Common bean ( Phaseolus vulgaris ) is the major food legume worldwide, making it an important target for novel approaches of genetic analysis. This study evaluated the use of ethyl methane sulfonate (EMS) for the generation of a mutant population for targeted induced local lesions in genomes (TILLING) in common bean. TILLING is a powerful reverse genetics approach that uses a large mutant population for identification of mutants in loci of interest. Based on overall survival, development, and yield of treated seed, 40 m m EMS was found to be an appropriate concentration for the generation of a mutant population in common bean genotype BAT 93. Higher concentrations of EMS resulted in survival rates of less than 10% and lower concentrations resulted in the generation of fewer mutants. Based on TILLING results from other species, a population of 5000 lines is estimated to be sufficient for saturation of the common bean genome. Phenotypic mutation frequencies and the isolation of targeted mutations in the BAT 93 mutant population indicate that mutagenesis was effective.

Common bean (Phaseolus vulgaris) is the most widely grown grain legume for human consumption and a major protein and mineral Source ill East Africa and Latin America. It is also a simple diploid species with a small genome (650 Mb). Despite its nutritional and economic importance and tractable genome. P. vulgaris has a paucity of mutant resources compared to other crops, making it difficult to perform genetic screening in the species. In this review we discuss recent Studies on mutagenesis that aim to produce large-scale, mutagenized populations for generalized trait screening, as well as previous EMS (ethyl methane sulfonate) and gamma radiation Mutants that were developed for biological nitrogen fixation or plant morphology traits. Mutant stocks in this crop will allow researchers to Conduct both forward (systematic phenotypic screening and reverse genetics (such as TILLING, or Targeting Induced Local Lesions In Genomes) experiments aimed at understanding the genes involved in various traits. including abiotic and biotic stress tolerance, grain quality. and nutritional Value, as well as genes involved in symbiosis with Rhizobia. Thus. mutant stocks will be important for gene discovery and creating novel variability. In this review. we highlight applications of mutation breeding for nutritional quality improvement of common bean. giving examples of seed protein, mineral content, and tannin accumulation traits.

A Serve-Ag Research, PO Box 690, Devonport, Tas 7310 B Queensland Department of Primary Industries, GPO Box 46, Brisbane, QLD 4001 C Department of Primary Industries, Water and Environment, Devonport, PO Box 303, Tas 7310 D Natural Resource Sciences, Department of Natural Resources and Mines, 80 Meiers Road, Indooroopilly Qld 4068 E Biological Crop Protection, 3601 Moggill Rd, Moggill, Qld 4070 F CSIRO Land and Water, Davies Laboratory,PMB, PO Aitkenvale, Townsville, Qld 4814 G NSW Agriculture, Yanco Agricultural Institute, PMB Yanco 2703, NSW

The effect of a change of tillage and crop residue management practice on the chemical and microbiological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0–5 cm of soil, in organic C, total N, electrical conductivity, NH4-N, NO3-N, soil moisture holding capacity, microbial biomass and CO2 respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0–5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.

Fatty acid methyl ester (FAME) analysis performed on the spores of four arbuscular mycorrhizal (AM) fungi (Glomus coronatum, Glomus mosseae, Gigaspora margarita and Scutellospora calospora) showed 16:1ω5c to be the dominant fatty acid present. In addition, spores of Gi. margarita contained large quantities of 18:1ω9c and three 20-C fatty acids (20:1ω9c, 20:2ω6c and 22:1ω9c) that were not present in the spores of the other two species. Addition of a known number of spores of each AM species to soil demonstrated that the spore fatty acids could be readily detected and quantified against the background of soil fatty acids. Addition of different combinations and quantities of spores to soil gave the expected ratios of the marker fatty acids in the soil FAME profiles. The results confirm the use of 16:1ω5c as a marker fatty acid for AM fungi in controlled environments and suggest that 18:1ω9c, 20:1ω9c, 20:2ω6c and 22:1ω9c could be used as possible markers for the detection of Gi. margarita.

Vance, C. P., Reibach, P. H. and Pankhurst, C. E. 1987. Symbiotic properties of Lotus pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium sp. ( Lotus ). Symbiotic properties of root nodules were evaluated in glasshouse‐grown Lotus pedunculatus Cav. cv. Maku inoculated with either a fast‐growing Rhizobium loti strain NZP2037 or a slow‐growing Bradyrhizobium sp. ( Lotus ) strain CC814s. Although the nodule mass of plants inoculated with NZP2037 was twice that of plants inoculated with CC814s, the yield of NZP2037 shoots and roots was 50% that of CC814s shoots and roots. Nodules induced by Bradyrhizobium fixed substantially more N than nodules induced by R. loti. Glucose requirements [mol glucose (mol N 2 fixed) ‐1 ] of nodules induced by CC814s and NZP2037 were 7.1 and 16.6, respectively. Nodule enzymes of carbon and nitrogen assimilation reflected the disparity of the two sym‐bioses. Xylem sap of the symbiosis with the higher yield contained a higher concentration of asparagine [9.86 μmol (ml xylem sap)‘] than did the lower yielding symbiosis [5.80 umol (ml xylem sap)“’]. Nodule CO 2 fixation was directly linked to nodule N assimilation in both symbioses. The results indicate that the difference between the two symbioses extend to nodule N and C assimilation and whole plant N transport. The data support a role for host plant modulation of bacterial efficiency and assimilation of fixed N.

Cosmids containing a nodulation gene from Rhizobium loti NZP2037 were isolated using a 12.8 kb nod:: Tn5EcoRI fragment from the Nod- mutant strain PN233, as a hybridisation probe. A physical map of the nod region was established using the enzymes EcoRI and HindIII and the site of insertion of Tn5 in PN233 determined. Site-specific exchange of the cloned nod:: Tn5 fragment demonstrated that Tn5, and not an indigenous insertion sequence, was responsible for the nod mutation in PN233. The nod cosmids isolated complemented the Nod- phenotype of strain PN233 but restoration of the Fix phenotype was variable suggesting a need for marker rescue to occur before nitrogen fixation occurred.

The symbiotic effectiveness of Rhizobium trifolii strains isolated from an established white clover (Trifolium repens L.)/ryegrass pasture over a 3-year period ranged from 24 to 130% of the effectiveness of a commercially recommended strain NZP561 (PDDCC 2163) when tested on ‘Grasslands Huia’ white clover. An effective field strain (NZP564) was superior to NZP561 in establishment and persistence in this soil. Soil inoculation using liquid inoculants gave better establishment and persistence of both NZP561 and NZP564 than seed inoculation. Keywords: Rhizobium trifolii Trifolium repens L.nitrogen fixationinoculationantibiotic resistance

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.

Light and electron microscopic studies have established that the endophyte of root nodules of Discaria toumatou is an actinomycete resembling those of other Frankia-induced actinorhizal nodules. The prokaryotic micro-organism has septate hyphae of 0.4–0.7 µm diam., and forms spherical vesicles of approx. 4 µm diam. in the infected plant cells. These vesicles contain complete and incomplete septa, granulated regions, electrondense regions, and prominent nucleoids. No sporangia or spores were seen in the nodules. Both the hyphal and vesicular forms of the endophyte are surrounded by a polysaccharide capsule. The infected host cells contain a single, large, lobed nucleus, many large plastids and mitochondria, and a few microbodies. Keywords: anatomyactinomyceteroot nodule Discaria toumatou Frankia actinorhizal nodulesultrastructurecytologyhyphaerootsnitrogen fixationNew Zealand