Embryo Rescue in Wide Crosses in Arachis. 1. Culture of Ovules in Peg Tips of Arachis hypogaea

Interspecific hybridization in Arachis is restricted by earlyembryo abortion for many cross-combinations. Rescue of youngembryos in vitro within a week after fertilization is necessaryto recover these embryos before they abort. Peg tips, with theovule and embryo tissues, of A. hypogaea L. cv. ‘NC 6’,were cultured to compare ovule growth, callus production andpeg elongation. Tissues were collected 1, 2, 3 and 4 d afterself-pollination, after which peg meristems were removed fromhalf the pegs and cultured on five media combinations. One-day-oldpegs had significantly (P = 0.01) more ovule growth than oldertissues. Presence of the meristem had a greater inhibition toovule growth for 2- to 4-d pegs than for 1-d-old pegs. Significantlymore callus was produced on 4-d pegs than younger tissues, andkinetin had the greatest stimulatory effect on callus. Elongationof pegs with the meristem attached was observed most often inmedia with high sucrose levels. The observations indicate thatvery young ovules can be grown in vitro, and techniques maybe applicable to rescue of young embryonic tissues of Arachis. Ovule culture, interspecific hybridization, Arachis hypogaea, peanuts     

In Vitro Shoot Regeneration from Callus, Leaf Axils and Petioles of Sugar Beet (Beta vulgaris L.)

Procedures are described for producing axillary shoots fromseedling apices and adventitious shoots from petioles and leaf-derivedcallus of sugar beet cultivars. The rate of adventitious shootregeneration from petioles was influenced by temperature, BAPconcentration of the medium, and the time in culture of theseedling apices from which the petioles were excised. Petiolesectioning confirmed that adventitious shoots originated inthe sub-epidermal parenchyma. Two distinct types of callus wereproduced from leaf explants, but only white friable callus wascapable of shoot development. This callus developed from browntissue and was composed of thin-walled cells with dense cytoplasmand prominent nuclei. Green compact callus with thick-walledlignified cells developed from green tissue, but did not produceshoots. Successful seed sterilization and shoot regenerationfrom petiole explants and callus was cultivar-dependent. Adventitiousshoots were rooted and successfully transplanted to pottingcompost under glasshouse conditions. Key words: Adventitious shoots, axillary shoots, callus, sugar beet (Beta vulgaris L.)     

The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

Abnormalities in Chrysanthemum Regenerated from Long Term Cultures

Plants regenerated from leafy callus of Chrysanthemum morifoliumRamat. maintained for 9 years (LC plants) were compared to thoseregenerated from leafy callus maintained for 1 month (SC plants)in order to assess the effects of long-term culture. LC plantsdiffered morphologically from those derived from the 1-monthculture. Aberrant forms, proliferation of apical buds, variableleaf shapes and stunted growth were observed in 15 per centof the LC plants. The remaining 85 per cent were characterizedby excessive growth of lateral shoots. Flowers were smallerand irregularly formed in most LC plants. Although the growthrates of plants in both LC and SC groups were similar, floweringwas delayed considerably in the LC plants. Application of GA₃or IAA had no effect in restoring normal form. Genetic instability,chimeral rearrangement and residual hormone effects are offeredas possible explanations for the abnormalities observed. Tissue culture, Chrysanthemum morifolium, phenotypic variability     

CELL POPULATION KINETICS IN CALLUS TISSUES OF CULTURED PEA ROOT SEGMENTS

Two callus tissues, one composed of diploid and the other of a mixture of diploid and polyploid cells, were derived by culturing 1-mm pea root segments; the mixed callus tissues were obtained by incorporation of kinetin in the culture medium. The callus tissues were used to determine (a) if cell proliferation was altered with the change in cell constituents of a callus; (b) the rate at which polyploid cells increased after kinetin stimulation; (c) the nature of the mitotic cycle in the diploid and mixed polyploid callus tissues; and (d) if the mitotic cycle changed as the tissue aged. Histological, cytological, radioisotope, and radioautographic analyses were made on callus tissue ranging from 1 to 4 days old. The results indicated that gross morphological changes were associated with the anatomical location of the proliferative cells. They showed that the percentage of polyploid division figures after stimulation by kinetin increased rapidly during the first 6 to 7 days in culture and then continued to increase at a much reduced rate. Cell counts revealed that cell proliferation in the mixed callus tissue was initially delayed when compared with the diploid tissue, but that after the delay was overcome cell number increased in each in similar manner. Analysis of the number of DNA-synthesizing cells showed that their percentage was highest during the first 2 days of culture and then leveled off at a value of about 10%. Mitotic cycle analysis indicated that it could be accurately measured only in the younger diploid callus tissues and that it increased in variability with increased age.     

