ABSTRACT
Background: Ferulago angulata [Known in Iran as Chavir] is an endangered species with Anti fugal and anti-bacterial activity that can act as a natural preservator
Objective: Evaluation the effect of in vitro culture conditions and plant growth regulators on somaclonal variation and the phytochemical content of in vitro regenerated plants
Methods: Induced calli were transferred to Murashige and Skoog [MS] medium supplemented with 1-Naphthaleneacetic acid [NAA] and 6-Benzylaminopurine [BAP] for regeneration stage. The essential oils were extracted by Clevenger apparatus and the yield and composition of essential oils were assayed by GC/Mass. Inter-Simple Sequence Repeat [ISSR], Start codon Targeted [SCoT] and CAAT Box Derived Polymorphism [CBDP] markers were used to assess somaclonal variation
Results: The highest callus formation was obtained via radicle explant in MS medium supplemented with 2 mg/L 2,4-D and 1mg/L BAP. Maximum percentage of regeneration was occurred via derived calli from hypocotyl in MS medium supplemented with 0.1 mg/L BAP and 0.5 NAA. The phytochemical assay revealed a considerable differences between natural habitats and in vitro regenerated plants. The percentage of ?-Pinene in plants derived from natural habitats and in vitro conditions were 27 and 1.53 respectively. The yield of essential oils were 2.26 and 0.64 [ml/100g D.M.] in natural habitats and in vitro regenerated plants respectively. The result of PCR assay indicated genetic variation between tissue cultured samples
Conclusion: The results indicated that in vitro culture conditions had a considerable effect on genome and metabolome of Ferulago angulate
ABSTRACT
The genetic diversity among plants derived from tissue culture is called somaclonal variation, which provides a valuable source of genetic variation for the improvement of medicinal plants. Objective: The present study was conducted to investigate the efficiency of molecular markers in detection of somaclonal variation and to assess the importance of DNA methylation in occurrence of genomic changes. The genomic DNA of a normal plant and eight abnormal regenerated plants from calluses cultured in different conditions were extracted using modified Delaporta method. The AFLP procedure was performed with application of two different double digestion methods using restriction enzymes. The digested fragments were ligated to appropriate adaptors and amplification was carried out using appropriate primers. Also percentage and component of essential oil were indicated by GC/MS analysis. Analysis of banding patterns showed high differences in amount of polymorphism detected between two different double digestion methods. According to the results of cluster analysis based on the Jaccard's similarity coefficient, all tested plants divided into two main group. While the first group contained only normal sample, other abnormal samples were placed in the second group. Phytochemical analysis showed that the important secondary metabolites such as Limonene, Fenchone, Estragole ,Anethole didn't produce in invitro culture condition. In contrast some metabolites like Cineol, Terpineol, 2,4 Decadienal produce just in invitro culture. The results indicated that the used method has the potential to be used for assessment of somaclonal variations in regenerated plants. Additionally, considering characters of served enzymes in this study, phenotypic variations in abnormal plants that are resulted from somaclonal variation can be related to genome methylation
ABSTRACT
Fennel [Foeniculum vulgar] is a medicinal plant species in the Apiaceae family with culinary and medicinal uses. This study was conducted to evaluate the effects of enzymatic digestion of PCR product in improvement of the efficiency of RAPD markers. Nine RAPD primers were used to amplify the genomic DNA of fifteen accessions of Fennel. Following amplification, a part of PCR products was digested with two restriction enzymes [EcoRl and MseI]. Both of digested and undigested PCR products were separated on agarose gel electrophoresis. The accessions were grouped by cluster analysis and polymorphic information content index was calculated for each marker. Also percentage and component of essential oil were indicated by CC/MS analysis. The comparison of banding patterns of digested and undigested PCR products revealed that digestion of RAPD-PCR product using a four base cutter enzyme such as Mse I shows a higher level of polymorphism as compared to standard RAPD. Cluster analysis based on data obtained by modified RAPD classified accessions more suitable as compared to standard RAPD data. There was no correlation between genetic diversity and metabolic yield. Restriction enzymes have enormous potential to improve the efficiency of RAPD markers in evaluation of genetic diversity across genome