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.

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Salinity of cultivable land is a growing global concern that has been affecting the yields of major crops worldwide such as pigeon pea. In the current study, transgenic pigeon pea plants were developed using an in-planta Agrobacterium-mediated genetic transformation method wherein OsRuvB, a rice DNA helicase gene, was incorporated to induce salt tolerance in pigeon pea plants. Transformation efficiency of 35.7% was achieved with stable insertion of OsRuvB in transgenic lines. When subjected to salinity stress induced by 75 mM NaCl increase in chlorophyll content, relative water content, peroxidase and catalase activity in transgenic lines was observed over the wild type plants. Membrane injury index and lipid peroxidation were significantly reduced in transgenic lines. Proline and Total Soluble Sugar content were enhanced in both transgenic plants and wild type strains. It was inferred that transgenic lines were tolerant to salinity stress and tolerance may be imparted through an alternative unknown pathway. ARTICLE HISTORY

Lectin receptor-like kinases (LecRLKs) play crucial roles in regulating plant growth and developmental processes in response to stress. In transcriptional gene regulation for normal cellular functions, cis-acting regulatory elements (CREs) direct the temporal and spatial gene expression with respect to environmental stimuli. A complete insightful of the transcriptional gene regulation system relies on effective functional analysis of CREs. Here, we analyzed the potential putative CREs present in the promoters of rice LecRLKs genes by using PlantCARE database. The CREs in LecRLKs promoters are associated with plant growth/development, light response, plant hormonal regulation processes, various stress responses, hormonal response like ABA, root-specific expression responsive, drought responsive, and cell and organ specific regulatory elements. The effect of methylation on these cis-regulatory elements was also analyzed. Real-time analysis of rice seedling under various stress conditions showed the expression levels of selected LecRLK genes superimposing the number of different CREs present in 5' upstream region. The overall results showed that the possible CREs function in the selective expression/regulation of LecRLKs gene family and during rice plant development under stress.

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Sensing stress and activating the downstream signaling pathways is the imperative step for stress response. Lectin receptor‐like kinase (LecRLK) is an important family that plays a key role in sensing stress conditions through lectin receptor and activates downstream signaling by kinase domain. We identified the role of OsLecRLK gene for salinity stress tolerance and hypothesized its role in Na+ extrusion from cell. OsLecRLK overexpression and downregulation (through artificial miRNA) transgenic lines were developed and its comparison with wild‐type (WT) plants were performed overexpression transgenic lines showed better performance, whereas downregulation showed poor performance than WT. Lower accumulation of reactive oxygen species (ROS), malondialdehyde and toxic ion, and a higher level of proline, RWC, ROS scavengers in overexpression lines lead us to the above conclusion. Based on the relative expression of stress‐responsive genes, ionic content and interactome protein, working model highlights the role of OsLecRLK in the extrusion of Na+ ion from the cell. This extrusion is facilitated by a higher expression of salt overly sensitive 1 (Na+/K+ channel) in overexpression transgenic line. Altered expression of stress‐responsive genes and changed biochemical and physiological properties of cell suggests an extensive reprogramming of the stress‐responsive metabolic pathways by OsLecRLK under stress condition, which could be responsible for the salt tolerance capability.

RuvB, a member of AAA+ (ATPases Associated with diverse cellular Activities) superfamily of proteins, is essential, highly conserved and multifunctional in nature as it is involved in DNA damage repair, mitotic assembly, switching of histone variants and assembly of telomerase core complex. RuvB family is widely studied in various systems such as Escherichia coli, yeast, human, Drosophila, Plasmodium falciparum and mouse, but not well studied in plants. We have studied the transcript level of rice homologue of RuvB gene (OsRuvBL1a) under various abiotic stress conditions, and the results suggest that it is upregulated under salinity, cold and heat stress. Therefore, the OsRuvBL1a protein was characterized using in silico and biochemical approaches. In silico study confirmed the presence of all the four characteristic motifs of AAA+ superfamily-Walker A, Walker B, Sensor I and Sensor II. Structurally, OsRuvBL1a is similar to RuvB1 from Chaetomium thermophilum. The purified recombinant OsRuvBL1a protein shows unique DNA-independent ATPase activity. Using site-directed mutagenesis, the importance of two conserved motifs (Walker B and Sensor I) in ATPase activity has been also reported with mutants D302N and N332H. The OsRuvBL1a protein unwinds the duplex DNA in the 3' to 5' direction. The presence of unique DNA-independent ATPase and DNA unwinding activities of OsRuvBL1a protein and upregulation of its transcript under abiotic stress conditions suggest its involvement in multiple cellular pathways. The first detailed characterization of plant RuvBL1a in this study may provide important contribution in exploiting the role of RuvB for developing the stress tolerant plants of agricultural importance.