Cytoplasmic Male Sterility in Barley : VIII. LIPOXYGENASE ACTIVITY AND ANTHER AMINO NITROGEN IN THE msm1-Rfm1a SYSTEM

The lipoxygenase (LOX) activity was determined in almost isogenic types of barley (Hordeum vulgare L.): normal cv. Adorra, cytoplasmic male sterile (msm1), and msm1 barley with restored fertility, heterozygous for the Rfm1a restorer gene. The LOX activity was lowest in male steriles in the leaf tissue studied at the anthesis stage. The LOX activity in developing anthers was higher than in leaf tissue, and decreased during degeneration of the sterile anthers.     

Analysis of the Maize dicer-like1 Mutant,fuzzy tassel,Implicates MicroRNAs in Anther Maturation and Dehiscence

Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.     

The floral biology ofTricyrtis latifolia Maxim. (Liliaceae)

The floral biology ofTricyrtis latifolia was investigated at four sites: Nigorigo, Kurumijima and Hikagedaira-san, Gifu Prefecture, and Jöge, Miyagi Prefecture. The flowers open fully by 3 a.m., and anthesis begins at about 8 a.m. Most pollen is taken away by bumblebees by 10 a.m. The distal parts of the trifid style-branches (each branch is further bifid) and the immature stigmata are above the anthers in the first day flowers. The distal parts of the style-branches with their extension curve downward and are positioned under the anthers on the morning of the second day and the stigmata are fully matured. The flowers wilt by the third day. The primary pollinators ofT. latifolia wereBombus diversus andB. honshuensis in three sites; only the former was a primary pollinator at the Jōge site. Bees forage the first and second day flowers, and their backs contact the anthers and stigmata of the second day flowers while the bees are sucking nectar. Breeding experiments showed thatT. latifolia was fairly self-compatible. The avoidance of autogamy and the possibility of geitonogamy are discussed.     

Functional analysis of OsTUB8, an anther-specific β-tubulin in rice

Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. β-tubulins (TUB), which are one of the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. OsTUB8 that was expressed in rice anthers was characterized with a multi-level approach. At the protein level, OsTUB8 was expressed mainly in anthers compared to callus, root, leaf sheath and leaf blade. In situ hybridization and GUS fusion analysis revealed that OsTUB8 was expressed in vascular bundles of anther filaments and in pollen. OsTUB8 expression was lower in the anthers of GA-deficient mutants, ‘Tanginbozu’ and ‘Akibrarewaisei’, compared to those of their respective wild types. Transgenic rice expressing OsTUB8 in an antisense orientation were suppressed in the amount of seed set upon maturity. Antisense-transgenic rice plants were 20–60% shorter compared to the vector-only control. These results suggest that OsTUB8 might be differentially expressed in rice anthers due to the action of GA, and involved in the processes of vegetative growth and seed set in rice.     

Evaluation of insecticidal activity of the essential oil of Allium chinense G. Don and its major constituents against Liposcelis bostrychophila Badonnel

Water-distilled essential oil from the dried bulbs of Allium chinense (Liliaceae) was analyzed by gas chromatography–mass spectrometry (GC–MS). Eighteen compounds, accounting for 98.4% of the total oil, were identified and the main components of the essential oil of A. chinense were methyl allyl trisulfide (30.7%), dimethyl trisulfide (24.1%), methyl propyl disulfide (12.8%) and dimethyl disulfide (9.6%) followed by methyl allyl disulfide (3.4%) and methyl propyl trisulfide (3.6%). The essential oil exhibited contact toxicity against the booklice (Liposcelis bostrychophila) with an LC₅₀ value of 441.8 μg/cm² while the two major constituents, dimethyl trisulfide and methyl propyl disulfide had LC₅₀ values of 153.0 μg/cm² and 738.0 μg/cm² against the booklice, respectively. The essential oil of A. chinense possessed strong fumigant toxicity against the booklice with an LC₅₀ value of 186.5 μg/l while methyl allyl trisulfide (LC₅₀ = 90.4 μg/l) and dimethyl trisulfide (LC₅₀ = 114.2 μg/l) exhibited stronger fumigant toxicity than methyl propyl disulfide (LC₅₀ = 243.4 μg/l) and dimethyl disulfide (LC₅₀ = 340.8 μg/l) against the booklice. The results indicated that the essential oil and its major constituents have potential for development into natural insecticides or fumigants for control of insects in stored grains.     

