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     

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.     

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     

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.     

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.     

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.     

Root system development of Lotus corniculatus L. in calcareous sands with embedded finer-textured fragments in an initial soil

Background and aims

In post mining landscapes as in the Lusatian region (Brandenburg, Germany), Pleistocene coarse-textured, sandy sediments are used for soil rehabilitation and land reclamation. The homogeneously-appearing initial soils are characterized by finer-textured soil clumps (fragments) of different sizes that are embedded in a sandy matrix. These soils with typical local-scale heterogeneity may serve as a model for studying how spatially-distributed soil fragments may be utilized by pioneering plant species. The aim of this study was to gain insight into the physical and chemical properties of sandy matrix and fragments that could possibly explain why embedded fragment may act as hot spots for root growth.

Methods

In 2009, three soil monoliths of dimension 50 cm?×?50 cm?×?50 cm that were exclusively vegetated by Lotus corniculatus L. planted in 2008 were studied. Each layer of 10 cm was sampled successively using a cubic metal frame with 10 cm edge length (25 samples per layer each with a volume of 1 l). The samples were analyzed for root biomass, root lengths and diameter, and for chemical and physical properties of sandy matrix and fragments.

Results

Bulk density, water contents, total carbon, total nitrogen, and plant available calcium contents were higher for the fragments compared to the sandy matrix. The roots of L. corniculatus were heterogeneously distributed in the monoliths. The root density distributions for the 1 L samples indicated a positive influence of fragments on directed root growth. Fragments embedded in the sandy matrix were found to be strongly penetrated by roots despite their relatively high bulk density. The presence of fragments also led to an increased root biomass in the sandy matrix in the direct vicinity of fragments. Such direct effects on root development were accompanied by more indirect effects by locally-elevated moisture and nutrient contents.

Conclusion

The results suggest that finer-textured fragments embedded in coarser-textured sediments, can have favorable effect on plant and root development during the initial stages of establishment of vegetation cover. The fragments can act as water and nutrient hot spots to improve supply of pioneering plants especially in coarse-textured soil. The existence of small-scale heterogeneities owing to incomplete sediment mixing e.g., in soil reclamation, could be generally important for controlling the speed and direction of early plants-establishment, for instance, in the succession of post-mining areas.     

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)     

Amylase Activity in Taproots of Medicago sativa L. and Lotus corniculatus L. Following Defoliation

Although the patterns of starch metabolism in taproots of alfalfa(Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatusL.) have been characterized, little is known regarding the activitiesof starch-degrading enzymes in taproots of these species. Ourobjective was to determine how defoliation influences starchdegradation and activities of amylases in taproots of alfalfaand birdsfoot trefoil. In Exp. 1, amylolytic activities andstarch concentrations in taproots of defoliated and undefoliatedplants were compared on days 0, 3, 7, 10, and 14 after defoliation.Taproot starch concentrations declined in defoliated plants,while increasing in taproots of undefoliated plants. Exoamylaseactivities in taproots of defoliated plants did not change withdefoliation, while endoamylase activities increased 2-fold indefoliated alfalfa and 50% in defoliated birdsfoot trefoil plantswhen compared to undefoliated plants. In Exp. 2, activity andisoform complement of amylases were monitored during seedlingdevelopment. High endoamylase activity was found in taprootsof both species at all samplings. In contrast, exoamylase accumulatedin taproots of alfalfa, but not birdsfoot trefoil, in a patternsimilar to starch accumulation. As in Exp. 1, defoliation increasedendoamylase, but not exoamylase activity in taproots of bothspecies. Taproots of both species contained one major and twominor endoamylase isoforms, but the electrophoretic mobilityof these isoforms differed between species. Activities of allisoforms, as indicated on starch-gel blots, increased in responseto defoliation. These results indicate that defoliation increasesactivity of taproot endoamylases, whose activity is associatedwith taproot starch degradation. Key words: Starch degradation, alfalfa, birdsfoot trefoil, enzymes     

Regeneration of plants from hypocotyl protoplasts of rapessed (Brassica napus L. var. oleifera) cultivars

Protoplast cultures were prepared from hypocotyls of ten spring rapeseed cultivars. Protoplasts from all genotypes tested formed calli, and shoots were regenerated from calli of nine of the genotypes at frequencies varying from 15 to 76%. The regenerating cultivars fell into a high regenerating group (>60% and a low regenerating group <25%).     

Efficient plant regeneration from hypocotyl protoplasts of broccoli (Brassica oleracea L. ssp. italica Plenck)

We have assessed the capacity of cultured protoplasts from two tissue sources of several commercially-grown broccoli cultivars to regenerate plants. A procedure that employs hypocotyl protoplasts and a culture medium with a high NAA:2,4-D auxin ratio was developed. The procedure permits highly efficient formation of colonies that regenerate shoots at frequencies of 8–17% with two of the four cultivars tested. The time required for the development of plants from protoplasts was 8–11 weeks. No mtDNA rearrangements were observed among any of 17 analysed regenerants. Double-stranded RNAs were detected in mitochondrial DNA (mtDNA) preparations of some, but not all, regenerants of one of the cultivars.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - ZR zeatin riboside - CF colony formation - mtDNA mitochondrial DNA     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.     

Isolation and culture of protoplasts ofCapsicum annuum L. and their regeneration into plants floweringin vitro

Summary Axenic shoot cultures ofCapsicum annuum cv.California Wonder were used as the source for isolation of protoplasts from mesophyll cells. Protoplasts underwent sustained mitotic activity and proliferated to form callus masses on NT or DPD medium enriched with 2,4-D, NAA and BAP each at 1 mg/l level. The callus could be differentiated into whole plants on the differentiation media and plants floweredin vitro under long day conditions.     

Chloroplast-DNA variation in the genus Lotus (Fabaceae) and further evidence regarding the maternal parentage of Lotus corniculatus L.

 To resolve the maternal parentage of the tetraploid Lotus corniculatus, restriction-site variation of chloroplast DNA (cpDNA) was studied in several accessions of that species, in the four putative parental diploid species, L. tenuis, L. alpinus, L. japonicus and L. uliginosus, and in four phylogenetically more distant diploid species, L. hispidus, L. edulis, L. ornithopodoides and Tetragonolobus maritimus var. siliquosus. Evidence of cpDNA maternal inheritance was obtained by using reciprocal controlled crosses between plants of L. corniculatus and natural tetraploid individuals of L. alpinus showing very distinct restriction patterns. Interspecific cpDNA variation in the eight Lotus species and T. siliquosus was analysed by comparing cpDNA fragment patterns produced by five restriction endonucleases and totalling 304 distinct fragments. Genetic differentiation in cpDNA was very high between the L. corniculatus group and L. hispidus on the one hand, and the three other species on the other hand. Sixteen restriction-site mutations and eight length polymorphisms were identified among the five species of the L. corniculatus group and L. hispidus, Lotus uliginosus, L. alpinus and L. japonicus showed at least six DNA changes with regard to the molecule of L. corniculatus. Accordingly, these species should be excluded as maternal progenitors of L. corniculatus. Conversely, the cpDNA of L. tenuis differed from that of L. corniculatus by only two small-length mutations. As also suggested previously from an analysis of several nuclear markers, the results reported here show decisively that L. tenuis may be considered as the most probable maternal ancestor of L. corniculatus. Received: 23 February 1997/Accepted: 28 February 1997     

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara)

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA3 gibberellic acid - NAA -naphthaleneacetic acid - PAS periodic acid Schiff - PCM protoplast culture medium - PCV packed cell volume