Action of 2,4-DB and dalapon on the symbiotic properties ofLotus corniculatus (birdsfoot trefoil)

Summary Field trials carried out in 1965 and 1966 showed that 2,4-DB, alone or in combination with dalapon, reduced nodulation and tended to decrease the efficiency of nitrogen fixation in birdsfoot trefoil. Dalapon appeared to enhance the inhibitory action of 2,4-DB on nodulation. No obvious cytological differences could be detected in the nodules or in the isolated bacteroids of field-treated and untreated plants. Under growth chamber conditions, 2,4-DB drastically reduced trefoil growth and nodulation particularly in treatments where the herbicide came directly in contact with the plants. It appears that the reduction in nodulation and nitrogen fixation is a result of plant damage and abnormal root growth caused by 2,4-DB application.Autoradiographs indicated that the translocation of the herbicide was rapid, with detectable concentrations observed in young leaves, leafveins, roots, and nodules 12 hours after leaf-feeding of 2,4-DB-1-C¹⁴. The radio-activity appeared to accumulate with time (up to 5 days) in the growing root tips and nodules. Fractionation of excised nodules from trefoil plants demonstrated the presence of radioactivity in the cell debris, bacteroids, 29,000g pellet, plant ribosomes, and the soluble portion. The greatest accumulation of radioactivity occurred in the soluble fraction.The degradation of 2,4-DB and 2,4-D in trefoil was demonstrated by the evolution of C¹⁴O₂ from non-nodulated and aseptically growing plants leaf-fed with 2,4-DB-1-C¹⁴ or 2,4-D-1-C¹⁴.4-(2,4-dichlorophenoxy) butyric acid.2,2 dichloropropionic acid.     

Male sterility in birdsfoot trefoil (Lotus corniculatus L.)

Summary This paper describes six male steriles in birdsfoot trefoil. Data on the following characters were recorded on these plants and on two male fertile plants: chromosome number, presence of pollen at tripping, percentage of irregularly shaped anthers, percentage of anthers with acetocarmine stainable pollen inside, maximum range of percentage of pollen fertility among anthers within a flower presenting pollen, and percentage of pod setting with selfing. All male steriles showed a somatic chromosome number typical of the species. All were indehiscent and complete control of pollination occurred in all but one. They differed in percentage of irregular anthers, percentage of anthers with stainable pollen inside, and range of percentage of pollen fertility among anthers within a flower. Indehiscence in flowers having some pollen inside the anthers was probably due to lack of internal pressure. It is concluded that these genotypes can be of value in breeding birdsfoot trefoil.The study was financed by the EEC Agrimed project Reperimento e valutazione di germoplasma di leguminose foraggere utilizzabili nell'impianto di prato-pascoli in ambiente mediterraneo e studio dei problemi connessi con la produzione delle loro sementi and was conducted at Istituto di Miglioramento Genetico Vegetale, Università degli Studi, Borgo XX Giugno, Perugia, Italy     

Isolation,culture and regeneration of protoplasts from birdsfoot trefoil (Lotus corniculatus)

A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Green cotyledons from in vitro grown seedlings were preplasmolyzed in CPW salts containing 13% mannitol (CPW 13 M) for 1 h prior to the enzyme treatment. The enzyme formula consisted of 2% (w/v) Onozuka Cellulase R-10, 1% (w/v) Macerase and 0.1% (w/v) Pectolyase Y-23 in CPW 13 M. This method produced high yields of viable protoplasts after purification. The procedure is reproducible and takes approximately 2.5 months from protoplast isolation to plantlet establishment in a greenhouse. More than 100 plantlets were grown in soil. Two somaclonal variants, a chimeric plant for chlorophyll production and an albino cell line, have been obtained by this procedure.     

Self-fertility problems in birdsfoot trefoil (Lotus corniculatus L.)

