The effects of Fungi on the Gametophytes of Pteridium aquilinum (L.) Kuhn

I. The prothalli have been shown to be convenient subjects forthe study of some fungal relationships with green plants. Purecultures have been established from all the populations examinedby germination of young spores, and it has been shown that duringdormancy the number of spores which can germinate in pure culturedecreases more rapidly than the number which can germinate incultures containing contaminant fungi. Previous reports of thestimulatory effect of fungi on prothallial growth and developmenthave been confirmed and extended. 2. Prothalli growing on mineral agar have been shown to be potentiallysusceptible to the attack of a wide variety of pathogens ofhigher plants, but only two naturally occurring species of fungipathogenic to prothalli have been found during an examinationof growth on a variety of soils from bracken-infested areasin the west of Scotland. 3. The aetiology of the disease incited by Botrytis cmerca inprothalli has been shown to be similar to that of this diseasein higher plants. The pathogen is some-times restricted to lesionsof limited size around the sites of successful infections andthis is associated with changes in the cell walls of the host.A protopectinase solution prepared from a culture of germinatingspores by Brown's method induced symptoms in the prothallialcells similar to those induced by the attack of the fungus.The cell-attacking properties of the solution were greatly reducedwhen the protopectinase content was inactivated by low heatfor a short time, but they were not entirely destroyed. Thisconfirms previous indications that more than one complex maybe involved in the attack, but since the second moiety demonstratedis at least partially thermolabile the evidence does not supportjudgement of its relative significance in the attacking process.     

Effect of Light on Alkaloid Accumulation in Cell Cultures of Nicotiana Species

The effect of light on alkaloid accumulation in a range of cellcultures of tobacco was determined. Cell suspension culturesof Nicoriana rabacwn L. cv. Wisconsin-38 with differing degreesof photosynthetic activity, callus cultures of N. glauca Graham,root cultures of N. rustica L. and shoot cultures of N. tabacumwere used. The alkaloid content of green illuminated cultureswas greatly reduced compared with non-green cultures grown inthe dark, but decreased accumulation did not correlate withincreasing photosynthetic activity. The accumulation of allof the major alkaloids was affected, regardless of the speciesof tobacco used. Transfer of N. glauca callus from the darkinto the light caused a decrease in alkaloid accumulation, whilemoving cultures from the light into the dark resulted in anincrease in alkaloid content. In root cultures light causeda reduction in growth, which affected alkaloid synthesis. Inshoot cultures there were only traces of alkaloid detectable,regardless of whether or not cultures were illuminated. Lightappeared to cause a non-photosynthetic suppression of alkaloidaccumulation in visibly undifferentiated cultures, and thiseffect was modified in visibly differentiated cultures. Key words: Nicoriana spp, tobacco, alkaloid accumulation, cell culture     

Effect of Light and NaCI Salinity on the Growth of Callus Cultures of Ipomoea pes-caprae (L.) R. Brown and Ipomoea batatas (L.) Lam

Callus cultures of Ipomoea pes-caprae and I. batatas were establishedon MS medium containing 10^–5 M 2,4-D and 10^–8 Mbenzyladenine. Ipomoea pes-caprae calli exhibited green pigmentationand grew better in the light than in darkness. Callus tissuesof I. batatas showed a pale-yellow colouration and they grewat the same rate in light as in dark conditions. I. pes-capraeand I. batatas callus cultures were sensitive to the presenceof 60 mM NaCl in the culture medium, the growth of the formerbeing more sensitive in light than in darkness. The significanceof the responses of I. pes-caprae callus cultures in relationto the mechanism of salt tolerance is discussed. Ipomoea batatas, Ipomoea pes-caprae, sweet potato, railroad vine, callus cultures, salinity, light     

Assimilation numbers in cultures of marine phytoplankton

During exponential growth in batch culture, assimilation numbersof eleven algal species ranged from 1.6–20.8, with a meanvalue of 5.3 g C/g Chlorophyll a/hr. The highest assimilationnumber of 20.8 g C/g Chlorophyll a/hr was observed in Coccolithuspelagicus, due to the relatively low concentration of chlorophylla/cell. The assimilation number declined from exponential tostationary phase in batch cultures for ten algal species, butincreased with age in batch culture in Amphiprora paludasa (abenthic diatom). The assimilation number declined with decreasinggrowth rate in nitrate-limited chemostat cultures of Phaeodactylumtricornutum and in iron-limited chemostat cultures of Phaeodactylumtricornutum and Isochrysis galbana.     

