Irradiated Callus Research Articles

Effect of Gamma Irradiation on Callus of Handeuleum (Graptophillum pictum L. Griff) Kalimantan and Papua Accession

Handeuleum (Graptophyllum pictum L.Griff) is a medicinal plant that is widely used as a traditional medicine for its healing properties, hence it should be developed as one of the leading Indonesian medicinal plants. The combination of tissue culture and gamma irradiation are two common methods to improve vegetatively-propagated cultivars. This experiment aims to determine the effect of gamma irradiation dose-rate on the callus culture of the two handeuleum accessions. Callus generated from proliferation were irradiated with gamma rays (0 Gy, 15 Gy, 25 Gy, and 35 Gy) and then grown on regeneration medium containing BAP (4.44 μM and 8.88 μM) and TDZ (4.44 μM and 8.88 μM). The result of the experiment shows that RD50 was at19.25 Gy. The interaction among the Papua accession, gamma irradiation dose-rate of 15 Gy, and regeneration media 4.44 μM BAP produced the highest weight of callus subculture 1 and 2. Irradiation dose-rate of 25 Gy on Kalimantan accession resulted in the highest relative fresh weight growth of irradiated callus. The treatment with a 25 Gy dose-rate produced brownish-white callus and produced the highest value of variability in the relative growth rate of irradiated callus. 
 Keywords: accession, irradiation dose-rate, purple leaves, variability

In-vitro selection of sugarcane (Saccharum officinarum L.) putative mutant for drought stress

In-vitro selection through tissue culture followed by field testing is one of the breeding methods to improve sugarcane varieties with drought-tolerant. This research aimed to obtain putative mutant sugarcane physically mutated using gamma-ray irradiation that passed the in vitro drought selection using PEG-selecting agents. This study was conducted from March to December 2016 using embryogenic calli of PSJT 941, PS 862, and BL varieties. The experimental design was the factorial randomized complete design with two factors, i.e. irradiation dose (0, 5, 10, 15, 20, 25, 30, 35 Gray) and PEG concentration (0, 10, 20%). The irradiated calli selected by PEG media had changed in color from yellowish-white to brownish and blackish and indicated the growth inhibition. The higher the dosage of irradiation and the concentration of PEG, the more calli with growth inhibition. Some putative callus mutants successfully passed in vitro selection using 10 and 20 % PEG, which showed tolerance to drought stress. The appropriate irradiation dose to produce tolerant mutants is 5 – 30 Grays for PS 862 and BL varieties, and 5-25 Grays for PSJT 941. To recognize the mutant response further, it is necessary for in-vivo selection at the greenhouse and in the field.

Cell line selection through gamma irradiation combined with multi-walled carbon nanotubes elicitation enhanced phenolic compounds accumulation in Salvia nemorosa cell culture

The current study focused on improving the production of phenolic acids in the Woodland Sage cell suspension culture (CSC) through attaining high-yielding cell lines and carboxyl functionalized multi-walled carbon nanotubes (MWCNT-COOH) elicitation. The leaf-derived callus was irradiated at different doses of gamma irradiation 10 to 100 Gy. The maximum content of rosmarinic acid (RA), salvianolic acid B (SAB), ferulic acid (FA), and cinnamic acid (CA) was recorded in callus cultures irradiated with 70 Gy, which was 18.53, 5.21, 1.9, and 7.59 mg/g DW, respectively. The CSC that established from 70 Gy γ-irradiated calli accumulated 1.7-fold RA more higher irradiated callus culture. The CSC elicited with various concentrations of MWCNT-COOH in range 25 to 100 mg/l significantly increased fresh weight (FW), dry weight (DW), and phenolic acid contents of cells. The highest FW with 268.47 g/l and DW with 22.17 g/l was obtained in 100 mg/l MWCNT-COOH treatment. The RA, SAB, CA and FA content of CSC treated with 100 mg/l MWCNT-COOH were 13-fold, 14.2-fold, 20-fold, and 3- fold higher than wild S. nemorosa plant at flowering stage, respectively. The antioxidant activity of cultures significantly enhanced with both gamma and MWCNT-COOH based on DPPH and FRAP assay. Our results showed that the combination of cell line selection and MWCNT-COOH elicitation significantly improved the production of secondary metabolites in Woodland Sage, which is useful for large-scale production of phenolic compounds. Our findings revealed that cell suspension culture established from γ-irradiation S. nemorosa callus which elicited with Multi-walled carbon nanotubes accumulated much higher phenolic compounds compared to the wild plant.

