High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into20–40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml^–1or 1 mg ml^–1 have been tested. With 1 mg ml^–1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml^–1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation     

Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

The involvement of ethylene in in vitro maturation of mid-binucleate pollen of Nicotiana tabacum

The role of ethylene during in vitro maturation of Nicotianatabacum pollen from the mld-binucleate (MB) stage was analysedby the addition of aminooxyacetic acid (AOA), aminoethoxyvinylglycine(AVG), CoCl₂ and AgNO₃ to the maturation medium (AMGLu). Anincrease in ethylene production was obtained in both isolatedpollen and pollen surrounded by sporophytic tissue during insitu maturation. in vitro maturation of pollen was inhibitedby AOA and AVG; ACC and ethrel were able to overcome this inhibitoryeffect. Cyclohexylamine (CHA) reverted the inhibition provokedby both Ag⁺ and Co²⁺ The results reported in this paper indicatethat ethylene is one of the factors implicated in in vitro maturationof MB pollen of Nicotiana tabacum. Key words: Nicotiana tabacum, maturation, germination, pollen, ethylene     

Photochemical Apparatus Organization in the Diatom Cylindrotheca fusiformis: Photosystem Stoichiometry and Excitation Distribution in Cells Grown under High and Low Irradiance

The photochemical apparatus organization in the thylakoid membraneof the diatom Cylindrotheca fusiformis was investigated in cellsgrown under high and low irradiance. High light (HL, 200µE.m^–2.s^–1)grown cells displayed a relatively low fucoxanthin to chlorophyll(Chl) ratio, a low photosystem (PS) stoichiometry (PSII/PS I=1.3/1.0)and a smaller photosynthetic unit size in both PS I and PS II.Low light (LL, 30µE.m^–2.s^–1) grown cells displayeda 30% elevated fucoxanthin content, elevated PS II/PS I=3.9/1.0and larger photosynthetic unit size for PS II (a change of about100%) and for PS I (by about 30%). In agreement, SDS polyacrylamidegel electrophoresis of thylakoid membrane polypeptides showedgreater abundance of PS I, RuBP carboxylase and ATP synthasepolypeptides in HL cells. In contrast, LL grown cells exhibitedgreater abundance of light-harvesting complex polypeptides.Assuming an efficiency of red (670 nm) light utilization of1.0, the measured efficiency of blue (481 nm) light utilizationwas 0.64 (HL cells) and 0.72 (LL cells). The lower efficiencyof blue versus red light utilization is attributed to the quenchingof absorbed energy by non-fucoxanthin carotenoids. Differencesin the efficiency of blue light utilization between HL and LLgrown cells are attributed to the variable content of fucoxanthin.The results support the hypothesis of a variable Chl a-Chl c-fucoxanthinlight-harvesting antenna associated with PS II and PS I in Cylindrotheca. (Received February 10, 1988; Accepted April 6, 1988)     

Use of a Disarmed Ri Plasmid Vector in Analysis of Transformed Root Induction

Nicotiana glauca, N. tabacum, Solanian dulcamara and S. nigrumwere transformed by Agrobacteriun rhizogenes strain BN1010 (TL^–TR⁺).The TR-DNA stimulated agropine-positive root induction and wastransformation competent in the absence of the TL-DNA. An unusualpattern of root induction was seen when stem explants were inoculatedwith this strain; occasionally, agropine-positive roots wereinduced at the inoculation sites, but prolific agropine-negativeroots were formed in profusion down the stems. The utility ofBN1010 as an efficient co-integrating vector was demonstratedby the separate transfer of a fragment containing rol ABC (BN1010::pEM15) and of a chimeric nopaline synthase-kanamycin resistancegene (BN1010:: Neo) into plants. Root cultures of S. dulcamaratransformed with BN1010:: Neo had an unusual, positively geotropicphenotype. Strain BN1010:: pEM15 (rol ABC⁺D^–TR⁺) incitedmore roots down stem explants than strain A4T. This indicatesthat rol D may act to suppress agropine-negative root productionin N. glauca and N. tabacum. Key words: Agrobacterium rhizogenes, TL-DNA, TR-DNA, disarmed Ri vector, transformed roots, Nicotiana glauca, N. tabacun, Solatium dulcamara, S. nigrum     

