Recovery of Totipotent Cells and Plantlet Production from Daylily Protoplasts

The release of protoplasts by means of enzymes from a totipotentcell suspension of a diploid daylily (Hemerocallis cv. ‘AutumnBlaze’), their collection and distribution in plasticculture vessels and their subsequent regeneration is described.Attention is given to aggregation and to the different formsof growth during the initial stages of culture. The methodsfor the subsequent multiplication of the regenerated cells intomorphogenetically competent compact clusters and the manipulationof these clusters to yield embryo-plantlets are also outlined.The implications of all this in terms of potential for the geneticmodification of daylily is discussed. Hemerocallis, cloning, protoplasts     

Growth and Maintenance of an Embryogenic Cell Culture of Daylily (Hemerocallis) on Hormone-free Medium

Callus cultures of the diploid daylily (Hemerocallis) clone‘Autumn Blaze’ were initiated and maintained inhormone-containing nutrient medium. At various times (from 6weeks to 1 year) after being initiated, hormone-derived cultureswere evaluated for their ability to be maintained and to multiplyon hormone-free medium at low pH (between pH 4 and 4.5). Cultureshad to be exposed to hormone-containing medium for at least12 weeks before they could be maintained on hormone-free mediumat low pH. The transition to maintainability on low pH hormone-freemedium included the production of many aberrant embryonal forms('neomorphs'). However, all hormone-derived cultures testedconsisted entirely of preglobular stage proembryos (PGSPs) after12–24 weeks on low pH hormone-free medium. PGSP cultureshave been maintained and multiplied as such for over 1 yearon low pH hormone-free medium. PGSPs continue their developmentinto various somatic embryo stages when cultured on hormone-freemedium buffered at pH 5.8. The production of well-formed somaticembryos was greatly enhanced when PGSPs were plated on activatedcharcoal impregnated filter papers that were placed on top ofthe agar surface. The gross morphology and histology of thePGSPs and stages of somatic embryo development are presented.The work shows that the ability of hormone-free medium at lowpH to permit PGSP multiplication without development into laterstages of embryo development is not restricted to carrot. Hemerocallis cv, ‘Autumn Blaze’, daylily, somatic embryogenesis, hormone-free medium, tissue culture     

Plantlet Production from Morphogenetically Competent Cell Suspensions of Daylily

Cell suspensions of the diploid daylily cultivar Autumn Blazewere produced from larger masses of tissue by culture in thebasal medium of Murashige and Skoog supplemented with 10 percent v/v coconut water and 2 mg 1^–12,4–D. By drasticallylowering the level of 2,4–D, followed by transferral toa modified White's or Schenk and Hildebrandt medium, clustersgrow and ultimately give rise to embryonic structures. A finalperiod in a semi-solid medium stimulates shoot and root growthto the point where successful transplanting of plantlets tosoil is assured provided safeguards to prevent ‘dampingoff’ and desiccation are taken. Normal plantlet formationmay be arrested in the formation of neomorphs which do not seemper se to be capable of further development but they can giverise to morphologically normal plantlets after they are stimulatedto form callus which, in turn, is given an appropriate sequenceof stimuli. Hemerocallis, daylily, totipotent cells, micropropagation, tissue culture.     

Interspecific Hybridization of Trifolium repens with T. hybridum Using In Ovulo Embryo and Embryo Culture

In ovulo embryo culture followed by culture of excised immatureembryos produced interspecific hybrids between Trifolium repensL. (white clover) and autotetraploid T. hybridum L. (alsikeclover). Ovules containing hybrid embryos were excised 12–14 dafter pollination and cultured on Nitsch (1951) medium supplementedwith 15% young cucumber juice for 5–6 d. Embryos weresubsequently excised and transferred to hormone-free EG medium,a medium suitable for the culture of immature embryos. A total of 118 hybrid seedlings were obtained from 1978 reciprocalpollinations. All seedlings produced showed various chlorophylldeficiencies, either totally albino or albino with green sectors.Transmission electron microscope studies were carried out toinvestigate plastid development in embryos and seedlings. Someembryos produced only callus. Plants were regenerated from sevencalli. Two semi-albino plants survived transfer to soil, andone plant produced flowers. Backcrosses to T. repens producedone green plant. Hybridity is supported by analysis of morphological characters,karyotype and the gel electrophoretic separation of leaf isozymes. Pollen irradiated with 40 Gy of gamma rays was also used forpollinations. Results indicate that in certain cases ionizingradiation might be useful in overcoming hybrid inviability. Trifolium repens, Trifolium hybridum, clover, interspecific hybridization, in ovulo embryo culture, irradiation     

Pronounced karyological divergence of the North American congeners Sphaerium rhomboideum and S. occidentale (Bivalvia: Veneroida: Sphaeriidae)

Chromosome sets of two North American sphaeriid species, Sphaeriumrhomboideum Say, 1822 and S. occidentale Lewis, 1856, were studiedusing conventional Giemsa staining and karyometric analysis.Pronounced karyological divergence of congeners was revealed.The diploid number of 2n = 44 was reported for S. rhomboideumand this is the first record of a diploid species in the highlypolychromosomic Nearctic sphaeriid fauna. The karyotype wascharacterized by medium-sized and small chromosomes, which decreasedin size gradually from 5.77 to 1.9 µm. Biarmed chromosomeswith medially and submedially located centromeres predominated,but six pairs of subtelo-telocentric elements were also observedin the karyotype. The estimated mitotic chromosome number forS. occidentale ranges from 189 to 213, but most of the cellsexamined contained about 204–209 chromosomes. A firstattempt to karyotype a polyploid sphaeriid was made. It wasrevealed that the comparatively large and middle-sized chromosomescould be grouped in four, so the karyotype presumably evolvedthrough tetraploidization. The small chromosomes formed thelarge fraction, about 137. Due to their similar and indistinctmorphologies, it was impossible to arrange them into subgroupswith confidence. Revealed karyological characteristics are discussedwith reference to the existing phylogenetic interpretationsof the evolutionary history of the Sphaeriinae. (Received 8 November 2006; accepted 25 June 2007)     

Chromosomal Variation in Crocus vernus Hill (Iridaceae) Investigated by in situ Hybridization of rDNA and a Tandemly Repeated Sequence

The physical localization of three tandemly-organized repetitiveDNA sequences was investigated byin situ hybridization to metaphasechromosomes of 11 Crocus vernus accessions. The sequences includedwere the 18S–25S rDNA, the 5S rDNA and a tandemly-repeatedsequence cloned from C. vernus(clone pCvKB8). Ten 2n = 8 karyotypesfrom accessions ranging across the Alps and the Pyrenees couldbe interpreted as variations of a standard karyotype. Polymorphismswere found involving size of the satellite chromosomes, extra5S rDNA sites, and extensive differences in size and numberof pCvKB8 loci. The 2 n = 16 type did not correspond to anypossible tetraploid derived from the 2 n = 8 types. Copyright2000 Annals of Botany Company Evolution, phylogeny, Crocus vernus Hill (Iridaceae), in situ hybridization, chromosomal polymorphism, karyotype evolution, repetitive DNA     

SHORT COMMUNICATION Factors Affecting Protoplast Culture ofCucumis melo'Green Delica'

Hypocotyls, cotyledons and etiolated half-expanded leaves ofCucumismelo‘Green Delica’ were used as explants for protoplastisolation and culture. Protoplasts isolated from cotyledonsand etiolated half-expanded leaves cultured in Durand, Potrykusand Donn (DPD) medium supplemented with 0.9 µMbenzylaminopurine(BAP), 3.6 µM2,4-dichlorophenoxyacetic acid (2,4-D) and1% sucrose, using the agarose bead culture method, were ableto form cell walls and subsequently go through cell division.Pretreatment of half-expanded leaf explants in the dark for14 d provided the best material for protoplast isolation andcell division. Approximately one third of protoplasts from etiolatedhalf-expanded leaves formed microcolonies. For hypocotyl protoplasts,none of the treatments used were suitable to induce cell division.There was no significant difference between sucrose, glucose,and sucrose plus glucose, in culture media on the plating efficiencyof leaf protoplasts ofC. melo‘Green Delica’; however,bigger colonies were formed in media supplemented with 1% sucrose.No shoot or whole plant regeneration was achieved. However,the methods reported here provide further information onC. meloprotoplastculture.Copyright 1998 Annals of Botany Company Cucumis melo,protoplast culture, 2,4-D, BAP, yeast extract, casein hydrolysate.     

