Rhizobium leguminosarum strains forming nitrogen-rich nodules inPisum sativum

The nodules which developed on the roots ofPisum sativum after inoculation withRhizobium leguminosarum bv.viciae strains showing a Hup⁺ symbiotic phenotype and/or a high relative efficiency of electron transfer to dinitrogen (RE) had a high nitrogen content of their tissue. In comparison with the nodules initiated by a strain possessing the Hup⁻ symbiotic phenotype or by an indigenous soilRhizobium population, the nitrogen-rich root nodules contained up to four times more nitrogen (9.2% of dry mass). In the nitrogen-rich nodules, total amino acid, and especially Ala, Lys and Phe contents were significantly increased. The nitrogen-rich nodule symbiotic phenotype (Nrn) was well expressed, irrespective of pea cultivars and host plant age. If a strain showing the Nrn phenotype was applied in double-strain inocula, a significant correlation was found between increasing rates of the strain and increasing percentage nitrogen content of nodule tissue in the nodulated plants.     

Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L.

Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.     

Somatic embryogenesis and plant regeneration inPisum sativum L.

Somatic embryogenesis was induced in immature zygotic embryos of pea (Pisum sativum L.), synthetic auxins α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) being used. Only one (line HM-6) of 46 genotypes tested exhibited good potential for somatic embryogenesis. 2,4-D was found as the best somatic embryo inductor. Three different ways of somatic embryo conversion have been described. Plantlets from individual somatic embryos were micropropagated as somaclones and subsequently rooted. A sterile morphological mutant has been found within a group of fertile plants of T₀-generation. Sufficient amount of T₁-seeds is available for somaclonal variation studies.     

The effect of ethrel on nodulation inPisum sativum L.

Summary Nodulation of inoculated pea plants was severely inhibited when ethrel (2-chloroethane phosphonic acid) was applied to the roots. The inhibition was evident at ethrel concentrations of 2 ppm and above. Ethrel applied to the foliage at 10 ppm also depressed nodulation. At least part of the effect of ethrel on nodulation came from its inhibition of root growth. Nodulation was inhibited on half of a divided root system treated with ethrel, while there was no inhibition on the neighbouring untreated half.     

Regulation of the nitrate reductase inPisum sativum andtriticum aestivum by irradiation

Nitrate reductase (NR) activity of bothPisum sativum L. cv. Bonneville andTriticum aestivum L. cv. Sonalika seedlings was influenced by the phytochrome system. Short durations of “red” irradiation (R) increased extractable levels of NR whereas subsequent short “far-red” irradiation (FR) partially inhibited the R modulated increases. Qualitatively, a negative correlation existed between thein vitro NR activities andin vivo phytochrome levels inPisum. “Blue” irradiation (B) also increased extractable levels of NR inTriticum. A partial action spectrum study made by exposing excised etiolated leaves ofTriticum and shoot apices ofPisum revealed a maximum increase in extractable NR activities and tissue nitrate level (inTriticum) at 656 nm. A partial action spectrum for the extracted enzyme ofTriticum indicated that at 700 nm the level of activity was increased (as compared to dark controls) more than by R (656 nm), FR (725 nm) or B (425 or 450 nm) irradiation, although all wavelengths used increased NR activity.     

Time dynamics of primary root branching inPisum sativum L.

Time-intervals in which the branching of primary roots occurs (the formation of lateral root primordia and the appearance of laterals on the surface of primary roots) were determined by a statistical method in several cultivars of pea. The same time-determination of the branching of the primary root in all studied cultivars was found indicating a genetic regulation of this process.     

Spiral feedback oscillations of growing hypocotyl with radicle inPisum sativum L.

Cinematographic records of longitudinal growth showed that hypocotyl with radicle inPisum sativum L. undergoes spiral oscillations during growth. This phenomenon can be characterized by the following time-space limits:

1. (1)
   Curvature of the hypocotyl with radicle takes place always in the zone of most rapid elongation (Fig. 2, 8).     
   
   

Role ofLLD, a new locus for leaflet/pinna morphogenesis inPisum sativum

Properties of a mutant at theLLD (LEAF-LET DEVELOPMENT) locus in peaPisum sativum L. are reported in this paper. Plants homozygous for the Mendelian recessive mutationlld bear leaves in which a few to many leaflets are incompletely developed. Opposite pinnae of rachis nodes often formed fused incompletely developed leaflets. Thelld mutation was observed to abort pinna development at almost all morphogenetic stages. Thelld mutation demonstrated high penetrance and low expressivity. The phenotypes oflld plants intl, tac, tl tac, tl af andtl af tac backgrounds suggested that LLD function is involved in the separation of lateral adjacent blastozones differentiated on primary, secondary and tertiary rachides and lamina development in leaflets. The aborted development of tendrils and leaflets inlld mutants was related to deficiency in vascular tissue growth. The morphological and anatomical features of the leaflets formed on atl lld double mutant permitted a model of basipetal leaflet development. The key steps of leaflet morphogenesis include origin of the lamina by splitting of a radially symmetrical growing pinna having abaxial outer surface, opposite to the vascular cylinder, through an invaginational groove, differentiation of adaxial surface along the outer boundary of split tissue in the groove and expansion of the lamina ridges so formed into lamina spans.     

