Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition

Cannabis sativa L. is used as medicine and narcotic in Lesotho. Phytochemical composition and total phenolics content (TPC) for hexane, chloroform, ethyl acetate and methanol extracts of aerial parts of C. sativa were determined. Ethyl acetate extract (0.1875, 0.375 and 0.75 mg mL-1) and methanol extract (0.75, 1.5 and 3.0 mg mL-1) were evaluated for cytotoxicity, genotoxicity and modulation of cyclophosphamide (CP, 1.25 mg mL-1)- and ethylmethane sulphonate (EMS, 0.25 mg mL-1)-induced genotoxicity using Allium cepa root meristem assay. CP or EMS did not reduce mitotic index (MI) of cells, hence not cytotoxic when compared with negative control using the t-test (p>0.05), but genotoxic. Both extracts were genotoxic with methanol extract also being cytotoxic. Genotoxicity was the number of aberrant cells per 100 mitotic cells. Modulatory effect (ME) was obtained by comparing mutagen-induced genotoxicity with mixture-induced genotoxicity and expressed as the number of units of mutagen-induced genotoxicity that equalled the mixture-induced genotoxicity. ME was either positive or negative and significant only if ME = ≥ 2. Both extracts were genotoxic with methanol extract also being cytotoxic. Aberrations observed were sticky chromosomes, c-metaphase, anaphase and telophase bridges, chromosome fragments and laggards. Mixture of methanol extract with CP or EMS was more genotoxic (+ME range = 1.61-11.89) than the mutagen or extract alone which suggested synergistic interaction. Mixture of ethyl acetate extract with CP induced insignificant +ME. Mixture of ethylacetate extract with EMS was significantly more genotoxic (+ME = 2.20) than EMS only at high extract concentration. The methanol and ethylacetate extracts of C. sativa were not anti-genotoxic to CP- or EMS- induced genotoxicity. TPCs for hexane, chloroform, ethyl acetate and methanol extracts were 39831.46, 2544.94, 2438.20 and 56601.12 mg GAE/gram dry weight respectively. The differences in the cytotoxicity and MEs of the extracts were attributed to differences in phytochemical composition of extracts.

KERAGAMAN GENETIKA VARIAN ABAKA YANG DIINDUKSI DENGAN ETHYLMETHANE SULPHONATE (EMS)

