Abstract
We have developed a rapid and simple RT-PCR based method to check the integrity of chimeric genes within plasmid constructs for plant transformation. It exploits the Agrobacterium tumefaciens-mediated transient expression of plasmid constructs in plant tissue. Total RNA was isolated from tobacco leaves co-cultivated for 3 days with Agrobacterium tumefaciens harbouring the plant transformation vectors constructed in our laboratory. First strand cDNA synthesis with oligo(dT) primers generate a pool of cDNA that was used for PCR amplification with primers specific for each of the genes present within the constructs. PCR amplification reactions were successful for all chimeric genes tested, thus confirming their intactness and suitability to be used for stable plant transformation.
Similar content being viewed by others
References
An G (1985) High efficiency transformation of cultured tobacco cells. Plant Physiol. 79: 568–570
Bartels D, Hanke C, Schneider K, Michel D & Salamini F (1992) A desiccation related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. EMBO J. 11: 2771–2778
Capell T, Escobar C, Lui H, Burtin D, Lepri O & Christou P (1998) Over-expression of the oat arginine decarboxylase cDNA in transgemc rice (Oryza sativa L.) affects normal development patterns in vitro and results in putrescine accumulation in transgenic plants. Theor. Appl. Genet. 97: 246–254
Hellens RP, Edwards EA, Leyland NR, Bean S & Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42: 819–832
Kapila JR, De Rycke R, van Montagu M & Angenon G (1996) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. 122: 101–108
Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
dos Santos, D.M.M.F., de Sousa Araújo, S., Duque, A.S.R.L. et al. Reverse transcription-PCR assay to verify gene integrity within plasmid constructs for plant transformation. Plant Cell, Tissue and Organ Culture 74, 293–296 (2003). https://doi.org/10.1023/A:1024093105154
Issue Date:
DOI: https://doi.org/10.1023/A:1024093105154