Embryoid and plantlet formation from leaf segments of Dactylis glomerata L.

Summary The purpose of this investigation was to demonstrate callus induction and plantlet formation from cultured leaf segments of 12–15 week-old Dactylis glomerata L. (orchardgrass) plants. Flat half-leaf sections, approximately 2–3 mm square, from the three innermost (youngest) leaves were isolated and individually plated serially beginning at the leaf base on a solid SH medium containing 30 M of 3,6-dichloro-oanisic acid (dicamba). Callus formed on leaf sections from all 50 plants used in the study. After transfer to SH medium with 1 M dicamba, plantlets formed from leaf sections of 9 of the 50 plants. In most cases plantlets formed from embryogenic callus but in a few cases embryoids formed directly on the leaf surface without an intervening callus state. These developed into plantlets when transferred to low auxin medium. The response for both callus and plantlet formation decreased with increasing distance both spatially and temporally from the shoot apex. Histological examination of embryogenic callus revealed the presence of non-zygotic embryos in various stages of development. The results provide further support for compentency (if not totipotency) of Gramineae leaf cells.     

Autopolyploidy in Dactylis glomerata L.: further evidence from studies of chloroplast DNA variation

Summary Chloroplast DNA variation has been used to examine some of the maternal lineages involved in the evolution of the intraspecific polyploid complex, Dactylis glomerata L. Diploid (2x) and tetraploid (4x) individuals were collected from natural populations of the subspecies glomerata (4x), marina (4x) and lusitanica (2x), as well as from sympatric 2x/4x populations of the Galician type. Digestion of their ctDNA with 11 restriction endonucleases revealed enough variation to characterise three ctDNA variants, designated MBMK, MBmK and mBMK. The distribution of these ctDNA variants reflects different stages in their spread among the populations. The MBMK ctDNA variant predominated at both ploidy levels in subspecies glomerata, lusitanica and marina, and in recent tetraploid Galician/glomerata hybrids. The MBmK variant was detected in a single tetraploid individual and probably results from a relatively recent mutation. Fixation of the mBMK minority variant in the diploid and tetraploid Galician populations adds to the evidence concerning the possible origin of the Galician tetraploids. It means that the Galician diploids were maternal ancestors of the tetraploids. This result complements evidence from earlier studies based on morphology or biochemical markers, and reduces the likelihood that the tetraploids arose by hybridisation between an ancient Galician diploid and an alien tetraploid. It is, however, consistent with a true autopolyploid origin of the tetraploids.     

Anticytokinin effects on in vitro response of embryogenic and nonembryogenic genotypes of Dactylis glomerata L.

The effect of triazine and carbamate type of anticytokinins on in vitro response of embryogenic and nonembryogenic genotypes of orchardgrass (Dactylis glomerata L.) was studied. Both compounds stimulated callus production. Anticytokinins influenced both the efficiency of somatic embryogenesis and frequency of embryoid formation.     

Successful crosses between Festuca arundinacea Schreb. and Dactylis glomerata L.

Summary Five F₁ plants have been obtained after extensive crossing between different ecotypes or varieties of Festuca arundinacea Schreb. and Dactylis glomerata L. The success did not appear to depend on specific treatments (spraying with -aminocaproic acid or gibberellic acid or pre-pollination with killed pollen from the seed parent), but the crossability is limited to exceptional plants.F₁ hybrids showed characteristics of both the parents. In four hybrids various developmental disturbances were observed (low viability, aneusomaty, absence of development of inflorescences). Only one hybrid consistently showed 2n=35 chromosomes, good viability and growth, however, it was sterile. After clonal propagation, attempts for polyploidization were started.     

