Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Dynamics of biomass and N accumulation of alfalfa under three N fertilization rates

The dynamics of biomass and N accumulation following defoliation of alfalfa and the application of N fertilization has rarely been studied under field conditions, particularly in the seeding year. Our objectives were to determine the effect of N fertilization on the dynamics of biomass and N accumulation during the first regrowth of alfalfa in the seeding year, and to determine if a model describing critical N concentration developed for established stands could be used in the seeding year. In two separate experiments conducted in 1992 and 1993, the biomass and N accumulation of alfalfa grown with three N rates (0, 40 and 80 kg N ha^-1) were determined weekly. Maximum shoot growth was reached with 40 kg N ha^-1 in 1992, and maximum shoot growth was not reached with the highest N fertilization rate in 1993. Nitrogen fixation, root N reserves and soil inorganic N uptake when no N was applied were, therefore, not sufficient to ensure non-limiting N conditions, particularly when growth rates were the highest between 14 to 21 d after defoliation. Nitrogen fertilization increased shoot biomass accumulation in the first 21 d of regrowth, biomass partitioning to the shoots and shoot and taproot N concentrations. The model parameters of critical N concentration developed by Lemaire et al. (1985) for established stands of alfalfa were not adequate in the seeding year. The N requirements per unit of shoot biomass produced are greater in the seeding year than on established stands, and this was attributed to a greater proportion of leaves in the seeding year.     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Growth and nutrient concentrations of alfalfa and common bean as influenced by soil acidity

Growth and nutrient utilization of alfalfa (Medicago sativa L. cv. Arc) and common bean (Phaseolus vulgaris L. cv. Carioca) were studied in an acid soil adjusted to eight levels of soil acidity by lime addition. Application of lime significantly (P<0.05) increased shoot and root growth for both species. However, common bean was far less sensitive to soil acidity than alfalfa. Maximum alfalfa growth was obtained at a soil pH of 5.8 and maximum bean growth was achieved at pH 5.0. Root and shoot growth of both legumes was positively correlated (P<0.01) with soil pH, exchangeable Ca and exchangeable Mg and negatively correlated (P<0.01) with soil exchangeable Al. Common bean had a lower internal P requirement for maximum growth and was more efficient than alfalfa in taking up Ca and Mg. These characteristics would contribute to the favorable growth of common bean in acid-infertile soils.     

Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts

Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS absorption unit full scale - CHI chalcone isomerase (EC 5.5.1.6) - CHS chalcone synthase (EC 2.3.1.74) - C40H cinnamic acid 4-hydroxylase (EC 1.14.13.11) - CLE elicitor from Colletotrichum lindemuthianum - IFOH isoflavone 2-hydroxylase - IFS isoflavone synthase - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Growth and regenerability of long-term suspension cultures of the U.S. rice cultivar Mercury

Suspension cultures of the U.S. rice cultivar Mercury have been maintained in modified General Medium for more than 3 years. These suspensions have continued to have high and relatively stable regeneration rates. Two different explants, immature panicles and seeds, were compared during the development of these embryogenic suspensions. Initial formation of secondary embryogenic callus from immature panicles on induction medium was greater than that from seeds. Suspensions of these two cell lines, however, did not differ morphologically and maintained similar regeneration rates. After 5 months in culture the rates of regeneration began to decline. The suspensions were plated onto regeneration medium without growth regulators for 2 weeks and then embryogenic cells were manually selected and used to develop secondary suspensions. Through this simple rejuvenation procedure, the suspensions retained high and stable regeneration rates. Variability in suspension growth, however, was observed during the culture period. Slower growth occurred at weeks 13, 15, 27, and 29 and was associated with a decrease in regeneration rates. Reproductive fertility of regenerated plants remained high for 3.5 years but then declined.Abbreviations CH casein (acid hydrolysate) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog basal medium - SE standard error     

Transformation of sugar beet cell suspension cultures

Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500–4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (+) calluses was obtained when 50 or 100 mgl⁻¹ of kanamycin was applied 2 d after transformation for 3–5 wk, followed by either no selection or reduced levels of the antibiotic. PCR analyses of the GUS (+) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical β-glucuronidase (GUS) staining.     

