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.     

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.     

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.     

Polyploidy and habitat differentiation in Dactylis glomerata L. from Galicia (Spain)

Summary The microdistribution of diploid and tetraploid plants of Dactylis glomerata L. was examined and related to their immediate environment in several sites in central Galicia, where morphologically indistinguishable individuals of both ploidies grow in sympatry. The two related cytotypes differed in habitat preference. Diploids were mainly confined to the low-density forest-floor habitat in woodlands of mostly ancient origin, whereas tetraploids were widespread in varied habitats but clearly predominant in open areas, particularly in disturbed anthropic sites. The in situ comparison of plant performance showed that where plants of each ploidy were more common they produced more tillers, panicles and seeds. This habitat preference closely reflected differences in life-history characteristics. The tetraploids had an early and short flowering time almost always completed before the aestival drought, whereas the diploids began to flower several weeks later and flowered throughout the drought. Comparisons along artificial gradients of soil water availability and light transmittance indicated that the cytotypes had distinct physiological requirements which probably originated in metabolic and more general genetic differentiation and could be directly attributable to ploidy. Habitat differentiation increases the species' colonizing ability. It also amplifies divergence in reproductive strategy between diploids and tetraploids, which reduces ineffective crossing between cytotypes and thereby permits them to coexist in sympatry. The effect of hybridization at the polyploid level on the differentiation between cytotypes was assessed from the recent introduction of a foreign tetraploid entity into the study area. Hybridization between the two distinct tetraploids was found to increase habitat differentiation between the diploids and the tetraploids, but the major part of this differentiation is probably attributable to ploidy itself.     

Quantitative nuclear DNA changes inEleusine (Gramineae)

2C nuclear DNA amounts were determined in 30 collections belonging to 10 species ofEleusine. About a 2.5-fold variation in genome size is evident in the genus. The 2C DNA amount in the diploid species ranged from 2.50 pg inE. verticillata to 3.35 pg inE. intermedia. In contrast, the tetraploid species showed a range from 4.95 pg inE. africana to 6.13 pg inE. floccifolia. At intraspecific level 10 collections ofE. coracana, 6 ofE. indica, 4 ofE. africana, 2 ofE. tristachya, and 2 ofE. kigeziensis did not show any significant variation. However, 2 collections ofE. floccifolia, connected with polyploidy, displayed about 90% variation. Polyploid species showed approximately double the genome size of that of their corresponding diploids. An evolutionary increase in DNA amount is evident inE. coracana during the course of its origin and domestication fromE. africana.     

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.     

Photosynthesis of natural cocksfoot populations under water and salt stresses

Sampling of natural cocksfoot (Dactylis glomerata L.) populations was carried out on the O Morrazo peninsula in NW Spain, characterized by a strong moisture gradient. The plants were kept in greenhouse under standard conditions. Nevertheless, they differ in height of plants, length and width of flag leaves, panicle size, stomatal density and size as well as in flowering period. The effects of two levels of soil water deficit and two levels of salinity on photosynthetic rate were tested. One population was exceptionally well adapted to its original environment with great tolerance to water deficit and salinity     

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.     

Species relationships and genetic variation in the diploid wheats (Triticum,Aegilops) as revealed by starch gel electrophoresis

Twenty enzyme loci were examined in the diploid species ofTriticum andAegilops for allelic variation by starch gel electrophoresis. SectionSitopsis, including the five species,Ae. speltoides, Ae. lingissima, Ae. sharonensis, Ae. bicornis andAe. searsii form a close subgroup withAe. speltoides slightly removed from the others.T. monococcum s. lat., was found to be closest to the species of theSitopsis group.Ae. comosa, Ae. umbellulata andAe. uniaristata form a second subgroup withAe. caudata most closely related to these species.Ae. squarrosa appears almost equally related to all of the species, showing no special affinity for any one species group. Nineteen out of twenty loci examined were polymorphic with a mean of 6.7 alleles per locus. Species could be, for most loci, characterized by the presence of predominant alleles. A conspicious genetic characteristic ofTriticum-Aegilops is the sharing of these predominant alleles between species. Within species variation is characterized by a diffuse distribution of secondary alleles.     

