Transformation of Solarium and Nicotiana Species using an Ri Plasmid Vector

Davey, M. R., Mulligan, B. J., Gartland, K. M. A., Peel, E.,Sargent, A. W. and Morgan, A. J. 1987. Transformation of Solanumand Nicotiana species using an Ri plasmid vector.—J. exp.Bot. 38: 1507–1516. Five Nicotiana species (N. benthemiana, N. debneyi, N. occidentals,N. plumbaginifolia, N. tabacum) and three Solanum species (S.dulcamara, S. nigrum, S. tuberosum) were transformed by wild-typeand engineered Ri plasmids. Depending on the host plant, rootstransformed by Agrobacterium strain A4TIII with an Ri plasmidcarrying a chimaeric nopaline synthase-kanamycin resistancegene, were 3 to 40 times more resistant to kanamycin than rootstransformed by the wild-type plasmid of strain A4T. Similarly,plants regenerated from A4TIII-derived roots of N. debneyi,N. plumbaginifolia and N. tabacum were 8 to 16 times more resistantthan A4T plants, and survived at 400 µg cm³ of kanamycin.A4TIII plants of S. nigrum flowered in vitro at 600–1000µg cm³ of kanamycin. Transformed roots and most regeneratedplants synthesized Ri-speciflc opines, while DNA hybridizationconfirmed the presence of DNA homologous to that from wild-typeand engineered Ri plasmids in transformed plants of S. nigrum. Key words: Agrobacterium, Ri plasmid, transformed roots, plant regeneration, kanamycin resistance.     

Studies on Foliar Penetration: VIII. EFFECTS OF CHLORINATION ON THE MOVEMENT OF PHENOXYACETIC AND BENZOIC ACIDS THROUGH CUTICLES ISOLATED FROM THE FRUITS OF LYCOPERSICON ESCULENTUM L.

The effects of chlorine substitution on the movement of phenoxyaceticand benzoic acids through enzymatically-isolated cuticles ofLycopersicon fruits were determined by following the transferof each acid containing ¹⁴C from a donor to a receiver solution.This cuticle is characterized by an isotropic cutin matrix,within which patches of birefringent cuticular waxes are foundnear the outer surface. The outer, morphological surface isrelatively smooth while at the junction with the outer wallsof the epidermal cells there is extensive cuticular developmentextending down between the anticlinal walls. The epicuticularwax appears as a soft sheet-like covering of which the surfaceis relatively featureless. Chlorination of phenoxyacetic acid results in an enhanced transferacross the isolated cuticle. The order was 2,4,5- and 2,4,6-trichlorophenoxyacetic> 2,4- and 3,5-dichlorophenoxyacetic > 2-chlorophenoxyacetic> phenoxyacetic acid. Removal of the epicuticular wax resultedin greater permeability for all compounds; transfer of the morepolar acids was favoured. In contrast, chlorination of benzoicacid decreases passage through the cuticle; the rate is highestfor benzoic acid followed in descending order by 2-chlorobenzoic,2,4- and 2,5-dichlorobenzoic and 2,3,6-trichlorobenzoic acid.Chlorination also depresses the passage of both phenoxyaceticand benzoic acid through a dialysis membrane. The effects ofchlorination on the lipid solubility of both series of compoundsare discussed in relation to differences in transfer acrossthe cuticle.     

Discriminant analysis of Lepidopteran prey characteristics and their effects on the outcome of bird-feeding trials

Discriminant analysis was used to analyse the results of 348 bird-feeding trials conducted from 1982 to 1985 in Leverett, Massachusetts. Four size classes, seven appearance categories, and five larval host types, based on 163 species of moths and butterflies used as prey in two or more trials, were selected as predictor variables to discriminate between prey taken and not taken by birds. Discriminant analysis of individual feeding trials correctly classified 97.5 percent of prey taken or not-taken and ranked the predictor variables according to their relative importance in determining prey acceptability. Characteristics most acceptable to birds were: (1) large size, (2) bark-like appearance, (3) warning colouration, (4) woody generalist, (5) dead-leaf-like appearance, (6) woody specialist, and (7) medium size. Characteristics least acceptable to birds were: (1) small size, (2) mimetic appearance, (3) butterfly appearance, (4) herbaceous specialist food type, (5) black-and-white appearance, (6) extra large size, and (7) overall generalist feeder. A summary of the analyses includes a discriminant function based on lepidopteran characteristics that can be used to predict the prey acceptability of species not used in this study. A multiple regression analysis of prey taken revealed that size alone and larval host type combined with other prey characteristics were the most important variables in determining the selection of prey regardless of their abundance in the trials.     

Studies on Foliar Penetration: VII. FACTORS CONTROLLING THE PENETRATION OF CHLORIDE IONS INTO THE LEAVES OF PHASEOLUS VULGARIS

The pattern of penetration of chloride ions into the abaxialsurfaces of the primary leaves of Phaseolus vulgaris has severalfeatures in common with those previously recorded for 2,4-dichlorophenoxyaceticacid (2,4-D), 2,2-dichloropropionic acid (dalapon), and 4-amino-3,5,6-trichloropicolinicacid (picloram). In the dark the rate of entry of chloride ionsup to 24 h is constant, but in the light entry is at first slowand then more rapid. This acceleration does not occur at lowtemperature or when the tissue is treated with 3-(3,4-dichlorophenyl)-l,l-dimethylureaor phenylmercuric chloride. Neither does it take place at theadaxial surface or when the leaves are more mature. The most distinguishing feature of the pattern of penetrationof chloride ions is its dependence on external pH. In darknessentry is unaffected by changes in pH but in the light the rateof entry is increased as the pH falls, but this response isrestricted to young leaves. No binding of chloride seems tooccur within the tissue. These findings support the view that penetration of the abaxialsurface in young leaves of Phaseolus is largely determined bya membrane system. This system decreases in importance as theleaf matures and the overlying cuticle thickens. At the adaxialsurface the thicker cuticle is seemingly a major barrier topenetration even in very young leaves.     

