Impact of microgravity on radiobiological processes and efficiency of DNA repair

Three major plant bioassays, i.e., the Allium root anaphase aberration (Allium-AA), the Tradescantia-micronucleus (Trad-MCN) and the Tradescantia stamen hair mutation (Trad-SHM) tests, were utilized in soil solutions or shallow well water samples to determine the degree of their genotoxicity. Shallow well water samples were collected from five different farms, and soil solutions were extracted with distilled water or dimethyl sulfoxide (DMSO) from pesticide-contaminated (metolachlor, atrazine, extrazine, and 2, 4-D) and pesticide-free soil samples. Genotoxicity was expressed in terms of anaphase aberration (AA) frequencies in the Allium-AA test, in terms of micronuclei frequencies in the Trad-MCN test, and in terms of pink mutation events in the Trad-SHM test. On average, results of Allium-AA tests showed a 2.78-3.01 fold increase in anaphase aberration frequencies in contaminated soil solution samples and well water samples as compared with the negative control. Results of Trad-MCN tests showed a 1.66-4.75 fold increase of MCN frequencies in contaminated soil solution samples and shallow well water samples as compared with the frequencies of the controls. Results of Trad-SHM tests showed a 2.7-2.86 fold increase of pink mutation events in the contaminated soil solution samples over that of the controls. Control groups of the Allium-AA tests had an average of 0.75/1000 anaphase figures, and control groups of the Trad-MCN tests had an average of 3.2 MCN/100 tetrads, while control groups of the Trad-SHM tests had an average of 1.4 mutation events/1000 hairs. In general, soil solutions of DMSO extracts showed higher genotoxicity than that of distilled water extracts. Among these three plant bioassays, the Trad-MCN test has the highest efficiency. The highest toxicity, based upon the Trad-MCN test results, was found in the pesticide contaminated soil samples from Monroe's farm. Water samples from the Fountain Green/Bushnell area ranked second in genotoxicity.     

In situ monitoring with the Tradescantia bioassays on the genotoxicity of gaseous emissions from a closed landfill site and an incinerator

A dual monitoring system composed of the Tradescantia-Micronucleus (Trad-MCN) and Tradescantia-Stamen-Hair-Mutation (Trad-SHM) bioassays was utilized to monitor directly the genotoxicity of the gaseous emission at a closed landfill site and around an incinerator. Four of the commonly emitted gaseous agents from the landfill flare pipes, i.e. toluene, ethylbenzene, trichloroethylene and ethyltoluene were also evaluated for their genotoxicity in the laboratory. The in situ monitoring trips (360 km one way) were carried out by transporting the plant cuttings in a clean air box or in an air-tight plastic bag to the site and exposing these test cuttings for 5–7 h. The exposed plant samples were examined for micronuclei frequencies or the pink mutation rate after the appropriate recovery periods (24 h for MCN, 7–11 days for SHM). A total of 20 monitoring trips were made to the landfill, and 8 to the nearby surroundings (100–500 m from the chimney) of the incinerator site in a two year period. The major findings of the Trad-MCN test on the clastogenicity of the gaseous emission from the flare pipe of the landfill site showed positive responses or toxic effects in 6 out of 20 trips, and that from the incinerator showed positive responses in 5 out of the 8 trips. These positive responses were closely associated with the weather, i.e. low wind velocity, high temperature and relative humidity, and especially the distance from the chimney of the incinerator. The MCN frequencies and mutation rates of the Elementary School site (E. Sch) which is about 200 m from the fence of the landfill site were mostly negative, except the test results of three trips. Trad-SHM tests on the mutagenicity of gaseous emissions from the flare pipe of the landfill showed 12 positive responses out of 20 trials and 2 positives out of 4 trials from the incinerator gaseous emissions. The average mutation rate from 20 Trad-SHM monitoring trips is positive when the ANOVA and Dunnett's t-statistic were applied to the consolidated data. There is a significant (0.01) difference between the lab control and the gas exposed groups, and between the field control and gas exposed groups. Results of the Trad-SHM test at the E. Sch. site were mostly negative except for one trip. In general, micronuclei frequencies and mutation rates of the field control groups were relatively higher than those of the lab controls. The Trad-MCN test on pure gases showed positive responses in all 3 repeated tests on toluene (50–892 ppm). The test results of ethylbenzene yielded positive responses at 172 ppm/min and 1549 ppm/min dosages and exhibited toxicity at higher concentrations. Trad-MCN tests on trichloroethylene and ethyltoluene yielded positive responses at around 100–200 ppm/min level. Three repeated Trad-SHM tests on toluene yielded no positive response at low concentrations (4.3–12.9 ppm).     

