Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

Relationships among pure cultured strains of Frankia based on host specificity

Fifty strains of Frankia were tested for their ability to nodulate six species of actinorhizal plants. Pure cultured strains were used to inoculate seedlings of Alnus glutinosa (L.) Gaertn., Alnus rubra Bong., Casuarina equisetifolia L., Elaeagnus angustifolia L., Hippophaë rhamnoides L. and Myrica cerifera L. in nutrient solution culture. From the results of this study, host inoculation groups among the actinorhizal plants were defined. Although overlap between host inoculation groups appears to be common, the results from this study did not support the view that Frankia strains are promiscuous. All Frankia strains tested in this study could easily be classified into four major host-specificity groups.     

Cultivar and pH effects on competition for nodule sites between isolates of Rhizobium in beans

Two experiments were carried out to evaluate the effect of acidity on bean-Rhizobium competition for nodule sites. SevenPhaseolus vulgaris host cultivars differing in acid-pH tolerance were grown in sand culture, and irrigated using a sub-irrigation system and nutrient solutions of pH 4.5, 5.0, 5.5, and 6.0. A mixed inoculant of two antibiotically markedRhizobium leguminosarum bvphaseoli strains CIAT899 (acid-tolerant) and CIAT632 (acid-sensitive) was used. The acid-tolerant CIAT899 dominated CIAT632 in nodule occupancy across all cultivars and pH treatments. Although several of the varieties had previously been identified as PH-tolerant, and these cultivars performed better than those reported to be acid sensitive, all showed a marked increase in nodulation and plant development when the pH was raised from 4.5 to 6.0. The second experiment using a modified Leonard jar system varied the inoculation ratio between CIAT899 and UMR1116 (acid-sensitive, inefficient in N₂-fixation) and contrasted nodulation response for the bean varieties Preto 143 (pH-tolerant) and Negro Argel (pH-sensitive) at 3 pH treatments (4.5, 5.5, 6.5). There was a significant effect of host cultivar, ratio of inoculation, and pH on the percentage of nodule occupancy by each strain. At low pH CIAT899 had higher nodule occupancy than UM1116 in the variety Negro Argel but had the same percentage of nodulation when the variety was Preto 143. Increasing the cell concentration of UMR1116 produced more inefficient nodules at all treatment combinations and reduced plant growth for both cultivars used.     

Rhizobium meliloti inoculation of alfalfa selected for tolerance to acid,aluminum-rich soils

Alfalfa (Medicago sativa L.) growth and nodulation in acid soil is reduced because the plant and its bacterial symbiontRhizobium meliloti cannot tolerate acid, aluminum-rich soil. A study was conducted to determine if a relatively acid-tolerant alfalfa germplasm combined with a relatively acid-tolerantR. meliloti strain could overcome these limitations. In a light room study, an acid-tolerant alfalfa germplasm inoculated with a more acid-tolerantR. meliloti strain produced greater top growth, nodule number and weight, and acetylene reduction values in an unlimed soil (pH 4.6) than the same germplasm inoculated with a relatively acid-sensitiveR. meliloti strain or an acid-sensitive germplasm inoculated with either a relatively acid-tolerant or acid-sensitiveR. meliloti strain.     

Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth ofCeanothus griseus varhorizontalis

Rooted cuttings ofCeanothus griseus varhorizontalis were irrigated with 0, 10, 20, 50, 75 or 100ppm nitrogen as NH₄NO₃ for eight weeks prior to inoculation with infectiveFrankia. After inoculation, half of the plants for each treatment nitrogen level continued to be irrigated with the preconditioning nitrogen level and half were given no more supplemental nitrogen. For plants continuously receiving nitrogen, nodule initiation (nodule number) was inversely correlated with increasing supplemental nitrogen levels, and suppressed above 50 ppm N. Leaf nitrogen above 2% in continuous-N plants correlated with greatly reduced or suppressed nodulation. Plants maintained after inoculation without supplemental nitrogen showed influence of the prior nitrogen treatment on nodulation. Preconditioning at 50 ppm and above greatly reduced the number of nodules formed. The evidence suggests that stored internal nitrogen can regulate nodulation.Plant biomass accumulated maximally when nodulation was suppressed, at 75 and 100 ppm supplemental N applied continuously. Internode elongation during the nodulation period occurred only on nodulated plants, or in the presence of supplemental N (10 ppm and above).     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions