Effect of Abiotic Stress on The Accumulation of Some Metabolites in Ruta graveolens (In Vitro)

      Structural and functional characterization of environmental stress- induced genes has contributed to a better understanding of how plants respond and adapt to different abiotic stresses by using DDRT-PCR, with the aim of increasing salinity and drought tolerance in Ruta graveolens by employing plant tissue culture technique. A significant increase in the mean response % for callus induction in leaf and stem explants of Ruta was achieved using Murashige and Skoog (1962) medium (MS) supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and Kinetin (Kin). The highest response for callus induction on leaf explants reached 96.3% at 1.5 mg/l 2,4-D and 0.5 mg/l Kin, while for stem explants reached 100% at 1 mg/l 2,4-D and 1.0 mg/l Kin. Callus proliferation was recorded at 1.0 mg/l 2,4-D and 0.5 mg/l Kin. The highest shoot regeneration was recorded at 1.5 mg/l BA and 0.5 mg/l  NAA. Highest shoot length was achieved at 0.5 mg/l GA . Shoots were transferred onto a half strength MS medium supplemented with 0.1 mg/l IBA achieving 100% rooting. Many experiments were carried out for screening R. greaveolens calli tolerance for drought using mannitol and saline water using direct and gradual screening methods. In vitro callus cultures were exposed for both direct and stepwise screening to different concentrations of mannitol (0.0 – 300 g/l). Direct screening revealed a
significant decrease occurred in the mean of callus fresh weight, while the highest callus fresh weight was recorded at 240 g/l mannitol reached 188 mg after 12 weeks. Gradual screening recorded maximum callus  fresh weight at 300 g/l mannitol reached 281 mg after the 5 th  reculture. In vitro callus cultures were exposed to both direct and stepwise concentrations of saline water (5.0 – 30.0 dS.m ). Direct screening revealed a significant decrease in the mean of callus fresh weight, while a significant increase in callus fresh weight was recorded in response to 25 dS.m  of saline water reached 233 mg after twelve weeks compared with the control at 5.0 dS.m  of saline water. Gradual screening recorded maximum value of callus fresh weight at 30 dS.m  saline water reached 669 mg after the 5 reculture. The highest calli regeneration % recorded 85.03% for callus cultures exposed gradually up to 300 g/l mannitol, while reached 69.63% when exposed gradually to saline water. Porline concentrations increased
in regenerated shoots derived from callus cultures subjected to both direct and gradual concentrations of mannitol and saline water. Gallic acid is markedly increased in all stressed plantlets subjected to both direct and gradual selection, but the direct exposure to saline water at 25 dS.m recorded the highest value reached 22.6 mg/g fwt. Rutin content in shoots regenerated from direct exposure of calli to 25 dS.m
 saline water recorded the highest value reached 311 mg/g fwt. Direct exposure to saline water and gradual exposure to mannitol for calli increased the concentrations of xanthotoxin in the regenerated shoots reached 12.3 and 11.3 mg/g fwt respectively. Highest concentration of bergapten (2.29) mg/g fwt was recorded in shoots regenerated from calli exposed to 240 g/l mannitol. The gain in salt tolerance in plantlets regenerated from calli treated previously with 25 dS.m  saline water directly and 30 dS.m saline water gradually increased to 75% and 80% respectively after resubjected to 25 and 30 dS.m
 saline water for 40 days respectively.While the gain in drought tolerance in plantlets regenerated from calli treated previously with 240 g/l mannitol directly and 300 g/l mannitol gradually increased upto 70% and 72% respectively after reculture on a medium containing 240 g/l mannitol and 300 g/l mannitol for 40 days respectively. Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was used as a powerful technique for analyzing differences in gene expression between drought and salinity stressed and unstressed (control) plantlets of R. graveolens. A new band with different molecular sizes were observed in the primers OPA-01, OPA-08, OPA-11, OPA-17 and OPA- 15 indicating the expression of a new genes amplified under stress conditions or may due to already existing genes.