Induction of Solute Release from Nicotiana tabacum Tissue 10

For determination of the effects of polymyxin B, polymyxin E,or ethylenediamine tetra-acetic acid (EDTA) on plant cell membranes,the rates at which three solutes, K⁺, P₁, and sugar, leakedfrom treated tissue culture cell suspensions of Nicotiana tabacumwere measured. The kinetics of leakage from cells treated witheither of the polymyxins was biphasic, whereas kinetics forcells treated with EDTA was monophasic. Only K⁺ leaked frompolymyxin-treated cells during the first phase, and all threesolutes leaked during the second phase. The slower first phaseis interpreted as leakage of K⁺ from the Donnan free space andcytoplasm, and the faster second phase as the leakage of solutesfrom the vacuole. The monophasic kinetics of EDTA treatmentindicated that solutes were leaking simultaneously from cytoplasmand vacuole. Of the divalent cations tested, only Ca⁺⁺ and Mn⁺⁺counteracted the effects of polymyxin and EDTA. Ca⁺⁺ even restoredP¹ and sugar uptake. Addition of Mg⁺⁺ or Sr⁺⁺ to polymyxin-treatedcells did not stop solute leakage but actually enhanced theleakage rates. A model is presented that suggests that polymyxinor EDTA induces solute leakage by forming pores in plant cellmembranes. The effects of divalent cations on membranes oncethe pores are formed are also discussed. Key words: Polymyxin, EDTA, Nicotiana tabacum, Solute leakage     

Computer-assisted Video Image Analysis of Spatial Variations in Membrane-associated Ca2+ and Calmodulin during Pollen Hydration, Germination and Tip Growth in Nicotiana tabacum L.

Video images of the distributional pattern of membrane-associatedcalcium (Ca²⁺) and calmodulin (CaM) have been documented andanalysed during pollen hydration, germination and tip growthin Nicotiana tabacum. Digitization of fluorescence microscopeimages of chlorotetracycline (CTC) and fluphenazine (FPZ)-fluorescenceemissions reveal that there is a maximum concentration of membrane-associatedCa²⁺ and also CaM in the vicinity of germination apertures ofhydrated pollen. With the onset of germination relatively higheramounts of Ca²⁺ and CaM were found to regionalize towards theaperture through which the pollen tube would emerge Both shortand long growing pollen tubes manifest tip-to-base Ca²⁺ andCaM gradients which are disturbed in non-growing tubes. Tubegrowth and the Ca²⁺-gradient were significantly affected byvanadate and verapamil suggesting that both a vanadate-sensitiveCa²⁺-transport system and verapamil-sensitive Ca²⁺ channelsare involved in maintaining Ca²⁺ homeostasis during pollen germinationand tube growth. The possible interactions of Ca²⁺ and CaM withdifferent cytoskeletal proteins modulating organelle movementare also briefly discussed. Image analysis, calcium, calmodulin, Nicotiana tabacum L., pollen germination, pollen tube, tip growth, Ca²⁺-channels, Ca²⁺ transport ATPase     

Structure of N-glycans on the S3- and S6-stylar self-incompatibility ribonucleases of Nicotiana alata

Self-incompatibility is a mechanism developed by many plantsto prevent inbreeding. The products of the selfincompatibility(S)-locus in the styles of solanaceous plants are a series ofglycoproteins with ribonuclease activity. In this study, wereport on the N-glycans from the stylar selfincompatibilityS₃- and S₆-ribonucleases of Nicotiana alata, which were enzymicallyreleased and fractionated by high-pH anion-exchange HPLC. Atotal of 14 N-glycans were identified and characterized by acombination of electrospray-ionization mass-spectrometry, ¹H-NMRspectroscopy, chemical degradation, and methylation analyses.This pattern of N-glycosylation is much more complex than thatpreviously found on the N.alata S₁- and S₂-RNases each of whichcontained only four N-glycans. N-glycan Nicotiana alata ribonuclease selfincompatibility     

Development of Jobacco Carpel Primordia in vitro

The state of determination of the two emergent carpel primordiaof Nicotiana tabacum was tested. Carpel rudiments were excisedand cultured singly or in pairs. The basal medium was that ofLinsmaier and Skoog, supplemented with 1.0 mg 1^–1 kinetin.Over the ensuing 4 week period, whole differentiated pistilsformed from the pairs and half pistils grew from the singlecarpels. It is concluded that these emergent organs show a certaindegree of autonomy and that they may have been determined atthe time of isolation. Nicotiana tabacum L, tobacco, carpel, organ determination, tissue culture, morphogenesis     