Changes in endogenous polyamines during the formation of somatic embryos from isogenic lines of Dactylis glomerata L. with different regenerative capacities

The endogenous levels of polyamines (PAs) in leaf-base explants isolated from plants of two isogenic lines of Dactylis glomerata L., differing in their competence for somatic embryogenesis, were compared. Leaf-bases isolated from plants with a high level of competence for somatic embryogenesis (HEC) contained four times the level of polyamines compared to those isolated from plants with a low level of competence for somatic embryogenesis (LEC). When the levels of individual polyamines in the HEC and LEC lines were compared, leaf-bases from plants of the HEC line had much lower PUT/SPD ratios than those from the LEC line. When changes in the levels of PAs were monitored during the first 28 d of culture, on a medium which promotes initiation of somatic embryogenesis, leaf-base cultures from plants of the HEC line showed a 50% increase in the levels of PAs during the first 7 d of culture, after which time levels began to decline. By day 21, levels had dropped below those found in freshly isolated leaf bases. While PUT and SPM levels increased by about 30%, the greatest increase was shown by SPD, which increased by more than 100% during the first 7 d of culture, before declining. In contrast much smaller changes in PA levels were found when leaf-bases from plants of the LEC line were cultured.     

Morphological variation and distribution of cytotypes in the diploid-tetraploid complex of the genus Dactylis L. (Poaceae) from Algeria

Morphological and cytological characters were analysed among thirty populations of the genus Dactylis L. from Algeria, to understand its infraspecific diversity. Principal Component Analysis using quantitative characters allowed discriminating the tetraploid populations into two subspecies, marina (Borrill) Greuter, localised on sea cliffs, and hispanica (Roth) Nyman, very widespread. Some individuals of these latest populations formed a distinct group, identified as subsp. glomerata Hayek. Three diploid taxa are described in the literature: subspecies santai Stebbins & Zohary, castellata Parker & Borrill and mairei Stebbins & Zohary that are considered as prevalent in Algeria, distributed in Tellian Atlas, in forest ecosystems within mesophytic habitats. Canonical Discriminant Analysis on natural populations and on experimental cultures showed two main groups: the first group corresponds to subspecies mairei, with a narrow distribution; the second one exhibits a wide morphological variability and belongs to santai type. Based on this study, a key to aid in identification of the subspecies is presented.     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

A histological study of tissue proliferation,embryogenesis, and organogenesis from tissue cultures ofDactylis glomerata L.

Summary Histological information is presented on the origin of initial tissue proliferation and on embryogenesis and organogenesis in sub-cultured tissue derived from mature orchardgrass (Dactylis glomerata L.) embryos. Embryos were plated on an LS agar medium containing 20 M 2,4-D. Examination of cultures between 96 and 144 hours after plating showed parenchyma proliferation originating primarily from the coleorhiza, especially the basal portion. Within 28 days after plating, the tissue showed various degrees and kinds of organized structures including lobed meristematic regions with vascular tissue. These are thought to develop ultimately into aerial roots which are common in grass tissue cultures. Subcultured tissue on 1.0 M 2,4-D medium showed somatic embryos completely isolated from the tissue mass suggestingde novo embryogenesis. Organogenesis was evident by shoot apical meristems existing on the surface of the tissue mass without attached roots and root meristems without shoots within the tissue. The observations are discussed in relation to the anatomy of the grass embryo.This research was supported in part by the Competitive Research Grants Office of the U.S. Department of Agriculture under Agreement No. 5901-0410-9-0331-0.     

Polyploidy and aspartate-transcarbamylase activity in Hippocrepis comosa L.

The DNA content of plants which were sampled in natural di-, tetra- and hexaploid populations of Hippocrepis comosa L. was estimated and the aspartate transcarbamylase activities of the corresponding cell-free extracts were compared. The amount of DNA is not exactly proportional to the number of genomes. The three kinds of populations do not differ in their aspartate transcarbamylase specific activity. While the enzyme properties are identical in the extracts derived from the diploid and hexaploid plants, the aspartate transcarbamylase present in the tetraploid cytotype shows a slightly lower affinity for one of its substrates and a significantly lower sensitivity to the feedback inhibitor UTP which is still observed after partial purification. These properties might be related to the previously reported greater ability of the tetraploid cytotype to adapt to a variety of biotopes.Abbreviations ATCase aspartate transcarbamylase - CAP carbamylphosphate - EDTA ethylenediaminetetracetic acid - Tris trihydroxymethylaminomethane - AMP adenosine monophosphate - ATP adenosine triphosphate - CMP cytidine monophosphate - CTP cytidine triphosphate - UMP uridine monophosphate - UTP uridine triphosphate     