Developmental characteristics of floral organs and pollen of Chinese cabbage (Brassica campestris L. ssp. chinensis)

Morphological and cytological studies are complementary approaches to understand the molecular mechanisms that regulate floral developmental pathways. To better understand abnormal mutant phenotypes in floral development, we conducted detailed observations and investigations of the morphology, cytology, and cell ultrastructure of wild-type Chinese cabbage (Brassica campestris L. ssp. chinensis Makino and syn. B. rapa ssp. chinensis) flowers when they developed from primordia to anthesis. First, we measured bud and organ length with a stereo microscope and observed the developmental status and characteristics of the floral organs using a scanning electron microscope; then we made thin slices of anthers to observe the developmental stage and characteristics of pollen using an optical microscope; and finally, we made super-thin slices of anthers to observe the ultrastructure of pollen during its development with the aid of a transmission electron microscope. In this study, the floral developmental continuum was divided into 17 stages based on significant changes in the shape of floral primordia, and the pollen developmental continuum was divided into 14 stages based on the developmental characteristics. The results could provide the morphological basis for further research on the molecular mechanisms that regulate development of the floral organs and/or pollen of Chinese cabbage and their allied species.     

Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens

Thymus longicaulis C. Presl is a small aromatic perennial herb used as a traditional remedy for cold, flu and cough. Composition of the essential oil of T. longicaulis from Croatia and its in vitro antimicrobial activity against the most common respiratory pathogens were evaluated. The yield of essential oil obtained by hydrodistillation from aerial plant parts was 1.2%. According to the GC-MS analysis, a total of forty one compounds (99%) were identified. Thymol (46.3%), ??-terpinene (16.2%), thymyl methyl ether (11.4%), and p-cymene (9.4%) were the main components. Antimicrobial activity of the essential oil against six clinically isolated bacterial and yeast strains was determined using standard disc agar diffusion method and microdilution broth assay. The essential oil exhibited antimicrobial activity towards all tested respiratory pathogens. The most sensitive strains were Haemophilus influenzae and Streptococcus pneumoniae(MIC=0.78 mg/mL), while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Our results indicate that T. longicaulis essential oil could be effective against clinically relevant respiratory pathogens which have the ability to develop resistance to antimicrobial drugs.     

New C9-polyacetylenes from the essential oil of the highly endangered species Baccharis palustris Heering (Asteraceae)

The essential oil composition of the aerial parts from Baccharis palustris Heering (Asteraceae), a highly endangered species, was analyzed by GC/qMS, GC/HRMS-TOF, FT-IR, UV–vis and NMR spectroscopy using 1D and 2D techniques. To our knowledge, this is the first report on the essential oil composition of B. palustris, as well as the first phytochemical study on this plant species. The volatile extracts were obtained from different phenologicals stages of plant, at flowering stage (FS) and vegetative stage (VS), where 51 components were identified accounting for 96.3 % and 99.0 % of the oil, respectively. The oil was rich in polyacetylene compounds (> 75 %), being the main components identified the new natural C₉-polyacetylenes 1-nonene-3,5-diyne (1, here named as baccharisdiyne) (52.7–65.0 %), 1,7(Z)-nonadiene-3,5-diyne [2, 7(Z)-dehydro-baccharisdiyne] (14.4–17.8 %), and 1,7(E)-nonadiene-3,5-diyne [3, 7(E)-dehydro-baccharisdiyne] (1.5–2.4 %). In addition, the known polyacetylenic compounds (Z)-lachnophyllum acid methyl ester (4) (4.3–5.3 %) and (E)-lachnophyllum acid methyl ester (5) (0.2 %) were also identified. Moreover, GC/qMS analysis allowed the identification of other 46 components in the essential oil samples, mainly mono- and sesquiterpenoids. In B. palustris, C₉-polyacetylenes probably derive biogenetically from a C₁₀ precursor: baccharisdiyne (1) would be derived from either or both lachnophyllum methyl ester geometric isomers (4 or 5), by enzymatic hydrolysis followed by decarboxylation. Dehydro-baccharisdiynes (2 and 3) could be produced by a similar pathway starting from the corresponding matricaria acid methyl ester isomers, one of which was tentatively identified at trace-level in B. palustris essential oil.     

