K., ShireenShireenS. Hassan, Osama2019-10-222019-10-2220081. Armstrong, C.L., C.E. Green and R.L. Phillips, 1991. Development and availability of germplasm with high Type II culture formation response. Maize Genet. Coop. Newslett., 65: 92-93. 2. Aitchitt, M., C.C. Ainsworth and M. Thangavelu, 1993. A rapid and efficient method for the extraction of total DNA from mature leaves of the date palm (Phoenix dactylifera L.). Plant Molecular Biology Reporter, 11(4): 317-319. 3. Assem, S.K., N. Borg and H.A. El-Itriby, 2006. Agrobacterium-mediated stable transformation of maize inbred lines using immature embryos and a standard binary vector system. Egypt. J. of Genet. Cyt., 35: 173-186. 4. Assem, S.K., 2001. Callus production and plant regeneration in Egyptian maize genotypes. Arab J. of Biotech., 4(2): 247-256. 5. Barbu, V. and F. Dautry, 1989. Northern blot normalization with 28S rRNA oligonuclotide probe. Nucleic Acid Res., 17: 7115. 6. Bodin, L., P. Beanue and M. Loriot, 2005. Determination of Cytochrome P450 2D6 (CYP2D6) Gene Copy Number by Real-Time Quantitative PCR. J Biomed Biotechnol. 3: 248-253. 7. Bubner, B. and I.T. Baldwin, 2004. Use of realtime PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep., 23: 263-271. 8. Callaway, A.S., R. Abranches, J. Scroggs, G.C. Allen and W.F. Thompson, 2002. High throughput transgene copy number estimation by competitive PCR. Plant Mol Bio Rep, 20: 265-277. 9. Chiang, P.W., W.J. Song, K.Y. Wu, J.R. Korenberg, E.J. Fogel, M.L. Van Keuren, D. Lashkari and D.M. Kurnit, 1996. Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance. Genome Res, 6: 1013-1026. 10. Chu, C.C., C.C. Wang, C.S. Sun, C. Hsu, K.C. Yin, C.Y. Chu and F.Y. Bi, 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci. Sin., 18: 659-668. 11. Dai, S., P. Zheng, P. Marmey, S. Zhang, W.Z. Tian, S.Y. Chen, R.N. Beachy and C. Fau, 2001. Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol. Breeding, 7: 25-33. J. Appl. Sci. Res., 4(4): 408-414, 2008 413 12. El-Itriby, H.A., S.K. Assem, Hussein, H.A. Ebtissam, F.M. Abdel-Galil and M.A. Madkour, 2003. Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and biolistic particle delivery system. In Vitro Cell Dev Biol., 39(5): 524-531. 13. Fink, L., W. Seeger, L. Ermert, J. Hanze, U. Stahl, F. Grimminger, W. Kummer and M.R. Bohle, 1998. Real time quantitative RT-PCR after laser-assisted cell picking. Nature Medicine, 4: 1329-1333. 14. Frame, B., H. Zhang, S. Cocciolone, L. Sidorenko, C. Dietrich, S. Pegg, S. Zhen, P. Schnable and K. Wang, 2000. Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency. In vitro Cell Dev Biol Plant, 36: 21-29. 15. Frame, B.R., H. Shou, R. Chikwamba, Z. Zhang, C Xiang, T. Fonger, S.E. Pegg, B. Li, D. Nettleton, P. Pei and K. Wang, 2002. Agrobacterium-mediated transformation of maize embryos using a simple binary vector system. Plant Physiology, 129: 13-22. 16. Higuchi, R., C. Fockler, G. Dollinger and R. Watson, 1993. Kinetic PCR: Real-time monitoring of DNA amplification reactions. Nature Biotechnol., 11: 1026-1030. 17. Ingham, D.J., S. Beer, S. Money and G. Hansen, 2001. Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotech,31: 132-134. 18. James,V.A., C. Avarrt, B. Worland, J.W. Snape and P. Vain, 2002. The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet, 104: 553-561. 19. Kohli, A., M. Leech, P. Vain, D.A. Laurie and P. Christou, 1998. Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA., 95: 7203-7208. 20. Li, Z., J. Hansen, Y. Liu, R. Zemetra and P. Berger, 2004. Using real-time PCR to determine transgene copy number in wheat. Plant Mol Bio Rep, 22: 179-188. 21. Livak, K.J. and T.D. Schmittgen, 2001. Analysis of relative gene expression data using realtime t quantitative PCR and the 2- C method. Methods, 25: 402-408. 22. Livak, K.J., S.J. Flood, J. Marmaro, W. Giusti and K. Deetz, 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 4: 357-362. 