ODAP Content Research Articles

Contents of total protein and β‐N‐oxalyl‐l‐α,β‐diaminopropionic acid (ODAP) in a collection of Lathyrus cicera of the Bank of Plant Germplasm of Cuenca (Spain)

AbstractOne hundred and thirty‐four accessions of Lathyrus cicera from the Bank of Plant Germplasm of Cuenca (BGV‐Cuenca, Spain) have been analysed for total protein and β‐N‐oxalyl‐l‐α,β‐diaminopropionic acid (ODAP) contents in order to identify those with a suitable nutritional quality for animal feeding. There was a large variability in both traits, the content of total protein ranged from 22.95% to 30.98% with a mean value of 27.62%, and the ODAP from 0.09% to 0.30% with a mean value of 0.17%. The accessions from Castilla y León and Castilla‐La Mancha showed higher percentages of protein and ODAP as compared with those from Andalucía, Greece and Extremadura. Nevertheless, total protein and ODAP contents showed a low positive correlation (r = 0.396, P = 0.01). Several accessions showed a content of ODAP similar to that of the variety ‘Chalus’ and two accessions seemed very suitable for animal feeding because of their high content of protein and low ODAP.

Potentiation of neurotoxicity of Lathyrus sativus by manganese: Alterations in blood–brain barrier permeability

Environmental factors have been speculated to play an important role in potentiating the neurotoxicity of Lathyrus sativus (LS). Hence, blood–brain barrier permeability and neurotoxicity studies were carried out in manganese- and LS-exposed animals. Dietary feeding of LS (80%) plus Mn (0.4 mg/100 g diet) for 90 days to guinea pigs showed significant (p < 0.05) decrease in brain nucleotidase and ATPase activities when compared to control or LS alone treated groups. Combined treatment of LS and Mn showed a significant (p < 0.05) decrease in neuronal aryl hydrocarbon hydroxylase (36–40%), ethoxyresorufin-O-deethylase (40–45%), glutathione-S-transferase (27–31%), and quinone reductase (24–25%) activities when compared to control and LS alone treated animals. Lipid peroxidation, a marker for membrane damage, was found to be relatively more enhanced (58–141%) along with significant (p < 0.05) depletion of GSH levels in LS+Mn-treated animals when compared to control, Mn alone, and LS alone treated groups. The neuronal catalase activity of lathyrus plus Mn-treated animals showed a pronounced decrease (37–49%) when compared to control, Mn, and lathyrus alone treated groups. On the contrary, glutathione peroxidase in brain of Mn and lathyrus alone treated animals indicated a respective increase (p < 0.05) of 18% and 20%, while the combined effect of lathyrus plus Mn exhibited an increase of almost 50% when compared to control guinea pigs. Single parenteral administration of Mn (15 mg/kg b.wt) to guinea pigs followed by single oral intubation of β-N-oxalyl-L-α, β-diamino propionic acid (ODAP, 75 mg/guinea pig) resulted in a significant increase (143%) in neuronal ODAP content. ODAP (50 mg/kg,iv) treatment to mice pretreated with MnCl2 (10 mg/kg b.wt for 3 days or 40 mg/kg b.wt for 1 day), caused an enhancement in blood–brain barrier (BBB) permeability (129–196%), while ODAP and Mn alone showed relatively less enhancement (66–87%). The lumbar region of LS+Mn showed a number of vacuolated areas of variegated size and chromatolytic neurons, along with a few degenerated neurons. These results suggest that Mn may potentiate the neurotoxicity of lathyrus/ODAP by altering the BBB permeability.

Genetic variability of some quality traits in Lathyrus spp. germplasm

Sixty-six accessions representing eighteen species of the genus Lathyrus collected from different geographic regions were evaluated for variations of quality traits (100 seeds weight, ash, total seed proteins and 3-(-N-oxayl)-L-2,3 diaminopropoinc acid – ODAP contents). High variability of ODAP levels was exhibited at both inter-specific and intra-specific levels. This variability was attributed to genetic and environmental factors. No significant correlation was found between ODAP and each of total protein content, ash content and 100 seeds weight. Cluster analysis of C.V. (Coefficient of variance) values for each accession identified the sixty-six accessions into eight groups. The most promising accession for breeding programs was L. sativus from Tunisia. This accession has good grain quality due to relatively low ODAP level and high protein content. The variations of protein content, ash content and 100-seeds weight were also discussed.