Isozymes as molecular markers for diploid and tetraploid individuals ofNajas marina (Najadaceae)

The genetic variability of five natural populations ofNajas marina L., i.e. one diploid of subsp.marina (Europe), two of subsp.intermedia (Europe) and both a diploid (C. Africa) and a tetraploid (Middle East) of subsp.armata, has been estimated by means of electrophoretic studies. These populations differ in their morphology and karyotype. Emphasis is placed on the characteristics and status of a tetraploid cytotype from Merkaz Sappir (Israel). Almost all the variation observed is expressed in seed alcohol dehydrogenase (ADH). The differences are in a unique allele of theAdh-2 locus and in the formation of novel heteromeric isozymes.Adh genes in seeds can be used as a marker for the autotetraploid character. The other enzyme systems tested failed in this respect. The genetic variability based on 23 loci is rather low. Nevertheless, the autotetraploid population has a higher or equal ratio of polymorphic loci than the related diploids. Cluster analysis illustrated not only thatNajas marina subsp.marina has diverged much from subsp.intermedia and subsp.armata, but also showed the difference between the latter two taxa, as well as the intermediate position of the autotetraploid population.     

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.     

The flavonoids in leaves of diploidTriticum species (Gramineae)

Leaf flavonoids have been identified in seven species ofTriticum, all of which have been considered at one time as putative parents of the cultivated tetraploid and hexaploid wheats. The major constituents are apigenin- and luteolin-based glycosylflavones, some of which contain various O-glycosidic attachments at the 6-position. Four tricin glycosides are present in minor amount, as is free tricin. The flavonoid patterns link togetherT. searsii, T. speltoides andT. squarrosa, on the one hand, andT. monococcum, T. boeoticum, T. thaoudar andT. urartu, on the other. These results indicate that the first three taxa are more likely to be diploid ancestors to the hexaploidT. aestivum than the latter four species.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

Zinc tolerance in populations ofDeschampsia cespitosa (Gramineae) beneath electricity pylons

Patterns of zinc tolerance were examined in eightDeschampsia cespitosa (L.)Beauv. populations from normal and zinc-contaminated soils, using solution culture methods. Zinc-tolerant populations have evolved beneath pylons, and their tolerance patterns (degree, variance, heritability) vary. Tolerance is genetically based in all of them. In contrast to the cases of other species previously reported, some within-population differences are considerable, and may be due to both heterogeneity of the soils zinc contents, various gene combinations and gene recombinations between genotypes. One control population contains many tolerant plants, an original result which is discussed.     

Giemsa C-banded karyotypes of diploid and tetraploidHordeum bulbosum (Poaceae)

The similar-looking basic genomes ofHordeum bulbosum (2x and 4x) have five rather similar metacentric, one submetacentric, and one satellited choromosome. C-banding patterns are characterized by one or two centromeric, or juxtacentromeric, small to larger bands in most chromosomes, by bands at the nucleolar organizers, by small or very small telomeric bands, and by the nearly complete lack of intercalary bands. Banding pattern polymorphism is widespread. Banding patterns supported by chromosome morphology enable identification of homologues, and discrimination between non-homologues inH. bulbosum (2x). The C-banded karyotype ofH. bulbosum (4x) supports an autopolyploid origin, but it was possible to identify only homologues of submetacentrics and SAT-chromosomes.     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

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.     

Isozyme gene duplication in diploid and tetraploid potatoes

Summary Isozyme techniques allow the study of gene redundancy in different ploidy levels of potato (Solanum tuberosum). In tetraploid potatoes all isozyme loci are duplicated. No sign of structural or regulatory divergence was found, as is expected due to their tetrasomic inheritance patterns. In addition to this genetic redundancy, produced by a relatively recent polyploidization event, some additional redundancy was found for at least three enzymes even in diploid groups and species. These older duplicate genes show structural and regulatory divergence, indicating they appeared by a separate polyploidization event far in the past. Their common origin is still recognizable by both their expression in the same subcellular compartment and by the dimerizing ability of the isozymes they encode. To account for the present chromosome number x = 12 of the Solanaceae family, the most frequently found among the species, a hypothetical polyploidization event is proposed.     

Phylogenetic relationships in theSideritis leucantha group (Lamiaceae)

Six closely related taxa of the sect.Eusideritis of the genusSideritis (S. leucantha, S. pusilla, S. flavovirens, S. granatensis, S. biflora andS. osteoxylla) are analysed to elucidate their phylogenetic relationships and position within the sect.Eusideritis. Meiotic behaviour, karyotype features, size and fertility of pollen grains, DNA amounts and seed protein profiles are reviewed. A polyploid origin of the group (from x = 7) and the further diversification through dysploidy and chromosome repatterning is postulated.S. osteoxylla is apparently of hybrid origin.     

Relationships between five species ofLolium (Poaceae)

Morphological analysis and electrophoresis of seed proteins of fiveLolium species disclosed that they form two distinct groups corresponding to those recognized from compatibility data.Lolium temulentum andL. remotum of the self-pollinated group were shown to be distinct but closely related species. Morphological intergradation and high similarities between protein profiles ofL. perenne, L. multiflorum andL. rigidum (cross-pollinated species) suggest little genetic differentiation between these taxa. This implies that treatment at the infraspecific level might better accomodate the data.