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

中国西南横断山区的野生鸭茅资源

中国西南横断山区广泛分布野生鸭茅资源,包括鸭茅亚种和喜马拉雅鸭茅亚种。二者的分布范围相近,但在形态及物候发育等方面存在明显差异,鸭茅亚种的主要农艺性状优于喜马拉雅鸭茅亚种。两个亚种在人工控制条件下可以杂交形成三倍体后代,但自然状态下尚未发现二者杂交的确切证据。近年来,中国加强了对两个鸭茅亚种的基础研究,但从保护和利用研究角度出发,开展的工作较少。从来源角度分析,喜马拉雅鸭茅亚种为当地起源类型,鸭茅亚种为外来归化种的可能性较大。由于喜马拉雅鸭茅面临鸭茅亚种的竞争威胁,因此应加强中国喜马拉雅鸭茅亚种野生资源的保护和开发利用研究。     

Ultrastructural characterization of somatic embryos regenerated from NaCl selected and nonselected calli ofDactylis glomerata

Summary Calli were induced from leaf expiants of aDactylis glomerata L. (orchardgrass) genotype which has a high capacity for somatic embryogenesis. After 7 months culture on SH medium containing NaCl, a line was selected which was tolerant to 200 mM NaCl. When both selected and nonselected calli were maintained for 56 days on media containing 0 to 300 mM NaCl, the selected line showed significantly higher regeneration capacity than nonselected calli when placed on media containing more than 50 mM NaCl. Ultrastructural features of control somatic embryos not exposed to the salt were compared to those from nonselected and selected embryos cultured on 200 mM NaCl medium. In the presence of NaCl there were changes in the appearance of cell walls and mitochondria, accumulation of lipids and a higher degree of vacuolation in cells of nonselected embryos compared to control and selected embryos.     

Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojecticle bombarded leaf cells

Young leaf tissue of orchardgrass (Dactylis glomerata L.) was placed on Schenk and Hildebrandt medium containing 30 μM dicamba. Microprojectiles coated with DNA containing the selectable bar gene (Basta? tolerance) and the reporter gene uidA coding for β-glucuronidase (GUS), both driven by the maize ubiquitin promoter (Ubi1), were propelled into the tissue with a particle inflow gun. Transient GUS expression was observed as blue spots of various sizes on leaf segments. Somatic embryos staining entirely blue were also produced, and embryos germinated on medium containing 3.0 mg 1^–1 bialaphos. Leaves of 67 putative transformed plants were painted with 0.1% Basta. Ten showed no reaction, and 6 showed only a localized response. Cultured leaf segments from tolerant plants also produced somatic embryos that expressed GUS. The genetic transformation was confirmed by Southern blot hybridization and PCR analyses of T₀ plants and by PCR analyses of somatic embryos produced from T₀ plants. Received: 9 April 1997 / Revision received: 11 May 1997 / Accepted: 3 June 1997     

鸭茅种质资源多样性研究进展

鸭茅是世界范围内广泛栽培的一种重要禾本科牧草,具有高产、优质、耐荫性强等特性。我国的野生鸭茅资源十分丰富,分布广泛,基因资源优良,但有关鸭茅的研究报导相对较少。为了更好地开发利用这一优良的牧草资源,本从形态学、细胞学和分子水平对国内外鸭茅种质资源多样性的研究进行了综述,并对今后我国的鸭茅研究工作提出了建议。     

Extraction, structural characterisation and evaluation of hydroxycinnamate esters of orchard grass (Dactylis glomerata) as substrates for polyphenol oxidase

Polyphenol oxidase (PPO) activity has been reported in orchard grass (Dactylis glomerata); however, to date, no endogenous substrates have been identified. In the present study, we report the isolation and structural elucidation of PPO substrates in this species. The free phenol fraction was extracted, separated by reverse-phase chromatography and six potential substrates, including two hydroxycinnamate esters, were identified by UV spectrometry, electrospray ionisation-tandem mass spectrometry (LC-ESI-MSⁿ) and 1D and 2D NMR analyses (¹H NMR, ¹³C NMR, DEPT, COSY, HMQC and HMBC). Furthermore, three caffeoylquinic acids (3-CQA, 4-CQA and 5-CQA) were identified by comparison of their spectral data (ESI-MS) with those of known compounds and literature data. Five of these compounds were demonstrated to be substrates for orchard grass PPO.     