Micropropagation of Albuca bracteata and A. nelsonii — Indigenous ornamentals with medicinal value

In this study, two species from the genus Albuca (Hyacinthaceae) with ornamental and medicinal properties were micropropagated. Adventitious bulblets of Albuca bracteata were cut into quarters and used as explants to examine the effect of temperature (10, 15, 20, 25, 30 or 35 °C), carbohydrates (glucose, fructose or sucrose at 0, 87.5, 175, 262.5 or 350 mM) and hormones (BA, mTR, NAA, IAA, GA₃, ABA or methyl jasmonate each at 0, 0.1, 1.0 or 5.0 mg/L) on the induction and growth of bulblets. Temperatures above 35 °C completely inhibited bulb formation, while induction at all other temperatures was high. Heaviest and largest bulbs formed at 20 °C. Low concentrations (87.5 mM) of all tested carbohydrates increased bulb induction compared to media without a carbohydrate source, while higher levels decreased bulblet induction. The cytokinins mTR and BA inhibited bulb induction, diameter and mass at moderate (1.0 mg/L) and high (5.0 mg/L) concentrations. GA₃, NAA and particularly IAA promoted bulblet induction, while ABA and methyl jasmonate had no significant effect on the induction or bulblet growth. Leaf material and young inflorescences of A. nelsonii were removed, decontaminated, and dissected into seven explant types: leaves, peduncles, pedicels, whole flowers, tepals, ovaries and anthers. These were placed on MS media without hormones, or containing 0.5 mg/L mTR, 0.5 mg/L NAA or 0.5 mg/L mTR + 0.5 mg/L NAA to establish which explant type and hormone combination promoted shoot formation. Some tepal and pedicel explants were capable of shoot production on media with both mTR and NAA, but peduncle explants produced the most shoots when mTR and NAA were both present in the culture medium. Flowers, leaves, ovaries and anthers were completely unresponsive, irrespective of medium composition. These techniques will aid the further horticultural development of these plants, and can be easily adjusted for other species within the genus to promote conservation.     

Geissorhiza melanthera sp., nov. (Iridaceae: Crocoideae) from the southern African winter rainfall region with comments on its pollination by the bee-fly Megapalpus capensis (Bombyliidae)

The new species Geissorhiza melanthera is a member of sect. Engysiphon of subgenus Weihea and is distinguished by its beige-yellow flowers with the bases of the tepals and throat of the perianth tube bright red. The unscented flowers are zygomorphic with unilateral and declinate stamens and style extended horizontally. Its most striking feature is the black anthers with latrorse dehiscence and a portion of the connective visible on the adaxial surface. Field observations on two separate days showed the only visitor to be the bee-fly Megapalpus capensis (Diptera: Bombyliidae), which landed on the anthers and then moved toward the perianth tube, inserting its proboscis and thorax into the upper perianth tube, evidently foraging for nectar. Pollen loads on the ventral body of flies were passively transferred to receptive style branches of flowers visited later. This is the first record in Iridaceae of potentially pollinating visits by Megapalpus or any bee-fly.     

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.     