Somatic embryogenesis on irradiated callus of garlic (Allium Sativum L.)

The research was done at the Tissue Culture Laboratory of Center for Application of Isotope and Radiation Technology, Jakarta. Callus derived from shoot tip of garlic cloves was exposed to gamma rays, then cut into sized of 1 mm3 and cultured in Gamborg medium enriched with plant growth regulators (PGRs) depends on the treatment. The PGRs applied are. The experiment was arranged in a randomized factorial design with 2 factors and 3 replications. The first factor was the doses of gamma rays (0, 10 and 20 Gy) and the second factor was different concentrations of PGRs (untreated, 1 and 2 ppm of thidiazuron (TDZ) and Zeatin). The result indicated that the application of 2 ppm TDZ or Zeatin promoted development on irradiated callus at the dose 10 Gy. The highest number of the shoot and the longest shoot were obtained from non-irradiated callus grown on medium enriched with 2 ppm TDZ, followed by 1 and 2 ppm Zeatin, 1 ppm TDZ and without PGRs. Irradiated callus at the dose 10 Gy grown on medium with 2 ppm TDZ was able to form a shoot, while other irradiated callusat the dose 29 Gy were not able to form the shoot.

Callus from Pyrostegia venusta (Ker Gawl.) Miers: a source of phenylethanoid glycosides with vasorelaxant activities

Pyrostegia venusta (Ker Gawl.) Miers is a plant used for the treatment of respiratory diseases, diarrhea, vitiligo, jaundice and to attenuate vomiting. However, the levels of the active principles exhibit great variation because of the type of soil, tissue, age and environmental conditions. An alternative to obtain a constant production of secondary metabolites is the plant tissue culture technology. In this work, callus from Pyrostegia venusta were exposed to 12.5 g L−1 polyethylene glycol, 50 g L−1 sucrose, or were irradiated with UV light to enhance the content of phenolic acids, flavonoids, and antioxidant activity. The biomass was doubled in the control and callus treated with PEG (240 mg dry weight, DW), and triplicated in the medium with 50 g L−1 sucrose (310 mg DW) in relation to the inoculum at day 21. The highest levels of phenolic acids and flavonoids were obtained in irradiated callus. Phenylethanoid glycosides, as verbascoside, isoverbascoside and leucosceptoside A were identified. The metabolites present in callus presented vasorelaxant activity (65 to 100%). The vasodilation was inhibited between 80 and 90%, in the presence of NG-nitro-l-arginine methyl ester, indomethacin or tetraethylammonium chloride but was not affected by atropine. Data suggest that vasorelaxation was mediated by nitric oxide, derivatives of arachidonic acid, and efflux potassium channels, and independent of muscarinic receptors. This is the first report that identified the metabolites present in P. venusta compact callus and described its vasorelaxant properties. It was possible to enhance the metabolites production in Pyrostegia venusta callus. The compounds identified corresponded mainly to phenylethanoid glucosides, which exhibited important vasorelaxant activity in isolated rat aorta rings.