Abscisic acid promotes accumulation of toxin ODAP in relation to free spermine level in grass pea seedlings ( Lathyrus sativus L.)

Interrelationship among abscisic acid (ABA) content, accumulation of free polyamines and biosynthesis of β- N-oxalyl- l-α,β-diaminopropionic acid (ODAP) was studied in grass pea ( Lathyrus sativus L.) seedlings under drought stress induced by 10% polyethylene glycol (PEG6000). Increase of ABA content occurred prior to that of ODAP and polyamine contents, and was found significantly positive correlation between ABA content and ODAP content. Addition of exogenous ABA increased ODAP content in leaves. On the other hand, pretreatment with α-difluoromethylarginine (DFMA), a polyamine biosynthesis inhibitor, significantly suppressed the accumulation of free putrescine (Put), free spermidine (Spd) and free spermine (Spm), which in turn inhibited biosynthesis of ODAP in well-watered leaves. Meanwhile, addition of exogenous Put alleviated DFMA-induced inhibition on the biosynthesis of Put and Spd, but did not affect the biosynthesis of Spm and ODAP in well-watered leaves. Same result was also achieved in drought-stressed leaves. Increasing accumulation of ODAP was significantly correlated with increasing Spm content ( R = 0.7957**) but not with that of Spd and Put. Therefore, it can be argued that ABA stimulated the biosynthesis of ODAP simultaneously with increasing the level of free Spm under drought stress condition.

Isozymes, protein and ODAP variability of grasspea (&lt;I&gt;Lathyrus sativus&lt;/I&gt; L.) in Ethiopia

Variability of grass pea landrace populations for isozymes, Oxalyl Diamino Propanoic acid ODAP) and protein contents were investigated following standard procedures to detect genetic variation. The result indicated that phenotypic polymorphism was observed for the three enzymes, the highest being for esterase. The degree of differentiation (FST) of the individual loci ranged from 0.031 (for ACP-3) to 0.784 (for AAT-2). The highest polymorphism was detected in populations collected from Gondar region indicating the need for priority germplasm collection and site selection for in situ conservation in this region. The ODAP and protein analyses also revealed presence of variability among the populations. Fortunately, four entries with low ODAP contents ranging from 0.14% to 0.18% were identified in populations collected from different regions indicating the variation of this trait within population and between regions. These lines with low ODAP content are obviously considered important for further breeding program. Key words/phrases: Ethiopia, grass pea , isozyme, Lathyrus sativus, ODAP ET: Ethiopian Journal of Science Vol. 27 (2) 2004: 153–160

Collection and evaluation of grasspea (Lathyrus sativus L.) germplasm of central Italy

Characterization of 16 grasspea (Lathyrus sativus L.) accessions collected in central Italy (Marche region) was performed for morphological and agronomic traits and for 3-(-N-oxalyl)-L-2,3 diaminopropionic acid (ODAP) content. Field trials, carried out in two locations, revealed high among-population genetic variation. In particular, an evident differentiation between commercial populations, cultivated by market-oriented farms, and household populations (not market-oriented) was found. Genotype x environment interaction was negligible. Medium-high values of ODAP content were observed following colorimetric and capillary electrophoresis analyses. A high positive correlation between the two methods was found (r = 0.83**), but the colorimetric values showed, on average, significant 14% lower ODAP values. This research represents a pre-breeding activity aimed at developing a breeding programme for the preservation and utilization of Italian grasspea germplasm within sustainable or organic agricultural systems.