Analysis of the volatile components of Lavandula canariensis (L.) Mill., a Canary Islands endemic species,growing in Australia

The essential oil of a Canary Islands endemic species, Lavandula canariensis (L.) Mill., gathered from Australia, was extracted by hydrodistillation and solid phase micro-extraction (SPME). The oils have been studied by GC and GC–MS. A total of 38 compounds have been identified in the leaf oil extracted by hydrodistillation, the principal components being carvacrol (23.6%), β-bisabolene (20.8%), (E,E)-α-farnesene (11.3%), β-caryophyllene (7.6%) and carvacrol methyl ether (7.3%), while the oil extracted by SPME showed carvacrol (42.6%) as the principal component with moderate amounts of (E,E)-α-farnesene (9.1%), β-bisabolene (7.5%), cis-hex-3-en-1-ol (5.6%) and carvacrol methyl ether (4.6%). SPME extracts indicated that carvacrol, cis-hex-3-en-1-ol and linalool were in greater concentration in the head space vapours than in the oil. This is the first report describing the essential oil composition of this species.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

Androecium development in the bromeliad Dyckia pseudococcinea L.B.Sm. (Pitcairnioideae-Bromeliaceae), an endangered species endemic to Brazil: implications for conservation

Dyckia pseudococcinea L.B.Sm. is endemic to the restingas of Maricá, State of Rio de Janeiro, in southeastern Brazil. However, since this area is under intense ecological stress, D. pseudococcinea is considered an endangered plant species. An important step toward the conservation of this species is establishing the developmental stage most favorable to in vitro propagation. Accordingly, the present study analyzed the androecium to establish the developmental stages of anthers, emphasizing anther wall development, androsporogenesis and androgametogenesis. The relationships between the size of flowers and anthers were also studied. Anther wall development follows the basic-type, while the tapetum, which is originated from the subepidermal layer (inner secondary parietal layer 2 and archesporial initials), follows the secretory-type. Androsporogenesis is successive and originates isobilateral and decussate tetrads. The oblate and monosulcate pollen grains exhibit microreticulate exine and are dispersed at the bicellular stage. At the vacuolated stage, the androspore (microspore) is still proliferating and not yet completely differentiated. Therefore, since the androspore has not yet lost its embryonic capacity, we suggest the preferential use of this developmental stage, which is present in flower buds 6.4–7.1 mm in length, in conservation protocols focused on the androgenesis of D. pseudococcinea.     

Floral anatomy of the Lecointea clade (Leguminosae, Papilionoideae, Swartzieae sensu lato)

Flower and inflorescence anatomy and morphology of Exostyles, Harleyodendron, Holocalyx, Lecointea, and Zollernia (Leguminosae, Lecointea clade) were studied. Features common to all genera but otherwise rare within the Leguminosae include: (1) the presence of phenolic compounds in the epidermal cells of the anthers and subepidermal cells of the bracteoles, sepals, petals, and ovaries (absent in Holocalyx balansae); (2) simple trichomes on the adaxial base of the bracteoles and on the surface of the calyx and ovaries; and (3) tapetum persisting until the androspores are formed. Other notable anatomical features are: (1) colleters on the adaxial bases of the bracts and bracteoles of Holocalyx balansae and Zollernia ilicifolia; (2) trichomes on the anthers of Harleyodendron unifoliolatum, Holocalyx balansae, Lecointea hatschbachii, Zollernia ilicifolia and Z. magnifica; (3) osmophores on the petals of Exostyles godoyensis; (4) asynchronous pollen development in the anthers of Holocalyx balansae and Zollernia magnifica; and (5) vascular bundles surrounded by lignified fibers in Harleyodendron unifoliolatum. These anatomical characters are discussed according to their possible phylogenetic implications.     

Effects of mutagens on androgenesis in some species of the genusNicotiana L.