ABSTRAK<br />Mutasi in vitro dengan perlakuan mutagen dapat digunakan untuk<br />meningkatkan keragaman genetika abaka melalui keragaman somaklonal.<br />Penelitian yang dilakukan bertujuan untuk: (1) menentukan konsentrasi<br />optimum EMS untuk induksi keragaman somaklonal dalam kultur kalus<br />embriogen abaka, (2) meregenerasikan bibit abaka varian dari kalus<br />embriogen yang diberi perlakuan EMS, dan (3) mengevaluasi tipe dan<br />frekuensi keragaman karakter kualitatif dan kuantitatif di antara populasi<br />tanaman mutan abaka yang diperoleh dari regenerasi kalus embriogen<br />yang diberi perlakuan EMS. Penelitian dilakukan mulai bulan Agustus<br />2003 sampai Agustus 2006 di Laboratorium Kultur Jaringan dan Kebun<br />Percobaan Karangploso, Malang pada Balai Penelitian Tanaman<br />Tembakau dan Serat, Malang (Balittas). Hasil penelitian menunjukkan<br />bahwa 0,6% EMS merupakan konsentrasi optimum karena pada<br />konsentrasi tersebut diperoleh keragaman somaklonal paling banyak.<br />Varian yang diperoleh menunjukkan karakter kualitatif dan kuantitatif<br />abnormal. Tipe varian tersebut umumnya bersifat negatif dan kurang<br />menguntungkan dibandingkan dengan populasi standar. Tipe dan frekuensi<br />keragaman kualitatif dan kuantitatif pada klon Tangongon berbeda dengan<br />klon Sangihe-1, mengindikasikan adanya pengaruh genotipe terhadap<br />keragaman somaklonal. Varian dari abaka klon Tangongon dengan<br />produksi serat tertinggi (161,0 g dan 154,0 g/tanaman) diperoleh dari<br />perlakuan EMS 0,3% (T1 28.1.1 dan T1 11.2.2), sedangkan dari klon<br />Sangihe-1, hasil serat tertinggi (35,0 g dan 40,0 g/tanaman) diperoleh dari<br />perlakuan EMS 0,6% (S4 28.1.0 dan S4 56.2.0). Produktivitas tersebut<br />lebih rendah dibandingkan dengan produksi serat tanaman kontrol klon<br />Tangongon (193,0 g/tanaman) dan Sangihe-1 (70 g/tanaman).<br />Kata kunci : Abaka, Musa textilis, keragaman somaklonal, EMS, muta-<br />genesis, in vitro, hasil, Jawa Timur<br />ABSTRACT<br />Genetic Variability of Abaca Variants Induced by<br />Ethylmethane Sulphonate (EMS)<br />In vitro mutation with mutagen treatment can be used to increase<br />the genetic variability of abaca by inducing somaclonal variation. The<br />objectives of the experiments were to (1) determine optimum concen-<br />tration of EMS to induce abaca somaclonal variation, (2) produce abaca<br />lines from EMS treated embryogenic calli and evaluate their performance<br />in the field, and (3) evaluate type and frequency of qualitative and<br />quantitative variant characters among regenerated abaca lines. This<br />experiment was conducted in Tissue Culture Laboratory and Karangploso<br />Experiment Station of Indonesian Tobacco and Fibre Crops Research<br />Institute (ItoFCRI) Malang from August 2003 to August 2006. The results<br />showed that EMS treatment on abaca embryogenic calli induced variation,<br />and the optimum EMS concentration was 0.6%. The variants exhibited a<br />number of abnormal qualitative and quantitative characters which were<br />generally negative characters since they showed lower value as compared<br />to control population. The presence of different types of qualitative and<br />quantitative variant characters was genotype dependent. Variants from<br />abaca clone Tangongon having the highest fibre yield (161.0 g and 154.0<br />g/plant) were obtained from 0.3% EMS treatment (T1 28.1.1 and T1 11.2.2<br />variants). While variants from abaca clone Sangihe-1 having the highest<br />fibre yield (35.0 g and 40.0 g/plant) were obtained from 0.6% EMS<br />treatment (S4 28.1.0 and S4 56.2.0 variants). The fibre yield of control<br />clones, Tangongon and Sangihe-1, were 193.0 g and 70 g/plant,<br />respectively.<br />Key words: Abaca, Musa textilis, induced mutation, somaclonal<br />variation, field evaluation, yield, East Java

Comparative biological sensitivity and mutability of chemo-mutagens in lentil (Lens culinaris Medik)

Present investigation was carried out using three different categories of chemical mutagens viz., ethylmethane sulphonate (EMS)–an alkylating agent, hydrazine hydrate (HZ)–a base analogue and sodium azide (SA) – a respiratory inhibitor on two varieties viz., Pant L-406 and Type-8 of lentil to study the immediate biological damage induced by the mutagens and to determine the sensitivity of biological material in question. Biological damage induced in M1 generation was estimated in terms of seed germination, seedling height and pollen fertility. A dose dependent reduction with increasing concentrations of the mutagens for all these parameters was observed in both the varieties. The inhibition was more severe at the highest concentration of all the three mutagens under study. Variety Type-8 was found to be more sensitive than the var. Pant L-406 with respect to the mutagens utilized. Reduction in seed germination, seedling growth and pollen fertility in M2 generation was reasonably less as compared to M1 generation.

Chemo-induced pod and seed mutants in mungbean (Vigna Radiata L. Wilczek)

Mungbean is one of the most important pulse crops due to its nutritive value and maintaining soil fertility through biological nitrogen fixation. Genetic variability is one of the pre-requisite for crop improvement. The present investigation was aimed at to enhance the genetic variability for three quantitative traits viz. pod length, number of seeds per pod and 100-seed weight in M2 and M3 generations of mungbean following mutagenesis with ethylmethane sulphonate (EMS), hydrazine hydrate (HZ) and sodium azide (SA). Mean pod length did not differ significantly in most of the mutagenic treatments in M2. However, significant improvement for the trait was exhibited with lower and moderate concentrations in M3 generation. The mean number of seeds per pod and 100-seed weight increased with lower and moderate concentrations of the mutagens in M2, whereas M3 generation showed a complete positive trend of shift. Long pod and bold seeded mutants may be exploited to increase the number of seeds per pod and seed size leading to increased yield potential. The genotypic coefficient of variation, heritability and genetic advance increased manifold in the treated population for all these traits suggesting that mutagen induced variability has the substantial scope to improve the mungbean crop.SAARC J. Agri., 15(2): 57-67 (2017)

api, A Novel Medicago truncatula Symbiotic Mutant Impaired in Nodule Primordium Invasion

