Genetic improvement of legumes using somatic cell and molecular techniques

Summary The merits and limitations of somatic cell techniques involving Agrobacterium-mediated transformation, direct gene transfer and protoplast fusion, are discussed in relation to the genetic improvement of forage and grain legumes. Whilst progress with legumes is limited compared to that with plants of other families such as the Solanaceae, the fact that many legumes are readily amenable to tissue culture now permits somatic cell techniques to be targetted to these species. Future development of the subject will necessitate close collaboration between molecular biologists and plant breeders to enable novel plants generated by in vitro technologies to be incorporated into conventional breeding programmes.     

The biotechnology of crop legumes

Summary The absence of variety-independent gene transfer methods for major agronomic species has, until now, limited the usefulness of recombinant DNA techniques to crop improvement programs. Until recently, only Solanaceous crops could be used to study fundamental and applied problems in plant sciences. During the past five years rapid advances in cell biology, in combination with the development of novel gene transfer methodology allowed utilization of the tools of plant molecular biology in conventional breeding programs. Cereal and leguminous species were considered to be recalcitrant to genetic manipulation. As a result of the development of direct DNA transfer methodology into organized tissue, we are now in a position to introduce any foreign gene into almost all of the major cereals and legumes. This can be achieved efficiently, often in a variety-independent fashion. The object of this review is to provide a comprehensive account of the state of the art in gene transfer for the cultivated leguminous crops. Important oilseed and feed species primarily in industrialized countries, as well as minor but equally important species for sustaining growth populations in developing countries will be examined. Advantages of the various gene transfer methods that were shown to be useful for specific crops, as well as limitations and problems associated with each crop and gene transfer method will be discussed. Data from field trials of transgenic legumes, where available, will be presented.     

Transfer of Grain Legume Nitrogen within a Crop Rotation Containing Winter Wheat and Winter Barley

In a crop rotation trial, conducted from 1985 to 1988 at TU-Munich's research station in Roggenstein, the transfer of grain legume nitrogen was evaluated in crop rotations containing fababeans and dry peas as well as oats (reference crop) and winter wheat and winter barley as following crops. The results obtained can be summarized as follows: Dinitrogen fixation by fababeans ranged from 165 to 240 kg N ha¹, whereas N₂-fixation by peas amounted from 215 to 246 kg N ha^?1. In all seasons the calculated N-balance where only grain was removed was positive, with a net gain being on average 106 (peas) and 84 (fababeans) kg N ha^?1. After the harvest of peas 202 kg N ha^?1 remained on the field on average over seasons (158 kg N ha^?1 in the above ground biomass and 44 kg N ha^?1 as NO₃-N in 0–90 cm depth). As compared to peas, fababeans left 41 kg N ha^?1 less due to smaller amounts of nitrogen in the straw. After oats very small amounts of residual nitrogen (33 kg N ha^?1) were detected. After the harvest of grain legumes always a very high nitrogen mineralization was observed during autumn especially after peas due to a close C/N-relationship and higher amounts of nitrogen in the straw as compared to fababeans. In comparison with fababeans, N-mineralization after the cultivation of oats remained lower by more than 50%. During winter, seepage water regularly led to a considerable decrease of soil NO₃-N content. The N-leaching losses were especially high after cultivation of peas (80 kg N ha ^?1) and considerably lower after fababeans (50 kg N ha^?1) and oats (20 kg N ha^?1). As compared to oats, a higher NO₃-N content in soil was determined at the beginning of the growing period after preceding grain legumes. Therefore, winter wheat yielded highest after preceding peas (68 dt ha^?1) and fababeans (60 dt ha^?1) and lowest after preceding oats (42 dt ha^?1). The cultivation of grain legumes had no measurable effect on yield formation of the third crop winter barley in either of the growing seasons.     

Screening techniques and improved biological nitrogen fixation in cool season food legumes

Dinitrogen fixation and legume productivity are greatly influenced through the interactions of legume host, Rhizobium, and the above- and below-ground environment. The benefits of improving legume N₂ fixation include reduced reliance on soil N, leading to more sustainable agricultural systems and reduced requirements for fertilizer N, enhanced residual benefits to subsequent crops, and increased legume crop yields. Most of the gains in N₂ fixation to date have been derived from management of legume cropping systems and through inoculation of legume seed with competitive and symbiotically effective rhizobia. Further gains are possible by developing plant cultivars with tolerance to soil abiotic factors, increased plant yield, and a broader and more effective matching of plant host and rhizobia. Techniques for screening material for superior N₂ fixation and examples of programs to increase fixed N, with attention to the major abiotic stresses, are discussed.     