The problems of Lotus corniculatus self-fertility are analyzed. The self-compatibility of hereditivity character, the efficacy of different methods of self-pollination (autogamy, geitonogamy within one plant and among plants of one clone), and usage of the mixture of self-compatible and self-incompatible pollen were studied.     

Prolific plant regeneration from protoplast-derived tissues of Lotus corniculatus L. (birdsfoot trefoil)

Protoplasts isolated enzymatically from seedling roots, hypocotyls and cotyledons of Lotus corniculatus L. produced callus which underwent prolific shoot regeneration. The rapidity and ease of recovering plants from protoplast-derived tissues makes this forage legume an attractive experimental system for genetic manipulation.     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

Genetic transformation of a pasture legume,Lotus corniculatus,L. (birdsfoot trefoil)

Summary A rapid procedure for introducing foreign genes inLotus corniculatus based on the induction of hairy roots byAgrobacterium rhizogenes was developed. Expression of chloramphenicol acetyltransferase and neomycin phosphotransferase II was revealed in transgenic plants. Southern blot hybridization was used to confirm the genetic transformation. The transgenic plants looked normal and did not show any morphological modification compared to the seed grown plants.     

Enhanced shoot regeneration from homogenized callus cultures of birdsfoot trefoil (Lotus corniculatus L.)

A homogenization and plating technique is described which increases the number of shoots produced and decreases the time required for plant regeneration from callus cultures of birdsfoot trefoil. A 2- to 15-fold increase in the number of plants recovered per gram of callus is observed depending on the genotype. Characterization of a sample of the regenerated plants indicated no differences between plants from homogenized versus nonhomogenized callus for traits such as time of first flower, number of branches per plant, pollen stainability, stomate length, and whole plant yield. The technique has proven useful for efficient recovery of plants from long-term cultures and cultures selected for herbicide tolerance where a 15-fold increase in plant regeneration was obtained.     

Diploidisation ofLotus corniculatus L. (Fabaceae) by elimination of multivalents

Lotus corniculatus L. (Fabaceae) is a natural tetraploid of probably hybrid origin, which regularly forms bivalents at metaphase I of meiosis. Whole-mount surface-spreading of synaptonemal complexes (SCs) under the electron microscope reveals that diploidisation of this spccies is achieved not by exclusive pairing of homologues during meiotic prophase, but by the elimination of multivalents in favour of bivalents before metaphase I. Observations show that 43% of multivalents are eliminated between zygotene and pachytene, presumably by dissolution and reassembly of SCs between homologous chromosomes. A further 63% are eliminated between pachytene and diakinesis, with a commensurate increase in the number of univalents. Elimination ensures few multivalents reach first metaphase and effectively diploidises this tetraploid.     

The development of sulfonylurea herbicide-resistant birdsfoot trefoil (Lotus corniculatus) plants from in vitro selection

Summary The use of information from flanking markers to estimate the position and size of the effect of a quantitative trait locus (QTL) lying between two markers is shown to be affected by QTLs lying in neighbouring regions of the chromosome. In some situations the effects of two QTLs lying outside the flanked region are reinforced in such a way that a ghost QTL may be mistakenly identified as a real QTL. These problems are discussed in the framework of a backcross using a regression model as the analytical tool to present the theoretical results. Regression models that use information obtained from three or more nearby markers are shown to be useful in separating the effects of QTLs in neighbouring regions. A simulated data set exemplifies the problem and is analysed by the interval mapping method as well as by the regression model.     

Analysis of quantitative traits connected with seed weight and flowering terms in birdsfoot trefoil (Lotus corniculatus L.) plants

The nature of inheritance of characters connected with the seed size (seed mass) and quality of the shoots (germination energy, seed viability, general shoot length, shoot mass, dry weight of shoots) in Lotus corniculatus L. was analyzed. The investigations were carried out on plants of three varieties: the wild form from the Krasnodar Region and local forms MF1 and MF3. The correlation analysis was carried out. The pattern of inheritance in the terms of plant flowering and length of shoots were studied.     

Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)

Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.Abbreviations BAP benzylaminopurine - KIN kinetin - NAA naphthaleneacetic acid - PAR photosynthetically active radiation Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2     

Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers

Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.     

Palatability in sheep and in vitro nutritional value of dried and ensiled sainfoin (Onobrychis viciifolia) birdsfoot trefoil (Lotus corniculatus), and chicory (Cichorium intybus)

Three temperate forages, sainfoin, birdsfoot trefoil, and chicory, characterized by elevated contents of plant secondary compounds, were compared to a ryegrass-clover mixture (control) in dried (Experiment 1) and ensiled form (Experiment 2) in their palatability and nutritional value. Palatability was measured in adult wethers (n = 6) allowed to choose between the familiar control forage and one of the three test plants. Palatability index was calculated from differences in intake of control and test plants measured after given times. Generally at first contact, palatability of the unfamiliar plants was low. Lag time until palatability index approached or exceeded a value of 100 was 2-5 d, but could not be related to the content of condensed tannins. Sainfoin had a high palatability, the highest content of condensed tannins (77.4 +/- 10.23 g/kg DM), a high content of duodenally utilisable crude protein (94.7 +/- 16.87 g/100 g CP), and a high content of metabolizable energy (9.5 +/- 0.38 MJ ME/kg DM), making this plant most promising for various purposes including anthelmintic action.     

The variability of critical species ofLotus L. in Iran and in the neighbouring countries I. the circle ofL. corniculatus L. andL. gebelia Vent.

The region of Iran, Iraq, Afghanistan and the neighbouring countries is important for some groups of the speciesLotus L., especially those of the circle ofL. corniculatus L. andL. gebelia Vent. The first group is represented by the speciesL. corniculatus L. with 4 subspecies (3 of which are important for this region), andL. tenuis Waldst. etKit. which here attains the eastern boundary of the continuous area of distribution, and by the eastern speciesL. krylovii Schischk. etSerg. andL. rechingeri Chrtková-?ertová. The second group is represented by the speciesL. gebelia Vent.,L. michauxianus Ser. in DC. andL. libanoticus Boiss. their areas of distribution covering mostly those regions. Most of the species show considerable variability within the species.     

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Some new taxa inLotus corniculatus L.

Four new taxa in the variable speciesLotus corniculatus L. has been described: var.alandicus Chrtková-?ertová, var.norvegicus Chrtková-?ertová, var.fallax Chrtková-?ertová and var.posoniensis Chrtková-?ertová.     

百脉根基因工程研究进展

牧草基因工程是近年来国内外研究的热点之一.针对农杆菌介导百脉根遗传转化原理、影响农杆菌介导百脉根遗传转化的重要因素、转基因技术在百脉根的生物固氮、抗逆性和品质改良以及生产可食性疫苗等方面的研究进行了全面综述,并就百脉根基因工程研究今后的主要发展方向进行了展望.     

Callus formation from root protoplasts of Quercus rubra L. (red oak)

Root protoplasts of Quercus rubra L. were isolated from 12 day old seedlings with an enzyme mixture containing Cellulase R1O + Rhozyme HP150 + Macerozyme R1O, supplemented with cysteine and bovine serum albumin.Protoplasts were purified by a Ficoll density gradient centrifugation and cultured at low density in a liquid medium. The modified woody plant medium, containing 2.2 M benzyladenine + 1.8; M zeatin + 5.3 M -naphthaleneacetic acid + 2.2 M dichlorophenoxyacetic acid, allowed sustained divisions and formation of microcalluses.Protoplast — derived microcallus developed into green and compact callus when transferred to an agarose solidified medium, supplemented with casein hydrolysate and indole 3-acetic acid (devoid of 2,4 dichlorophenoxyacetic acid) and placed under low illumination.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA benzyladenine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - MES 2-(N-morpholino) ethanesulfonic acid - MS medium Murashige and Skoog medium (1962) - NAA -naphthaleneacetic acid - WPM woody plant medium (Lloyd and Mc Cown, 1981)