Tradescantia bioassays for the determination of genotoxicity of water in the Panlong River, Kunming, People's Republic of China.

The Panlong river passes through Kunming City and receives a large quantity of municipal sewage and wastewater from industrial effluent. Along the river, 20 sites were selected to collect water samples to assess the genotoxicity using two Tradescantia assays, the micronucleus (Trad-MCN) and the stamen-hair-mutation (Trad-SHM) assays. The lowest frequencies in the Trad-MCN assay and the Trad-SHM assay are 3.19 MCN/100 tetrads and 1.32 M/1000 stamen hairs, respectively. In the water samples obtained from the Songhua Reservoir, the MCN frequencies and mutation rates are not statistically significantly different from the data found for the negative control (2.49 MCN/100 tetrads and 0.71 M/1000 stamen hairs). Among the other water samples, 19 in Trad-MCN assay and 17 in Trad-SHM assay show significantly higher genotoxicity than the control. The highest genotoxicity was in samples No. 19 for the MCN assay (8.73 MCN/100 cells), over three times higher than the negative control, and in sample No. 11 for the SHM assay (4.30 M/1000), six times higher than the negative control, and were about the same as for the positive control (10.0 mg/l NaN3, 9.28 MCN/100 tetrads and 7.44 SHM/1000 stamen hairs), respectively. The peak frequencies for the Trad-MCN assays were observed in the water samples obtained from the sites that were near industrial and municipal wastewater that ran into the river as effluent. In general, the frequency of MCN and SHM mutations increased where the river passed through Kunming. The Trad-MCN assay seemed more sensitive than that of the Trad-SHM assay in detecting genotoxicity of the river water pollution.     

The Tradescantia-micronucleus test on the genotoxicity of UV-B radiation.

Lanzhou city is located in north central China near inner Mongolia. The solar UV-B background radiation in this area is occasionally extremely high (8 microW/cm2). Such high background solar UV-B radiation could be attributed to the sporadic depletion of the ozone layer in the stratosphere. The excessive UV-B radiation is a potential hazard in the environment. This prompted the present study on the effect of UV-B radiation on the cytogenetic damage to pollen mother cells of the plant Tradescantia. The Tradescantia-micronucleus (Trad-MCN) test was used to determine the genotoxicity of UV-B radiation. In addition to the usual 10 h of solar emission of UV light a series of increasing dosages (2, 4, 6, 8 h) of artificial UV-B radiation was applied to Tradescantia (clone 3) plant cuttings. Inflorescences of the treated and control plants were fixed and used for preparation of microslides. Micronuclei frequencies were observed in the early tetrads to show the degree of genotoxicity. Results of two repeated experiments show a dose-related increase of MCN frequencies under normal sunshine days. In the third experiment conducted under a cloudy and rainy day and an extraordinary high solar UV-B background, the MCN frequencies were markedly higher than that of the negative control but did not show the clear dose response to the treatment as in the first two experiments. The Trad-MCN test has successfully detected the effect of artificial UV-B radiation over the solar UV-B background radiation.     

Influence of climatic conditions on the mutations in pollen mother cells of Tradescantia clone 4430 and implications for the Trad-MCN bioassay protocol

The present was study aimed at investigating the influence of relative humidity and temperature on spontaneous and pollution-induced mutation rates during exposure and recovery periods in the Trad-MCN test. Cuttings of Tradescantia clone 4430 were exposed to a negative control, to 4 mM maleic hydrazide (MH), and to a polluted water sample under varying conditions of air temperature and humidity in climatic chamber experiments. The relative humidity did not affect the spontaneous mutation rate in the clone investigated, but was negatively correlated with the frequency of pollution-induced mutations. Low temperature caused an increase in the number of micronuclei in the negative control, but no comparable response in polluted samples. At an extremely high temperature, signs of strong physiological damage and/or of a meiotic delay of pollen maturation were detected. When the temperature increased gradually and the extreme value was maintained only for short time, such detrimental effects were not observed. Subsequent treatment with high and low temperatures, by contrast, resulted in the highest MCN rates of all experiments. Our studies point to the possibility of producing irregular results of the Trad-MCN test if the influence of climatic factors has not sufficiently been considered.     