Nitrogen release from roots of alfalfa and soybean grown in sand culture

An enclosed root chamber containing sterile sand medium was used to study net nitrogen (N) release from actively growing root systems of ‘Saranac’ alfalfa (Medicago sativa L.) and ‘Fiskeby V’ soybean (Glycine max L. Merr.). Plants were inoculated with a rhizobial strain appropriate to each host, irrigated with N-free nutrient solution, and grown either to 85 or to 173 d after germination (alfalfa) or to physiological maturity (soybean). Alfalfa released 4.5% of symbiotically-fixed plant N into the root zone over its growth period; soybean released 10.4% of plant N. Root zone leachates were analyzed for total N and for amino acid and ammonium content. Significant ammonium-N release occurred from the alfalfa but not the soybean root system; little amino-N was released by root systems of either species. Shoot harvest and water deficit caused increased release of N from alfalfa roots. The results provide evidence that alfalfa and soybean released significant proportions of their N into the root zone, and indicate that while substantial ammonium-N was released from alfalfa roots, passive leakage of amino-N was not a primary mechanism for N release from root systems of either species. Cooperative investigation of USDA-ARS and the Minnesota Agric. Exp. Stn. (Scientific J. Series No. 16048). This research was supported in part by a Graduate School Fellowship to LSB from the Univ. of Minnesota.     

Microgametophytic selection in two alfalfa (Medicago sativa L.) clones

Summary Microgametophytic selection was investigated using two ecologically diverse autotetraploid clones of alfalfa. Several selection pressures (drying, aging, freezing, and high and low temperatures) were applied to microgametophytes at three stages of the life cycle, 1) during microsporogenesis, 2) post-anthesis, and 3) pollen tube growth. Pollen aging produced a progeny population with a greater mean plant size and a lower coefficient of variation than the control progeny. High temperature (29.5 °C) applied both during microsporogenesis and pollen tube growth resulted in progeny populations which were significantly taller and, in one case, had a larger leaf number than the control populations. In contrast, air dried pollen resulted in a progeny population which had significantly smaller character means and larger coefficients of variation than the control population. Also, low temperature (15 °C) during pollen tube growth yielded progeny with reduced branch number and a larger coefficient of variation than the control progeny. In cases where progeny derived from selected microgametophytes were found to differ from the control offspring, corresponding shifts in the reciprocal cross were not observed. For the temperature stress treatments, the lack of reciprocal differences may be related to the different temperature adaptations of the two ecotypes. These results suggest that microgametophytic selection can be effective in shifting the mean of the progeny generation; however, the results obtained will vary depending upon the selection pressure, stage of selection, and the parents used.     

Comparisons of alfalfa somaclonal and sexual derivatives from the same genetic source

Summary Tetraploid alfalfa (Medicago sativa L.) clones were derived from the same diploid genetic background by four different methods. A phenotypically superior clone was selected from each method and compared for herbage yield and fertility. The four methods and their best clones were: a) In vitro somatic chromosome doubling of one diploid hybrid (HG2-4x); b) selection within a two allele tetraploid synthetic population derived from HG2-4x (HAG); c) somaclonal variant selection from cell suspension culture of the diploid hybrid (NS1); and d) sexual polyploidization of a sibling hybrid (HXG). Clones HG2-4x, HAG, and NS1 were likely diallelic or monoallelic at all loci. Clone HXG was probably tetrallelic or triallelic at most loci. Experiments measured fertility, clonal herbage yield, and herbage yield of test cross progeny for each selected clone. Fertility rankings were HXG = HAG > NS1 > HG2-4x. Clonal herbage yield rankings were HXG = HAG > NS1 > HG2-4x. Test cross progeny herbage yield rankings varied depending on the tester, but, in general, HXG HAG NS1 HG2-4x. Overall the best clones from the sexual methods exceeded the best somaclonal variant which, in turn, was better than the chromosome doubled clone.     