Characterization of Italian and exotic Lathyrus germplasm for quality traits

The aim of the following work is to characterize quality traits (protein, ashes, 100 seed weight and ODAP content) of 117 accessions of Lathyrus, from either Italian or foreign countries. Significant differences among entries were estimated for all traits and accessions were classified into similar genetic groups. Principal component and hierarchical cluster analyses produced five different groups. In particular, the absence of a strong association among the investigated traits and their dominant independent roles in each separate principal component allows the achievement of a useful recombinant in breeding work. An interpretation of each cluster, based on the data obtained from the accessions, provides practical information to establish a subset of grass pea collection for further activities.

Variation and association of morphological and biochemical characters in grass pea (Lathyrus sativus L.)

Fifty grass pea land race populations of Ethiopia that were selected based on administrative regions and different altitude classes were used in this study to determine variability in morphological characters, Oxalyl Diamino Propanoic acid(ODAP) – a causative agent of lathyrism, and protein contents and their associations. Highly significant differences were observed among the populations for most of the morphological characters. Populations collected from Gondar region and the higher altitude group (>2550 m) showed the highest mean and coefficient of variation indicating the presence of high genetic diversity in this region and altitude group. ODAP analysis from single plants showed significant variation both within a population and between populations. Four individual plants with low ODAP contents ranging from 0.149% to 0.182% (range within safe level) were identified in populations collected from different regions, indicating the diversity and variation of this trait with in one population and between regions. These low ODAP lines are considered important for further breeding. The association of ODAP with grain yield, biomass, plant height and seed size was negative, suggesting that the selection of tall and late maturing varieties with large seed size and high grain yield potential will enable the development of varieties with low ODAP content.

Interspecific hybridisation of &lt;i&gt;Lathyrus sativus&lt;/i&gt; (Guaya) with wild Lathyrus species and embryo rescue

Lathyrus sativus is an economically important legume crop cultivated for food and forage in Asia and Africa. The use of this hardy drought tolerant and high yielding crop, however, has been limited because of a neurotoxin, _-N-oxalyl-L-_,_-diaminopropionic acid (ODAP) in the seeds. The objective of this study was to increase the genetic diversity of L. sativus by interspecific hybridisation with wild Lathyrus species devoid or with very low ODAP content. This would in turn assist to study the biochemical and genetic mechanisms which control the biosynthesis of ODAP. L. sativus was crossed with 12 wild Lathyrus species. Except for the reciprocal crosses of L. sativus and L. pseudo-cicera, others failed to develop viable seeds. In several of the interspecific hybrids, pod development was observed but embryos aborted during early stages of development. Embryo culture was attempted to rescue these immature embryos. The response of the interspecific hybrid embryos to in vitro culture varied. Cell proliferation and callus induction were observed in most of the combinations, and mature plants were regenerated from immature embryos of a cross between L. sativus (male parent) and L. cicera (female parent) both of which contain ODAP. However, it is expected that, with few modifications, this method will be useful in other interspecific hybrid combinations of Lathyrus. Key Words: Hybridisation, Lathyrus, lathyrine, Neurotoxin, ODAP (African Crop Science Journal: 2000, 8(2): 129-136)

Physiological and Biochemical Analysis of Lathyrus sativus Somaclones Derived from Root and Internode Explants

Low ODAP somaclones from root and internode explants were evaluated for physiological and biochemical parameters. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. All the somaclones during development had substantially lower ODAP content in leaves compared to parent P 24. Amongst somaclones, Bio R 202 had the maximum leaf area, biomass as well as photosynthetic rate. Harvest index and seed yield were highest in Biol 222 whereas ODAP content was the least in it.

Molecular Characterisation of Low ODAP Somaclones of Lathyrus sativus

Somaclones of Lathyrus sativus having low ODAP contents were characterised at molecular level with respect to soluble protein pattern, esterase and ADH isozyme pattern, Southern hybridisation of genomic and mitochondrial DNA with specific probes. In 10-day-old seedling esterase isozyme pattern showed the presence of an unique band of Rm 0.73 in Bio L-12 and 15-8-1 and the absence of a band of Rm 0.80 in 15-8-1, as compared to parent and other somaclones. Southern hybridisation of genomic DNA with cDNA clone 29 showed an extra band of 13.5 kb in L-56 and 15-8-1 only. Hybridisation of mitochondrial DNA with mitochondrial specific ati ATPase probe showed differences with the pattern from Bio R-15 being unique. Some of these differences observed will be useful as marker for their identification.

