Physical Mutagenesis Research Articles

Random mutagenesis of super Koji (Aspergillus oryzae): improvement in production and thermal stability of \u03b1-amylases for maltose syrup production

BackgroundAlpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto-oligosaccharides. It is an important enzyme generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought after properties for industrial use. The Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and safe for use in food industry. Hence, Koji strain’s development for the screening of potent mutants, hyper producer of thermostable α-amylases, with desired attributes is the need of the time.ResultsA process has been developed to improve super Koji (A. oryzae cmc1) strain through γ-rays treatment. The doses i.e. 0.60, 0.80, 1.00, 1.20 & 1.40 KGy gave more than 3.0 log kill. Initially, 52 Koji mutants resistant to 1% (w/v) Triton X-100 were selected. 2nd screening was based on α-amylases hyper production and 23 mutants were sorted out by measuring clearing zones index (CI). Afterwards nine potent mutants, resistant to 2-deoxy D-glucose, were screened based on CI. These were further analyzed for thermal stability and productivity of α-amylase under submerged conditions. The mutants’ M-80(10), M-100(6) & M-120(5) gave about four fold increases in α-amylases productivity. The half life of M-100(6) α-amylase at 55 °C was 52 min and was highest among the mutants. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia depicted that exposure to gamma rays increased rigidity of the mycelium. The potent Koji mutant M-100(6) was grown on soluble starch in 10L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1 protein. Growth kinetic parameters were: μ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1.ConclusionIt was concluded that the developed five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ-rays mediated physical mutagenesis. The developed thermostable α-amylases of super Koji mutantM-100(6) has immense potential for application in saccharification process for maltose syrup production. Moreover, the developed five step strain’s development process may be used for the simultaneous improvement in productivity and thermal stability of other microbial enzymes.

An Inverse Correlation Between the Production of Itaconic and Mevinolinic Acids in Aspergillus terreus Mutants

The high producer A. terreus ATWD 136 and the world A. terreus FRR 5360 wild strains were used to improve their productivity by physical mutagenesis. The production was decreased in all the mutants obtained from the mutagenesis of the FRR 5360 strain, while the mutants obtained from the ATWD 136 strain showed different responses in the production. Thus, they were grouped in two categories according to IA/MA production. The first category (three mutants) revealed that UV-irradiation-enhanced IA production, while the production of MA was decreased. The second category (14 mutants) showed an enhancement obtained in the MA production, while the production of IA was decreased. The mutant 16 was the best IA producer (threefold higher than the ATWD 136 parent strain), and the mutant 12 was the best MA producer (twofold higher than the ATWD 136 parent strain). Finally, RAPD–PCR confirmed the presence of different value of genetic variability between the wild A. terreus ATWD 136 and its hyper-producing mutants (M 12 and M 16). From the results, the authors concluded that there is an inverse correlation between level yield of IA and MA in mutated A. terreus strains.

Strain improvement for the production of antioxidant activity from Bacillus sp. by induced stress

In the present study, water samples were collected from different beaches in Chennai such as Marina beach, Elliott beach, VGP Golden beach and Kovalam beach to evaluate the best source for antioxidants. The bacteria were isolated on Starch Casein Agar media and screened for the antimicrobial activity. Among 11 isolates, 5 isolates showed antimicrobial potential which were further evaluated for the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity. Among 5 isolates, one sample showed significant DPPH scavenging activity with half minimal inhibitory concentration of (IC50)344.754µg/mL. The maximum antioxidant production was observed at pH 7 and at temperature of 37ºC with an IC50 of 188.66µg/mL and 293.76µg/mL respectively. The potent antioxidant producing strain was subjected for mutagenesis. In physical mutagenesis, the organism exposed for UV light for 25 minutes showed maximum antioxidant production with an IC50 of 133.55µg/mL. This mutant strain was then subjected for chemical mutagenesis with the addition of different concentrations of Ethidium bromide such as 10µL, 20µL, 30µL, 40µL and 50µL. The mutant strain obtained with the addition of 20µL Ethidium bromide (EtBr) showed significant antioxidant activity with an IC50 of 325.4µg/mL. The sample was purified by solvent extraction method and was evaluated for antioxidant production. The analyte was subjected to HPLC (High Performance Liquid Chromatography) analysis to avince the presence of antioxidants. The ethyl acetate extract showed the total phenolic content of 0.892 mg GAE/g of dry extract. It also showed the total flavonoid content of 0.522mg RE/g of dry extract.

