Recovery of amiRNA3-PARP1 transgenic maize plants using a binary vector having the biosafe PMI Gene
Positive plant selectable marker genes are commonly used in plant transformation because they not only enhance the frequency of generation transgenic tissues but are considered biosafe, unlike antibiotic or herbicide resistance genes. In this study, the binary vector pNOV2819-ubiamiRNA3PARP1, harbouring the phosphomannose isomerase (pmi) gene was developed and used in recovery of transgenic maize (Zea mays L.) plants containing the drought tolerance gene, amiRNA3-PARP1. The pre-amiRNA3-PARP1 and Tnos transgenes were sequentially PCR-cloned upstream the ubiquitine promoter in the Ubi/NC1300 plasmid. The pre-amiRNA3-PARP1 expression cassette was transferred into the pmi gene-containing pNOV2819 plasmid to produce the pNOV2819- ubiamiRNA3PARP1 vector. Transgenic IL3 and A188 plants containing pre-amiRNA3-PARP1 were generated through transformation with LBA4404 harbouring the pNOV2819-ubiamiRNA3PARP1 vector. The plants were confirmed transgenic by PCR. It is clear that the developed vectors are effective in recovery of amiRNA3-PARP1 transgenic tissues and plants containing the pmi gene, which has been shown to have no negative environmental or health effects.
Key Words: Agrobacterium transformation, amiRNA, PMI, Zea mays