Non-toxic Varieties Research Articles

Development of SCAR Marker Specific to Non-Toxic Jatropha curcas L. and Designing a Novel Multiplexing PCR Along with nrDNA ITS Primers to Circumvent the False Negative Detection

Jatropha curcas L., a multipurpose shrub, has acquired significant economic importance for its seed oil which can be converted to biodiesel an emerging alternative to petro-diesel. In addition to the commercial value, it is also having medicinal and even high nutritional value to use as animal fodder which is limited due to the toxicity. Development of molecular marker will enable to differentiate non-toxic from toxic variety of J. curcas in a mixed population and also for quality control since the toxic components of J. curcas has deleterious effect on animals. In the present study, the efforts were made to generate the specific SCAR marker for toxic and/or non-toxic J. curcas from RAPD markers. Among the markers specific for toxic and non-toxic varieties, four were selected, purified, cloned, sequenced, and designed primers out of which one set of primers NT-JC/SCAR I/OPQ15-F and R could able to discriminate the non-toxic with toxic Jatropha by giving expected 430 bp size amplification in non-toxic variety. Furthermore, novel multiplex PCR was designed using the nrDNA ITS primers to overcome the false negatives. Present work also demonstrates utility of the conserved regions of nrDNA coding genes in ruling out the artifacts in PCR-like false negatives frequently occur in SCAR due to various reasons. The specific SCAR markers generated in the present investigation will help to distinguish non-toxic from toxic varieties of J. curcas or vice versa, and isolated marker along with designed multiplex protocol has applications in quality control for selective cultivation of non-toxic variety and will also assist in breeding and molecular mapping studies.

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.

In vitro plant regeneration of non-toxic Jatropha curcas L.: Direct shoot organogenesis from cotyledonary petiole explants

Jatropha curcas, the energy plant has attained great attention in recent years because of its biodiesel production potential; however, oil and deoiled cakes are toxic. A non-toxic variety of J. curcas is reported from Mexico. A simple and efficient protocol has been developed for plant regeneration using cotyledonary petiole explants of non-toxic variety of J. curcas. The percentage of induction of shoot buds (59.11%), and the number of shoot buds (5.01) per explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 2.27 μM thidiazuron (TDZ). These induced shoot buds multiplied when subcultured on MS medium supplemented with 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BAP), and 5.5 μM α-naphthaleneacetic acid (NAA) for 4 weeks and subsequent elongation achieved on MS medium supplemented with 2.25 μM BAP and 8.5 μM indole-3-acetic acid (IAA). Shoots more than 2 cm long were harvested and cultured on MS medium containing different concentrations and combinations of IBA, IAA, NAA, and 0.25 mg L−1 activated charcoal, and 19.91% rooting was achieved in 15 μM IBA, 5.7 μM IAA, and 16.5 μM NAA after 4 weeks with more than 90% survival rate.

Solanaceous steroidal glycoalkaloids and poisoning by Solanum torvum, the normally edible susumber berry

Ingestion of immature, environmentally stressed, or cultivar-specific Solanum species (particularly the potato) has been previously associated with gastrointestinal and neurological symptoms caused by solanaceous steroidal glycoalkaloids (SGAs). We report on two geographically, temporally disparate outbreaks of poisoning by susumber berries ( Solanum torvum– Solanaceae) and on detection of alkaloids not present in non-toxic berries. Five family members in New York City participated in a traditional evening meal containing Jamaican susumber berries. All those consuming berries were symptomatic the following morning with varying degrees of gastrointestinal distress, dizziness, slurred speech, cranial nerve deficits, and ataxia. The most seriously afflicted patient developed hypertension, confusion, proximal upper extremity weakness, and hypercapnic respiratory failure requiring prolonged mechanical ventilation. A separate cohort of six patients in Toronto ate unripe Jamaican susumber berries. They presented 14 h post-ingestion with varying degrees of diarrhea, weakness, facial paralysis, slurred speech, ataxia, early hypertension, and proximal weakness. Two patients had ventilatory decompensation; one required intubation. Poisonous berries appeared indistinguishable from non-toxic varieties. We isolated solasonine, larger amounts of solamargine, and other steroidal glycoalkaloids in the toxic berry strains. S. torvum poisoning can produce significant neurological and gastrointestinal effects which appear to be mediated by SGAs present in the berries.