In vitro regeneration of gamma irradiated callus of artemisia annua and evaluation of increase artemisinin content by HPLC analysis

Gamma radiations are the efficient ionizing radiations which have potential to induce mutations in plants They also modify physiological characteristics of plant to create new mutants for large scale production of commercially important secondary metabolites In present study the effect of various low doses gamma irradiations and Gy on growth regeneration and elicitation of Artemisinin content from Artemisia annua was studied Callus culture was established on Murashige and Skoog medium supplemented with phytohormones Napthalein acetic acid and N benzylaminopurine mgL minus Effect of gamma doses on callus growth and elicitation of artemisinin content were investigated from M T M T M T M T and M T generation of in vitro callus culture In current study it was found that low dose of gamma irradiations are effective strategy to enhance the yield of artemisinin in A annua The radiation sensitivity test was performed which is based on the reduction in survival rate of callus culture after treatment with gamma irradiation at different doses from to Gy which showed the negligible effect till Gy but at and Gy it inhibited the callus growth and callus regeneration The detection and quantification of artemisinin content was performed by HPLC analysis The mobile phase for artemisinin elution was acetonitrile water v v at a flow rate of ml min minus and UV detection at nm Artemisinin concentration of unknown sample was calculated by using the Y equation y x R sup generated from standard curve The retention time of artemisinin in above solvent system was observed at min The higher accumulation of artemisinin content was observed in Gy dose micro g gm dry callus weight which is tenfold higher than non treated micro g gm dry callus weight The present study confirms that gamma radiation induced mutational changes in callus culture and it enhanced the artemisinin content

Microwave treatment can induce chrysanthemum phenotypic and genetic changes

Abstract The impact of microwaves on the DNA of plant cells is still ambiguous, but the usage of this electromagnetic radiation as a source of variation in mutation breeding could be very advantageous. The aim of the study was to examine the influence of microwave radiation on the in vitro regeneration and acclimatization efficiencies, as well as on the genetic and phenotypical variation of chrysanthemum ‘Alchimist’. Leaf explants with or without callus were subjected to microwave treatment for various periods and in different milieus. Microwaves were generated by a domestic magnetron at 2.45 GHz, 800 W·cm−2. Irradiation negatively affected shoot formation if applied for longer periods. On the other hand, it did not affect the rooting and acclimatization steps which were fully successful. Chrysanthemums produced from MW-treated explants had longer shoots with inflorescences of greater diameter and altered phenotypes. It was also noticed, that MW-treatment affected the generative phase by prolonging the bud colouration period. The evaluation of genetic variation was performed with the RAPD technique by using 10 primers for all regenerated shoot; a total of 116 genotypes were analysed. Approximately 22% of plants regenerated from MW-treated explants showed band profiles different from the reference control. Most plants (86%) with altered band profiles originated from irradiated callus. These plants also showed higher mean genetic distance coefficient. In conclusion, microwaves can be considered as an efficient and easy-to-access tool in mutation breeding of chrysanthemum ‘Alchimist’.

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.

Changes induced by gamma ray irradiation on biomass production and secondary metabolites accumulation in Hypericum triquetrifolium Turra callus cultures

The present study investigated the effect of gamma irradiation on biomass formation and yield of pharmacologically relevant secondary metabolites in callus cultures induced from different seedling parts (leaf, stem and root). Calli induction and maintenance were performed on MS medium supplemented with 0.5mgL−1 IAA and 0.4mgL−1 TDZ and all parameters were recorded after the third 21days successive subculture post-elicitation. Fresh callus biomass (FCB) expressed as growth index (Gi) was investigated and the higher Gi value (1109%) was recorded on callus culture from leaf explants and irradiated with 10Gy dose, as compared to untreated control (757%). H. triquetrifolium callus cultures exhibited quantitatively different phenolic compounds (p-OH-benzoic and chlorogenic acid, epicatechin) and naphtodiantrones (hypericin and pseudohypericin). The chromatographic analysis of callus extracts revealed that after gamma rays elicitation with 10Gy dose, the higher amount of p-OH-benzoic acid was recorded on calli from leaf explant (4.35mg 100g−1 DW material) while root callus recorded the highest amount of chlorogenic acid (12.91mg 100g−1) over the control (3.55mg 100g−1 and 10.22mg 100g−1, respectively). The 10Gy and 20Gy irradiation doses stimulate the epicatechin accumulation on calli from leaf and stem (126.39 and 148.80mg 100g−1) compared to the control samples (98.81 and 101.72 respectively).In this study, small amount of hypericin and pseudohypericin were identified and quantified on irradiated callus cultures initiated from stem and leaf of Hypericum triquetrifolium, but not in root calli. Callus induced from leaf and irradiated with 10Gy showed the higher amounts of hypericin and pseudohypericin content (0.29 and 4.00mg 100g−1 DW material). Phytochemical screening carried out on different elicited callus cultures variants reveal, for the first time, the stimulatory effects of gamma irradiation on the production of phenolic compounds and naphtodiantrones in Hypericum triquetrifolium Turra. The results of this study are interesting and offer an innovative approach of elicitation, not yet widely studied for the Hypericum genus.