Ground vegetation has a major role in element dynamics in an ash-fertilized cut-away peatland

In Finland, recycling of wood-ash as a fertilizer has been widely studied in recent decades in peatlands drained for forestry. Wood-ash is reported to promote tree growth without any significant negative impacts on the environment and could, therefore, be a suitable option on cut-away peatlands, which are estimated to increase by 30,000 ha over the next ten years. However, the environmental effects of ash-fertilization on cut-away peatlands or on ground vegetation are not well known. We studied the effects of wood- and peat-ash application on the nutrient content in soil and plants and on the heavy metal content in the vegetation of a cut-away peatland. Six treatments of different quantities and mixtures of wood-ash, peat-ash, biotite, or Forest PK-fertilizer were replicated in three plots. Both wood- and peat-ash fertilization ensured an adequate level of nutrients for the early establishment and growth of ground vegetation and birch seedlings in a cut-away peatland. Dense pioneer moss cover was important in trapping the heavy metals, such as cadmium, which are present in the ash. Ground vegetation of mosses and herbaceous plants also had a major role in the retention of the nutrients that could otherwise have leached from the open cut-away peatland area during the early stages of afforestation. Both wood- and peat-ash proved to be suitable for the initial fertilization of afforested cut-away peatlands, but in order to guarantee a sufficient long-term potassium supply for tree growth, an application of biotite with the peat-ash is recommended.     

全国小豆豇豆优异种质资源综合评价

将“六五”和“七五”期间各省、市初步选出的优异种质,包括单一性状和综合性状优异的种质资源,在“八五”期间进行再评价。安徽试点承担小豆优异种质产量鉴定、豇豆优异种质产量鉴定和豇豆优异种质资源观察鉴定圃等三项试验。经田间试验和统计分析,初步选出7—8个产量高、综合性状优良的小豆和豇豆品种,还选出一批具有矮生、早熟、大粒、多荚、蛋白质含量高的单项性状优良的种质资源,供育种、生物科学和生产上利用。     

豆科蔬菜种子贮藏蛋白的电泳分析

本研究采用ＳＤＳ－聚丙烯酰胺凝胶电泳，对豆科蔬菜９个属、１１个种及品种的种子贮藏蛋白进行比较分析，得出豆科的特征谱带及各属的特征谱带。其植株生长势越强，谱带条数越多，即所含蛋白亚组分越多；同属不同种间比较，得出蔓生菜豆进化程度较高，特有谱带６条；同种不同品种间迁移率相同的亚组分较多，但谱带宽窄、染色深浅等存在明显差异，表现出一些本品种的特征特性，采用生物化学分类法对蔬菜作物进行分类，改变了传统的形态学分类，也使植物分类技术向分子水平迈进，为豆类蔬菜育种提供一些参考。     

Nitrogen fixation and beneficial effects of some grain legumes and green-manure crops on rice

Studies were conducted on paddy soils to ascertain N₂ fixation, growth, and N supplying ability of some green-manure crops and grain legumes. In a 60-day pot trial, sunhemp (Crotalaria juncia) produced a significantly higher dry matter content and N yield than Sesbania sesban, S. rostrata, cowpeas (Vigna unguiculata), and blackgram (V. mungo), deriving 91% of its N content from the atmosphere. Dry matter production and N yield by the legumes were significantly correlated with the quantity of N₂ fixed. In a lowland field study involving sunhemp, blackgram, cowpeas, and mungbean, the former produced the highest stover yield and the stover N content, accumulating 160–250 kg N ha^-1 in 60 days, and showed great promise as a biofertilizer for rice. The grain legumes showed good adaptability to rice-based cropping systems and produced a seed yield of 1125–2080 kg ha^-1, depending on the location, species, and cultivar. Significant inter- and intraspecific differences in the stover N content were evident among the grain legumes, with blackgram having the highest N (104–155 kg N ha^-1). In a trial on sequential cropping, the groundnut (Arachis hypogaea) showed a significantly higher N₂ fixation and residual N effect on the succeeding rice crop than cowpeas, blackgram, mungbeans (V. radiata), and pigeonpeas (Cajanus cajan). The growth and N yield of the rice crop were positively correlated with the quantity of N₂ fixed by the preceding legume crop.     