Chosen species and cultivars(Nicotiana sanderae, N. glutinosa, N. otophora, N. paniculata, N. sylvestris, N. velutina, N. tabacum cv. Samsun, N. tabacum cv. Samsun — a dihaploid line, and an amphidiploid N.sylvestris x N tomentosiformis) were treated with adequate concentration of n-buthylmethane sulphonate. The anthers of M₁ plants were cultured on the medium of NITSCH 1969 plus 0.1 mg IAA/1. Special attention was paid to quantitative characteristics of androgenesis. Androgenesis occurred more often in the treated variants. The conclusions have been confirmed of a heterosis effect of adequate chemomutagen concentration at the diploid level (an increase in the number of androgenic anthers in the treated variants, and a decrease in the mean number of haploids per androgenic anther) in the broad range of the material. We have recorded a high androgenic ability in the amphidiploidN. sylvestris x N. tomentosiformis, and androgenesis inN. velutina for the first time.     

Histological Comparisons of Parasitism by Schistonchus spp. (Nemata: Aphelenchoididae) in Neotropical Ficus spp.

Syconia (enclosed infructescences) infested with host-specific species of Schistonchus (Aphelenchoididae) were collected from six species of Ficus (Moraceae) native to Florida or Panama. They were sectioned and histologically examined to assess the effects of parasitism. Parasitism by Schistonchus spp. was associated with hypertrophied cells, tissue necrosis, and the presence of an exudate in all species. Occasional hypertrophy of the outer epidermal cells occurred on seed florets, wasp florets, and on the endothecial cells of male florets in F. aurea (subgenus Urostigma) from Florida. Aberrations of the inner mesocarp occurred under the hypertrophied cells on seed florets. In F. laevigata (subgenus Urostigma) from Florida, Schistonchus sp. infested immature male florets and was associated with hypertrophy of endothecial cells, epidermal cells of the anther filaments, and anthers. Schistonchus sp. also caused aberrations of the anther filament, anthers, and pollen. Ficus poponoei (subgenus Urostigma) and F. glabrata (subgenus Pharmacosycea), both from Panama, had hypertrophied outer epidermal cells on seed florets. Ficus poponoei also had Schistonchus sp. within the pedicel of an aborted floret, with hypertrophy of the cortical parenchyma. Ficus trigonata (subgenus Urostigma) from Panama had hypertrophy of the outer epidermis of seed florets. When the outer epidermis on these florets was missing, the inner mesocarp was hypertrophied. Ficus maxima (subgenus Pharmacosycea) from Panama had hypertrophy on the outer epidermis of seed and aborted florets. Schistonchus spp. were not found in wasp larvae or pupae in any of the Ficus spp. examined. Hypertrophy was never observed in the absence of Schistonchus spp.     

Arsenic accumulation in Ocimum spp. and its effect on growth and oil constituents

A comparative evaluation of As accumulation and subsequent effects upon exposure to arsenite [As(III)] was performed in three species of Ocimum. Plants accumulated high amount of As (μg g^?1 dry weight; dw) (662 in O. tenuiflorum, 764 in O. basilicum and 831 in O. gratissimum at 100 μM As(III) after 10 days) with the order of accumulation being roots > stem > leaves. A significant reduction in plant height and biomass was observed. However, essential oil yield and major oil constituents, such as eugenol, methyl chevicol, and linalool, increased at lower As(III) concentrations [mostly up to 25 μM As(III)] in all three species. Positively, no detectable amount of As was found in oil of any species. The study proposes that Ocimum may be used as a phytoremediator and at the same time as a source of essential oils under proper regulation.     

Unsaturated fatty acid from seed oil of consolida regalis

Icosenoic acid [20:1(11)] was isolated from the seed oil of Consolida regalis. The seeds contain ca 30% of fatty oil which comprise ca 25% 20:1 (11). The structure of the fatty acid methyl ester having been isolated by column chromatography was determined by GC/MS, IR and ¹H NMR and was confirmed by HPLC analysis of the bromophenacyl ester derivative.     

Development of embryos and seedlings from pollen grains inLycium halimifolium Mill. in thein vitro culture

Anthers ofLycium halimifolium were grownin vitro at the following stages of development: tetrads, microspores and binucleate pollen grains. Pollen plantlets were obtained only from anthers inoculated at the stage of microspores. The growth of androgenic embryos was very slow. Mature embryos were formed in about 3% of the inoculated anthers. The largest number of embryos obtained from one anther was 4.