Irradiated Plantlets Research Articles

Genetic and phenotype recovery of Ananas comosus var. MD2 in response to ionizing radiation

Due to their sessile nature, plants are exposed to various environmental stressors such as exposure to high levels of harmful ultraviolet (UV), ionizing, and non-ionizing radiations. This exposure may result in various damages, ranging from DNA and chromosomal aberrations to phenotypic abnormalities. As an adaptation, plants have evolved efficient DNA repair mechanisms to detect and repair any damage caused by exposure to these harmful stressors to ensure their survival. In this study, the effects of gamma radiation (as a source of ionizing radiation) on clonal Ananas comosus var. MD2 was evaluated. The morphology and physiology of the clonal plantlets before and after exposure to gamma radiation were monitored at specific time intervals. The degree of genetic variation between the samples pre- and post-irradiation was also analyzed by using inter-simple sequence repeat (ISSR) markers. The resulting data revealed that the heights of the irradiated plantlets were significantly reduced (compared to control), but improved with the recovery period. Irradiated samples also exhibited relatively good photosynthetic efficiency that further improved as the plantlets recover. These observations were supported by the ISSR analysis, where the genetic dissimilarities between the irradiated samples and control were reduced by 0.1017, after 4 weeks of recovery. Overall, our findings suggested that the phenotype recovery of the clonal A. comosus var. MD2 plantlets was contributed by their ability to detect and repair the DNA lesions (as exemplified by the reduction in genetic dissimilarity after 4 weeks) and hence allow the plantlets to undergo phenotype reversion to normal plant stature.

MOLECULAR, PHENOTYPIC MARKER ASSAYS, AND RADIOSENSITIVITY TESTS OF GAMMA-IRRADIATED CELOSIA ARGENTEA

Random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and UPOV phenotype markers were used to study the DNA polymorphism in gamma-ray induced morphological mutants of Celosia argentea var. plumosa Hungarian variety ‘Arrabona.‘ In the experiments, the study determined the radio sensitivity and the genetic diversity of gamma radiation of C. argentea var. plumosa ‘Arrabona.‘ Seeds of C. argentea var. plumosa ‘Arrabona‘ were irradiated with gamma rays to increase their genetic diversity. The irradiation doses consisted of 0, 75, 150, 300, 450, and 600 Grays (Gy). The germination percentage, survival rate, and phenotype of irradiated plantlets underwent evaluation in the first (M1) and second (M2) generations. The investigation of genetic diversity used the ISSR and RAPD primers. Based on the results, the first-generation genetic distance increased as the doses increased. But the trend changed considerably through the generation due to the low condition and fertility of the high doses of gamma-irradiated plants. These individuals did not show at the next mutant generation, changing the population gene pool. In addition, open pollination has also changed genetic diversity. The RAPD and ISSR primers proved proper to evaluate the genetic diversity, nonetheless fewer direct connection occurred between the appearance and the used RAPD or ISSR markers. The LD50 dose between 150 and 300 Gray treatments and the radiation between 300450 Gray induced the median growth reduction in the mutant ‘Arrabona‘ population. Based on these results, the study concluded that both UPOV-based phenotyping and molecular marker analysis revealed appropriate for determining genetic divergence, but detecting greater genetic distance resulted in molecular markers.

Disclosing the Genetic Diversity of ‘Earlibrite’ Strawberry Mutant Induced by Gamma-ray Irradiation Using ISSR Markers

Strawberry plantlets of the ‘Earlibrite’ variety were irradiated with gamma rays to induce genetic variability for resistance to abiotic stress. This study aimed to identify the DNA polymorphism of ‘Earlibrite’ strawberry mutants using ISSR markers. For this purpose, a population of M1V0 strawberry plants was gamma-irradiated at 20 Gy. In addition, the irradiated plantlets were subcultured onto MS medium enriched with 0.13 M of 1-NAA and 2 M of 6-BAP. The genetic variation of the mutant was molecularly analyzed using the ISSR marker. Analysis was performed on M1V0 mutant plantlets. The results showed that 93.36% of 192 scoreable bands produced by the ISSR markers were polymorphic. An average of PIC values was 0.48 per primer with a range of 0.40 (ISSR_56) to 0.50 (ISSR_22, ISSR_55, ISSR_58, ISSR_60, ISSR_61, ISSR_62 and ISSR_L10). The primer MI ranged from 2.38 (ISSR_62) to 4.85 (ISSR_21). The genetic variation of mutant individuals ranged from 2.74% to 87.04% according to the Jaccard similarity index. Cluster analysis using ISSR markers grouped the ‘Earlibrite’ strawberry mutant genotypes into five distinct groups. It is concluded that 20 Gy gamma-ray induced mutation produces strawberry mutants and that ISSR was a valuable marker for mutant identification.