Soya Bean Seed Growth and Maturation In vitro without Pods

Immature Glycine max (L.) Merrill seeds, initially between 50and 450 mg f. wt, were grown and matured successfully in vitro.Excised seeds were floated in a liquid medium containing 5 percent sucrose, minerals and glutamine in flasks incubated at25 °C under 300 to 350 µE m^–2 s^–1 fluorescentlight. During 16 to 21 d in culture, seeds grew to a matured. wt of 100 to 600 mg per seed at an average rate of 5 to 25mg d. wt per seed d^–1 depending on initial size. Growthrates were maximal during the first 8 to 10 d in vitro but declinedwith loss of green colour in the cotyledons. Seed coats rupturedwith rapid cotyledon expansion during the first 2 d in culture.Embryos were tolerant to desiccation and 80 to 90 per cent germinatedif removed from culture before complete loss of green colour.The growth of excised seeds in vitro exceeded the growth ofseeds in detached pods, but when windows were cut in pods topermit direct exposure of seeds to the medium, seed growth wascomparable. Glycine max (L.) Merrill, soya bean, seed culture, seed growth, seed maturation, germination     

Cytoevolution, Phylogeny and Taxonomy in Epacridaceae

Chromosome numbers in Epacridaceae were collated from variousauthors and superimposed on to recent cladograms for the family.The results strongly indicate that reduction in chromosome numberoccurs with evolutionary advancement. Thus Epacridaceae, incommon with ‘Ericaceaesens. strict .’ and ‘Vacciniaceaesens.strict. ’, has evolved from a primitive karyotype of atleastx =12 and probablyx =13. This conclusion dispels earlierpaleopolyploid models based on a primitivex =6 in the family.Other aspects of cytoevolution, such as chromosome number asa generic character especially in cytologically advanced generaof the Styphelieae, are discussed. Cytoevolution; cladistic models; primitive character states for chromosome number; dysploid falls; cytotaxonomy; Epacridaceae; Richeoideae; Epacridoideae; Epacrideae; Styphelieae; Ericaceae     

Karyotype, DNA Content and Meiotic Behaviour in Five South American Species ofVicia(Fabaceae)

This paper presents the karyotype, DNA content and meiotic behaviourof five species ofViciafrom Argentina (V. macrogramineaBurk.,V.gramineaSM.,V. epetiolarisBurk.,V. pampicolaBurk. andV. nanaVog.).All the species have the same chromosome number and karyotypeformula (2n=14; 6m+4st+4t). Each species, however, displaysa characteristic number and position of the nucleolar organizerregion (NOR) and different sizes of the respective satellites,confirmed by Ag-NOR banding. Moreover, significant differenceswere found in the total chromosome volume (TCV) and DNA contentof the species. Positive correlations between DNA content andTCV, and between DNA content and type of life cycle were alsofound. TCV and DNA content are lower inV. nana(annual) and higherinV. macrograminea(biennial–perennial). The material displayedmarked karyotypic orthoselection, with similar karyotypes inall studied species, even when the overall chromosome size varied.Evolutionary changes in DNA amount are proportional to the relativelength of each chromosome arm, maintaining karyotypic uniformity.Significant differences were found between the meiotic behaviourofV. gramineaand that of the other species.V. gramineahas alower frequency of ring bivalents and chiasmata per cell, andalso has a lower interstitial chiasma frequency. In general,the results are congruent with the morphological data reportedfor these species.Copyright 1998 Annals of Botany Company. Viciaspecies, karyotype, orthoselection, nuclear DNA content, NOR banding, meiotic behaviour.     