Response to Waterlogging and Differing Sensitivity to Divalent Iron and Manganese in Clones of Dactylis glomerata L. derived from Well-drained and Poorly-drained Soils

Clonally propagated plants of Dactylis glomerata derived froma well-drained, heavily grazed cliff habitat (clone L) and froman undergrazed poorly-drained soil (clone A) were tested forwaterlogging tolerance in soil-culture. Water-logging did notaffect the two clones differentially, a result, which contrastedstrongly with that of a previous experiment in which simulatedgrazing (clipping to 20 cm) unexpectedly caused clone A to beless tolerant of waterlogging than clone L. Maximum leaf andleaf sheath length was reduced more by water-logging in cloneL than in clone A (P < 0.05). In solution-culture when providedwith factorial combinations of 0.5, 5 and 50 mg dm^–2 ofFe²⁺ and Mn²⁺ the shoot dry weight yield of the dry-soil clonewas reduced more than that of the wet-soil clone by 50 mg Fedm^–3 irrespective of Mn²⁺ concentration (P < 0.01)but the reduction of growth was less at higher Mn²⁺ concentrations.Fifty milligrams of Mn²⁺ dm^–3 reduced the growth of thedry soil clone but increased the growth of the wet soil clonewith Fe²⁺ at 5 mg dm^–2 (P < 0.05). Iron at 0.5 mg dm^–2was suboptimal for shoot growth of both clones at any levelof Mn²⁺ and caused more severe leaf chlorosis in the wet soilclone. Leaf tissue of clone L contained more iron than thatof clone A after waterlogging (P < 0.01) but in solutionculture, though increasing iron from 0.5 to 50 mg dm^–3almost doubled leaf iron content (P < 0.001), the interactionClones x Mn x Fe just failed to reach significance at P <0.05. The manganese content of leaf tissue from the two clonesvaried differently in response to solution manganese (Clonesx Mn P < 0.01), clone A showing a slightly greater increaseof manganese content at high solution concentration. Iron at50 mg dm^–3 suppressed Mn uptake (Mn x Fe, P < 0.001)in both clones. The two clones thus show marked environmentaladaptation to the chemistry of wet and dry soils. Dactylis glomerata, Cocksfoot grass, Orchard grass, waterlogging, iron, manganese, toxicity, deficiency, ecotypes     

Embryogenesis and Plantlet Formation through Direct Culture of Isolated Pollen of Nicotiana tabacum cv. Samsum and Nicotiana rustica cv. Rustica

Pollen embryos and plantlets of Nicotiana tabacum cv. Samsunand Nicotiana rustica cv. Rustica were obtained through directpollen culture without prior treatment or prior culture of anthersor buds. Isolated pollen was cultured first in a medium withoutsucrose, then transferred into Nitsch's H medium containing2% sucrose and 5 mM glutamine. The optimum medium for the initialculture was water and the optimum period of culture was ca.6 days when binucleate pollen was used. ¹ Present address: Friedrich Miescher Inst., P.O.B. 273, CH-4002Basel, Switzerland. (Received January 18, 1982; Accepted March 19, 1982)     

Inhibition of the Chloroplast Photochemical Reactions by Treatment of Wheat Seedlings with Low Concentrations of Cadmium: Analysis of Electron Transport Activities and Changes in Fluorescence Yield