Horticultural Performance of Field Grown Somaclones and Irradiated Strawberry (Fragaria × Ananassa Duch.)Variants

In the present investigation horticultural performances of field grown somaclones and gamma irradiated strawberry were studied. The somaclones performed better with regard to vegetative and reproductive characters while axillary buds irradiated with lower dose of gamma rays (10 and 30 Gy) were more vigorous in terms of number of leaves and petiole length. Whereasaxillary buds irradiated with higher dose of gamma rays (50 and 60 Gy) showed abnormal fruit development, reduced growth, small leaf area and fruit size. A total of six variants were obtained on screening the somaclones and irradiated plantlets on the basis of phenotypic characters, fruiting characters and Single Sequence Repeat (SSR) markers. The frequency of variants was higher (3.22%) in soamclones than in irradiated plants (2.75%) obtained from axillary buds. These finding would be helpful in providing a powerful technological tool for the improvement of clonally propagated crops. Bangladesh J. Bot. 51(3): 555-563, 2022 (September)

Microwave Radiation as an Inducer of Secondary Metabolite Production in Drosera rotundifolia In Vitro Plantlets.

In this study, low-intensity microwave irradiation (frequency of 2.45 GHz; 26.3 kW m-3 power density) for 0, 5, and 30 s was tested for the first time on Drosera rotundifolia in vitro plantlets to explore its effect on the production of highly valued phenolic compounds. Analysis of the extracts obtained from irradiated plantlets revealed time-dependent increases in the levels of photosynthetic pigments, particularly the carotenoids, whereas symptoms of growth decline were not observed. Similarly, the highest total antioxidant capacity and total phenolic and flavonoid contents were detected in 30-s-irradiated plantlets. High-performance liquid chromatography analysis revealed that the content of the bioactive phenolics 5-hydroxy-7-methylnaphthalene-1,4-dione (1), 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (2), and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one (3), which are considered to be responsible for the pharmacological properties of this species, was around twofold higher in 30-s-irradiated plantlets than in controls. Nevertheless, the accumulation of 5-hydroxy-2-methylnaphthalene-1,4-dione (4), which was present only in trace amounts in the plant roots, decreased by 30% upon microwave irradiation. The results indicate that microwave treatment acts as an effective inducer of the production of phenylpropanoid compounds, which opens up new opportunities for its use in biotechnological applications.

Induced Mutations Using Gamma Ray and Multiplication of Plantlet through Micro Cross Section Culture of Banana (Musa acuminata cv. Berangan)

The conventional breeding approach in banana was slow predominantly because of the limited genetic diversity, infertility and polyploid condition of the species. Induced mutation is one of the promising tools to create new varieties for the improvement of banana. While vitro propagation technique ensures a rapid and in an organized manner for production of banana. Thus, induced mutation using gamma ray in combination with in vitro culture micro cross section culture was conducted to evaluate the effects of gamma radiation on growth and development of plantlet of Musa acuminata cv. Berangan. Tissue cultured banana plantlet was used as the source of explant. The plantlet was cut into halved and radiated using gamma ray with the dose of 0 (control), 20, 25, 30, 35, 40, 45, 50, 55, 60 and 80 Gy. The highest survival rate (23.33%) was recorded in treatment 10 Gy while the lowest survival (0.33%) was in treatment 80 Gy. The lethal dose (LD50) which had caused 50% mortality to the irradiated material was found to be 37 Gy. The morphological study on the growth of in vitro irradiated plantlets showed that 25 Gy induced a stimulation effect on the number of shoot produced (5.56), root number per explant (8.5) and root length (37.36 cm) compared to untreated banana explants (0 Gy). Hence, this study introduces the in vitro multiplication of gamma irradiated culture system for future breeding of a new varieties of banana.