Nitrogen Utilization in N-limited Barley During Vegetative and Generative Growth: III. POST-ANTHESIS KINETICS OF NET NITRATE UPTAKE AND THE ROLE OF THE RELATIVE ROOT SIZE IN DETERMINING THE CAPACITY FOR NITRATE ACQUISITION

Barley (Hordeum vulgare L., cvs Golf, Mette, and Laevigatum)was grown under nitrogen limitation in solution culture untilnear maturity. Three different nitrogen addition regimes wereused: in the ‘HN’ culture the relative rate of nitrate-Naddition (RA) was 0·08 d^–1 until day 48 and thendecreased stepwise to, finally, 0·005 d^–1 duringgrain-filling; the ‘LN’ culture received 45% ofthe nitrogen added in HN; the ‘CN’ culture was maintainedat RA 0·0375 d^–1 throughout. Kinetics of net nitrateuptake were measured during ontogeny at 30 to 150 mmol m^–3external nitrate. V_max (which is argued to reflect the maximuminflux rate in these plants) declined with age in both HN andLN cultures. A pronounced transient drop was observed just beforeanthesis, which correlated in time with a peak in root nitrateconcentration. Similar, but less pronounced, trends were observedin CN. The relative V_max (unit nitrogen taken up per unit nitrogenin plants and day) in all three cultures declined from 1·3–2·3d^–1 during vegetative growth to 0·1–0·7d^–1 during generative growth. These values are in HN andLN cultures 15- to more than 100-fold in excess of the demandset by growth rates throughout ontogeny. Predicted balancingnitrate concentrations (defined as the nitrate concentrationrequired to support the observed rate of growth) were below6·0 mmol m^–3 in HN and LN cultures before anthesisand then decreased during ontogeny. In CN cultures the balancingnitrate concentration increased during grain-filling. Apartfrom the transient decline during anthesis, most of the effectof ageing on relative V_max can be explained in terms of reducedcontribution of roots to total biomass (R:T). The loss in uptakeper unit root weight is largely compensated for by the declinewith time in average tissue nitrogen concentrations. The quantitativerelationships between relative V_max and R:T in ageing plantsare similar to those observed for vegetative plants culturedat different RAs. The data support the contention that the capacity for nitrateacquisition in N-limited plants is under general growth control,rather than controlled by specific regulation of the biochemicalpathway of nitrate assimilation. Key words: Barley, nitrogen concentration, root: total plant biomass ratio, V_max     

Micropropagation of Betula celtiberica

Plantlets were successfully regenerated from shoot segmentsof Betula celtiberica excised from young seedlings. Initiationand elongation of multiple shoot-buds were obtained after 20d culture in MS-modified medium plus BAP 0.6 mg l^–1 followedby 20 d culture in the same medium in the presence of a reducedBAP concentration (0.1 mg l^–1). Rooting was achieved 7d after having transplanted the isolated shoots to fresh medium,supplemented with IBA (0.2 mg l^–1). Betula celtiberica, birch, micropropagation, organogenesis     

Initiation and Growth of Callus and Cell Suspensions of Theobroma cacao L

Callus and suspension cultures of Theobroma cacao L., initiatedfrom immature cotyledons of beans from pods harvested 120–130days after pollination were established. A modified B-5 or Murashige—Skoogagar medium sustained growth of callus without loss of vigourafter each sub-culture. A 15-fold weight increase occurred duringthe 4 week culture periods at 30 ± 1 °C. Coconutwater improved callus growth substantially. The optimum hormonalconcentrations for growth of suspensions were 0.5 mg 1^–1of 2, 4-dichlorophenoxyacetic acid and 0.1 mg I^–1 of kinetinin a Murashige—Skoog basal medium liquid medium. The optimumtemperature for growth of suspensions was 25–30 °C.The cell number and cell mass of suspensions increased 20-foldin 14 days. No organogenesis or embryogenesis was observed. Theobroma cacao L., acao, cell culture, suspension culture, tissue culture.     