Exploring the clastogenic effects of air pollutants in S?o Paulo (Brazil) using the Tradescantia micronuclei assay.

This study was designed to determine the clastogenicity of particulate matter (aerodynamic diameter smaller than 10 microm) in the urban polluted air in the city of S?o Paulo. The Tradescantia-micronucleus (Trad-MCN) assay was used throughout this study to evaluate the clastogenicity of the extracts of the particulate matter. Tradescantia pallida (Rose) Hunt. cv. purpurea, an indigenous cultivar, was used in the Trad-MCN assay. The efficacy of this plant material for the Trad-MCN assay was validated with dose-response studies using formaldehyde and beta radiation. Dose-response curves were established with these known mutagens. The extracts of the PM10 particles at concentrations between 5 and 50 ppm induced a dose-related increase in MCN frequencies. The results indicate that T. pallida is equally sensitive to mutagens as the standard Tradescantia clone 4430 or 03 and the particulate matter in the urban air are clastogenic to the chromosomes of this plant. Inhalation of these particles by urban dwellers may affect their health by inducing similar genetic damage.     

Somatic embryogenesis from immature peach palm inflorescence explants: towards development of an efficient protocol

Various factors affect the induction of somatic embryogenesis in peach palm (Bactris gasipaes Kunth). Among these, both the type and level of auxins had the greatest influence on in vitro responses, although the genotype and the developmental stage of the explants also influenced results. Younger inflorescences were more competent to respond to SE induction than more mature inflorescences and the use of a pre-treatment with 2,4-D (200 μM) in liquid MS culture medium also increased the embryogenic capacity, and diminished the development of flower buds. Higher oxidation rates were observed in explants maintained on 2,4-D-supplemented culture medium, while on 300 μM or 600 μM Picloram and Dicamba lower oxidation rates were observed. The progression from floral meristem to flower bud occurred at high frequency when low concentrations of auxins were used, independent of the type. Higher concentrations of Picloram or Dicamba reduced or even inhibited flower bud development. Picloram also enhanced the embryogenic induction rate more than 2,4-D and Dicamba, and among the concentrations evaluated 300 μM Picloram enhanced induction for both genotypes, with significant differences between genotypes. The best combination of variables used the least mature inflorescence (Infl1) from genotype I with the 2,4-D pre-treatment and 300 μM Picloram to generate 5 embryogenic calli from 18 explants; 26 embryos were obtained on average from each embryogenic callus. From these, eighteen embryos converted to plantlets and six of these survived transfer to the greenhouse.     

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice)

Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/l Dicamba and 1 mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.Abbreviations BM Basic medium - 2.4 -D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-2-methoxy benzoic acid - Picloram 4-amino-3,5,6-trichloropicolinic acid     

Sister chromatid exchanges induced in human lymphocyte chromosomes by trifluralin, atrazine and simazine

Many epidemiological and experimental "in vivo" studies have proved in recent years the carcinogenic properties of herbicides. In order to evaluate the "in vitro" action on the human DNA of Trifluralin, Atrazine and Simazine (active principles of herbicides Treflan and Fogard S respectively) the authors have studied the rates of SCE in cultures of human lymphocytes exposed to different concentrations of a solution 1 ppm of the substances. Trifluralin and Simazine, but not Atrazine, increase SCE per cell, with statistical significance, in the cultures with the highest concentrations of these substances. (SCE per cell: Trifluralin 5.27 +/- 1.38, Simazine 5.09 +/- 1.19, Control 3.51 +/- 1.14).     

Proficiency of the Tradescantia-micronucleus image analysis system for scoring micronucleus frequencies and data analysis.

