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

Abstract

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 T 1 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.

Rice lectin receptor‐like kinase provides salinity tolerance by ion homeostasis

“…(a) SOS1 , (b) NHX1 , (c) NHX2 , (d) CHX11 , (e) CAX , (f) TPC1 , (g) CatB , (h) SOD , (i) APX7 . The method (Passricha et al, ) was used to measure the relative expression of the respective genes. Values are means ± standard deviations from three independent measurements [Color figure can be viewed at wileyonlinelibrary.com]…”

“…Real-time PCR method was used for the selection of homozygous plants in T 1 generation (Passricha, Saifi, Khatodia, & Tuteja, 2016). In this method, DNA of T 0 plant with single-copy insertion was used as reference.…”

Segregation of rol Genes in Two Generations of Sinningia speciosa Engineered Through Wild Type Rhizobium rhizogenes

“…Based on the single copy, single locus nature of Reg1, copy number segregation in a 1:2:1 ratio for 2:1:0 copies, respectively, was expected. The increase in copy number is related to plants which are homozygous for the locus of insertion (Passricha et al, 2016). The obtained genotypic segregation ratios of 3:21:26 and 2:15:27 deviate significantly from theoretically expected ratios, with marked underrepresentation of the rol ++ plants.…”

A quick, easy and cost-effective in planta method to develop direct transformants in wheat

“…Genotypic frequencies of wheat transgenic lines harboring a single copy of the transgene were determined through analysis of seedling mortality using Chi-squared test. As wheat is a self-pollinated crop, the heterozygous lines and double insertion of transgenes were identified through mortality rate 3:1 and 15:1, respectively (Passricha et al 2016). The transgenic lines with 100% plant survival were considered as single homozygous transgenic lines with single copy of transgene.…”