Effects of growth medium, temperature, salinity and seawater source on the growth of Gymnodinium catenatum (Dinophyceae) from Bahia Concepcion, Gulf of California, Mexico

Laboratory studies were performed to determine the effect oftemperature, salinity, seawater sources and culture media onthe vegetative growth of clonal cultures of Gymnodinium catenatumisolated from Bahía Concepción, Mexico. Theseisolates were heterothallic and isogamous. Exponential growthrates of G. catenatum in f/2 with different selenium concentrationsand soil extract and GSe media were moderate. Maximum cell yieldswere obtained in GSe and f/2 media with selenium (10^–8and 10^–7 M), while in f/2 medium with soil extract cellyields were considerably lower. The highest percentage of longchains was found in f/2 media supplied with selenium (10^–8M). The optimal temperature range for growth was 11.5–30°C,with the highest growth rates between 21 and 29°C. The rangeof salinity tolerated by G. catenatum changed with seawatersource. With seawater from Vineyard Sound (Massachusetts, USA),G. catenatum grew at salinities from 15 to 36, with an optimalgrowth rate obtained at salinities between 26 and 30. With seawaterfrom Bahía Concepción, this species toleratedsalinities from 25 to 40, with optimal growth at salinitiesbetween 28 and 38. Ecophysiological measurements reported hereare consistent with the environment of the bay, which has limitedinput of humic materials from runoff and high salinity and temperature.These data, when viewed with data from studies of globally distributedG. catenatum, demonstrate the ability of this species to livein a broad array of habitats.     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. II. Anther and Pollen Development In Vivo and In Vitro

The various pathways of pollen development were investigatedin cultured anthers of Lolium temulentum, Festuca pratensisand the L. multiflorum x F. pratensis hybrid ‘Elmet’.In all three, development from the vegetative cell was the predominantpathway of pollen callus development. However, there were characteristicdifferences in the behaviour of the generative cell. In L. temulentumit remained attached to the pollen wall and degenerated, whereasin F. pratensis it divided. In ‘Elmet’ it detachedfrom the pollen wall and remained undivided. Both polarizedand unpolarized partitioned calluses were observed. Developmentof the fusion product of the vegetative and generative nucleiwere also observed in anthers of L. temulentum. Anomalous grainswere not found to be major source of pollen calluses. Sections of anthers of L. temulentum were used to investigatethe origin of S pollen grains, the small pale-staining grainswhich denote pollen dimorphism. Such grains form out of contactwith the tapetum and are therefore determined before or duringmeiosis (i.e. before harvest of anthers for culture). Sectionswere also used to demonstrate the influence of the durationof pretreatment on the development of the middle layer of theanther wall. Festuca pratensis, Lolium temulentum, Lolium x Festuca, anther culture, haploid, microspore, pollen     

Fungal Associations: III. The Role of Pectic Enzymes on the Synergistic Relation between Rhizoctonia solani Kuhn and Fusarium solani Snyder and Hansen, in the Rotting of Potato Tubers

The greatest activity of protopectinase obtained from the growthof Rhizoctonia solani and Fusarium solani on autoclaved potatoplugs occurred at pH 6.5, and greatest activity of the ‘lossof viscosity’ enzyme was found at 6–5 for Rhizoctonia,and between 6.5 and 8.3 for Fusarium. Protopectinase enzymeobtained from double infections of the Fusarium spp. with Rhizoctonia,or by mixing the enzymes of individual Fusarium spp. with Rhizoctoniaenzyme, were more active than the enzymes from single inoculations.Cylindrocarpon radicicola enzyme was more active when obtainedfrom a pure culture than from double infection. Similarly, mixingthis enzyme with the enzyme of Rhizoctonia reduced its activity.The evidence indicated that the protopectinase of Rhizoctoniawas similar to that of Cylindrocarpon and differed from thatof the Fusarium spp. Using paper partition chromatography, two bands from Rhizoctoniacrude enzyme had a stimulatory effect on Fusarium enzyme, whileonly one band from Fusarium enzyme stimulated Rhizoctonia enzyme. The purified enzyme of Rhizoctonia degraded pectin to galacturonicacid. Fusarium pure enzyme degraded pectin to an intermediatestage. A mixture of the two enzymes degraded pectin to galacturonicacid, without the intermediate stage formed by Fusarium alonebeing detected. The role played by pectic enzymes upon the synergistic relationof Rhizoctonia solani and Fusarium solani on rotting potatotubers is discussed.     