Regenerasi Tanaman dari Kalus Tebu yang Diiradiasi Sinar Gamma pada Medium dengan PEG 6000

ABSTRACT<br /><br />Sugarcane cultivars tolerant to drought stress are needed for specific location. In vitro selection can be used to obtain plants tolerant to drought stress through regeneration of callus in the culture medium. The purpose of this study was to obtain regenerants from irradiated callus on the stress medium. The experimental design was a completely randomized design with two factors. The first factor was the regeneration medium (RG) added with PEG 6000, i.e., 0%, 5%, 10% and 15%, while the second factor was the dose of gamma ray irradiation i.e. 0 krad, 5 and 10 krad, and 15 krad. There were 12 treatments, each treatment was repeated 18 times and each repetition was a culture bottles planted with three clums of callus 10 mm diameter. RG medium was the MS medium added with 0.5 mg L-1 BAP, Kinetin 0.1 mg L-1 and IBA 1.0 mg L-1. The higher the concentration of PEG, the less regenerants were produced. Regenerants could be generated from the selection medium PEG up to 15%. Several shoots still produced from callus irradiated with 10 krad. Selection medium with PEG up to 10% decreased the precentage of albino regenerants. The gamma ray irradiation dose and the concentration of PEG significanly affected the number of roots produced.<br /><br />Keywords: drought tolerant, in vitro, polyethylene glycol, plantlets, albino<br /><br />

Production of artemisinin in shoot cultures of Artemisia cina irradiated callus

Artemisinin is an antimalaria agent that is only found in Artemisia sp . medicinal plant. This compound is occurred at very low level in that plant. Irradiated callus of Artemisia cina have been conducted by the dose of 40 Gy to improve artemisinin content in shoot cultures. Regeneration of irradiated callus to shoot cultures formation has been successful in Murashige & Skoog (MS) medium combined with Naphtalene Acetic Acid (NAA) 1 mg/g, Benzyl Amino Purin (BAP) 2 mg/L and 15 % coconut water. Ten mutant lines of shoot cultures have been analyzed for the artemisinin content. The artemisinin content of mutant lines were higher than the original plant. The highest artemisinin content is 5 mg/g found in mutant line number 404, this line also has wider leaf area than original plant. Key word : artemisinin, Artemisia cina , shoot cultures, irradiated callus

Androgenesis Induced in <i>Nicotiana alata</i> and the Effect of Gamma Irradiation