Induced mutagenesis in Cicer arietinum by the application of EMS and Gamma rays with special reference to the cytological studies

The present study was conducted to analyze the chemical and physical mutagenesis on Cicer arietinum with special reference to cytological studies. In this regard, CO – 4 variety of chick pea was subjected to different concentration of gamma rays (20, 30, 40, 50 and 60kR) and EMS (10, 20, 30, 40 and 50mM) for inducing mutation. The M1 plants exposed to mutagen produces a clear cut difference from the untreated control. The root mitotic studies reveal a wide range of chromosomal aberrations such as stickiness, laggards, bridges and some other precocious movement. The percentage of abnormal cell increased with dosage in both mutagens; and 50mM EMS showed more chromosomal aberrations when compared to gamma rays.

Improvement of Secondary Metabolites from Phyllanthus odontadenius against Malaria by Mutagenesis

Aims: Majority of deaths in children aged under 5 years are due to Plasmodium falciparum malaria. Malaria deaths in children decreased but malaria remains a major killer of children, taking the life of a child every 2 minutes. This study aims to investigate the increasing of the in vitro antiplasmodial activities by mutagenesis techniques using gamma-rays (Cs-137) or sodium azide (NaN3) as utagens. It will allow the importance of mutagenesis use as tools for improvement of secondary metabolites against malaria parasites using chemical or physical mutagens. Study Design: Laboratory experiment tests : identification of plant material, immersion of seeds in SA (sodium azide) solutions or irradiation by Gamma-rays (Cs-137) of P. odontadenius seeds for improvement of secondary metabolites against malaria parasites, in vitro culture of seeds followed by the in situ culturing of plantlets for obtaining material of study, phytochemical screening of Phyllanthus odontadenius aerial parts to determine the change of compounds in comparison to controls, in vitro antiplasmodial tests for the determination of SA concentrations or those of gammarays doses which killing 50% of malaria parasite populations (IC50). Place and Duration of Study: Department of Biotechnology and Molecular Biology, Department of Biochemistry: General Atomic Energy Commission, Regional Nuclear Studies Center of Kinshasa, P.O. Box. 868 Kinshasa XI, Democratic Republic of the Congo (DRC). National Institute of Biomedical Research (NIBR) at Kinshasa/Gombe (DRC). Laboratory of UMR-MD3, Aix-Marseille University, Faculty of Pharmacy/Marseille, France. The experiments were conducted from Junuary - December 2010; August and December 2011 and during May and September 2012 until July 2014. Methodology: Aerial parts of plants M1 and M2 from Gamma-rays irradiation of P. odontadenius seeds or from immersion of P. odontadenius seeds in sodium azide solutions used as biological material for the in vitro antiplasmodial analysis. The in vitro antiplasmodial activities assays on clinical isolates of P. falciparum and on chloroquine-resistante P. falciparum strain K1 was determined using microscopic method, the isotopic micro-test method and using HRP2-based ELISA assay. Results: Gamma-rays (Cs-137) increased (multiplied) the in vitro antiplasmodial activities from 2.48 up to 7.6 in comparison to control. Thus, the in vitro antiplasmodial activities were improved or exceeded from 147.57% up to 660% than those of control plant. SA had increased (multiplied) the in vitro antiplasmodial activities from 1.24 up to 10.15 comparing to the control plants. The antiplasmodial activities in vitro were exceeded compared to the control plants from 24.43% up to 915%. Conclusion: Treatment of P. odontadenius seeds by Gamma-rays or by SA give plants high in vitro antiplasmodial activities. Values of in vitro antiplasmodial activities varried from 1.24 (147.57%) to 10.15 (915%). 125, 150 and 225 Gy of Gamma-rays (Cs-137) for physical mutagenesis and 10.15 and 17.5 M of SA solutions for the chemical mutagenesis could be used for improving in vitro antiplasmodial activities against P. falciparum (clinical isolates of P. falciparum or chloroquine-resistant P. falciparum strain K1). Thus, plants extracts from treated seeds have justified the usefulness of mutagens in plant breeding particularly in the increasing production of secondary metabolite against malaria parasite.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants.