Responses of Marchantia in Aseptic Culture to Well-known Auxins and Antiauxins

Responses of young thalli of Marchantia nepalensis to ten well-knownauxins and antiauxins in aseptic culture are described. Effectsof one concentration of an auxin or an antiauxin were studiedin both liquid and solid cultures. The normal growth of inoculated thalli was inhibited but callus-liketissue was produced on them by the highest concentration (I.Omg./I.) of almost all the growth substances except MH, TIBA,and 2, 4^–DNP. At a later period callus-like tissue producedby IAA, IBA, and IPA differentiated into new thalli but thatproduced by NAA, NOA, 2,4^–D, and TCPA only did so on beingtransferred to control medium. At lower concentrations inoculated thalli developed normallybut they were soon overgrown by daughter thalli. No protonemalphase comparable to that of mosses could be observed in thedevelopmental stage of a regenerating thallus. The early stageis characterized by a stable filamentous structure, normallyconsisting of two to three cells. Filamentous structures ofvarying number of cells were however produced in some treatments. Profuse rhizoids were produced by the highest concentrationof NOA, 2,4^–D, TCPA, IBA, and IPA. Certain growth substancesinhibited the formation of tuberculate but not of smooth rhizoidsin liquid but not in solid cultures. Also tuberculate rhizoidswith feebly developed pegs were produced in certain liquid culturesonly and even in control liquid culture. More gemma-cups wereproduced in liquid cultures. Germinated gemmae with rhizoidswere invariably present in solid but not in corresponding liquidcultures. Germinated gemmae within gemma-cups were frequentlyfound. The significance of these results is discussed.     

Experimentally Synthesized Plant Chimeras I. In vitro Recovery of Nicotiana tabacum L. Chimeras from Mixed Callus Cultures

Experiments were conducted to develop techniques for synthesizingchimeras between plants of known genotype by utilizing in vitrotechniques Chimeral calli composed of green and albino tobaccocells were obtained by initiating callus tissue from mixturesof albino and green cotyledons, hypocotyls, callus culturesand cell suspensions The most effective mixing of genotypesoccurred when callus was derived from mixed filtered cell suspensionsUpon shoot regeneration, chimeral calli yielded 1317 non-chimeraland four chimeral plants Chimeras may have arisen as a resultof experimental procedures or possibly from spontaneous chromosomalabnormalities since leaves of some albino control plants occasionallyproduced small green islands of cells Explanations for the recoveryof a high percentage of non-chimeral shoots are presented Tobacco, callus cultures, cell suspensions, tissue culture, shoot apical meristems, somatic-crossing over     

Organogenesis in Cell Suspension Cultures of Atropa belladonna L. and Atropa belladonna Cultivar lutea Doll

Callus cultures have been initiated from excised cultured rootsof Atropa belladonna and A. belladonna cultivar lutea and usedto establish suspension cultures in a synthetic culture medium(referred to as SSM) containing 2.0 mg/1 -naphthaleneaceticacid (NAA). Visible cellular aggregates develop in these suspensioncultures and when such aggregates are cultured in an NAA-omittedmedium, large numbers of roots arise superficially on the aggregates.Root development is enhanced by incorporating into the auxin-freemedium either tropic acid or I-naphthoxyacetic acid and boththese substances can promote root initiation in the presenceof levels of NAA which alone suppress organogenesis. The aggregatesalso give rise under appropriate conditions of culture to shootsand to embryo-like structures. The nature and frequency of suchmorphogenesis in the aggregates is dependent not only upon thecomposition of the culture medium but upon the number and thelength of previous culture passages through which the callusand suspension cultures have been propagated in the SSM. Theultimate loss of morphogenetic potential in serially propagatedcallus is discussed. The structure of the aggregates in SSM and in the media promotingroot initiation is described. Examination of the aggregatesfor their content of alkaloids indicates that the major belladonnaalkaloids can only be detected in cultures forming roots.     

Cytological Analysis of Seedling Roots, Transformed Root Cultures and Roots Regenerated from Callus of Swainsona galegifolia (Andr.) R. Br.

The stability of chromosome number was investigated in culturesof roots from Swainsona galegifolia. Roots from germinated seedsor plants grown in vitro when cultured in liquid medium howed90% or more cells with the diploid number of 2n = 32. The remainingcells showed aneuploidy mostly below 32. The stability of chromosomenumbers was not affected by transformation with Agrobacteriumrhizogenes although when roots were transformed with A. rhizogenesLB 9042 the range of chromosome numbers in the few aneuploidcells present was higher than in roots for which strain A4 wasused. In contrast, roots regenerated from callus had only 15%of cells with 2n = 32 and showed a modal number of 18. Six rootcultures established from individual roots regenerated fromcallus showed a wide variation in number (8–83). Fivecultures had a modal number around 18, the sixth, a modal numberof 39 which is above the diploid number. The implication ofthe results for the production of secondary metabolites fromroot culture is discussed. Key words: Agrobacterium rhizogenes, callus cultures, chromosome number, root cultures, Swainsona galegifolia     