Genetic introgression between tetraploidDactylis glomerata subspp.reichenbachii andglomerata in the French Alps. Insight from morphological and allozyme variation

The morphology, phenology and allozyme polymorphism in seven tetraploidDactylis glomerata populations growing in the French Alps were studied. Based on habitat and morphological characteristics, two of the populations could be classified asD. glomerata subsp.reichenbachii, which is known to comprise diploid and tetraploid plants growing exclusively on dolomite, on south-facing meadows. This subspecies has been previously recorded in the Italian and Swiss Alps but not in the French Alps. The remaining five populations were morphologically intermediate but more similar to the cosmopolitanD. glomerata subsp.glomerata. On the basis of allozyme variation, all populations were more related to subsp.reichenbachii than to the cosmopolitan subsp.glomerata, suggesting that gene flow has occurred between the two tetraploid subspecies. The possibility that selection has acted differently on morphological and allozyme characters is discussed.     

Phylogenetic relationships and gene flow between sympatric diploid and tetraploid plants ofDactylis glomerata (Gramineae)

Phylogenetic relationships between sympatric, morphologically indistinguishable diploid and tetraploid plants ofDactylis glomerata L. (Gramineae) in Galicia (Spain) were assessed using allozyme markers for 6 distinct systems. The study exploited recent introduction in Galicia and subsequent hybridization of an alien 4xDactylis subspecies possessing distinct allozymes from those of all the native plants. Opportunities for gene exchanges between the ploidies were estimated from in situ observations of flowering, examination of progenies in 2x/4x natural and experimental crosses, and enzyme analyses. Results show a high genetic similarity between the Galician diploids and tetraploids, which possess peculiar alleles in common. Although the ploidy levels usually have distinct flowering periods, interploidal crosses do occasionally occur. Gene flow is likely much more important from the diploid to the tetraploid level. A good genetic intermixing occurs between the Galician and the alien tetraploid entities which have simultaneous flowering. Autopolyploidization of the diploids followed by various rates of hybridization is proposed as one very probable origin of natural tetraploids inDactylis.     

Embryogenic cells in Dactylis glomerata L. (Poaceae) explants identified by cell tracking and by SERK expression

 Single mesophyll cells in leaf explants of Dactylis glomerata L. (Dactylis) that were competent to form somatic embryos directly or through callus were identified by semi-automatic cell tracking. These competent cells were a subpopulation of small, isodiametric, cytoplasm-rich cells located close to the vascular bundles. Using whole mount in situ hybridization, we showed that a similar subpopulation of cells expressed the Somatic Embryogenesis Receptor-like Kinase (SERK) gene during the induction of embryogenic cell formation. In both leaf explants and suspension cultures, a transient pattern of SERK gene expression was found during early embryo development, up to the globular stage. In later embryo stages, SERK mRNA was present in the shoot apical meristem, scutellum, coleoptile and coleorhiza. Received: 14 May 1999 / Revision received: 27 August 1999 / Accepted: 8 September 1999     

An acidic esterase as a biochemical marker for somatic embryogenesis in orchardgrass (Dactylis glomerata L.) suspension cultures

 The isoenzyme pattern of esterases (EC 3.1.1.2) secreted into the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension cultures during defined stages of somatic embryogenesis was compared with that of non-embryogenic suspension cultures during unorganised cell proliferation. Isoelectric focusing revealed the presence of 7–14 predominantly acidic isoforms. Comparison with the corresponding cell-wall isoenzyme pattern showed minor, mainly quantitative differences. The pattern of intracellular soluble esterases did not change markedly during somatic embryo development. A unique esterase whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 36 kDa and pI=3.8 was detected only in embryogenic cultures at very early stages of development. Since this isoform appeared long before morphological changes had taken place, it could possibly be used as a biochemical marker for embryogenic potential in D. glomerata L. suspension cultures. Received: 6 June 2000 / Revision received: 17 July 2000 / Accepted: 17 July 2000