Nicotiana alata anthers cultured on different modified media based on MS, MT and N were used to obtain haploid plants through direct and indirect ways. The haploid plants resulting on MS medium ranged from 52% - 80%, on MT medium ranged from 32% - 52% and on N medium ranged from 28% - 44%. Accordingly, the best medium used for haploid induction was MS supplemented with 0.2 mgl-l NAA + 0.5 mgl-l KIN. On the other hand, MS medium supplemented with 0.4 mgl-l NAA + 0.5 mgl-l KIN or 1.0 mgl-l BAP + 0.5 mgl-l NAA were the best mediums for callus induction and plant regeneration, respectively. Morphologically, the leaf size, stem highest and diameter, flower size and diameter, anther length and number were about 67% of the diploid plants growth. Irradiated anthers with doses of 0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy caused reducing the number of haploid plants with increasing gamma radiation dose. For the haploid plants irradiated with same doses, the mortality percentage of bud survival was increasing with increasing gamma radiation dose. The irradiated callus with doses of 0, 5, 10, 15 and 20 Gy was affected negatively on growth rate and morphology. Proline content in irradiated plantlets increased with increasing gamma radiation dose. As well, total soluble protein content was increased with gamma irradiation up to 10 Gy. However, the higher doses caused a severe decrease of total soluble proteins. The production of proline and total soluble proteins in haploid plants were 48.6% and 69.5%, respectively comparing with diploid plants.

Androgenesis Induced in <i>Nicotiana alata</i> and the Effect of Gamma Irradiation

Nicotiana alata anthers cultured on different modified media based on MS, MT and N were used to obtain haploid plants through direct and indirect ways. The haploid plants resulting on MS medium ranged from 52% - 80%, on MT medium ranged from 32% - 52% and on N medium ranged from 28% - 44%. Accordingly, the best medium used for haploid induction was MS supplemented with 0.2 mgl-l NAA + 0.5 mgl-l KIN. On the other hand, MS medium supplemented with 0.4 mgl-l NAA + 0.5 mgl-l KIN or 1.0 mgl-l BAP + 0.5 mgl-l NAA were the best mediums for callus induction and plant regeneration, respectively. Morphologically, the leaf size, stem highest and diameter, flower size and diameter, anther length and number were about 67% of the diploid plants growth. Irradiated anthers with doses of 0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy caused reducing the number of haploid plants with increasing gamma radiation dose. For the haploid plants irradiated with same doses, the mortality percentage of bud survival was increasing with increasing gamma radiation dose. The irradiated callus with doses of 0, 5, 10, 15 and 20 Gy was affected negatively on growth rate and morphology. Proline content in irradiated plantlets increased with increasing gamma radiation dose. As well, total soluble protein content was increased with gamma irradiation up to 10 Gy. However, the higher doses caused a severe decrease of total soluble proteins. The production of proline and total soluble proteins in haploid plants were 48.6% and 69.5%, respectively comparing with diploid plants.

Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane (Saccharum officinarum L.)

Gamma ray-induced in vitro mutagenesis and selection for salt (NaCl) tolerance were investigated in sugarcane (Saccharum officinarum L.). Embryogenic callus cultures were irradiated (10 to 80Gy) and subjected to in vitro selection by exposure of irradiated callus to NaCl (0, 50, 100, 150, 200, and 250mmolL−1). Increasing NaCl concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+ and K+ concentration. Higher accumulation of proline and glycine betaine was observed in NaCl stressed callus irradiated at 20Gy. Na+ concentration increased and K+ concentration decreased with increasing salt level. Irradiated callus showed 50–60% regeneration under NaCl stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%, number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

Gamma radiation, in vitro selection for salt (NaCl) tolerance, and characterization of mutants in sugarcane (Saccharum officinarum L.)

Gamma-ray-induced mutagenesis and in vitro selection of sugarcane (Saccharum officinarum L. Lam.) plants tolerant to NaCl was achieved using embryogenic calli of sugarcane var. Co740. Sensitivity to gamma radiation and NaCl was studied in vitro in embryogenic calli followed by characterization and evaluation of mutant clones. The results indicated that callus growth and plant regeneration were significantly affected by radiation dose (0, 10, 20, 30, 40, 50, 60, 70, or 80 Gy 60Co gamma rays) as well as NaCl exposure (0, 50, 100, 150, 200, or 250 mM). Salt tolerance was achieved by culturing irradiated callus on selection medium with different NaCl concentrations through stepwise in vitro selection. Salt-selected embryogenic callus lines were then analyzed for proline, glycine betaine, Na+, and K+ contents. Salt-selected plants were grown to maturity, and their agronomic performance was evaluated under normal and saline conditions. Twenty-four mutant clones were characterized for proline, glycine betaine, Na+, and K+ contents. The mutant clones exhibited improved sugar yield with increments in Brix%, number of millable canes, girth, and yield. The results suggest that in vitro culture and induced mutagenesis offer an effective way to enhance the genetic variation in sugarcane.