The Tradescantia-micronucleus (Trad-MCN) bioassay is an efficient short-term test for genotoxicity of pollutants. In order to increase the efficiency and to standardize the micronucleus (MCN) scoring process, an automated scoring system was developed using the principle of image analysis in computer science. This assemblage is called the Tradescantia-micronucleus image analysis (Trad-MCNIA) system. The MCN frequencies scored by this system were compared with those scored by human observation for its proficiency. A set of low MCN frequency (around 5 MCN/100 tetrads) slides prepared from a control group, a set of medium MCN frequency (around 20 MCN/100 tetrads) slides prepared from sodium azide treated plant cuttings and a set of high MCN frequency (around 50 MCN/100 tetrads) slides prepared from X-ray treated materials were used for this study. In the low MCN frequency slides, the Trad-MCNIA system scored about the same value as human observation. In the medium and high frequency slides, MCN frequencies scored by the system were lower than those scored by human observers. This discrepancy was corrected by increasing the power of the objective of the microscope in the system. The MCN frequencies scored by the system attained 90% congruity with those scored by human observers after the correction. The scoring speed of the system was about 3.5 times as fast as that by human observers, and the data could be statistically analyzed immediately after the data scores were recorded. Further improvements can be made by upgrading the video camera and the computer speed.     

Genotoxicity biomonitoring of sewage in two municipal wastewater treatment plants using the Tradescantia pallida var. purpurea bioassay

The genotoxicity of untreated and treated sewage from two municipal wastewater treatment plants (WTP BN and WTP SJN) in the municipality of Porto Alegre, in the southern Brazilian state of Rio Grande do Sul, was evaluated over a one-year period using the Tradescantia pallida var. purpurea (Trad-MCN) bioassay. Inflorescences of T. pallida var. purpurea were exposed to sewage samples in February (summer), April (autumn), July (winter) and October (spring) 2009, and the micronuclei (MCN) frequencies were estimated in each period. The high genotoxicity of untreated sewage from WTP BN in February and April was not observed in treated sewage, indicating the efficiency of treatment at this WTP. However, untreated and treated sewage samples from WTP SJN had high MCN frequencies, except in October, when rainfall may have been responsible for reducing these frequencies at both WTPs. Physicochemical analyses of sewage from both WTPs indicated elevated concentrations of organic matter that were higher at WTP SJN than at WTP BN. Chromium was detected in untreated and treated sewage from WTP SJN, but not in treated sewage from WTP BN. Lead was found in all untreated sewage samples from WTP SJN, but only in the summer and autumn at WTP BN. These results indicate that the short-term Trad-MCN genotoxicity assay may be useful for regular monitoring of municipal WTPs.     

Genotoxicity of 2,4- and 2,6-dinitrotoluene as measured by the Tradescantia micronucleus (Trad-MCN) bioassay

The phytogenotoxicity of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) was assessed using the Tradescantia micronucleus (Trad-MCN) bioassay. Tradescantia cuttings bearing young inflorescences were exposed for 6h to 2,4- or 2,6-DNT amended water solutions up to their respective solubilities. The nominal concentrations were 0, 1.9, 3.8, 7.5, 15, 30, 60, 100, 150, 200mg/l of 2,4-DNT, and 0, 7.5, 15, 30, 60, 90, 120, 180mg/l of 2,6-DNT. Each treatment was repeated three or four times. Chemical concentrations in test solutions were analyzed prior to and after the exposure. Cadmium chloride (0-20mM) was used as the positive control. Micronuclei (MCN) were scored in the tetrad-stage pollen mother cells. The MCN frequency (%), i.e. the number of micronuclei scored in 100 tetrads, was the measurement endpoint. Results indicated that both 2,4-DNT and 2,6-DNT were genotoxic with the minimum effective dose (MED) of 30 and 135mg/l, respectively. Longer exposure (30h) without recovery time at 150mg/l of 2,4-DNT and 180mg/l of 2,6-DNT did not induce significantly higher MCN frequencies.     

Tradescantia-micronucleus assay for the assessment of the clastogenicity of Austrian water.

Seven water samples collected from Vienna and Salzburg areas in Austria were tested for their clastogenicity with the Tradescantia-micronucleus (Trad-MCN) assay. There was no indication of clastogenic activity in two drinking water samples; likewise, samples from two major rivers (Danube and Salzburg) and of a river that received effluents from a paper mill also gave negative results. Urban river water as well as ground water samples which were collected near an industrial waste dump site caused a statistically significant and dose dependent increase of the MCN frequencies.     