Comparison of Glycolipids and Plastids in Callus Cells and Leaves of Alnus and Betula

The fine structure of plastids and fatty acid composition ofglycolipids (e.g. monogalactosyl diacylglycerides, MGDG; digalactosyldiacylglycerides, DGDG) in callus cells of Alnus glutinosa,A. incana and Betula pendula cultured in light was comparedwith that in intact leaves. The tissues were qualitatively verysimilar but a rather high amount oflignoceric acid (24:0) wascharacteristic for the callus of A. incana. This fatty acidwas found only in trace amount in other tissues. Linolenic (18:3)and palmitic (16:0) acids are the most abundant (25–65%and 17–27% respectively) fatty acids in all tissues studied.The proportion of 18:1 and 18:2 was much higher in the calluscompared with corresponding intact leaves, which are especiallyrich (48–65%) in 18:3. In callus cultures a higher proportion(17–19%) of linoleic acid (18:2) is found in both Alnusspecies than in the two callus strains of Betula (9–12%). All leaf and callus samples contained esterified steryl glycosidesand two cerebrosidelike spots in thin-layer chromatography,but they were more prominent in callus cultures than in leaves.The callus cells have plastids with rather well developed thylakoidswhich explains the similarity of the main glycolipid components(MGDG and DGDG) to that of leaves. (Received April 23, 1984; Accepted August 17, 1984)     

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     

Meiotic Chromosome Pairing, Isozyme Analyses and Ferritin Expression in a Red Clover Mutant Capable of Somatic Embryogenesis

Red clover genotypes capable of regenerating plantletsin vitrofromnon-meristem-derived callus are rare. A previous study identifieda pair of near isogenic lines which were derived from a singleseed but differed in regenerative ability. The callus-derivedplants of this clone were highly regenerative when reintroducedto culture whereas the epicotyl-derived plants produced non-regenerativecallus cultures. The objective of the present study was to observemeiotic chromosome pairing and to compare isozyme profiles andferritin gene expression in regenerative and non-regenerativeplants and cultures from the clone. Meiotic cells exhibitednormal homologous chromosome pairing. Starch gel zymograms fromglasshouse-grown regenerative (F49R) and non-regenerative (F49M)plants failed to show somaclonal variation for alcohol dehydrogenase,glutamate dehydrogenase, esterase or peroxidase. Isoelectricfocusing of callus cultures from regenerative and non-regenerativeplants revealed that regeneration was accompanied by a reductionin staining intensity and numbers of peroxidase bands comparedto non-regenerative cultures. A unique cathodic peroxidase band(pI 7.6) was associated with non-regenerative cultures. Ferritinexpression was greater in callus than in fresh petiole tissue.Ferritin expression remained high in non-regenerative calluscultures but declined in regenerative cultures as regenerationprogressed.Copyright 1999 Annals of Botany Company Trifolium pratenseL., red clover, ferritin, isozymes, meiosis, peroxidase, somatic embryogenesis.     

Changes in Lipid Composition during Callus Differentiation in Cultures of Oilseed Rape (Brassica napus L.)

The lipid composition of different callus cultures of Brassicanapus varied according to their state of differentiation. Photomixotrophiccallus was characterized by the ability to synthesize relativelyhigh levels of triacylglycerol (TAG) which was rich in oleate.Glycosyldiacylglycerols were also detected. In contrast, heterotrophiccallus was found to possess high proportions of membraneousphospholipids which were rich in palmitate, linoleate, and linolenate.Moreover, the lipid content was considerably less than thatof photomixotrophic callus. Caulogenesis was achieved in bothtypes of callus strains and the lipid composition of the regeneratedleaves contained a much higher proportion of chloroplast glycosyldiacylglycerolsand thus resembled more those of the parent plant. Some callientered a senescent phase whereby there was considerable degradationof the constituent membrane lipids. Senescent callus also exhibiteda high proportion of polyploid nuclei. In this study we havebeen able to cause large changes in the morphology of calluscultures. These morphological changes were accompanied by significantalterations in the quality and quantity of acyl lipids. In photomixotrophiccells the lipid changes resembled those seen for developingseed tissues where high rates of TAG deposition are accompaniedby an altered fatty acid pattern. Thus, the selection of differentcallus types should be of use for investigations of the regulationof lipid biosynthesis under controlled culture conditions.     

