Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l -1 paromomycin to select for pBI121 transformants and 200 mg l -1 cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycinresistant roots appeared on 45-50% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l -1 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro was achieved on B5 medium containing 1.0 mg l -1 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834.
Potato (Solanum tuberosum L.) an agro-economically important food crop in the world, is sensitive to many fungal pathogens including Rhizoctonia solani (AG-3), the causal agent of stem and root rot diseases. Chitinase and glucanase are cell wall degrading enzymes which have been shown to have high antifungal activity against a wide range of phytopathogenic fungi. In the present study, plasmid pBIKE3 harboring a double-gene cassette containing the chitinase (chit42) and β-1,3-glucanase (bgn13.1) genes was constructed. In this construct, the chit42 gene is located between the CaMV 35S promoter and nos terminator derived from pBI121, while the bgn13.1 gene is downstream of a modified CaMV 35S promoter, followed by the nos terminator both of which were derived from the pRTL plasmid. Micro-tubers of potato plants (the Savalan cultivar) were transformed with the pBIKE3 construct via the Agrobacterium delivery system. Integration of these two genes into the potato genome and their expression at the transcriptional level was confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). The radial diffusion assay showed that the heterologous expressed chitinase and glucanase enzymes demonstrated antifungal activity on R. solani (AG-3).
Chitin is the main component of the cell wall of plant pathogenic fungi. Chitinase 42 (Chit42) from Trichoderma atroviride (PTCC5220) plays a significant role in the biocontrol activity of this fungus against fungal pathogens. This enzyme lacks a chitin binding domain (ChBD) which is involved in its binding to crystalline chitin. In this research, a chimeric chitinase (Chit42+ ChBD) containing a strong chitin binding capacity was constructed by fusing a ChBD from chitinase 18-10 to Chit42 both from isolate PTCC5220. The construct was cloned and overexpressed in Escherichia coli BL21 (DE3). The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the thermal and chemical stability of the chimeric chitinase, when compared with the native Chit42. In vitro assays indicated that the chimeric chitinase showed higher antifungal activity toward plant pathogenic fungi.
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