The effect of treatment of wheat plants with Cd²⁺ ions on thephotochemical activity of the primary leaves was examined. Threeday-old etiolated seedlings were treated with Cd²⁺ ions for24 h in dark, and after this treatment the plants were grownin the light until the primary leaves were fully developed.Cd²⁺ ions (30–120 µM) induced a significant decreasein activities of both photosystem II and photosystem I. Theextent of the decrease in PS II activity was much greater thanthat in the PS I activity. Analysis of changes in the fluorescenceyield of chlorophyll also indicated that Cd²⁺ ions drasticallyaffect the photochemistry of photosystem II. Cd²⁺ ions induceddecrease in the rates of photoreduction of 2,6-dichlorophenolindophenol even in the presence of the exogenous electron donor,hydroxylamine, both in Tris-treated and untreated chloroplasts.This result suggests that the site of inhibition is near thesite of donation of electrons by hydroxylamine. Treatment withCd²⁺ ions impairs the electron transport system on the reducingside of PS II. The decrease in the fluorescence yield of Chi is less than that in the evolution of O₂ mediated by oxidizedphenylenediamine. This difference may be a result of inhibitionon the reducing side of PS II. In addition to inhibition onthe reducing side, Cd²⁺ ions may affect the oxidizing side ofPS II. A comparative study of the rates of evolution of O₂ withp-benzoquinone and dichloro-p-benzoquinone as electron acceptorswas performed since the halogenated benzoquinones have beenshown to accept electrons from both active and inactive centersof photosystem II while some of the benzoquinones accept electronsonly from active centers. The results suggest that Cd²⁺ ionsinduced a marginal increase in the number of inactive reactioncenters in PS II. Analysis of light-saturation-kinetics of theevolution of O₂ catalysed by PS II indicates a reduction inthe size of the antennae as well as in the concentration ofthe active (-type) reaction centers of PS II. Thus, the Cd²⁺-inducedeffects on the photochemistry of PS II involve changes on thereducing side of PS II as well as possible changes in the sizesof the populations of active and inactive centers. Thus, short-termexposure to Cd²⁺ ions during establishment of seedlings hasa severely detrimental effect on photochemical activities inchloroplasts. (Received October 17, 1990; Accepted July 3, 1991)     

The Role of Glucose on Flower Bud Formation in Thin-Layer Tissue 12Nicotiana tabacum L.

The effect of glucose on flower bud formation was studied inthin-layer tissue cultures of epidermal strips from flower stalksof Nicotiana tabacum L. cv. Samsun. A minimum concentration of 30 mol m^–3 glucose in the MS-mediumcontaining 1.0 mmol m^–3 of both NAA and BA was necessaryfor flower bud formation. With 150 mol m^–3 glucose a minimumstay of 10 d was required for optimal flower bud formation. Withholding glucose for a limited period at different time intervalsafter the onset of culture caused a delay in flower bud formationand did not affect previous development on glucose. The resultsindicated that competence for flower bud initiation is not restrictedto the early stage of culture. The process may start at anytime later at the appropriate glucose concentration. However,for both optimal initiation and further development of flowerbuds the presence of a metabolizable sugar is required. Incubationof the tissue on glucose is associated with higher respirationrate. Key words: Flower formation, Glucose, mannitol, Nicotiana tabacum, Respiration, tissue culture     

Hormonal regulation of genetic tumor induction: Cytokinin and abscisic acid

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens? N. langsdorjfii were grown aseptically on nutrient mediumin a controlled environment chamber. At regular intervals theincidence of tumor formation was scored and plants were harvested.Total cytokinin activity was determined by means of the cucumbercotyledon bioassay, while ABA activity was measured by radioimmunoassay.A close correlation between onset of tumor formation and elevationin endogenous cytokinin activity was demonstrated, but no correlationwas observed between onset of tumorigenesis and change in thelevel of ABA. In addition, exposure of plants to exogenous ABAdid not alter therate of tumor formation. These results arediscussed in relation to the trigger mechanism for tumor inductionin the Nicotiana system. ¹A preliminary report of some of this work was presented atthe American Society of Plant Physiologists meeting in June1978. ³Present address: Department of Biology, West Virginia University,Morgantown, West Virginia 26506, U.S.A. (Received March 10, 1979; )     

The Composition of Phloem Exudate and Xylem Sap from Tree Tobacco (Nicotiana glauca Grah.)