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

Productivity of γ-Linoleic acid by oleaginous fungus Cunninghamella echinulata using a pulsed high magnetic field

Abstract γ-Linolenic acid (GLA) plays a crucial role in brain function and normal growth and development. High percentage of the population cannot effectively produce GLA, resulting in the use of GLA dietary supplements. Several oleaginous fungi can produce low level of GLA. The present study aimed to improve the production of GLA using physical mutagenesis. Spore suspensions of Cunninghamella echinulata were treated with pulsed high magnetic field. A total of 64 strains were isolated and screened using Nile red staining and fluorescence detection. Three mutants were selected after fermentation screening. GLA yields of Mut-29, Mut-15, and Mut-64 increased by 46.2%, 23.1%, and 19.2%, respectively, compared with that of the parent strain. Biomass, lipid content and yield, GLA content, and GLA yield of Mut-29 increased significantly (P

Novel Mutations in Oleoyl‐PC Desaturase (ahFAD2B) Identified from New High Oleic Mutants Induced by Gamma Rays in Peanut

To diversify the sources of the high‐oleate trait in peanut (Arachis hypogaea L.), the broad‐based interspecific derivative with inherent resistance to major foliar diseases, GPBD‐4, was subjected to chemical (ethyl methanesulfonate) and physical (γ rays) mutagenesis, resulting in significant variation in fatty acid profile of mutant progenies as tested by gas chromatography in advanced (M4) generations. From these mutant progenies, two stable high‐oleic (>70%) mutant lines, GM 4‐3 and GM 6‐1, were chosen for molecular characterization of ahFAD2B by cloning and sequencing. We report here the identification of two novel mutations (1085A‐G and 1111G‐A) in oleoyl‐PC desaturase (ahFAD2B) of high‐oleic mutant line GM 6‐1 and the latter single transition 1111G‐A in another high‐oleic mutant line GM 4‐3. The deduced amino acid sequence comparison of the parental genotype, GPBD‐4, with normal‐oleic (∼50%) and high‐oleic (>70%) mutant lines indicated the common change from glycine to serine at 372 from start codon due to the common single‐nucleotide polymorphism (SNP, 1111G‐A) observed in both the high‐oleic mutant lines. Another amino acid change from glutamate to glycine was also found in the mutant line GM 6‐1 because of the additional SNP it had at 1085A‐G in the coding region of ahFAD2B. These novel mutations identified in the new high‐oleic mutant lines could be used for marker‐assisted selection for the high‐oleate trait, and these foliar disease‐resistant, high‐oleic mutant lines could serve as additional valuable genetic resources for oil quality improvement in future peanut breeding programs.

In vitro physical mutagenesis of giant reed (Arundo donax L.)

AbstractGiant reed (Arundo donax L.) is a C3 perennial, warm‐season, rhizomatous grass of emerging interest for bioenergy and biomass derivatives production, and for phytoremediation. It only propagates vegetatively and very little genetic variation is found among ecotypes, basically precluding breeding efforts. With the objective to increase the genetic variation in this species, we developed and applied a mutagenesis protocol based on γ‐irradiation of in vitro cell cultures from which regenerants were obtained. Based on a radiosensitivity test, the irradiation dose reducing to 50% the number of regenerants per callus (RD50) was estimated at 35 Gy. A large mutagenic experiment was carried out by irradiating a total of 3120 calli with approx. 1×, 1.5× and 2× RD50. A total of 1004 regenerants from irradiated calli were hardened in pots and transplanted to the field. Initial phenotypic characterization of the collection showed correlated responses of biomass‐related quantitative traits to irradiation doses. Approx. 10% of field‐grown clones showed remarkable morphological aberrations including dwarfism, altered tillering, abnormal inflorescence, leaf variegation and others, which were tested for stability over generations. Clone lethality reached 0.4%. Our results show for the first time that physical mutagenesis can efficiently induce new genetic and phenotypic variation of agronomic and prospective industrial value in giant reed. The methodology and the plant materials described here may contribute to the domestication and the genetic improvement of this important biomass species.

Enhancing carotenoid production in Rhodotorula mucilaginosa KC8 by combining mutation and metabolic engineering

Rhodotorula mucilaginosa has been considered as a potential industrial yeast due to its unicellular and fast-growing characteristics, and its ability to produce carotenoids, including torularhodin. However, its low total carotenoid production limits its commercial application. In this study, mutation breeding and metabolic engineering were employed to enhance carotenoid production in the R. mucilaginosa strain KC8. After chemical–physical mutagenesis, R. mucilaginosa K4 with a 67% greater concentration of carotenoids (14.47 ± 0.06 mg L−1) than R. mucilaginosa KC8 (8.67 ± 0.07 mg L−1) was obtained. To further enhance carotenoid production, gene HMG1 encoding the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was introduced from another yeast, Saccharomyces cerevisiae, and overexpressed in R. mucilaginosa K4. The carotenoid production of HMG1-gene-overexpression transformant G1 reached 16.98 mg L−1. To relieve the feedback inhibition of ergosterol, and to down-regulate ergosterol synthesis, ketoconazole, an ergosterol synthesis inhibitor, was added at a concentration of 28 mg L−1. The carotenoid production of the transformant G1 reached 19.14 ± 0.09 mg L−1, which was 121% higher than in R. mucilaginosa KC8. This suggests that a combination of chemical–physical mutagenesis, overexpression of the HMG1 gene, and adding ketoconazole is an effective strategy to improve carotenoid production.