Molecular characterization and identification of markers for toxic and non-toxic varieties of Jatropha curcas L. using RAPD, AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).

Natural Variation in Toxicity of Wheat: Potential for Selection of Nontoxic Varieties for Celiac Disease Patients

Natural Variation in Toxicity of Wheat: Potential for Selection of Nontoxic Varieties for Celiac Disease Patients

Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic Jatropha curcas L.

Protocols for in vitro propagation of non-toxic variety of J. curcas through axillary bud proliferation and direct adventitious shoot bud regeneration from leaf segments have been established. Shoot bud proliferation from axillaries was assessed on an initial basal Murashige and Skoog (MS) salt medium supplemented with different concentrations of benzyladenine (BA), kinetin and thidiazuron (TDZ) followed by subculture to medium with 4.4-8.9 μM BA. Regardless of the concentration of BA in the subculture medium, shoot multiplication rate was optimum (10–12.3) with primary culture on medium supplemented with 2.3–4.5 μM TDZ. Efficient adventitious shoot regeneration from leaf tissues was achieved with culture on medium with 8.9–44.4 μM BA + 4.9 μM indole-3-butyric acid (IBA) followed by transfer to medium supplemented with 8.9 μM BA + 2.5 μM IBA. Similarity index between toxic Indian variety and the non-toxic variety based on 435 RAPD markers was 96.3%. Crossing studies followed by phorbol ester quantitation revealed that outcrosses with toxic J. curcas do not affect the phorbol ester content of seeds borne on the non-toxic variety.

Natural Variation in Toxicity of Wheat: Potential for Selection of Nontoxic Varieties for Celiac Disease Patients

Celiac disease (CD) is an intestinal disorder caused by T-cell responses to peptides derived from the gluten proteins present in wheat. Such peptides have been found both in the gliadin and glutenin proteins in gluten. The only cure for CD is a lifelong gluten-free diet. It is unknown, however, if all wheat varieties are equally harmful for patients. We investigated whether wheat varieties exist with a natural low number of T-cell-stimulatory epitopes. Gluten proteins present in public databases were analyzed for the presence of T-cell-stimulatory sequences. In addition, wheat accessions from diploid (AA, SS/BB, and DD genomes), tetraploid (AABB), and hexaploid (AABBDD) Triticum species were tested for the presence of T-cell-stimulatory epitopes in gliadins and glutenins by both T-cell and monoclonal antibody-based assays. The database analysis readily identified gluten proteins that lack 1 or more of the known T-cell-stimulatory sequences. Moreover, both the T-cell- and antibody-based assays showed that a large variation exists in the amount of T-cell-stimulatory peptides present in the wheat accessions. Sufficient genetic variation is present to endeavor the selection of wheat accessions that contain low amounts of T-cell-stimulatory sequences. Such materials may be used to select and breed wheat varieties suitable for consumption by CD patients, contributing to a well-balanced diet and an increase in their quality of life. Such varieties also may be useful for disease prevention in individuals at risk.