Isolation of flower color and shape mutations by gamma radiation of Chrysanthemum morifolium Ramat cv. Youka

We investigated the effect of radiation damage on in vitro mutation induction in chrysanthemum. White petals of chrysanthemum (Chrysanthemum morifolium Ramat cv. Youka) were selected to induce mutation by gamma radiation. Calli produced were irradiated with gamma rays at 0, 10, 15 and 20 Gy. We found that the plants from the irradiated calli were different from control plants in number of leaves, leaf length & width, number of flowers, flower diameter, petiole diameter and petiole length after transplanting into the greenhouse for almost 70 days. Three mutants in flower color and shape were found in 15 Gy-treated plants. First type of mutant (M.1) has tubular petals. The second (M.2) and third (M.3) ones both have yellow flowers, while one of them has spooned shaped ray florets similar to the original cultivar and the other one has flat shaped florets. Semi-quantitative RT-PCR showed that most of carotenoid-biosynthesis related genes, except for violaxanthin deepoxidase (VDE) and lycopene e-cyclase (LCYE), showed similar expression patterns in petals of the original ‘Youka’ and its mutants (M2 & M3). VDE and LCYE results showed high expression levels in M3 and M2 & M3 respectively, comparing with the control. On the other hand, expression patterns for VDE were similar in control and M2. These yellow mutants were maintained vegetatively and proved to be true-to-type in one successive generation. It can be concluded that gamma radiation with 15 Gy dose can be used for in vitro induction of flower color and shape mutations of chrysanthemum cv. Youka.

Application of a low dose of gamma rays in wheat androgenesis

Gamma-rays of dose of 5 Gy using a 60 Co source was applied in a single wheat cross to study the effect on anther culture (AC) parameters. The female parent was a 6U g /6D mono-substitution line of cv. Trakia with resistance to pow- dery mildew and leaf rust, while the male parent was cv. Aglika, characterized as one of the best local varieties with high productivity and grain quality. Callus induction, plant regeneration, green and albino plants from the control (normal anther culture, 0 Gy) designated C, were compared with parameters studied in three other groups, derived from anthers irradiated with 5 Gy (designated AI), calli irradiated with 5 Gy (designated CI) and anthers and calli irradiated consecu- tively with 5+5 Gy (combined treatment group, designated ACI). Anther culture response of the four F1 group of plants was intermediate between the parents, with a slightly depressive effect of the dose irradiation in the AI and ACI groups. In general, the effect of the 5 Gy dose of irradiation on androgenesis was estimated to be negative for at least one group of haploid plants (AI), derived from irradiated anthers. Green plants were mostly reduced in the combined treatment group originating from irradiated calli and anthers.

Irradiation effect on in vitro organogenesis, callus growth and plantlet development of Gerbera jamesonii

The present work was carried out to study the effects of gamma irradiation on in vitro growth of explants, callus and the formation of shoots and plantlets. Irradiation is known to exhibit or inhibit the differentiation of cells and growth of plants in vitro, which helps in producing new plant varieties. Gamma irradiation is one of the physical mutagens that are widely used for mutation breeding. A gradual decline was observed in the number of shoots regenerated from irradiated petiole explants compared to control. Numbers of shoots regenerated from irradiated petiole explant cultured on Murashige & Skoog medium supplemented with 2.0 mg L-1 BAP and 0.5 mg L-1 NAA was reduced to 6.6±0.9 from 7.5±0.4 (control) when explants were exposed to 20 Gray of irradiation dose. Similar observation was reported on effects of gamma irradiation on in vitro propagated plantlets. Gradual decline was observed based on plant height as the dose of gamma irradiation increased. A significant decline was observed in the fresh weight of irradiated callus compared to control. In this case, growth responses of callus were strongly influenced by the radiation dose. The fresh weight of callus was reduced to 76.4±2.2% compared to 89.7±0.5% of control when callus tissues were exposed to 20 Gy.

