Gamma Irradiation of Embryogenic Callus Cultures and In vitro Selection for Salt Tolerance in Sugarcane (Saccharum officinarum L.)

Radiation induced mutagenesis followed by in vitro selection was employed for salt tolerance in popular Indian sugarcane （Saccharum officinarum L.） cv. CoC-671. Embryogenic calli were gamma irradiated and exposed to different levels of NaCl （42.8, 85.6, 128.3, 171.1,213.9, 256.7, 299.5, or 342.2 mM）. The relative growth rate （RGR） decreased progressively with increasing salt stress and was the least with a salt stress of 256.7 mM （0.25±0.009）, almost 10 fold lesser than the control. The RGR was significantly lower in 85.6 mM and higher salt stressed calli than the control. The survival percent also decreased, with an increase in NaCl concentration. In case of 10 and 20 Gy irradiated calli, regeneration was observed up to 85.6 mM NaCl selection, medium, whereas, higher treatments （128.3 mM and beyond） exhibited browning initially. However, in the subsequent subcultures, regeneration was obtained in the case of 10 and 20 Gy irradiated calli on 128.3 and 171.1 mM NaCl selections. Higher dose of gamma irradiation （40 Gy） also showed regeneration, but only with 85.6 mM NaCI selection. The unirradiated calli regenerated the highest number of plantlets followed by 10 and 20 Gy irradiated calli on salt selection. A total of 147 plantlets were selected from different salt levels. The salt selected plants are being tested for their field performance.

In Vitro Selection and Identification of Drought-Tolerant Mutants in Sweetpotato

In vitro selection of drought-tolerant mutants in sweetpotato cv. Lizixiang was studied by using PEG6000 as selection stress. Embryogenic suspension cultures were cultured in MS medium containing 035% PEG6000 and 2 mg L 1 2,4-dichlorophenoxyacetic acid (2,4-D). The results indicated that 30% PEG6000 can be used for the optimal selection stress of drought-tolerance. Embryogenic suspension cultures irradiated with 80 Gy gamma-ray were cultured in MS medium containing 30% PEG6000 and 2 mg L 1 2,4-D and 20 drought-tolerant cell aggregates were obtained. These cell aggregates were transferred to solid MS me- dium supplemented with 2 mg L 1 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 1 mg L 1 abscisic acid (ABA), resulting in the germination of somatic embryos. In this study a total of 18 regenerated plants were obtained. The regenerated plants were transplanted in a greenhouse and 11 lines were formed. The analysis on drought treatment of seedlings, water retaining capacity of leaves and coefficient of drought-tolerance showed that 3 lines had significant drought-tolerance in comparison with the control plants.

Effect of Gamma Irradiation and Selection with Fungus Filtrate(Rhizoctonia solani Kuhn)on the in Vitro Culture of Common Bean(Phaseolus vulgaris)

The present investigation was undertaken to study the effect of gamma irradiation (dose from 10 to 100 Gy) and in vitro selection with fungus filtrate as selecting agent (concentration from 20% to 100%) on the susceptibility of the common bean to Rhizoctonia solani. The best results were found with a dose of 20 Gy or a concentration of 20% of fungus filtrate applied separately. These conditions were used to evaluate the combined effect of both approaches in a second experiment. The combined effect of irradiation and then selection adversely affected growth (height and roots) and survival of the in vitro plants. It may not be necessary to combine the variation generated by irradiation with the selection technique. For future assays we propose the application of: 1) gamma radiation, thereby inducing not only mutants with pathogen resistance, but also with other agronomic traits of interest. Later in the subculture MV4 potential fungus-resistant mutants will be evaluated in the field;or 2) selection pressure using fungus filtrate during three subcultures, which may be sufficient to induce the variation necessary to obtain in vitro plants resistant to fungus.

SELECTIVE TOXICITY OF RHODAMINE 123 ON CARCINOMA CELL IN VITRO

The selective toxicity of the mitochondria-specific cationic fluorescent dye rhodamine 123 (Rh-123) on He La cells in culture was studied. In this report, we demonstrate that with continuous exposure, Rh-123 markedly inhibited the growth of HeLa cells but had little effect on normal human kidney fibroblasts. With continuous exposure to Rh-123, the growth rate, colony forming ability, anl mitotic index of HeLa cells were decreased. The mechanism of toxicity of Rh-123 on HeLa cells was investigated by EM and enzyme cytochemistry stain. The mitochondria of carcinoma cells were the main targets for the inhibitory action of Rh-123, since they selectively accumulated the dye. At the dosage of Rh-123 which was toxic to HeLa cells, the structure and function of mitochondria were disrupted, as the mitochondria-related enzymes, i.e., ATPase, LDH and SDH were inhibited. The possible mechanism of the action of Rh-123 on HeLa cells is briefly discussed.

In vitro Selection for Fusarium Wilt Resistance in Gladiolus

Cormels pieces of four Fusarium susceptible Gladiolus cultivars （Friendship, Peter Pears, Victor Borge and Novalux） formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85-95% germination after breaking dormancy of 8 weeks at 4℃. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum.