Agrobacterium-mediated genetic transformation of ajowan (Trachyspermum ammi (L.) Sprague): an important industrial medicinal plant

Abstract

Biotechnology-based breeding methods can improve useful industrial medicinal plants in a faster way. Tissue culture-based and in planta Agrobacterium-mediated gene transformation methods were applied to enhance drought and salinity tolerance in ajowan medicinal plant using betaine aldehyde dehydrogenase (BADH) gene. The pBI121 binary vector harboring kanamycin resistance gene (nptII) and BADH gene in its T-DNA region was applied to transfer BADH gene. Different concentrations of the selective antibiotic were tested on 15 day-old hypocotyl segments and 75 mg/L kanamycin was identified as the minimum inhibitory concentration to screen for transformed cells of ajowan. The different parameters of gene transformation including Agrobacterium optical density (OD), Agrobacterium strain, Agrobacterium killing antibiotic, acetosyringone concentration, and inoculation duration were tested during tissue culture-based Agrobacterium-mediated transformation. The OD600 = 0.6-0.8 of LB4404 strain of Agrobacterium × 160 mg/L timentin × 250 μmol/L acetosyringone × 30 min inoculation duration were identified as the best gene transformation parameters. Successful integration and expression of transferred gene were confirmed in T0 plants using polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) methods. Five transgenic lines with 2.94% transformation efficiency were achieved by regular tissue culture-based Agrobacterium-mediated gene transformation method, whereas the dip flora in planta transformation method has led to one transgenic line with 1.42% transformation efficiency. Bioassay analysis revealed enhanced drought and salinity tolerance in transformed plants in comparison with non-transformed plants. The morphological characteristics of plant height and seedling fresh weight of BADH-transformed plants were improved under salinity stress. Under drought stress, the proline content and relative water content of transgenic plants were more than non-transformants. The results of gas chromatography mass spectrometry (GC–MS) revealed significant increase in thymol content of BADH-transformed plants (55.07%) compared with non-transformed plants (39.20%) under drought stress condition. The obtained transgenic plants are cultivable in unfavorable environments to give an acceptable level of useful secondary metabolites. The optimized protocol is applicable for gene transformation of other medicinal plants of Apiacea family for different purposes.