Spent medium analysis for liquid culture micropropagation of <Emphasis Type="Italic">Hemerocallis</Emphasis> on Murashige and Skoog medium

Residual nutrients from Murashige and Skoog medium were analyzed following a 5-wk multifactor experiment. Plant density, sugar concentration, and plant growth regulators (benzyladenine and ancymidol) were examined using four genotypes of daylily (Hemerocallis) to determine which factors most influenced nutrient use. Active nutrient uptake was observed for 11 nutrients (potassium, sodium, copper, phosphorus, iron, calcium, magnesium, manganese, boron, sulfur, and zinc) with lower concentrations in spent medium than in the tissue water volume (fresh-dry mass expressed as mL H₂O). Two patterns of nutrient use were visualized by correlative analysis of nutrient uptake. Greatest growth lowered plant nutrient concentrations of potassium, sodium, phosphorus, iron, and copper in all genotypes, and luxuriant uptake was indicated with least growth. Potassium, sodium, iron, and copper concentrations in plant dry matter were equal to or exceeded what is observed in vigorously growing nursery plants. However, phosphorus concentration in plant dry matter was low enough to be considered deficient when compared to Hemerocallis plants in nursery production. With a second group of nutrients (calcium, magnesium, manganese, and boron), the genotype, “Barbara Mitchell” lacked active uptake and was deficient. Calcium concentration was low in all plants compared to Hemerocallis grown under nursery conditions (“Barbara Mitchell” was the lowest concentration) despite active uptake by the other three genotypes—“Brocaded Gown,” “Mary’s Gold,” and “Heart of a Missionary.” Magnesium concentration in these three genotypes was low enough in vessels with greatest growth to question its adequacy at high densities. Increased sucrose in medium reduced the dry matter concentrations of all tested nutrients. Plant growth regulators had less impact on nutrient use than genotype and plant density. Nutrient uptake may be an important physiological component of genotypic variation.     

Regeneration of Plants from Protoplasts of Arabidopsis thaliana L. cv. Columbia (C24), Via Direct Embryogenesis

This report describes a protocol for the regeneration of fertileplants from mesophyll protoplasts of Arabidopsis thaliana raceColumbia (C24). Regeneration was rapid and reproducible. Theprotocol is especially novel in that a large proportion of regeneratingprotoplasts regenerated via direct somatic embryogenesis. Protoplastsisolated from in vitro-grown plants entered sustained divisionafter 3–5 d in culture medium and over a period of severaldays 6–22% of protoplasts underwent at least one celldivision. Approximately 2–16% of these protoplasts continuedto divide and after 3 weeks in culture had formed macroscopiccolonies, of which 70–80% were regular embryo-like structures.Four weeksafter release from the alginate culture matrix andtransfer to solid medium in the light, 68–88% of thesestructures had produced well-developed shoots. Shoots couldbe maintained in culture or established in peat blocks. Theregenerated plants were fertile. Key words: Arabidopsis thaliana, protoplast, regeneration, embryogenesis, dicamba     

Callus Culture of Sainfoin (Onobrychis viciifolia) and Plant Regeneration through Somatic Embryogenesis

Callus of sainfoin (Onobrychis viciifolia Scop.) was initiatedfrom stem and root explants which were obtained from seedlingsgrowing in vitro, on Linsmaier Skoog (LS) medium supplementedwith 1 mg l^–1 2, 4-D and 1 mg l^–1 BA or only 1 mgl^–1 BA, and the Vacin and Went medium without hormones.Somatic embryos were formed on LS medium containing 1 m l^–1BA. Embryos developed into complete plants on filter paper saturatedwith hormone-free LS medium. Onobrychis viciifolia, somatic embryogenesis, callus culture, plant regeneration