Southern blot test for12/20/2023 ![]() ![]() ![]() 1995) and most recently digital droplet PCR (ddPCR Hindson et al. 1992), thermal asymmetric interlaced‐PCR (TAIL‐PCR Liu et al. Southern blot analysis (Southern, 1975), quantitative polymerase chain reaction (qPCR Higuchi et al. Here, decreasing the number of generations between the initial transformation and identification of homozygous lines with stable expression has greatest value. Knowledge of the transgenic locus structure and zygosity status is particularly important in plants, including all crops, which have a life cycle of months and sometimes years. Rapid and reliable evaluation of transgenic allele(s) for copy number and homozygosity is a vital step in utilizing these transformation events, so that homozygous lines with stable transgene expression are obtained for accurate testing. In parallel, there is considerable interest in introducing new pathways and modifying existing pathways in plants to produce new or improved bioproducts (Clemente & Cahoon 2009 Rogers & Oldroyd 2014). The use of stable transformation of plants to test a range of hypotheses has been increasing rapidly and has been suggested a key element in addressing future security of food supply as well as in adapting to global change (Khush 2005 Hibberd et al. A protocol for this application of ddPCR to large plant genomes is provided. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. This shows the importance of routine T‐DNA copy number estimation. ![]() Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. Comparison of segregation analyses and ddPCR of T 1 progeny from 26 T 0 plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Stable transformation of plants is a powerful tool for hypothesis testing. ![]()
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