Effects of Benzyladenine on in vitro ‘Hom Rangsi’ Rice and Induction of Aluminum Acid Tolerance Lines by Gamma Irradiation

‘Hom Rangsi’ was the non-photoperiod aromatic mutant rice which derived from fast neutron radiation KDML 105. ‘Hom Rangsi’ seeds were cultured on MS solid medium without any supplemented for a week. And then, all explants were placed on MS (Murashige and Skoog, 1962) medium supplemented with 0, 5, 10, 15, 20, 25 mg/L BA (benzyladenine) for multiple shoot induction. The optimal concentration of BA for induced multiple shoot induction of ‘Hom Rangsi’ line was MS + BA 25 mg/L, the highest number of shoots were 5.38 shoot/seed. The following experiment was done, irradiated ‘Hom Rangsi’ seeds with 0, 100, 200, 300, 400 Gy gamma ray which cultured on MS solid medium supplemented with 400 mg/L Al3+ pH 2.9 were selected for acid tolerance lines. After six weeks cultured, the survivals of irradiated plantlets were 86.32, 77.78, 58.95, 58.95, 21.87% and the height of irradiated plantlets were 8.4, 8.3, 6.7, 6.6, 6.1 cm respectively without any shoot budding. All survival plantlets were transferred to suitable MS + BA 25 mg/L medium which discovered from the first experiment for multiple shoot budding. After six weeks cultured, the maximum of 5.24 shoots/plantlet were found from 300 Gy irradiation significantly and followed by 400, 200, 0 and 100 Gy irradiation treatments which gave 4.55 and 4.41, 4.37 and 4.31 shoots/planlet respectively.

THE GENETIC VARIATION ASSESSMENT OF IN VITRO IRRADIATED TOMATO (Lycopersicon esculentum Mill) BY SCoT AND ISSR MARKERS

Tomato (Lycopersicon esculentum Mill.) are considered major and important globally vegetable crops and in Egypt in particular. Tissue culture techniques have encouraged the utilization of mutation methods in crop improvement. The mutation induction in vegetative crops through tissue culture may be the optimal method to improve these crops. Induced genetic variation in tomato plantlets by using gamma radiation and identified these changes through SCoT and ISSR markers. Egyptian tomato cultivar Idkawy explant was cultured onto MS medium supplemented with 0.2 mg-l BAP. The resulted plantlets were irradiated with γ radiation doses (50, 100, 150, 200 or 250 Gy). The survival, growth rate, and mean of shoot length were decreased with increasing gamma radiation dose. The irradiated plantlets survival percentages were ranged from 78.75% to (50 Gy) and 18.75% to (250 Gy), whereas, the shoot length decreased by a rate of 2.71 cm for the dose (50 Gy) and 1.2 cm for dose (250 Gy). Genetic diversity was evaluated by SCoT and ISSR markers using ten primers for each. It was noticed that the polymorphism percentage mean of SCoT marker (60.53%) is higher than the ISSR marker (39.6). The PIC values average for both markers SCoT and ISSR were 0.429 and 0.347, as well, MI values were 0.345 and 0.156, respectively. On the other hand, the effective no. of alleles (Ne), Nei’s genetic diversity (H) and Shannon’s information index (I) parameters, it was found that the dose 100 Gy caused the highest genetic variation compared with other doses using SCoT marker, however, in ISSR marker was dose of 150 Gy the highest dose for induced genetic variation. The obtained results demonstrate that SCoT marker was more accurate and efficient than ISSR marker for distinguishing and genetic variation analysis of irradiated tomato plantlets. The relationships within treatments were assessed through cluster analysis (UPGMA) based on SCoT and ISSR analysis.

Pengaruh Iradiasi Sinar Gamma terhadap Pertumbuhan Stek Tunas Apikal dan Aksilar Kopi Arabika

<p><em>The Arabica coffee is predominantly self-pollinated plants thereby contributing to low genetic diversity. The effort to increase the genetic diversity of Arabica coffee through crossing strategy is time-consuming, and induce mutation is necessary to enhance the rate of genetic variation. The aims of this study were to observe the effect of gamma-ray irradiation on the growth of apical and axillary bud cuttings and to determine the value of LD<sub>50</sub> on apical cuttings and Arabica coffee axillaries. The study was conducted at the Tissue Culture Laboratory, Industrial and Beverage Crops Research Institute, from January to December in 2018.. The planting material that was irradiated was Arabica coffee plantlets resulting from somatic embryogenesis propagation. Irradiation is carried out at the National Nuclear Energy Agency. The irradiated plantlets were cut and subcultured onto MS medium without growth regulators, 30 g L<sup>-1</sup> sucrose, and 2.5 g L<sup>-1</sup> phytagel were added. The design used a completely randomized design with 10 replications. The treatments tested were the dose of gamma-ray irradiation (0, 10, 20, 30, 40, and 50 Gy). The results showed that gamma-ray irradiation had an effect on all observed parameters. The mortality percentage of apical shoot cuttings began to be found at 30 Gy, while axillary cuttings at 20 Gy increased with an increasing dose of gamma-ray irradiation. The number of shoots and leaves varied between irradiation doses on both apical and axillary cuttings. The LD50 value of apical shoot cuttings was 36.80 Gy, while axillary cuttings were 22.24 Gy</em></p>

