Investigating Heavy Metals Tolerance in Ruta graveolens In vivo and In vitro

Many experiments were carried out to investigate the ability of Ruta graveolens to tolerate heavy metals in vitro and to investigate molecular bases of heavy metals tolerance. Callus was induced on stem segment explants cultured on Murashige and Skoog (MS) medium supplemented with different concentrations  of 2,4-Dichlorophenoxyacetic acid (2,4-D) (0.0, 0.75, 1.0 or 1.25 mg/l) and kinetin (kin) (0.0, 0.75, 1.0 or 1.25 mg/l).The combination of 1 mg/l 2,4-D plus 1 mg/l kin and 1.25 mg /l 2,4-D plus 1.00 mg /l kin gave the highest % callus induction  reached 100% and 93% respectively. Callus was aintained on MS medium supplemented with 1 mg/l 2,4-D and 0.5 mg/l kin. Callus pieces initiated on stems explants were subcultured into maintenance medium supplemented with manitol at the concentration 300 mg/l to induce drought stress or with saline water at an electrical conductivity (EC) 30 ds.m -1 for inducing salt stress. Drought and salt tolerant calli were selected and subjected to heavy metals (Zn, Cd or
Fe) at different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 or 3.0 ppm). Results showed that callus fresh weight decreased with increasing metals concentrations. Tolerance index was assessed at different metals concentrations and stress durations. The highest values were recorded under metals concentration 1 ppm after 63 days except in case of salt tolerant calli as it showed its highest tolerance index value after 21 days under Cd stress.Atomic absorption spectrophotometer 5000 was used for estimation metal concentrations inside leaf tissues of plants grown on typical soil and those of stressed and non-stressed callus. Results showed that R. graveolens cultures stand for moderately high concentrations of heavy metals (7100 Zn, 440 Fe and 95 €g/g Cd). Heavy metals tolerant calli were selected and shoots were regenerated on 1/2 MS medium supplemented with Benzyl adenine (BA) (0.0, 2.5 or 3.0 mg/l) and Naphthalene acetic acid NAA (0.0 or 0.5 mg/l). BA at concentration 2.5 mg/l gave the highest % shoot
formation after 8 weeks. Shoot elongation and multiplication was achieved on the same regeneration medium enriched with 0.5 mg/l Gibberellic acid (GA3). An examination of regenerated plants for their tolerance to heavy metals was achieved by culturing on MS medium supplemented with heavy metals at concentration 1 ppm. Healthy elongated shoots were cultured on1/2 MS medium supplemented with 0.1 mg/l Indole butyric acid (IBA) to initiate rooting. PCR analysis was performed to investigate molecular basis of heavy metals tolerance in R. graveolens. 1-aminocyclopropane-1carboxylic acid (ACC) synthase gene (ACS2) of sweet orange (Citrus sinensis) was selected to design tow primer pairs for PCR assay, so that to detect the presence of the gene in R. graveolens plant in control and heavy
metal treated plants. The primers were named Pp600 and Pp400. Analysis of PCR product exhibit that control sample produced a PCR product with a molecular size ~ 600 bp using Pp600 primers pair and a PCR product with a molecular size ~ 400 bp using Pp400 primers pair confirming the presence of ACS2 gene. The Effect of abiotic stresses salt, drought and heavy metals on ACS2 gene was also investigated. Results showed that all treatments produced a PCR product with a molecular size ~ 600 bp using specific primers pair Pp600 except in case. Results indicated that samples produced a PCR products with variant molecular sizes using specific primers pair Pp400. Samples under drought and heavy metals (Fe, Zn, and Cd) stresses gave a product with a molecular size ~ 400 bp. R. graveolens
under salt and Fe stress produced a PCR product ~ 213 bp. Samples under salt and Zn stresses which produced a PCR product with a molecular size ~226 bp. Field experiment was carried out to investigate plant tolerance to heavy metals in vivo. It is concluded from the current study, the possibility of increasing Ruta tolerance to heavy metals and then exploitation such tolerant plant for re-vegetation of salt and drought affected lands. The presence of tolerance gene was confirmed.