Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay

Abstract

The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100 % for a single locus and 92.3 % for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection.

A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice

“…The sequencing results were analyzed using Sequence Scanner software (Applied Biosystems). Real-time qPCR was performed to determine transgene copy number in homozygous lines by following previously described method ( Wang et al, 2015 ). The single-copy rice endogenous gene RBE4 ( Wang et al, 2015 ) was used as a reference and the Hpt gene was used as probe.…”

Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13

“…As quantifying trait generation during breeding can be difficult, SHERLOCK will enable trait heterozygosity to be determined, as well as facilitating the detection of trait stacking. 29 While we used a thermal heating block for these assays, a simple heating device combined with a simple lateral flow reader may enable on-site and portable monitoring of traits or pathogens in future applications. Technologies for the rapid detection of nucleic acids have a multitude of applications in agriculture, and while we have demonstrated the use of SHERLOCK for rapid and portable trait detection in plants during breeding, the same principles could be applied to numerous other contexts in agriculture.…”

SP-LL-37, human antimicrobial peptide, enhances disease resistance in transgenic rice

“…Amplification conditions were as follows: denaturation at 95℃ for 10 min, followed by 50 cycles of amplification (95℃ for 10 sec, 60℃ for 30 sec) and cooling at 40℃ for 10 sec. All samples were tested through qPCR for both 5' and 3' regions of the ribosomal RNA gene for selection of single copy transgenic plants, the method used in this study was described by Wang et al [16]. …”