23. McGarvey, P. and J.M. Kaper, 1991. A simple and rapid method for screening transgenic plants using PCR. Biotechniques, 11: 428-432. 24. Meyer, P., 1998. Stabilities and instabilities in transgene expression. In: Lindsey K. (ed) Transgenic Plant Research. Harwood Academic, Amesterdam, 263-27. 25. Molestina, R.E. and A.P. Sinai, 2005. Host and parasite-derived IKK activities direct distinct temporal phases of NF- B activation and target gene expression following toxoplasma gondii infection. J. Cell Sci., 118: 5785-5796 26. Paule, M.R. and R.J. White, 2000. Survey and summary: transcription by RNA polymerase I and III. Nucleic Acids Res., 28: 1283-1298. 27. Schmidt, M.A. and W.A. Parrott, 2001. Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Rep, 20: 422-428. 28. Schmittgen, T.D., B.A. Zakrajsek, A.G. Mills, V. Gorn, M.J. Singer and M.W. Reed, 2000. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem, 285: 194-204. 29. Shou, H., P. Bordallo and K. Wang, 2004. Expression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize. Journal of Experimental Botany, 55: 1013-1019. 30. Song, P., C.Q. Cai, M. Skokut, B.D. Kosegi and J.F. Petolino, 2002. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS™ derived transgenic maize. Plant Cell Rep, 20: 948-954. 31. Srivastava, V., O.D. Anderson and D.W. Ow, 1999. Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA, 96: 11117- 11121. 32. Wang, X.S., W. Hunter and P. Aine, 2000. Isolation and purification of functional total RNA from woody branches and needles of skit and white spruce. BioTechniques, 28: 292-296. 33. Winer, J., C.K. Jung, I. Shackel and P.M. Williams, 1999. Development and validation of real-time quantitative reverse transcriptasepolymerase chain reaction forhttps://t.ly/AJ6p8MSA Google ScholarIn transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene. Thus, making estimation of transgene copy number and determination of the expression levels an important area of genetically modified crop research. The analysis of transgenic plants can be as important as the transformation process itself. After producing the primary transformants, it is very important to decide which plants contain the transgene and in how many copies[7]. While multiple copies are useful for over-expression experiments, single or low copy transformation events are preferred for most applications because they are stable over several generation of subsequent breeding[24]. The analysis of transgene integration has been recently relied on PCR-based methods, which require only small amounts of plant material and are easily automated for high-throughput quantita tion . Conventional PCR is commonly used for screening large numbers of putative transformants for transgenic sequences[23] and as an alternative to marker selection for segregation analysis[18]. However, conventional PCR analysis is plagued by false positives from contamination because a band with the size of the desired product is counted as signal, regardless of the intensity. Transgene copy number has traditionally been estimated by Southern analysis, although recently other methods, including comparative genomic hybridization, fluorescence in situ hybridization, multiplex amplifiable probe hybridization and microarray have been applied to copy number estimation. Unfortunately, all of these methods are laborious and time-consuming, require considerable amount of DNA from fresh or frozen samples and often involve the use of hazardous radioisotopes[34]. Real time PCR (ABI Prism 7700 Sequence Detection System) has been recently established as a rapid and sensitive technique for precise quantitation[13]. With real-time PCR as a quantification tool, it is possible to rapidly analyze large numbers of putative transformants from high-throughput transformation procedures[7]. Real-time PCR was initially used in plant research as a highly specific and sensitive detectionenUniversity for Real time PCRmaize transformationAgrobacterium tumefacienscopy number, biolistictransgene expressionArticle