Digitoxin Biosynthesis in Isolated Mesophyll Cells and Cultured Cells of Digitalis

In the laminae of Digitalis, most of the digitoxin present isfound in the mesophyll. A new method for determining the amountof digitoxin biosynthesis using a digitoxin antibody was devisedto estimate this activity in isolated mesophyll cells and culturedcells. Isolated mesophyll cells showed significant activity,which suggests that the site of biosynthesis and the accumulationof cardenolides in a lamina of Digitalis is mainly in the mesophyllcells. Of five liquid cultures of D. purpurea; green shoot-formingcultures, white shoot-forming cultures, root-forming cultures,undifferentiated green cells and undifferentiated white cells,the green shoot-forming cultures had the highest activity. Thewhite shoot-forming cultures had about one-third the activityof the green shoot-forming cultures, and the other three cultureshad very low activity. No stimulatory effect of light was foundduring the 48-h incubation. (Received January 19, 1984; Accepted June 8, 1984)     

Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

Factors Influencing Morphogenesis in Excised Roots and Suspension Cultures of Atropa belladonna

Excised root cultures of Atropa belladonna L. and A. belladonnavar. lutea Döll, established from liquid-grown seedlingsand from callus, when allowed to continue growth without subculturefor several weeks, spontaneously initiate shoot buds from smallnodules of callus which arise at the cut ends of the roots.The frequency and rapidity of formation of such buds is dependentupon the number of passages through which the roots have beensubcultured. The morphogenetic expression of callus cultures and of suspensioncultures derived from them is influenced not only by the timethat the root cultures have been maintained in culture but bythe composition of the medium used for callus initiation andsubsequent culture. The presence of elevated levels of ammoniumions in these media favours the development of incipient plantsrather than roots. Cultures have thus been obtained in whichthe predominant form of morphogenesis is embryogenesis (as establishedin a subsequent paper by Konar, Thomas, and Street, 1972).     

Electronic Measurement of Plant Cell Number and Size in Suspension Culture

The rapid and accurate determination of cell number and sizeis very desirable for obtaining data on growth kinetics andchanges in the physiological state of a culture. The CoulterCounter has been widely applied to bacterial and animal cellsuspension cultures to determine electronically cell numberand cell size. The application of such techniques to plant cellsuspension cultures is discussed in the following paper anddemonstrated for cultures of Pauls Scarlet Rose (Rosa sp.) andsoybean (Glycine max L.). A new formula for coincidence correctionsis suggested.     

Pollen-Derived Haploids of Nicotiana Knightiana, N. raimondii, and N. attenuata

Haploid plantlets were obtained in large numbers in three diploid,24-chromosome species of Nicotiana by culture of anthers ator just past the first pollen mitosis. The three species wereN. Knightiana, N. raimondii, and N. attentiata. Efficiency ofhaploid production varied from about 10 per cent in N. attenuatacultures to 30 and 38 per cent respectively in cultures of N.raimondii and N. Knightiana. H-medium without hormones and standardcultural conditions were used. N. Knightiana appeared to beespecially suitable for haploid studies on account of its highplantlet productivity, low chromosome number, and distinctivekaryotype.     

Comparison of the flowering behavior of the long-day plant Lemna gibba G3 from different laboratories

In an effort to determine the cause for the wide discrepanciesin the level of flowering response reported for the long-dayplant Lemna gibba L., strain G3, cultures of L. gibba G3 wereobtained from the laboratories of W. S. Hillman (G3-H), R. Kandeler(G3-K), Y. Oota (G3-O) and and A. Pieterse (G3-P) and comparedto the L. gibba G3 (G3-C) from this laboratory. Under continuouslight all cultures gave FL% values of 77 or above, and on a9L:15D short-day treatment, all cultures were completely vegetative.However, on daylengths of 10 to 12 hr, small but statisticallysignificant differences were obtained for the different cultures.The critical daylength curves for G3-G, which showed the shortestcritical daylength, and G3-K, which showed the longest criticaldaylength, differed by approximately one hour. Salicylic acidtreatment caused flower promotion in each culture, but statisticallysignificant differences were obtained between some of the culturesin their response to salicylic acid. It is concluded that the large discrepancies in the floweringresponses of L. gibba G3 that have been reported are due primarilyto differences in culturing methods and counting proceduresin the different laboratories. However, the results also indicatethat there may be distinct cultures of L. gibba G3 that exhibitsmall physiological and/or genetic differences that would makeprecise quantitative comparison between different laboratoriesvery difficult. (Received January 23, 1979; )     