Inheritance of neurotoxin (ODAP) content, flower and seed coat colour in grass pea (Lathyrus sativus L.)

A strong epidemiological association is known to exist between the consumption of grass pea and lathyrism. A neurotoxin, β-N-Oxalyl-L-α, β-diaminopropanoic acid (ODAP) has been identified as the causative principle. This study was undertaken to investigate the mode of inheritance of the neurotoxin ODAP, flower and seed coat colour in grass pea. Five grass pea lines with low to high ODAP concentration were inter-crossed in all possible combinations to study the inheritance of the neurotoxin. Parents, F1 and F2 progenies were evaluated under field condition and ODAP analyzed by an ortho-phthalaldehyde spectrophotometric method. Many of the progenies of low x low ODAP crosses were found to be low in ODAP concentration indicating the low ODAP lines shared some genes in common for seed ODAP content. The F1 progenies of the low ODAP x high ODAP crosses were intermediate in ODAP concentration and the F2 progenies segregated covering the entire parental range. This continuous variation, together with very close to normal distribution of the F2 population both of low x low and low x high ODAP crosses indicated that ODAP content was quantitatively inherited. Reciprocal crosses, in some cases, produced different results indicating a maternal effect on ODAP concentration. Blue and white flower coloured lines of grass pea were inter-crossed to study the inheritance of flower colour. Blue flower colour was dominant over the white. The F2 progenies segregated in a 13:3 ratio indicating involvement of two genes with inhibiting gene interactions. The gene symbol LB for blue flower colour and LW for white flower colour is proposed.

Physiological and Biochemical Analysis of Somaclones Derived from Leaf Explants of Lathyrus sativus

Low ODAP somaclones have been evaluated for physiological and biochemical parameters especially in relation to attributes that lead to increased biomass production. All the somaclones during development had substantially lower ODAP content in leaves as compared to parent P24. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. Somaclone Bio L12 had the highest whereas parent P24 and Bi0164 had the least leaf area. Harvest index was the highest and biomass production was the lowest in the Bio 164. Bio L08 gave the highest seed yield. Photosynthetic rates were also higher in Bio L12, although no significant positive correlation was observed in leaf photosynthesis and seed yield. The differences in physiolpgical and biochemical parameters indicate the possibility of development of high yielding genotypes. The results in present investigation show differences in photosynthetic rate, leaf characteristics, seed yield and ODAP content among somalones and parent. Somaclones with extremely low ODAP content with variability in leaf morphology and photosynthetic rate is indicative of variation induced during plant tissue culture.

A comparative study of the O‐phthalaldehyde method for the neurotoxin 3‐N‐oxalyl‐l‐2,3‐diaminopropanoic acid as modified by various laboratories

AbstractThe optimization of the quantitative photometric estimation of the neurotoxin 3‐N‐oxalyl‐L‐2,3‐diaminopropanoic acid (ODAP) after hydrolysis and reaction with o‐phthalaldehyde (OPT) reagent is described.Six modifications of the OPT procedure (as practiced in different laboratories) and the original Rao method were compared in estimating the ODAP content of two cultivars of Lathyrus sativus. Considerable variation in the ODAP content of the same sample was observed even when determined by the same procedure in some cases. The desired reproducibility was observed with the procedure of the Agriculture Canada Research Station, Mordsen, Manitoba, Canada (Campbell method). The use of 0.5 M tetraborate as buffer further significantly improves the reproducibility. The Campbell modification includes a number of controls which reduce variation. The present study recommends the Campbell modification of the OPT method but with tetraborate buffer as standard procedure for the screening of the toxicity of L. sativus cultivars.

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B5 medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R1 generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R2 generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.