by morphological, physiological and ecological characteristics were in full agreement with those determined by RAPD analysis. The phylogenetic tree derived from the RAPD results was similar to that of the traditional classification. The data acquired from this study could be used for identification and classification of other orchid genera and oriental cymbidium.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.