The composition of xylem sap and exudate from stem incisionsof Nicotiana glauca Grah. was compared in detail. Exudationfrom stem incisions occurred over a 5 min period in certainplants, enabling collection of 5–30 µl of sap. Therate of exudation showed an exponential decline. Exudate hada high dry matter content (170–196 mg ml^–1) andhigh sugar (sucrose) levels. Xylem sap had a low pH (5.8) andexudate a pH of 7.9. Glutamine dominated the amino compoundsin xylem sap and exudate, and K⁺ was the major cation. Totalamino compounds in stem exudate reached 10.8 mg ml^–1 whereasxylem sap contained much lower levels (0.28 mg ml^–1).All mineral elements and amino compounds with the exceptionof calcium were more concentrated in stem exudate than in xylemsap. Sucrose was labelled heavily in stem exudate following pulsingof an adjacent leaf with ¹⁴CO₂. A concentration gradient ofsugar (2.1 bar m^–1) was recorded for stems. Levels ofsucrose, amino compounds and K⁺ ions in stem exudate showeda diurnal periodicity. Each commodity reached maximum concentrationat or near noon and minimum concentration about dawn. The evidencesuggests that exudate from stem incisions of N. glauca is arepresentative sample of solutes translocated in the phloem. Nicotiana glauca Grah., phloem sap, xylem sap, sucrose, amino compounds, mineral ions     

14C Fixation and Translocation in two Clones of Sugarcane with Contrasting Rates of Sucrose Uptake in vitro

The rates of ¹⁴CO₂ fixation and translocation of ¹⁴C labelledassimilates were measured in field experiments at two timesof the day in two sugar-cane clones known to have differentrates of sucrose uptake in vitro but the same weight of leafper unit weight of cane. The rate of ¹⁴CO₂ fixation and the velocity and rate of translocationwere significantly greater at both times in the clone with thehigher rate of sucrose uptake in vitro. The velocities of translocationwere 2.18 and 2.36 cm/min^–1 for the clone with high sucroseuptake and 1.46 cm min^–1 at both times in the clone withlow uptake. It is suggested that among sugar-cane clones the ability oftheir canes to store sugar may play a part in determining theirrates of photosynthesis and translocation.     

Visualization of Membrane Calcium and Calmodulin in Embryo Sacs in situ and Isolated from Petunia hybrida L. and Nicotiana tabacum L.

We have used chlorotetracycline (CTC) and fluphenazine (FPZ)as fluorescent probes to visualize the distributional patternsof membrane calcium (mCa²⁺) and the Ca²⁺-receptor protein calmodulin(CaM) in various cell types of unfixed living isolated and unisolatedembryo sacs of Petunia hydrida L. and Nicotiana tabacum L. Ourresults indicate that in the young embryo sacs of Petunia, bothsynergids and the central cell sequester relatively higher amountsof mCa²⁺ and CaM than the egg cell and the antipodals. Much of the mCa²⁺ in the synergids is polarized in its distributionin that the mCa²⁺ is higher towards the micropylar end of thesynergids. Interestingly, in the mature embryo sacs of Petuniaonly one of the two synergids and the egg cell proper manifesta higher level of mCa²⁺. In vivo only one of the synergids inthe young as well as in the mature embryo sacs if Nicotianaconsistently show higher mCa²⁺. In the mature embryo sacs of Petunia the level of CaM is almostuniform in all the cell types except that one of the synergidsand the three antipodal cells show a slightly higher level ofCaM. The possible implications of these findings in the late eventsof vectorial orientation of pollen tube tip, pollen-tube-synergidinteractions and sperm delivery mechanism are discussed.Copyright1993, 1999 Academic Press Membrane-Ca²⁺, calmodulin, living embryo sacs, Petunia, Nicotiana, pollen tube-synergid interaction     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Light-Harvesting for Photosynthesis in Four Strains of the Red Alga Porphyra yezoensis Having Different Phycobilin Contents

The light-harvesting system of photosynthesis was studied infour strains of Porphyra yezoensis differing in their phycoerythrin(PE) content; the red strain, richer in PE than the wild strain,and the green and the yellow strains, poorer in PE. Specialattention was given to possible alteration of pigment systemin response to PE content, especially in the green and the yellowstrains. The relative quantum yields of Chi a fluorescence at–196°C and O² evolution were compared. Four strains commonly showed a low yield in pigment system II(PS II) fluorescence on Chl a excitation. The yield was as lowas in those algae in which PS II has only a small portion ofChl a as the light harvester. Measurement of O₂ evolution gavethe same results. Results indicate that the functional compositionof Chl a system remains unaltered in four strains with differentPE content. PS II in the green and the yellow strains reflectsa reduction in the size of light-harvesting components, suggestingthat pigmentation in these strains is fixed genetically as asun-type. ⁴ On leave from University of Washington, Seatle, Washington,U.S.A. (Received September 18, 1982; Accepted December 25, 1982)