Diversity of the diatom genus Pseudo-nitzschia Peragallo in the Quoddy Region of the Bay of Fundy, Canada

Representative species of the genus Pseudo-nitzschia are well established in the Bay of Fundy and are frequent contributors to the phytoplankton community of the Quoddy Region. This manuscript documents for the first time the specific diversity of this genus in the Quoddy Region. At least seven species of Pseudo-nitzschia are present in our samples, five of which represent new records for the region. These new records include: Pseudo-nitzschia americana, Pseudo-nitzschia fraudulenta, Pseudo-nitzschia multiseries, Pseudo-nitzschia pungens and Pseudo-nitzschia subpacifica. The rarest and least known species is P. americana, while the most morphologically diverse one is Pseudo-nitzschia pseudodelicatissima (and P. pseudodelicatissima-like morphologies) known from the area since the 1930s, and includes both toxic and non-toxic varieties. Therefore, morphometrics, taxonomic discussion and distribution patterns are more completely documented for these two species. P. subpacifica is the most recent addition to the local diversity of the genus. Pseudo-nitzschia seriata, earlier reported from the Bay of Fundy, was not observed in our samples. The presence of Pseudo-nitzschia delicatissima is confirmed, although a comparison with the narrow needle-shaped taxa reported earlier in the previous century is not possible. Five of the seven Pseudo-nitzschia species are now known to produce the neurotoxin domoic acid in various regions throughout the world, with P. pseudodelicatissima producing domoic acid in the Bay of Fundy [Mar. Ecol. Prog. Ser. 67 (1990) 177]. Although analysis of morphology continues to be the most reliable method for species identification, surprisingly little is known about the specific range of natural morphological variability outside Europe. Here, we present morphological data for species detection in the Bay of Fundy and emphasize the differences between the Bay of Fundy populations and those from elsewhere.

Coeliac disease—a meeting point for genetics, immunology, and protein chemistry

Coeliac disease is caused by a genetically determined, specific immune response to antigens present in wheat gluten. This immune response may be focused on a limited region of the alpha gliadin component of gluten, and previous studies have suggested that the generation of epitopes for recognition by CD4+ T cells requires deamidation of the protein by tissue transglutaminase. However, it had not previously been shown that candidate epitope peptides could be generated from gluten in vivo, or that these epitopes were selective products of physiological digestion of gluten by tissue transglutaminase. Lu Shan and colleagues (Science 2002; 297: 2275-79) have recently shown that a 33-mer peptide containing known peptide epitopes is generated by digestion with intestinal enzymes in vivo and in vitro, producing a highly stimulatory antigen for CD4+ T cells. The resulting peptide is resistant to further digestion of a gliadin by intestinal brush border enzymes and is a highly specific substrate for deamidation by tissue transglutaminase. Because the 33-mer peptide is not present in cereal proteins that do not cause coeliac disease, Shan and colleagues suggest that generation of this peptide in vivo underlies the specific association between gluten, immune responsiveness, and tissue transglutaminase in coeliac disease. In addition, the 33-mer peptide can also be produced by digestion of gluten by bacterial prolyl endopeptidases, suggesting possible future strategies for generating non-toxic varieties of gluten. WHERE NEXT? It is now important to determine whether this 33-mer peptide and this pathway accounts for all immune recognition of wheat gluten in coeliac disease, and to explore if the tissue transglutaminase homologues found in other organisms can be used to produce non-toxic preparations of wheat.

Assessment of lectin activity in a toxic and a non-toxic variety ofJatropha curcas using latex agglutination and haemagglutination methods and inactivation of lectin by heat treatments

Lectin activity in a toxic (Cape Verde) and non-toxic (Mexico) variety of Jatropha curcas seed meal was investigated using latex agglutination and haemagglutination assays. Lectin activity expressed as reciprocal of the minimum quantity (in mg) of Jatropha meal per ml of the assay mixture which produced agglutination with the latex beads was 2·88 ± 0·57 and 1·71 ± 0·00 (mean ± SD, n = 3) for the toxic and the non-toxic varieties, respectively, which did not differ significantly (P > 0·05), while with the haemagglutination assay these values were 102 and 51, respectively, and differed significantly (P < 0·05). The lectins were inactivated by heating and moist heat was more effective than dry heat. The results suggest that lectins may not be responsible for short term toxicity caused by consumption of raw Jatropha meal. © 1998 SCI.

Comparative evaluation of non-toxic and toxic varieties of Jatropha curcas for chemical composition, digestibility, protein degradability and toxic factors

Four varieties of Jatropha curcas which originated from Nicaragua (Cape Verde and Nicaragua toxic varieties cultivated in Managua), Nigeria (a wild variety from Ife; toxicity unknown) and Mexico (a wild non-toxic variety collected from Papantla) were studied. The average seed weight was 0.69, 0.86, 0.53 and 0.65 g for Cape Verde, Nicaragua, Ife-Nigeria and non-toxic Mexico varieties, respectively. The kernel to shell ratio in seeds was relatively similar (62.7:37.3 for both Cape Verde and Nicaraguan, 60:40 for Ife-Nigerian and 63.5:36.5 for non-toxic Mexican). The shell of the seeds composed mainly of fiber (>83% neutral detergent fiber) and very little crude protein (CP < 6%). The kernels were rich in CP (22.2–27.7%) and lipid (53.9–58.5%). The meal (defatted kernels) had a CP content of 57.3, 61.9, 56.1 and 64.4% for Cape Verde, Nicaragua, Ife-Nigeria and non-toxic Mexico varieties, respectively, and about 90% of this CP was true protein. The pepsin insoluble nitrogen was from 5.5 to 7%. The amino acid composition of meals from Cape Verde, Nicaragua and non-toxic Mexico varieties was similar. The levels of essential amino acids except lysine were higher than that for the FAO reference protein. The meal from the toxic variety (Cape Verde) did not have any anti-fermentative activity on rumen microbes. The estimated digestible organic matter (DOM) and metabolizable energy (ME) for the shells were low (26.2–27.1% and 2.4–2.8 MJ kg −1), whereas these values for Jatropha meals were 77.3–78.4% and 10.7–10.9MJ kg −1. For commercially available (heat-treated) soyabean meal, DOM and ME were 87.9% and 13.3MJ kg −1, respectively. The in-vitro rumen degradable nitrogen (% of total nitrogen) for meals from Cape Verde, Nicaragua, Ife-Nigeria and non-toxic Mexico varieties was 43.3, 37.7, 38.7 and 28.9, respectively, and for the soyabean meal it was 80.9%. Tannins, cyanogens, glucosinolates and amylase inhibitors were not detected in meals of any of the four varieties. A small amount of tannins were present in shells (2.0–2.9% as tannic acid equivalent). High levels of trypsin inhibitor activity (18.4–26.5 mg trypsin inhibited g −1), lectin (51–102; inverse of the minimum amount in mg ml −1 of the assay which produced haemagglutination in presence of 10 mM Mn 2+) and phytate (7.2–10.1%) were observed in the meals. The concentrations of phorbol esters in kernels of Cape Verde, Nicaragua and Ife-Nigeria varieties were 2.70, 2.17 and 2.30mg g −1, whereas kernels of non-toxic Mexican had a very low level (O.11 mg g −1) of phorbol esters.