Indigenous rhizobia associated with native shrubby legumes in Taiwan

Root-nodulating bacteria were isolated and characterized from seven native shrubby legumes growing in Taiwan. Phenotypic characteristics measured included growth rates in various media, colony morphology, and tolerances to extremes of temperature, salt and pH. The isolates were very diverse phenotypically. Among the 83 isolates that were screened, the majority were fast-growing rhizobia. Twenty eight strains tolerated high concentration of salt (4.5% NaCl) and grew well between temperatures of 37 and 45 °C. The majority of the strains also tolerated extreme pH in their medium from 3.5 to 12. All strains formed nitrogen fixing nodules, and the highest activity was detected in the legume Hedysarum crinita L. PCR restriction fragment length polymorphism (PCR-RFLP) and sequencing of the small subunit ribosomal RNAs revealed that the majority of the isolates belonged to the genera Rhizobium, Bradyrhizobium and Agrobacterium. Only a single strain represented the genus Sinorhizobium. In addition, a strain related to Burkholderia from the β-class of the Proteobacteria (CC-CC-5) was found within nodules of the legume Catenaria caudatum. The study contributes to the understanding of symbiotic nitrogen fixation in selected wild legumes that are native to Taiwan and provides insights into the distribution of nodulating and nitrogen-fixing bacteria from other distinct lineages.     

Root-nodule bacteria from indigenous legumes in the north-west of Western Australia and their interaction with exotic legumes

Bacteria were isolated from root-nodules collected from indigenous legumes at 38 separate locations in the Gascoyne and Pilbara regions of Western Australia. Authentication of cultures resulted in 31 being ascribed status as root-nodule bacteria based upon their nodulation of at least one of eight indigenous legume species. The authenticated isolates originated from eight legume genera from 19 sites. Isolates were characterised on the basis of their growth and physiology; 20 isolates were fast-growing and 11 were slow-growing (visible growth within 3 and 7 d, respectively). Fast-growers were isolated from Acacia, Isotropis, Lotus and Swainsona, whilst slow-growers were from Muelleranthus, Rhynchosia and Tephrosia. Indigofera produced one fast-growing isolate and seven slow-growing isolates. Three indigenous legumes (Swainsona formosa, Swainsona maccullochiana and Swainsona pterostylis) nodulated with fast-growing isolates and four species (Acacia saligna, Indigofera brevidens, Kennedia coccinea and Kennedia prorepens) nodulated with both fast- and slow-growing isolates. Swainsona kingii did not form nodules with any isolates. Fast-growing isolates were predominantly acid-sensitive, alkaline- and salt-tolerant. All slow-growing isolates grew well at pH 9.0 whilst more than half grew at pH 5.0, but all were salt-sensitive. All isolates were able to grow at 37 °C. The fast-growing isolates utilised disaccharides, whereas the slow-growing isolates did not. Symbiotic interactions of the isolates were assessed on three annual, one biennial and nine perennial exotic legume species that have agricultural use, or potential use, in southern Australia. Argyrolobium uniflorum, Chamaecytisus proliferus, Macroptilium atropurpureum, Ononis natrix, Phaseolus vulgaris and Sutherlandia microphylla nodulated with one or more of the authenticated isolates. Hedysarum coronarium, Medicago sativa, Ornithopus sativus, Ornithopus compressus, Trifolium burchellianum, Trifolium polymorphum and Trifolium uniflorum did not form nodules. Investigation of the 31 authenticated isolates by polymerase chain reaction with three primers resulted in the RPO1 primer distinguishing 20 separate banding patterns, while ERIC and PucFor primers distinguished 26 separate banding patterns. Sequencing the 16S rRNA gene for four fast- and two slow-growing isolates produced the following phylogenetic associations; WSM1701 and WSM1715 (isolated from Lotus cruentus and S. pterostylis, respectively) displayed 99% homology with Sinorhizobium meliloti, WSM1707 and WSM1721 (isolated from Sinorhizobium leeana and Indigofera sp., respectively) displayed 99% homology with Sinorhizobium terangae, WSM1704 (isolated from Tephrosia gardneri) shared 99% sequence homology with Bradyrhizobium elkanii, and WSM1743 (isolated from Indigofera sp.) displayed 99% homology with Bradyrhizobium japonicum.