Assessing zygosity in progeny of transgenic plants: current methods and perspectives

Homozygosity is highly desirable in transgenic plants research to ensure the stable integration and inheritance of transgene(s). Simple, reliable and high-throughput techniques to detect the zygosity of transgenic events in plants are invaluable tools for biotechnology and plant breeding companies. Currently, a number of basic techniques are being used to determine the zygosity of transgenic plants in T₁ generation. For successful application of any technique, precision and simplicity of approach combined with the power of resolution are important parameters. On the basis of simplicity, resolution and cost involved, the available techniques have been classified into three major classes which are conventional methods, current methods and next generation methods. Conventional methods include antibiotic marker-based selection and the highly labor intensive Southern blot analysis. In contrast, methods such as real time PCR, TAIL PCR and competitive PCR are not only cost effective but rapid as well. Moreover, methods such as NGS, digital PCR and loop-mediated isothermal amplification also provide a cost effective, fast and not so labor intensive substitute of current methods. In this review, we have attempted to compare and contrast all the available efficient methods to distinguish homozygous plants in progeny of transgenics. This review also provides information of various techniques available for determining zygosity in plants so as to permit researchers to make informed choices of techniques that best suit their analyses. More importantly, detection and subsequent selection of homozygous individuals is central for facilitating the movement of transgenic plants from the laboratory to the field.