Growth and Proline Content of Irradiated in Vitro Shoots of Ubi Kuning Cassava Genotype Cultured at Different Temperatures

Cassava (Manihot esculenta) is an important crop to food security under climate change due to its various tolerance mechanism under stress conditions. However, the sustainable growth of cassava in the field depends on many factors especially temperature. The objective of the research was to investigate the growth performances and proline contents of irradiated Ubi Kuning at dosage of 10 Gy, cultured in Murashige Skoog (MS) hormone-free solid medium for 4 weeks at three different temperature treatments i.e 25oC, 28oC and 30oC. Each treatment consisted of 3 clone explants with 5 replicates. Results show that growth performances of irradiated plantlets were better compared to that of non-irradiated plantlets in terms of plant height and number of leaves at all temperature tested. The best growth performances were obtained from irradiated plantlets grown under 30oC. The proline content of irradiated Ubi Kuning was high when they were grown under 25 oC and 30oC, implying that these plantlets had the possibility to tolerant to lower and higher-temperature condition. This study is initially useful to find out the growth ability of irradiated Ubi Kuning in response to lower and higher temperature.

Molecular Diversity Analysis of Two Irradiated Potato Varieties in vitro Revealed by Random Amplified Polymorphic DNA

Potato buds cvs. Spunta and Valor were cultured in vitro on MS solid medium supplemented with 0.2 mg -1 BAP. The resulting plantlets were irradiated with gamma radiation doses 10, 20, 30 and 40Gy. Irradiated single node pieces were transferred onto fresh MS with BAP. Plantlets survival percentage calculated after eight weeks, gamma radiation caused decrease in the survival percentage of micropropagated buds in both Spunta and Valor cultivars ranging from 91.4 % to 28.5% in cv. Spunta and 93.3% to 30.47% in cv. Valor, comparing with the values of the two non-irradiated cultivares 95.2% and 96.2% respectively. Microtubers produced from irradiated plantlets were decreased with increasing gamma radiation doses with changes in size and numbers. The estimated proline content in irradiated plantlets was increased with increasing gamma radiation dose. The genomic DNA of non-irradiated (control) in two cultivars and eight radiation treatments was amplified with 10 RAPD primers that generated 54 polymorphic bands. The highest number of genetic similarity was 0.9672 showed between irradiated plantlets with dose 20 and 30Gy in cv. Valor. However, the highest genetic distance was 0.3995 observed between irradiated plantlets with dose 20Gy in cv. Valor and 30Gy in cv. Spunta. The dendrogram generated by cluster analysis distinguished the irradiated plantlets genetically.

Growth and Proline Content of Irradiated in Vitro Shoots of Ubi Kuning Cassava Genotype Cultured at Different Temperatures

Cassava (Manihot esculenta) is an important crop to food security under climate change due to its various tolerance mechanism under stress conditions. However, the sustainable growth of cassava in the field depends on many factors especially temperature. The objective of the research was to investigate the growth performances and proline contents of irradiated Ubi Kuning at dosage of 10 Gy, cultured in Murashige Skoog (MS) hormone-free solid medium for 4 weeks at three different temperature treatments i.e 25oC, 28oC and 30oC. Each treatment consisted of 3 clone explants with 5 replicates. Results show that growth performances of irradiated plantlets were better compared to that of non-irradiated plantlets in terms of plant height and number of leaves at all temperature tested. The best growth performances were obtained from irradiated plantlets grown under 30oC. The proline content of irradiated Ubi Kuning was high when they were grown under 25 oC and 30oC, implying that these plantlets had the possibility to tolerant to lower and higher-temperature condition. This study is initially useful to find out the growth ability of irradiated Ubi Kuning in response to lower and higher temperature.