Some aspects of relation between nitrogen metabolism inPisum sativum and mineral nutrition

Summary Plants ofPisum sativum grown in water culture were subjected to deficiencies in the macronutrients N, P, K, S, Ca, Mg, substitution of ammonium for nitrate and change in pH to pH 3 or 9. Free amino acids, soluble nitrogen, protein and glutamate dehydrogenase activity of shoots were measured for all nutrient types. Changes in amino acid concentrations correlated well with changes in glutamate dehydrogenase. It is considered whether this enzyme is indicative of the nutritional state of plants.     

Host genes inPisum sativum L. conferring resistance to EuropeanRhizobium leguminosarum strains

Summary Using primitive and wild pea plants from Afghanistan, Iran and Turkey, three host genes were detected, which confer resistance to nodulation by Rhizobium strains of cultivated peas from Europe. A dominant gene Sym 1 controls temperature-sensitive nodulation in pea cv. Iran. Another gene Sym 2 confers general resistance to a large number of European Rhizobium strains at all temperatures used. The degree of dominance of the latter gene is dependent on the Rhizobium strain used. A third gene Sym 4 is responsible for specific resistance to a single Rhizobium strain.     

The absorption and translocation of zinc-65 inPisum sativum in relation to fruit development

Summary The specificity of zinc accumulation by the first pod ofPisum sativum, variety Alaska, from the leaf in whose axil this pod is borne has been demonstrated. The secondpod receives a relatively insignificant amount of this transported zinc. The distribution pattern is somewhat similar to that previously found for phosphorus but differs in that zinc is translocated from the first bloom-node leaf 2 days prior to anthesis. At this stage no phosphorus is being translocated from this leaf. The preferential accumulation of zinc by the first pod reaches a maximum about 5 days after full bloom and the magnitude of this accumulation, relative to the aerial vegetative parts of the plant, remains essentially constant as the pod approaches maturity. The zinc accumulated by the ovules increases gradually beginning at about the 5th day after full bloom and is accompanied by a decrease in zinc retained by the carpel.Paper No. 4979, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

The cytology of cotyledon cells and the induction of giant polytene chromosomes inPisum sativum

Summary The cotyledon cells ofPisum sativum have high DNA contents. By appropriate culture techniques, some of these cells can be triggered into division. Two types of dividing nuclei were seen. Firstly those that were polyploid with metaphases containing chromosome numbers ranging in value from 4 x to 32 x. Included among these were unexpected numbers equivalent to 12 x and 14 x. Secondly there were cells containing giant polytene chromosomes and these progressed from prophase to a metaphase where the polytene chromosomes separated into constituent single chromosomes.     

A cytochemical investigation of the host-parasite interface inPisum sativum infected by the downy mildew fungusPeronospora pisi

Summary A cytochemical study involving enzymic digestions, chemical extractions and specific staining methods was made of the host-parasite interface in downy mildew infected pea plants. Enzymic hydrolysis revealed both the penetration and extrahaustorial matrices to have a proteinaceous component, possibly glycoprotein, while the extrahaustorial matrix had cellulose as an additional constituent. Intense silver proteinate staining of both matrices following a prolonged incubation in thiocarbohydrazide indicated the presence of complex carbohydrates. Carbohydrates were confirmed as constituents of both matrices following the complete suppression of silver staining using the aldehyde blocking agents dimedone and sodium borohydride. Both matrices also exhibited a marked affinity for phosphotungstic acid. Following acetylation a complete elimination of phosphotungstate staining resulted, again indicating carbohydrate as a constituent of both matrices. Digestion of the fungal cell wall using an enzyme which hydrolyses -1,3-glucans showed that these carbohydrates are important in its construction. However such enzyme treatment did not affect the structural integrity of either the penetration or extrahaustorial matrix. The extrahaustorial membrane exhibited negligible staining with phosphotungstic-chromic acid treatment, while the host plasmalemma showed a positive but variable staining reaction. A very intense staining reaction characterized the fungal plasmalemma after staining with either phosphotungstic-chromic acid, phosphotungstic acid or silver proteinate.     

Relationships between nitrate level,nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO₃-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - ax ^b) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO₃-R activity correlated well with thein vitro activity up to the 50 ppm NO₃-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.