Approaches towards the enhanced production of Rapamycin by Streptomyces hygroscopicus MTCC 4003 through mutagenesis and optimization of process parameters by Taguchi orthogonal array methodology.

The present research was conducted to define the approaches for enhanced production of rapamycin (Rap) by Streptomyces hygroscopicus microbial type culture collection (MTCC) 4003. Both physical mutagenesis by ultraviolet ray (UV) and chemical mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine (NTG) have been applied successfully for the improvement of Rap production. Enhancing Rap yield by novel sequential UV mutagenesis technique followed by fermentation gives a significant difference in getting economically scalable amount of this industrially important macrolide compound. Mutant obtained through NTG mutagenesis (NTG-30-27) was found to be superior to others as it initially produced 67% higher Rap than wild type. Statistical optimization of nutritional and physiochemical parameters was carried out to find out most influential factors responsible for enhanced Rap yield by NTG-30-27 which was performed using Taguchi orthogonal array approach. Around 72% enhanced production was achieved with nutritional factors at their assigned level at 23 °C, 120rpm and pH 7.6. Results were analysed in triplicate basis where validation and purification was carried out using high performance liquid chromatography. Stability study and potency of extracted Rap was supported by turbidimetric assay taking Candida albicans MTCC 227 as test organism.

Mapping and application of the twin-grain1 gene in rice.

The map-based cloning and application of a flower organ number gene twin - grain1 provide great potential for improving seed production in hybrid rice. A new germplasm for high-yield rice breeding, the twin-grain1 (tg1) mutant with more than one grain in a glume, was obtained from the Zhejing 22 rice variety via physical mutagenesis. The mapping results showed that TG1 is allelic to FLORAL ORGAN NUMBER2 (FON2)/FLORAL ORGAN NUMBER4 (FON4), a flower organ number gene located at 88.7cM on chromosome 11. The novel tg1 gene allele was introgressed into the cytoplasmic male sterility (CMS) line Zhejing 22A, giving rise to a new CMS line Zhejing 22-tg1A. The Zhejing 22-tg1A line showed enhanced glume opening and stigma exsertion, which increased the outcrossing rate in hybrid rice. A small-scale hybrid rice seed production test demonstrated that the grain yield of the Zhejing 22-tg1A/Zhejinghui 5 line was significantly increased compared to that of the Zhejing 22A/Zhejinghui 5 line. The plot yield evaluation of the F1 hybrid lines showed a higher yield for the Zhejing 22-tg1A/Zhejinghui 5 line than that of the Zhejing 22A/Zhejinghui 5 line. The results implied great potentials for the tg1 gene inhybridrice breeding.

Mutagenesis and Biotechnology Techniques as Tools for Selecting New Stable Diploid and Tetraploid Olive Genotypes and Their Dwarfing Agronomical Characterization

In Olea europaea L. subsp. europaea, tetraploid genotypes do not exist in nature. Herein, we report the first example of selection of tetraploid olive plants, obtained by combining physical mutagenesis and biotechnology techniques. Stable tetraploid (4n) and diploid (2n) genotypes were isolated in vitro through shoot-tip fragmentation of two mixoploid mutants derived from the gamma irradiation of self-incompatible Leccino and self-compatible Frantoio cultivars. In this study, the stable mutants FRM5-4n, FRM5-2n, LM3-4n, and LM3-2n were characterized in the field for vegetative and reproductive behavior with the aim to use them as varieties or dwarfing rootstocks. The stable 4n genotype of Leccino acquired self-fertility whereas the 4n Frantoio maintained it. A high and constant yield was showed by LM3-2n during 9 years of observation, maintaining the same oil quality as the Leccino wild type (wt). Moreover, the LM3-2n acquired the capacity to be intercompatible with the diploid mutant Leccino dwarf (LD) and with the Leccino wt. This acquired property would allow for a reduction of heterozygosity in the offspring, if crossed with each other for some generations and with the Leccino wt, because it is a sort of self-fertilization. When used as rootstocks, both 4n and 2n Leccino mutants proved to be very effective for reducing the scion size of the high-vigor Canino cultivar, which is well known for its excellent extravirgin oil. Finally, it was demonstrated that the self-grafting of vigorous cultivar caused a reduction in plant size, thus suggesting that it is possible to produce semidwarf plants from vigorous genotypes to consider them in high-density olive orchards.

Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23g lipids/g cell dry wt and 0.75mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41g lipids/g CDW and 0.69mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

Effect of induced physical and chemical mutagenesis on the cytological behaviour of Sorghum (Sorghum bicolor (L.) Moench)

Effect of induced physical and chemical mutagenesis on the cytological behaviour of Sorghum (Sorghum bicolor (L.) Moench)

Isolation of desirable induced morphological mutations in elite lines from a population ofAbelmoschus esculentus(L.) Moench by gamma rays and EMS

Induced mutation often produces abnormalities which cause morphological alterations in external form of plants including colour, shape, size, etc. In the present study, evaluation was performed during summer and kharif seasons of 2010–11 on Abelmoschus esculentus. Seeds of two elite varieties (GO-2 and GJO-3) were subjected to physical (gamma rays) and chemical (ethyl methane sulphonate; EMS) mutagenesis to find out the frequency and spectrum of induced desirable morphological mutations. In M2 generation, 12 different morphological mutations were induced in both the varieties. It was found that the spectrum of these mutations and their frequency varied with the type and dose of mutagens and the genotype used. Gamma radiations induced more number of morphological mutants as compared to EMS treatments in both the varieties. The highest mutation rate (11.19%) was detected in 40 kR gamma rays treatment followed by 0.25% EMS treatment in GO-2. In general, gamma irradiation treatments seemed to be superior in producing greater frequencies over all doses for both the varieties GO-2 (19.17%) and GJO-3 (16.33%).

Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation

This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO4, pretreated S. spontaneum, K2HPO4, (NH4)2SO4 and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.

Induced Physical Mutagenesis and its Effect in Cytological Behavior of Ashwagandha (<i>Withania somnifera</i> (L.) Dunal)

The mitotic effect of physical mutagen gamma rays was observed in the root tip cells of Ashwagandha. The Chromosome analysis has been showed as an important tool for establish variability of the plant seed by use of physical mutagen gamma rays. The gamma rays have of low wavelength and high penetrable power. The plant has tremendous medicinal values and it is known from ancient times. The dry and well matured seeds of ashwagandha were irradiated with different doses of gamma rays viz., 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 KR respectively. The chromosome number of control plant is 2n = 48. The gamma rays affect the normal cytological behavior of ashwagandha species. The chromosomal aberrations increase with increase in the doses of gamma rays to optimum level of 30KR, because it causes changes in the chromosome structure, cellular structure and metabolism of plants. The chromosome aberration like, Sticky metaphase, Precocious moment chromosome, Fragments, Anaphasic bridge, Anaphasic laggard, Telophasic laggard. The present investigation was carried out to study the cytogenetic analysis of the species Withania somnifera. The chromosomal aberration increases with increase in the doses to optimum level (50 KR) of physical mutagen gamma rays.

Induced Physical Mutagenesis and its Effect in Cytological Behavior of Ashwagandha (<i>Withania somnifera</i> (L.) Dunal)

The mitotic effect of physical mutagen gamma rays was observed in the root tip cells of Ashwagandha. The Chromosome analysis has been showed as an important tool for establish variability of the plant seed by use of physical mutagen gamma rays. The gamma rays have of low wavelength and high penetrable power. The plant has tremendous medicinal values and it is known from ancient times. The dry and well matured seeds of ashwagandha were irradiated with different doses of gamma rays viz., 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 KR respectively. The chromosome number of control plant is 2n = 48. The gamma rays affect the normal cytological behavior of ashwagandha species. The chromosomal aberrations increase with increase in the doses of gamma rays to optimum level of 30KR, because it causes changes in the chromosome structure, cellular structure and metabolism of plants. The chromosome aberration like, Sticky metaphase, Precocious moment chromosome, Fragments, Anaphasic bridge, Anaphasic laggard, Telophasic laggard. The present investigation was carried out to study the cytogenetic analysis of the species Withania somnifera. The chromosomal aberration increases with increase in the doses to optimum level (50 KR) of physical mutagen gamma rays.

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation.