M. Lalonde

Restriction enzyme digestion patterns of Frankia plasmids

SummaryAfter the initial screening of more than 200Frankia strains, the plasmid DNA observed in eight Frankiae was analyzed.In situ lysis was performed to obtain an estimate of their copy number and molecular weight. Four plasmid classes were distinguished, 7–9, 18–20, 30–35 and 50–55 kb. Twelve plasmids were thus analysed with restriction enzymes to determine their plasmid restriction patterns.While someFrankia plasmids with comparable molecular weights were found to be heterologous in their restriction enzyme pattern, an 8 kb plasmid found in bothFrankia sp. ArI3, isolated fromAlnus rubra andFrankia sp. CpI1 isolated fromComptonia peregrina showed undistinguishable ‘fingerprints’. Furthermore, an 18 kb plasmid found in the same two strains, also showed homologous restriction enzyme patterns. However, the copy numbers of the two ArI3 plasmids were higher than those of the CpI1 plasmids.Similarly, strains ACN1AG,

Requirements for in vitro propagation of seven nitrogen-fixing Alnus species

TX41_{b^--- }^{AC} and TX38_{b^--- }^{AC}

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens

, isolated fromAlnus crispa all contained a 50 kb plasmid, and the three plasmids were found upon restriction analysis to be undistinguishable.In one strain, ARgX17c isolated fromAlnus rugosa, it was found through restriction enzyme analysis that two plasmids of a similar molecular weight were in fact heterologous.The possible origin of the homologous plasmids and their potential as specificFrankia markers to be used in ecological studies are discussed.

Performance of in vitro propagated Alnus glutinosa (L.) Gaertn. clones inoculated with Frankiae

In 1978, nothing could have been said about the genetics of a microorganism which had never been isolated except by Pommer 7~ who, in an unnoticed report, described an actinomycete now recognized as being a true Frankia. But in the six years that have elapsed since Callaham et aL 19 isolated CpIt in pure culture the question of the genetics of Frankia has arisen for many reasons. The economic importance of biological nitrogen fixation is well established especially with regard to leguminous plants for the agriculture of less developed countries which cannot afford the intensive use of fertilizers required for the sustenance of their rapidly growing populations. Similarly, forestry in many areas faces the problem of clear-cut or burned land on which it takes as long as fif ty years for the natural succession to replenish the timber to an economically profitable stage, due in large part to nutrient removal from the sites 4 . Non-legumes or actinorhizal plants associated with the appropriate Frankiae provide the main input of biologically-fixed nitrogen in temperate regions and they are already being used for the re-afforestation of mine spoils in Pensylvania 22 , to establish rapid plant covers on dam dykes in New Quebec 32 to serve as nurse trees for the production of highly valuable black walnut in Illinois 33 or Douglas fir on the West Coast 4 , and in general, as windbreaks, highway landscaping, for land reclamation and as ornamental species 3~ . In Africa and the coastal regions of China, Casuarina or filao is being planted on an enormous scale to stabilize the coastal sand dunes and protect the agricultural fields lying behind it from being buried under metres of sand (Dommergues and Zhao, pers. comm.). But other tree species are more important economically than alder or filao, for pulp and paper production or furniture and house building. The actinorhizal symbiosis is quite different in its interactions from that of Rhizob ium with legumes 37 . For instance, Frankia has a much more varied host spectrum, being in symbiosis with t9 genera o f dicotyledonous plants belonging to seven families 1 . Fundamental

Gene transfer by electroporation in betulaceae protoplasts: Alnus incana

Studies on the in vitro propagation of Alnus crispa, A. glutinosa, A. incana, A. japonica, A. rubra, A. sinuata and A. viridis indicated interspecific as well as intraspecific variations in their requirements for in vitro culture. The WPM and Blaydes media supported, respectively, growth of A. glutinosa and A. crispa but not that of both species, while the MS medium induced equal or significantly better growth than WPM and Blaydes media for both species. The optimum type and concentration of sugar to be used in the multiplication medium varied with species. Only A. glutinosa showed good growth on sucrose while glucose was optimum for all other species but at different concentrations. All species rooted in 3 weeks on half-strength MS medium including 1 μM IBA. All clones of A. glutinosa and A. rubra rooted 100%, whereas “easy-to-root” and “difficult-to-root” clones were observed in the other species. In the rooting medium, glucose promoted rooting of the “difficult-to-root” clones better than sucrose. Survival following transfer to an artificial substrate was 100% for all species. Nodulation tests using pure cultures of two Frankia strains showed 100% nodulation on all Alnus clones.

Detection of pectolytic activity andpel homologous sequences inFrankia

In 1978, nothing could have been said about the genetics of a microorganism which had never been isolated except by Pommer who, in an unnoticed report, described an actinomycete now recognized as being a true Frankia. But in the six years that have elapsed since Callaham et al. isolated CpI1 in pure culture the question of the genetics of Frankia has arisen for many reasons. The economic importance of biological nitrogen fixation is well established especially with regard to leguminous plants for the agriculture of less developed countries which cannot afford the intensive use of fertilizers required for the sustenance of their rapidly growing populations. Similarly, forestry in many areas faces the problem of clear-cut or burned land on which it takes as long as fifty years for the natural succession to replenish the timber to an economically profitable stage, due in large part to nutrient removal from the sites. Non-legumes or actinorhizal plants associated with the appropriate Frankiae provide the main input of biologically-fixed nitrogen in temperate regions and they are already being used for the re-afforestation of mine spoils in Pensylvania, to establish rapid plant covers on dam dykes in New Quebec to serve as nurse trees for the production of highly valuable black walnut in Illinois or Douglas fir on the West Coast, and in general, as wind-breaks, highway landscaping, for land reclamation and as ornamental species. In Africa and the coastal regions of China, Casuarina or filao is being planted on an enormous scale to stabilize the coastal sand dunes and protect the agricultural fields lying behind it from being buried under metres of sand (Dommergues and Zhao, pers. comm.). But other tree species are more important economically than alder or filao, for pulp and paper production or furniture and house building.

Selection and micropropagation of nodulating and non-nodulating clones of Alnus crispa (Ait.) Pursh

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine.

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

SummaryThreeAlnus glutinosa (L.) Gaertn. clones, obtained byin vitro propagation techniques, were inoculated with four strains ofFrankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.Shoot fresh weight, shoot height and collar diameter were measured 60 and 82 days after inoculation. Shoot fresh weight seems to be more sensitive and reliable than the other parameters. Nitrogenase activity, measured by the acetylene reduction assay, was assayed 78 days after inoculation and was consistent with the biomass measurements.Better growth was observed when type N strains were used. Significant growth differences were observed between clones AG-2 and AG-8 on the one hand and clone AG-4 on the other. Thus, the use of genetically defined host plants and microsymbionts permitted the demonstration of significant performance variation even among cloned plants from the same provenance (AG-4 and AG-8).The duration of the experiment influenced the results with differences becoming less significant with time. This might be caused by an external limiting factor such as the pot size, competition for light,etc. But it could also be indicative of differences in nodulation speed among the treatments.