Thyroid disease in the smaller hospital

Although the major problems of thyroid disease as encountered in the smaller hospital are relatively the same as those met in the larger institution, minor differences in the management of thyroid cases may result in major consequences. Critical judgment of the merit of any therapeutic measure predicates an intelligent evaluation of the ultimate benefit to the patient; and an appraisal of the results of a method of procedure is best derived from adequate and systematic follow-up studies. A thorough review of case records furnishes a complement of experience that can be attained in no other way. Such review combined with meticulous examination of surviving patients literally brings the thyroid surgeon to judgment. Provision for follow-up studies is lacking almost universally in smaller hospitals. Professional apathy notwithstanding, there are many obstacles which tend to preclude the issue of reviews from hospitals with fewer than 100 beds. Because of these faulty circumstances, much valuable knowledge is not forthcoming, morbidity and mortality data are inaccurate and there are many surgeons with an unwitting concept of their work and its results. The 205 cases reviewed in this study accumulated over a period of thirty years; and although most records were complete a number could not be utilized because much of the necessary information was not recorded. From the details available it was learned that: The diffuse forms of goiter in both the toxic and non-toxic groups have an earlier age incidence than the nodular forms. The diffuse toxic goiter appears predominently a decade later than the diffuse non-toxic variety. There is not an appreciable difference in the age incidence of the nodular non-toxic and the nodular toxic goiters. Malignancy of the thyroid gland is most common after the fourth decade of life and it usually follows a preexisting nodular goiter. As generally understood, and corroborated in these studies, females are more frequently affected by thyroid disease than males. The sexes are affected equally with cancer of the thyroid gland. Except for the examples of acute thyrotoxicosis, symptoms are of comparatively long duration, averaging approximately five years. Swelling is the most constant complaint. Pressure is complained of more commonly by toxic patients. Nervousness, palpitation and weight loss are not confined to toxic cases. Exophthalmos is approximately twice as frequent as gastrointestinal complaints in thyrotoxicosis. Evidence is inconclusive as to any deleterious effects resulting from the various anesthetic agents used in this series. Preliminary ligation of the thyroid arteries is not an absolutely safe procedure and is increasingly giving way to operative attacks on the gland in one or more stages. Subtotal thyroidectomy is the operation of choice, although single adenomatous nodules may be removed locally. Postoperative complications are usually of serious consequence and require immediate, often heroic, treatment. Complications markedly increase the period of hospitalization. The proper use of iodine pre- and postoperatively in toxic cases greatly reduces the incidence of complications and likewise shortens the period of hospitalization. The mortality rate in goiter surgery is higher in smaller hospitals. It is presumed that the mortality rate, 5.8 per cent, in this series closely approaches the average for thyroid cases in the smaller institution, a major consequence. The immediate surgical mortality rate in cancer of the thyroid gland is much higher than that of other surgical conditions of the gland. Surgery offers the greatest probability of cure in both non-toxic and toxic goiter; and should be supplemented with radiation therapy in the treatment of thyroid cancer.

Behaviour of Diphtheria Bacilli in Acidified Media, especially with reference to Virulence Tests

1. The growth of diphtheria bacilli in suger-free pure casein digest broth may be accelerated by the addition of dilute nitric, acetic and lactic acids, and delayed by certain monobasic fatty acids.2. The limiting acid zone for toxic and non-toxic varieties is unaffected by initial dilute acidifications by several acids, but incresaed by the addition of citric acid.3. The time periods and intensity of the reaction changes produced by diphtheria bacilli growing in a sugar-free casein digest medium are altered by the addition of certain acids. Toxic strains produce earlier and more progressive alkalinity when dilute acetic or nitric acid is included in the medium: and later and lessened alkalinity in the presence of butyric, formic, and propionic acids. Certain other typical acids do not exert any obvious action. Non-toxic strains are less affected by the selected acids, although butyric acid generally retards reaction changes.4. The time required for virulence testing is decreased, and the potency of the bacillus under examination is more evident when 1/200 η acetic or nitric acid is added to the culture medium. Acidified casein digest plus peptone broth yields practically the same results as acidified fermented veal peptone infusion. The acidification does not affect the metabolism of non-toxic strains.5. The disadvantages of unfermented meat infusions and the ascribed harmful effects of the fermentation products in fermented meat broths, may perhaps be avoided by the substitution of casein digest peptone medium. The advantages of the starting action of the glucose may be retained by adding acetic or nitric acid to the casein digest.6. Individual strains of diphtheria bacilli differ considerably in their capacity of altering the reaction of culture media, and in their toxic potency. It does not seem permissible to apply to newly isolated organisms all the published results obtained from cultures that have been repeatedly subcultured and established as toxin producers in nutrient bouillon. As a presumptive test in the virulence testing of bacilli just transferred from human tissues to artificial culture media the tendency of non-toxic strains to produce early formation of alkali in adequately buffered and adjusted unfermented veal broth may be thought worthy of extended trial.