Effect of Herbicides on Cellulose Decomposition by Sporocytophaga myxococcoides

The effects of 14 herbicides on cellulose decomposition by Sporocytophaga myxococcoides were studied by growing the organism in two different liquid cellulose-mineral salts media in the presence of the test agent and then quantitatively determining the residual cellulose. In the first medium, containing acid-hydrolyzed cellulose, anthrone reagent was used to determine residual carbohydrate. In the second medium, Gooch Pyrex fritted-glass crucibles were used to determine the amount of residual Whatman powdered cellulose. The herbicides used were Atrazine, Simazine, Dacthal, Diuron, Amiben, Banvel-D, Banvel-T,2,3,6-TBA, Dicryl, Maleic Hydrazide, Stam F-34, Zytron, Fenac, and Dalapon. Of these, Zytron appeared to be the most inhibitory to the test bacterium, and was the only compound exhibiting inhibition at a commonly used field rate of application (2 ppm).     

Influence of X-ray dose fractionation on the frequency of somatic mutations induced in tradescantia stamen hairs

X-rays were used to investigate the influence of dose fractionation on the induction of pink and colorless somatic mutations in stamen hair cells of Tradescantia clone 02. Inflorescences were exposed to a single acute dose of 60 rad, two acute doses of 30 rad, or three acute doses of 20 rad. The dose rate in all cases was 30 rad/min. Intervals between dose fractions were varied from 35 sec to 48 h and the mutation frequency was compared with that resulting after the single dose of 60 rad. The data show a reduction in mutation frequency for fractionation intervals longer than 15 and 6 min for pink and colorless mutations, respectively, but not for shorter intervals.One interpretation of the data predicts that pink mutation frequencies are reduced by 11% for fraction intervals of from 30 min to 6 h, and that colorless mutation frequencies are reduced by 24% for intervals of from 15 min to 6 h. The corresponding sparing effect of dose fractionation is equal to 6 rad for pink mutations and 9 rad at the colorless mutation endpoint. A calculation has been made which indicates that the percentages of the total repairable (presumably two-hit) damage that is repaired during fraction intervals up to 6 h, are 16 and 35% for pink and colorless mutations respectively.     

Genotoxic effects of volatile organic compounds in a chemical factory as evaluated by the Tradescantia micronucleus assay and by chemical analysis

The clastogenic effects of volatile organic compounds in the workplace air of a chemical factory were studied by means of the Tradescantia micronucleus (Trad-MCN) assay and chemical analysis. Sampling was performed at a chemical factory producing PVC film in Cheong-ju, South Korea. Inflorescences of Tradescantia BNL 4430 were placed for 2, 6, and 9 h at the height of 1.40 m at two locations in the workplace and one outdoor of the chemical industry. Air samplings were conducted in the same places and the collected tube samples were analyzed by automatic thermal desorption/gas chromatography/mass spectrometry (ATD/GC/MS). The frequencies of micronuclei in specimens exposed for 2 h in sites 1-3 were 6.13 +/- 0.47, 5.40 +/- 1.60, and 2.93 +/- 0.43 MCN per 100 tetrads, respectively. GC/MS analysis proved the presence of various volatile organic compounds such as trichloroethylene, toluene, ethyl benzene, (m, p, o)-xylene, styrene, 1,3,5-trimethyl benzene, and 1,2,4-trimethyl benzene. Mean values of toluene measured by 2 h sampling in sites 1-3 were 1946.6, 1368.3, and 340.1 microg/m3, respectively. The toluene concentrations in sites 1 and 2 were at least four to six times higher than that in site 3. The micronucleus frequencies increased with exposure time. In addition, there was a correlation between the micronucleus frequencies and toluene concentration in the air (R2 = 0.96). The results of this in situ monitoring proved the applicability of the Trad-MCN assay combined with chemical analysis for monitoring genotoxic chemicals in the work environment.     

Mutagenicity of drinking water detected by the Tradescantia micronucleus test

Spring Lake reservoir of Macomb, Illinois, is a typical model of the drinking water supply of some midwestern towns of the United States. Water samples collected periodically in 1980 and 1981 from this lake were tested for mutagenicity using the Tradescantia micronucleus (Trad-MCN) test, a highly sensitive mutagen-detecting bioassay. Water samples from 1981 were also analyzed chemically. The micronucleus (MCN) frequency peaked (12-14 MCN/100 tetrads) in mid-July in both years, as compared with the average frequency (5 MCN/100 tetrads) of the base-line control that was maintained in nutrient solution (prepared with distilled water and pure chemicals). Drinking water from the tap was tested in parallel with lake water, and its mutagenicity tended to fluctuate with the mutagenicity of the lake water.     

Mutagenicity of ground water (in the bore-holes) in Ararat Valley (Armenia) detected by Trad-SHM bioassay

The genotoxicity of ground water from four bore-holes of different depths (40-120m) in the Ararat valley (Armenia) used both for drinking and irrigation was investigated. The frequency of recessive somatic mutations was determined using the Tradescantia-stamen-hair-mutation (Trad-SHM) test. The Tradescantia clone 02 was used. The pink mutation events (PMEs) were increased by 3.18-6.81-fold in comparison with the control depending both on the depth of subterranean water location and the increase of Na(+) ion concentration in these water samples. The peak frequency was found in water from the 40-45m depth. The deeper the bore-holes, the lower the mutagenicity of water and the concentration of Na(+) ions. Different types of mutant sector arrangements and their frequencies changed depending on the subterranean water depth.     

Further yearly analyses of spontaneous pink mutant events in the stamen hairs of tradescantia clone BNL 4430 cultivated in the NSC growth chamber

In order to confirm the results obtained in the previous 1-year-term (December 12, 1998, through December 10, 1999) scorings and analyses of spontaneous pink mutant events (PMEs) in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating (NSC) growth chamber, similar scorings and analyses were continued for another 52-week period from December 11, 1999, through December 8, 2000. The environmental conditions were not changed, except for a minor modification in the method of supplying the nutrient solution used. During the scoring period, 732,128 stamen hairs with an average cell number of 24.90 cells were observed, and 2,368 PMEs were detected. The overall spontaneous somatic mutation frequency was 1.35 +/- 0.03 PMEs per 10(4) hair-cell divisions, which was significantly lower than the value of 1.56 +/- 0.03 determined in the previous 52-week period, and the frequencies were lower during April through September than in other months, the period showing lower frequencies lasting 1-month longer than in the previous year. The present results reconfirmed the occurrence of a clear seasonal variation in the spontaneous mutation frequency in the NSC growth chamber, and the lower overall frequency, probably related to the minor modification in supplying the nutrient solution, is helpful for conducting mutagenicity tests at low levels, offering a lower background level. The analyses of the sectoring patterns of all these PMEs showed that the most of the 203 cases of multiple (two to five) pink sectors observed in the same stamen hairs (scored as 253 PMEs for calculating mutation frequency) were the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells, agreeing with our previous study, and the significance of somatic recombinations in causing single PMEs was also reconfirmed.     

Evaluation of regeneration potential of mature embryo derived callus in Indian cultivars of barley (Hordeum vulgare L.)

A protocol for plant regeneration in Indian cultivars of barley (Hordeum vulgare L.) has been developed using mature embryo culture. The influence of various auxins 2,4-D (2,4-dichlorophenoxyacetic acid), Dicamba (3,6-dichloro-o-anisic acid) and Picloram (4-amino-3,5,6-trichloropicolinic acid) on the callus induction and subsequent plant regeneration revealed highest percent of callus induction form cultivar (cv) BL 2 on MSB₅ medium (MS salts + B₅ vitamins) supplemented with 6 mg l^?1 Picloram, but maximum number of shoot buds (6–13) were regenerated on MSB₅ medium containing 0.5 mg l^?1 Picloram. Regenerated shoots were rooted on half-strength MSB₅ medium. Plantlets were successfully transferred to soil and grown to maturity in greenhouse. The effect of copper sulphate revealed significant improvement in callus induction and plant regeneration when the concentration of CuSO₄ was increased to 3 μM (30 times higher than normal MS medium) for cv BL 2. Regeneration potential differed for different cultivars of barley used, with highest for cv BL 2 and lowest for cv BH 924. We conclude that the Indian barley genotypes exhibit plant regeneration from mature embryo cultures. The protocol has potential application in barley improvement through genetic engineering.