Effect of Method of Agar Addition on Post-autoclave pH of the Tissue Culture Media

Tissue culture media (MS, GB-5 and N6) were gelled with agarby employing different methods When agar was dissolved directlyin media by autoclaving at 120 °C for 1 mm followed by sterilization,the media pH dropped markedly — more so at the lower concentrationsof agar On the other hand, when agar was first dissolved indistilled water and added to the culture media the pH loss wasminimized considerably pH measurements carried out as a functionof time showed a gradual decline in media pH The media supplementedwith Panax ginseng callus became more acidic than the mediawith no callus Tentative reasons for the post autoclave pH fallassociated with various methods of agar addition are described Culture media, agar addition, post-autoclave pH, Panax gingseng     

Experimentally Synthesized Plant Chimeras 2. A Comparison of In vitro and In vivo Techniques for the Production of Interspecific Nicotiana Chimeras

In vitro and in vivo techniques were compared for synthesizingchimeras between Nicotiana glauca Grahm and N tabacum L Interspecificchimeral callus, produced from mixed callus cultures in vitro,was placed on media which favoured only N tabacum shoot formationNone of the 474 regenerated N tabacum shoots incorporated Nglauca cells into their meristems When chimeral callus was regeneratedunder hormonal conditions favouring simultaneous organogenesis,of 397 shoots, only non-chimeral shoots of both species aroseIn vivo, reciprocal splice grafts between species were decapitatedjust above the graft union and treated with or without auxin—lanolinpastes Auxin increased callus formation but inhibited adventitiousshoot formation Three of 209 adventitious shoots arising fromthe graft union were interspecific mericlinal chimeras whichwere later stabilized as periclinal chimeras All three chimerasformed when N glauca was the understock Two of the chimerasarose on untreated shoots which produced no visible callus,indicating that excessive callus formation may be unnecessaryfor multiple cell origin of adventitious shoots to occur Chimeras, tobacco, Nicotiana glauca, Nicotiana tabacum, tissue culture, graft chimeras, callus cultures     

Fine Structure of Parenchymatous and Differentiated Pinus radiata Callus

The fine structure of Pinus radiata D. Don callus before andafter differentiation into stem-like tissues has been examinedwith the electron microscope. In callus prior to differentiation(here called parenchymatous callus) the cells accumulate tanninsas they age and are quite distinct from the cells of differentiatedcallus. In the latter, cambium, phloem and xylem cells may beidentified by their general morphology and by their ultrastructuralfeatures. Differentiation into a true stem-like structure is,however, incomplete in that the tissues are not uniformly oriented,and parenchyma cells of the rays and phloem contain chloroplasts.The tracheids also show unusual differentiation in that borderedpits form over their entire surface and may be of two types.The reasons for these variations are discussed.     

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.     

Salt tolerance in Suaeda maritima (L.) Dum: A COMPARISON OF MITOCHONDRIA ISOLATED FROM GREEN TISSUES OF SUAEDA AND PISUM

Mitochondria were isolated from green leaves and stems of theglycophyte Pisum sativum and the halophyte Suaeda maritima.The preparations oxidized malate, succinate, and 2-oxoglutarateas well as externally added NADH. Acceptor control ratios wereabout 2.8 for mitochondria from Pisum and 1.8 for mitochondriafrom Suaeda oxidizing malate+pyruvate in 125 mM sodium chloride.The mitochondrial fraction was contaminated with chloroplastfragments which resulted in relatively low rates of oxygen uptakewhen these were expressed on a protein basis. The addition of sodium chloride at concentrations greater than200 mM considerably reduced the rates of oxygen uptake by bothspecies in the presence and absence of phosphate acceptor (ADP).Acceptor control ratios were reduced and there was a markeddecline in the ADP/O ratio. Sucrose at equivalent molar concentrationshad a much less drastic effect on the mitochondria. There was no significant difference in the effects of thesetwo solutes on mitochondria from the two species and the similarityof response is discussed in relation to the cytoplasmic ioncontent of the halophyte.     