Molecular Diversity Analysis of Two in vitro and Irradiated Potato Varieties Expressed by Random Amplified Polymorphic DNA

Potato buds cvs. ҅Valor’ and ‘Spunta’ were cultured in vitro on MS solid medium with 0.2 mg -1 BAP. The resulting plantlets were irradiated with gamma radiation doses 10, 20, 30, 40 and 50 Gy. Irradiated segments were transferred onto fresh MS with BAP and plantlets survival percentage was calculated after eight weeks. Gamma radiation caused the death of 3.8% to 81% in cv. ҅Spunta’ and 2.9% to 83.9% in cv. ҅Valor’. Microtubers produced from irradiated plantlets were decreased with increasing gamma radiation doses, with notable changes in shape, size and numbers. The proline contents in irradiated plantlets were steady increase with gamma radiation doses. The genomic DNA of the two cultivars and ten radiation treatments was amplified with 10 RAPD primers that generated 53 polymorphic bands. The highest number of genetic identity was 0.9672 showed between irradiated plantlets with 20 and 30 Gy in cv. ҅Valor’. However, the highest genetic distance was 0.3995 observed between irradiated plantlets with dose 20 Gy in cv. ҅Valor’ and 30 Gy in cv. ҅Spunta’. The dendrogram generated by cluster analysis distinguished the irradiated plantlets genetically.

Radiosensitivity Levels of In Vitro Cultured Celosia cristata Planlets by γ - Ray Irradiation

Plantlets of the ornamental plant Celosia cristata were irradiated with gamma rays to increase their genetic diversity. This study was aimed to establish the lethal levels of gamma radiation (LD20, LD30 and LD50) for C. cristata plantlets. The irradiation doses used were 0, 25, 50 and 75 Gy. The growth of irradiated plantlets was evaluated to the third generation. Irradiated C. cristata MV1 plantlets showed a decrease in growth, with plantlets irradiated at 75 Gy showing only 30% survival. Abnormal growth characteristics observed in the third generation plantlets included the shortening of internodes, and curling of leaves. LD50, LD30 and LD20 of C. cristata were 68.73 Gy, 46.68 Gy and 35.65 Gy, respectively.

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.

Morphological and biochemical responses of Oryza sativa L. (cultivar MR219) to ion beam irradiation

Heavy ion beam, which has emerged as a new mutagen in the mutation breeding of crops and ornamental plants, is expected to result in the induction of novel mutations. This study investigates the morphological and biochemical responses of Oryza sativa toward different doses of carbon ion beam irradiation. In this study, the dry seeds of O. sativa were irradiated at 0, 20, 40, 60, 80, 100, and 120 Gy, followed by in-vitro germination under controlled conditions. Morphological and biochemical studies were conducted to investigate the morphological and physiological responses of O. sativa towards ion beam irradiation. The study demonstrated that low doses (10 Gy) of ion beam have a stimulating effect on the height, root length, and fresh weight of the plantlets but not on the number of leaves. Meanwhile, doses higher than 10 Gy caused reductions in all the morphological parameters studied as compared to the control samples. The highest total soluble protein content [(2.11 ± 0.47) mg/g FW] was observed in plantlets irradiated at 20 Gy. All irradiated plantlets were found to have 0.85% to 58.32% higher specific activity of peroxidase as compared to the control samples. The present study also revealed that low doses of ion beam (10 and 20 Gy) had negligible effect on the total chlorophyll content of O. sativa plantlets while 40 Gy had a stimulating effect on the chlorophyll content. Plantlets irradiated between 40 to 120 Gy were shown to be 0.38% to 9.98% higher in total soluble nitrogen content which, however, was not significantly different from the control samples. Carbon ion beam irradiation administered at low to moderate doses of 10 to 40 Gy may induce O. sativa mutants with superior characteristics.

Effects of Acute Gamma Irradiation on Physiological Traits and Flavonoid Accumulation of Centella asiatica