Genetic approaches have proved to be extremely useful in dissecting the complex nitrogen-fixing Rhizobium–legume endosymbiotic association. Here we describe a novel Medicago truncatula mutant called api, whose primary phenotype is the blockage of rhizobial infection just prior to nodule primordium invasion, leading to the formation of large infection pockets within the cortex of noninvaded root outgrowths. The mutant api originally was identified as a double symbiotic mutant associated with a new allele (nip-3) of the NIP/LATD gene, following the screening of an ethylmethane sulphonate–mutagenized population. Detailed characterization of the segregating single api mutant showed that rhizobial infection is also defective at the earlier stage of infection thread (IT) initiation in root hairs, as well as later during IT growth in the small percentage of nodules which overcome the primordium invasion block. Neither modulating ethylene biosynthesis (with L-α-(2-aminoethoxyvinylglycine or 1-aminocyclopropane-1-carboxylic acid) nor reducing ethylene sensitivity in a skl genetic background alters the basic api phenotype, suggesting that API function is not closely linked to ethylene metabolism or signaling. Genetic mapping places the API gene on the upper arm of the M. truncatula linkage group 4, and epistasis analyses show that API functions downstream of BIT1/ERN1 and LIN and upstream of NIP/LATD and the DNF genes.

Selection against males in Caenorhabditis elegans under two mutational treatments

Within populations with mixed mating systems, selfing is expected to be favoured over outcrossing unless a countervailing process such as severe inbreeding depression is present. In this study, we consider the relationship between the expression of deleterious alleles and the maintenance of outcrossing in the nematode species, Caenorhabditis elegans . This species is characterized by an androdioecious breeding system composed of males at low frequency and self-fertilizing hermaphrodites that can only outcross via males. Here, we find that experimentally increasing the mutational load in four different isogenic wild isolates using 10 generations of Ethylmethane sulphonate (EMS) and UV irradiation mutagenesis significantly diminishes the cost of males. Males are maintained at higher frequencies in mutagenized versus non-mutagenized populations. Nevertheless, males still tend to be driven to low frequencies within isolates that are known to be prone to lose males. Further, we determine the viability effects of a single round of mutagen exposure and find that, for EMS, outcrossing overcomes the almost completely recessive and nearly lethal effects generated. We briefly interpret our results in light of current evolutionary theory of outcrossing rates.

The genetics, ultrastructure, and tubulin polypeptides of mebendazole-resistant mutants of Caenorhabditis elegans

Mebendazole-resistant mutants of Caenorhabditis elegans were isolated following mutagenesis of the wild-type strain, N2, with ethylmethane sulphonate. The mutants define a single autosomal gene and are allelic to ben-1. They grow, move, and reproduce normally in the presence of mebendazole and are cross-resistant to albendazole, cambendazole, fenbendazole, methylbenzimidazole carbamate, and thiabendazole. Accumulations of membrane-bound, electron-dense material associated with the nervous tissue and associated cells are seen in electron micrographs of L3, L4, and adult stages of N2 grown in the presence of mebendazole, but are not found in the mutants. The electron-dense material appears to be associated with the accessory cells of the neuropil rather than with the neurons. The tubulins from wild type, N2, and the mutants were characterised by two-dimensional electrophoresis followed by silver staining and Western blotting with monoclonal antibodies to α- and β-tubulin. A single isotype of α-tubulin is apparent in both N2 and the mutants. One major and three minor β-tubulin isotypes, bt-1 to bt-3, can be discerned in N2; the minor isotype bt-2 is absent in all the mutants. These findings suggest that the isotype bt-2 is the primary site of action of benzimidazole-based drugs in C. elegans.