A Method for in Vitro Storage of Lolium multiflorum Lam

A means of storing Lolium multiflorum Lam. stock plants in vitrohas been developed over a period of three years. Variousexplantsand cultureconditions were examined. Shoot tips 0.3–0.9mm long were found to be best for establishing storage culturesbecause they gave a high yield of plantlets in culture and importantpathogens are eliminated. For sub-culturing, after each annualcycle of storage at 2–4 °C, shoot tips, tiller buds,tiller bases and nodes can be used. Tiller buds were most abundantand best for increasing the number of stored plantlets whennecessary. There was no advantage of including an auxin in theculture medium for shoot tips and Murashige and Skoog's mediumwith 0.2 mg l^–1 kinetin has been adopted for initiating,sub-culturing and storing cultures. The optimum temperaturerange for regenerating plantlets was 20–25 °C. Lightwas necessary for a high rate of plantlet regeneration and lightquality and intensity affected their growth. Lolium multiflorum Lam., ryegrass, storage in vitro, tissue culture     

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.     

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     

Analysis of In Vivo Protein Synthesis and Histological Studies of Haustorial Formation in Root Cultures of Witch weed (Striga asiatica L Kuntze)

We recently described an in vitro approach that uses root culturesto study haustorial formation in Striga asiatica. Previous studieshave used haustoria formed on intact radicles of Striga seedlings.In vitro cultured roots formed haustoria that appeared morphologicallysimilar to those formed by Striga radicles, but were 5–10-foldlarger. In this study, we provide biochemical and histologicalevidence to support further the similarity of root culture haustoriato haustoria formed on radicles of seedlings. We examined invivo protein synthesis during haustorial development on rootcultures and radicles by 2-D PAGE. Four proteins increased inabundance in both root cultures and radicles after 6 h of haustorialinduction. All four proteins appeared transiently in root culturesand radicles, being more abundant at 6 h, and less abundantafter 24 h of haustorial induction. Only three of the four haustorial-specificproteins were more abundant in root cultures after 2 h of haustorialinduction; all four had decreased in abundance after 12 h ofhaustorial induction. Using light microscopic analysis we comparedthe ontogeny of root culture haustoria to that of haustoriaon radicles. These studies revealed that root culture haustoriaundergo developmental changes similar to those reported forradicle haustoria such as early expansion of cortical cells,the emergence of haustorial hairs from epidermal cells, andthe development of densely staining cells at the haustorialapex. In addition, these changes occurred within a similar time-frameand sequence in root culture and radicle haustoria. Finally,root culture haustoria were found to be capable of attachingto sorghum host roots. Key words: Striga asiatica L., Kuntze, haustoria, root cultures, proteins, histology, 2D-PAGE     

Morphogenesis in Yucca schidigera Roezl. Root Organ Cultures

Root development in suspension cultures of Yucca schidigerawas light-mediated. The green cultures consisted of roots, smalltissue aggregates and suspension cells. Roots possessed an apicalmeristem with a root cap, meristematic region and region ofdifferentiating tissues. Phloem, xylem vessels and tracheidsoccurred in discrete polyarch vascular bundles. Xylary wallthickening was reticulate, and endodermis and pericycle werepresent. Roots of intact Y. schidigera plants had a similardistribution of vascular tissues. Dark-grown cultures were cream-colouredand contained only lobed tissue aggregates and suspension cells. Yucca schidigera Roezl., tissue cultures, morphogenesis, root organ, light/dark     

The Establishments of Tissue 4

Callus cultures of 12 temperate grasses were established, somefor the first time, by incubating detached roots or whole seedlingson a Linsmaier and Skoog basal medium which contained 2, 4-dichlorophenoxyaceticacid (2, 4-D) at 1.0 mg 1^–1 as the only growth hormone.The callus, which developed in the pericycle of the roots andon the embryo of the seeds, was subcultured on the same medium;further growth of the callus varied from good in the case ofDactylis glomeraia, Agrostis tennis, Cynosurus cristatus, andPoa trivialis, to poor in several Lolium species and varieties.Most cultures developed root primordia which sometimes grewinto visible roots, but shoot primordia, none of which grewinto shoots, were found only in the callus of Lolium multiflorumvar. westerwoldicum. Cell suspension cultures were also readily established and maintainedusing the same culture medium. Most cultures contained a highproportion of round or oval cells, which ranged from 18 to 77µm in diameter or length, while many also had a significantproportion of larger, more elongated cells which varied in lengthfrom 46 to 182 µm. The cells of Dactylis glomerata werecharacteristically larger and more convoluted than the cellsof other grass species that were examined. The addition of kinetinat 0.1 mg 1^–1 to the 2, 4-D-containing culture mediumincreased the proportion of irregular-shaped cells and reducedthe dispersion of the cells, perhaps by improving cellular contactand adhesion; in some species, such as Agrostis tenuis and Phleumpratense, the presence of kinetin promoted the deposition ofstarch granules in cells.