Transformation of maize ( Zea mays L.) by Agrobacterium Tumefaciens and shoot apical meristem explant
Volume 9, Issue 1, Summer 2018, Pages 66-75
https://doi.org/10.52547/JCT.9.1.66
Khadijeh Bagheri, Roya Taghibeigloo, Bahram Maleki Zanjan
Abstract Aim: The aims of study were to investigate callogenesis and regeneration of the studied cultivars and also exploring the possibility of gene transfer into maize by using complete bud and stem apical meristem explants.
Material and methods: Complete bud and stem apical meristem of six cultivars were used as explants on MS medium + 5 mg.L-¹ 2,4-D. For regeneration, calluses were cultured in MS including 1 mg.L-¹ Kn and 10 mg.L-¹ BAP. According to the results of tissue culture, SC703 and SC704 cultivars were selected for gene transfer. Transformation of explants was done with LBA4404 strain of Agrobacterium tumefaciens containing pBI121 vector. To investigate the transgenic nature of the samples, PCR and histochemical assay were used.
Results: Callus induction was taking place after 30 to 40 days and the frequency of callus induction in six cultivars was 100%, in addition the highest rate of regeneration (90 %) was observed in stem apical meristem explants of SC704. In some cultivars, stem apical meristem explants were more suitable, although in other cultivars, complete bud explants were better. PCR results for NPTII gene and GUS histochemical assay showed that some calli are transgenic and GUS gene is expressed.
Conclusion: The results of callogenesis, regeneration and genetic transformation showed that SC 704 was better than the others.
Expression of active beta-glucuronidases enzyme in tobacco plant seeds
Volume 8, Issue 3, Winter 2018, Pages 231-241
https://doi.org/10.52547/JCT.8.3.231
Kh Bagheri, B Maleki Zanjani, M Mekanik
Abstract -
Aim: The purpose of this research was to design and prepare a suitable gene construct and transfer it to the tobacco plant and analysis of transgenic plants.
Material and methods: Seed-specific construct that containing Napin promoter, Ω sequence, GUS gene, and SAR sequence was prepared in pBI121 plasmid and proliferated in E.coli.. Then tobacco leaf explants were inoculated with LBA4404 agrobacterium strain by standard protocol. Selection of regenerated shoots also were performed in a selection media (co-culture medias + 25 mg/L Kan + 200 mg/L Cef). Transgenic plants were analyzed by PCR, RT-PCR and histochemical assay.
Results: Analysis of regenerated plantlets by using PCR and specific primers of nptII and GUS indicated that transfer of these genes to plantlets was successful. RT-PCR reaction results showed that nptII is transcribed in both tissues while GUS is transcribed only in the seed tissue. This finding was expected because Nos promoter (which controls the nptII transcription) is a constitutive and Napin is a seed specific promoter.. Expression and activity of Beta-glucuronidase enzyme in seeds of selected plants was confirmed by SDS-PAGE and histochemical assay.
Conclusion: The result of this research showed that designed gene construct was appropriate, because Napin promotes the expression of GUS gene in the seeds and Omega sequences have also been effective in increasing of transgene expression. In the fallowing, this construct can be used for the production of recombinant proteins by replacing valuable genes with GUS..