Nitrogen-fixing characteristics of alfalfa cultivars nodulated by representatives of an indigenousRhizobium meliloti serogroup

Studies were conducted to evaluate whether field-grown cultivars of alfalfa (Medicago sativa L.) nodulate differentially with members of a soil population ofRhizobium meliloti, and to determine the influence of the dominant nodule occupants on N₂-dependent growth of the same cultivars under greenhouse conditions. Nodules were sampled from four replicate plots of Vernal, Anchor, and Saranac alfalfa, and the isolates analysed serologically. Results from agglutination tests identified serogroup 31 as a dominant nodule occupant. A significant cultivar effect was observed, with a greater and more consistent occupancy rate by serogroup 31 across the replicates on Vernal (60%) compared to Anchor (24%) or Saranac (36%). The symbiotic effectiveness of the parent isolate of serogroup 31 was evaluated on each cultivar over four successive harvests in a greenhouse study. Significant cultivar x N source interactions for herbage dry weight resulted following the second harvest. Of the three cultivars, only inoculated Vernal responded with an increase in shoot dry weight and N₂ assimilated relative to N supplemented plants between harvests two and three. In separate greenhouse experiments, field isolates of serogroup 31 from nodules on Vernal produced homogeneous, effective responses both on Vernal and Anchor. In contrast, serogroup 31 field isolates from Anchor nodules were highly heterogeneous in effectiveness on the parent host, with poorly effective isolates being substantially more effective on Vernal. The data indicate that attention should be given to the potential impact of the indigenousR. meliloti population upon cultivar ranking at specific field locations, and also to strain-cultivar idiosyncracies when carrying out physiological sutidies of regrowth characteristics.Technical Paper No. 8716 of the Oregon State University Agricultural Experiment Station.     

Regeneration and characterization of plants obtained from anther cultures in Medicago sativa L

Summary The androgenic ability of four Medicago sativa L. genotype (Boynitza 5, Byala, 494, and 3815) was tested. Callus and organogenesis were induced in all lines studied. The percentage of anthers producing calluses and organogenesis showed wide variation (calluses—from 11% up to 77%; organogenesis—4.8% to 15.2%). It has been established that genotype, nutrient medium composition, and stage of pollen development considerably affected both callus production and organogenesis. Androgenesis in M. sativa could be achieved via callus and direct embryogenesis. About 500 morphologically different regenerants were obtained. Wide variability in chromosome number of regenerated plants was observed by cytological studies. Haploid, dihaploid, as well as mixoploid plants were obtained.     

Molecular divergence of alfalfa somaclones

Summary Plantlets were regenerated from alfalfa callus following passage through a tissue culture medium which contained gibberellic acid. A proportion of these plantlets showed obvious morphological variations. Leaflet, stem and petiole tissue of these plants were extracted to yield a soluble protein homogenate which was characterized by polyacrylamide gel electrophoresis. Over 18 individual protein bands were resolved and visualized by staining with coomassie blue G250. Electrophoretic gels from regenerated plantlets and from the parent plant were scanned spectrophotometrically and analyzed. The relative quantity of each of the proteins resolved from plants was correlated with proteins of other plants via the Pearson's product-moment correlation. Cluster analysis was then performed using these correlation coefficients to judge relatedness among somaclones and the parent plant. Two of 22 somaclones (9.1%) differed significantly from the parent and from the other somaclones judged by quantitative protein pattern variations. Three distinctive lineages through tissue culture produced plantlets. Using a discriminant analysis strategy somaclones could be grouped according to lineage with 80.8% accuracy based upon distinctions between protein electrophoretic patterns. Two of the somaclone lineage groupings showed no overlap with the parental grouping which indicated significant molecular divergence of these plantlets as judged by quantitative protein differences.     

Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA

Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l^–1 kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.Communicated by C.F. Quiros     

Growth and rosmarinic acid accumulation in callus,cell suspension,and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 M) and indole-3-acetic acid (IAA) (0 or 3 M). For root culture, hairy roots were cultured in B5 medium containing 2.7 M -naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 M TDZ and 3 M IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/ 100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/ 100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/ 100 mg dry weight) was obtained with 4% (w/ v) sucrose. Incorporation of 10 mg l^–1 phenylalanine in the medium increased RA yield in the roots to 4.68 mg/ 100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/ 100 mg dry weight, respectively.