Mechanism of Suppression of DOPA Accumulation in a Callus Culture of Stizolobium hassjoo

Mechanisms of suppression of 3,4-dihydroxyphenylalanine (DOPA)accumulation were investigated in a callus culture of Stizolobiumhassjoo. DOPA was detected in the callus but in a much smalleramount than in the intact plant, and its content changed duringculture. Biosynthesis of DOPA from labeled tyrosine in callus was confirmedby obtaining the constant specific radioactivity of the formedDOPA after co-crystallizing it four times with an authenticspecimen. The variation in the percentage of radioactivity incorporatedfrom labeled tyrosine into the ethanol-insoluble fraction wasa mirror image of that of the DOPA content during culture. Theincrease in incorporation of radioactivity from labeled tyrosineinto DOPA preceded that of the DOPA content. The rate of incorporationof radioactivity from labeled tyrosine into the ethanol-insolublefraction was lower in etiolated seedlings than in callus atevery stage of growth. However, the rate of incorporation ofradioactivity from labeled tyrosine into DOPA was about thesame in etiolated seedlings as in 19-day-old callus, which showedthe highest activity of DOPA synthesis during culture. The results obtained here indicate that the biosynthetic pathwayof DOPA from tyrosine operates in callus at any growth stageand that the shift of the metabolic flow of tyrosine from DOPAsynthesis to other pathways, e.g., protein synthesis, can explainthe change in DOPA content during callus culture, and partiallythe suppression of DOPA accumulation in callus. (Received February 4, 1981; Accepted May 18, 1981)     

Biosynthesis of furano-terpenes by sweet potato cell culture

Callus was induced from sweet potato root tissue on an agarmedium containing Heller's minerals, vitamins, 2,4-D, yeastextract and sucrose. Furano-terpenes were scarcely detectedin the callus. However, when the callus was transferred to aliquid culture medium and incubated with reciprocal shaking,furano-terpenes were rapidly produced mainly in the culturemedium. Furano-terpene production by the cell culture was suppressedby addition of Ceratocystis fimbriata spores or HgCl₂ to theculture medium. Yeast extract and sucrose in the culture mediumwere important for furano-terpene production. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity increased in the cells,followed by the production of furano-terpenes. The TLC patternof furano-terpenes produced by the cell culture was essentiallythe same as that produced by sweet potato root tissue infectedby C. fimbriata or treated with HgCl₂, but the quantitativeproportion of the individual furano-terpenes in the former differedmarkedly from that in the latter. (Received January 11, 1979; )     

Programmed cell death in Datura innoxia Mill. callus cultures cultivated in light and in dark

It has been established that dimedone in solid culture medium influencedthe growth of Datura innoxia Mill. callus tissues and theapoptotic processes of cells. This formaldehyde (HCHO) capture reagent appearsto modify the metabolism of plant cells, resulting in quantitative changes inthe apoptotic index (A_i). Apoptotic cells were detected insix-week-old callus tissues by the TUNEL reaction. The amount of TUNEL positivenuclei showed a characteristic spatial distribution. Enhanced DNA fragmentationwas observed in the cell layers close to the surface of the cultures. ElevatedA_i was determined in cultures grown in the dark compared to thetissues grown in the light. High doses of dimedone considerably decreasedapoptosis in tissue cultures under both light and dark conditions.     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Effects of Monochromatic Radiations on Growth of Pelargonium Callus Tissue

Pith callus tissues were grown under continuous blue (450 mµ),green (545 mµ), red (650 mµ), and ‘white’(full-spectrum) light, and in the dark for 22 days at 27±2°C at energy levels of 15,000 ergs cm^–2 sec^–1. Mean increases in fresh weight of tissues grown under ‘white’and blue light were significantly greater than those of tissuesgrown in green and red light and in the dark. Tissues grownin the dark yielded mean fresh weight increases significantlylower than tissues grown under blue, red, and ‘white’light. No significant differences were shown between blue and‘white’, red and green, and green and dark treatmentsrespectively. Cell differentiation occurred in all treatmentsonly to the extent of vessel element formation. There were nodifferences in degree of differentiation between treatments. It was proposed that the high-energy reaction of photomorphogenesiswas in operation in the Pelargonium callus tissue. The resultsindicated the presence in the tissue of high-energy photoreceptor(s).The use of high-intensity, incandescent illumination for experimentalprocedures approximating natural conditions of irradiation wasindicated as desirable for pith callus tissues of Pelargoniumzonale var. Enchantress Fiat.