Proline, sodium and potassium concentration changes in gamma rays and NaCl

In this study, the effects of gamma rays on changes of proline, sodium and potassium in potato calli (Solanum tuberosoum L.) cultivar White Desiree were investigated. After irradiation of calli by 60 and 80 Gy of gamma radiation, irradiated and non irradiated calli were transferred to a callus production medium containing 0, 30, 60, 90, 120 and 150 mM NaCl. After 26 days post treatment amounts of proline, Na+ and K+ were measured. The results showed that generally in each of gamma ray treatment, amounts of proline at 120 and 150 mM NaCl were significantly more than 0, 30, 60 and 90 mM salt. A significant difference observed only at 150 mM NaCl between irradiated and non irradiated calli. This result could be related to the proline role in osmotic adjustment at high concentration of salt. The amounts of sodium increased by increasing salt the concentration but the amount of potassium decreased. These results showed that the sodium influx inhibited the potassium uptake, and may be because of strong inhibitory effect of sodium on the potassium uptake system or efflux potassium from the cells. The Na+/K+ ratio decreased at 150 mM by 60 and 80 Gy of gamma radiation.

In vitro selection of salt tolerant variants following 60Co gamma irradiation of long-term callus cultures of Zoysia matrella [L.] Merr.

To study growth in the presence of NaCl, in vitro plantlets regenerated from callus of manilagrass (Zoysia matrella [L.] Merr.) were cultured on regeneration medium supplemented with or without 0.3 M NaCl. The results indicated that growth was significantly inhibited by NaCl, with the leaves becoming relatively shorter and thicker. The differences of in vitro plantlets grown under NaCl stress provided specific criteria for the selection of salt tolerant variants. The 6-year maintained calli were treated with different doses (0, 5, 10, 20, 40, 80, 100, 150, 200, 250, and 300 Gy) of 60Co γ rays. Regeneration rate and regeneration capacity of the calli were highest after treatment with 20 Gy 60Co γ rays, 27.08 and 91.67% respectively. When the irradiation dose was increased to 100 Gy, 10.42% of the calli developed shoots, but at 150 Gy, both regeneration capacity and regeneration rate declined significantly, and no shoot was observed after 6 weeks of regeneration. Therefore, 100–150 Gy is the most appropriate irradiation span for inducing somaclonal variation. The irradiated calli were selected in vitro for NaCl tolerance. Five NaCl tolerant variant lines, Ze1, Fv1, Te1, Tw1, Fr1, were selected on subculture medium supplemented with 0.35 M NaCl, then transferred to regeneration medium containing 0.25 M NaCl, and grown in a greenhouse. The dark green colour index (DGCI) was used to identify the amount of injury caused by NaCl treatment. This was significantly higher in four of the lines, Ze1, Fv1, Te1, Fr1 (30.88, 31.17, 30.45 and 37.70%, respectively) compared to the control line (CK), which was regenerated from calli subcultured monthly (27.39%), indicating that watering with NaCl caused less injury in these four lines. These lines had lower proline contents than CK under salt stress. The superoxide dismutase (SOD) activity was higher in Ze1 under control condition and its peroxidase (POD) activity increased significantly under salt stress. With Fr1 catalase (CAT) activity was higher under salt stress. The higher activity of these antioxidant enzymes may contribute to the enhanced salt tolerance of the four plant lines.