In the present study, two accessions of Centella asiatica (CA03 and CA23) were subjected to gamma radiation to examine the response of these accessions in terms of survival rate, flavonoid contents, leaf gas exchange and leaf mass. Radiation Sensitivity Tests revealed that based on the survival rate, the LD50 (gamma doses that killed 50% of the plantlets) of the plantlets were achieved at 60 Gy for CA03 and 40 Gy for CA23. The nodal segments were irradiated with gamma rays at does of 30 and 40 Gy for Centella asiatica accession ‘CA03’ and 20 and 30 Gy for accession ‘CA23. The nodal segment response to the radiation was evaluated by recording the flavonoid content, leaf gas exchange and leaf biomass. The experiment was designed as RCBD with five replications. Results demonstrated that the irradiated plantlets exhibited greater total flavonoid contents (in eight weeks) significantly than the control where the control also exhibited the highest total flavonoid contents in the sixth week of growth; 2.64 ± 0.02 mg/g DW in CA03 and 8.94 ± 0.04 mg/g DW in CA23. The total flavonoid content was found to be highest after eight weeks of growth, and this, accordingly, stands as the best time for leaf harvest. Biochemical differentiation based on total flavonoid content revealed that irradiated plantlets in CA23 at 20 and 30 Gy after eight weeks contained the highest total flavonoid concentrations (16.827 ± 0.02; 16.837 ± 0.008 mg/g DW, respectively) whereas in CA03 exposed to 30 and 40 Gy was found to have the lowest total flavonid content (5.83 ± 0.11; 5.75 ± 0.03 mg/g DW). Based on the results gathered in this study, significant differences were found between irradiated accessions and control ones in relation to the leaf gas. The highest PN and gs were detected in CA23 as control followed by CA23 irradiated to 20Gy (CA23G20) and CA23G30 and the lowest PN and gs were observed in CA03 irradiated to 40Gy (CA03G40). Moreover, there were no significant differences in terms of PN and gs among the irradiated plants in each accession. The WUE of both irradiated accessions of Centella asiatica were reduced as compared with the control plants (p < 0.01) while Ci and E were enhanced. There were no significant differences in the gas exchange parameters among radiated plants in each accession. Moreover, malondialdehyde (MDA) of accessions after gamma treatments were significantly higher than the control, however, flavonoids which were higher concentration in irradiated plants can scavenge surplus free radicals. Therefore, the findings of this study have proven an efficient method of in vitro mutagenesis through gamma radiation based on the pharmaceutical demand to create economically superior mutants of C. asiatica. In other words, the results of this study suggest that gamma irradiation on C. asiatica can produce mutants of agricultural and economical importance.

Effects of low-dose gamma irradiation on artemisinin content and amorpha-4,11-diene synthase activity in Artemisia annua L

Purpose: This research work aimed to create in vitro plantlet variants of Artemisia annua through gamma irradiation. The obtained variants were then evaluated for the correlation between their artemisinin content and enzyme activity of amorpha-4,11-diene synthase (ADS), the first enzyme of the artemisinin pathway.Materials and methods: Shoot tips from plantlets of A. annua were exposed to gamma rays at a low-dose range and transferred to hormone-free MS medium for in vitro cultivation. A dose-response curve and the value of 50% of lethal dose (LD50) were then obtained. This LD50 dose of gamma rays was used for treating another batch of shoot tips. The surviving plantlets after four subsequent subcultures were evaluated for their ability to accumulate artemisinin in correlation with the enzyme activity of ADS.Results: The dose-response curve showed that the LD50 value was at 8 Gray (Gy). The surviving irradiated plantlets from this dose treatment had artemisinin content ranging from 0.03–0.70% (w/w) of dry weight, comparing with only 0.18% present in the original non-irradiated samples. Their correlation coefficient between the ADS activity and the artemisinin content appeared to be R2 = 0.090 for all the 18 samples tested, although, selectively, more than half of these (11 samples) showed their R2 value of as high as 0.851.Conclusions: There was no correlation between the artemisinin content and ADS activity found in the whole population, but the correlation was observed in the main subpopulation of the irradiated A. annua plantlets.

Constituents of the root exudate of Avena fatua grown under far-infrared-enriched light

The constituents of the root exudate of plantlets of wild oats (Avena fatua) grown in the greenhouse under far-IR-enriched radiation were analysed for the first time by HPTLC and capillary GC, and further separated by medium-pressure liquid chromatography (MPLC; monitored by TLC) and characterised by GC-EIMS and 1H-NMR. This combination of methodologies permitted the study of root exudates under controlled conditions and is appropriate for examining a variety of ecophysiological/environmental effects in a short time period. In this instance, the response compounds of the irradiated plantlets were identified as linear and branched alcohols, linear and branched alkanes, a bicyclic monoterpene, mono- and bicyclic sesquiterpenes, and free and esterified fatty acids. An ester, isopropyl myristate, was the main component present, although the unusual occurrence of branched compounds, sesquiterpenoids and related compounds of isoprenoid metabolism is noteworthy. The effect of far-IR-enriched light on the production of these compounds is discussed. Copyright © 2000 John Wiley & Sons, Ltd.