The Uptake of Growth Substances: II. THE ABSORPTION AND ACCUMULATION OF 2:3:5-TRIIODOBENZOIC ACID BY THE ROOT AND FROND OF LEMNA MINOR

The absorption by Lemna minor of 2 : 3 : 5-triiodobenzoic acid(TIBA), labelled in the 2 position with iodine-131, has beeninvestigated. Under constant conditions of light (300 foot candles)and temperature (25?C.) the course of uptake by the whole plantis dependent on the concentration of TIBA in the culture solution(pH 5.I). Up to circa 1 mg./l. a high rate of uptake in thefirst 30 minutes is followed by a slower but steady accumulationduring the next 4.5 hours. When the concentration is increasedto 5 mg./1. uptake, at first rapid, falls off progressivelyso that after 1-2 hours the content of the tissues starts todecrease because TIBA moves out in to the external solutionfaster than it enters. The magnitude of this reversal from apositive to a negative rate of uptake,.partitularly for theroots, is even more pronounced at 10 mg/l. When plants, first placed in labelled solutions for 2 to 2.5hours are transferred to culture solution the loss of the labelledcompound to the external solution, especially from the roots,is very rapid : the decrease in root content may reach 90 percent. in the first 80 minutes. The initial rate of egress islittle affected either by the hydrogen ion concentration orthe presence of unlabelled TIBA in the external solution. Between5, 15, and 25? C. the temperature quotients range from 1.29to 1.70 for the root and 1.66-1.37 for the frond. Comparable experiments on the initial phase of uptake duringthe first 2o to 30 minutes demonstrate that (a) the rates aregreatly dependent on the external pH i.e. uptake is largelyin the molecular form and (b) between 5 and 15?C. the ratesare not temperature dependent but between 15 and 25?C. theyare: the quotients for roots and frond are 2.6 and 2.0. Comparisons of the course of uptake of normal plants and plantspreviously frozen in liquid air showed that at both I? and 25?C.'pre-frozen' fronds continue to accumulate TIBA and may containat the end of 5 hours 8 times as much as control fronds. Incontrast, the uptake by the pre-frozen roots is depressed. Theaddition of high (30-50 mg./l.) but not low (10 mg./1.) concentrationsof indole-3-acetic acid (IAA) to the external solution alsocauses a striking accumulation of TIBA in the frond but uptakeby the roots is not changed. Lastly, the presence of TIBA in the external solution whilenot affecting the uptake of cerium-144. in the first 2 hourssubsequently depresses uptake by the root but not by the frond. These findings are compared with the similar pattern of uptakefor z,4-dichlorophenoxyacetic acid described in the previouspaper. The nature of the processes controlling the inward andoutward flow of TIBA, their location within the cell and theirbearing on the physiological actions of auxins are discussed.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

Studies on Foliar Penetration: I. FACTORS CONTROLLING THE ENTRY OF 2, 4 - DICHLOROPHENOXYACETIC ACID

A technique, using leaf disks, has been developed to study thepenetration of isotopically labelled compounds into leaves underconditions where there is no appreciable change in the concentrationof the external solution and no subsequent translocation. Inthis preliminary survey, the leaves of Phaseolus vulgaris andColeus Blumei were employed to investigate the entry of 2,4-dichlorophenoxyaceticacid (2,4-D), labelled in the carboxyl group with ¹⁴C. Over3 days there is no loss of ¹⁴C to the atmosphere from treatedleaves of Phaseolus. The rate of penetration is enhanced when(a) the leaves are young, (b) the water status is lowered, (c)the temperature is raised (Q₁₀=2.3–2.8), and (d) a surface-activeagent is added to the external solution. Penetration is alsofavoured by a decrease in the pH, the relation indicating thatboth ions and molecules enter. Penetration is greater in thelight and prior illumination of the tissues positively affectsthe subsequent rate in the light, but not in the dark. In boththe light and the dark considerably more 2,4-D penetrates theabaxial surface of Phaseolus leaves. For Coleus an even greaterdifference between surfaces is found in the light but not inthe dark. For both species in the light the rates of entry intoboth surfaces are proportional to their respective stomataldensities. The simultaneous addition of indoleacetic acid tothe external solution caused more 2,4-D to enter Phaseolus leaves,but the addition of triiodobenzoic acid restricts entry. Therate of penetration remains constant over 24 hours and between0.1 and 200 mg./l. the rate is linearly related to concentration.Subsequent to entry, the 2,4-D is in a form which does not diffusefrom the tissue into buffer or exchange with unlabelled 2,4-D.Moreover, no outward movement takes place from treated tissuewhich has been frozen and thawed. These findings are discussedin relation to previous work on foliar penetration. It is concludedthat at least with Phaseolus penetration largely takes placethrough the guard cells and/or accessory cells.