CRISPR/Cas tool designs for multiplex genome editing and its applications in developing biotic and abiotic stress-resistant crop plants.

Crop plants are prone to several yield-reducing biotic and abiotic stresses. The crop yield reductions due to these stresses need addressing to maintain an adequate balance between the increasing world population and food production to avoid food scarcities in the future. It is impossible to increase the area under food crops proportionately to meet the rising food demand. In such an adverse scenario overcoming the biotic and abiotic stresses through biotechnological interventions may serve as a boon to help meet the globe's food requirements. Under the current genomic era, the wide availability of genomic resources and genome editing technologies such as Transcription Activator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered-Regularly Interspaced Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) has widened the scope of overcoming these stresses for several food crops. These techniques have made gene editing more manageable and accessible with changes at the embryo level by adding or deleting DNA sequences of the target gene(s) from the genome. The CRISPR construct consists of a single guide RNA having complementarity with the nucleotide fragments of the target gene sequence, accompanied by a protospacer adjacent motif. The target sequence in the organism's genome is then cleaved by the Cas9 endonuclease for obtaining a desired trait of interest. The current review describes the components, mechanisms, and types of CRISPR/Cas techniques and how this technology has helped to functionally characterize genes associated with various biotic and abiotic stresses in a target organism. This review also summarizes the application of CRISPR/Cas technology targeting these stresses in crops through knocking down/out of associated genes.

Next Generation dsRNA-Based Insect Control: Success So Far and Challenges.

RNA interference (RNAi) is a method of gene silencing where dsRNA is digested into small interfering RNA (siRNA) in the presence of enzymes. These siRNAs then target homologous mRNA sequences aided by the RNA-induced silencing complex (RISC). The mechanism of dsRNA uptake has been well studied and established across many living organisms including insects. In insects, RNAi is a novel and potential tool to develop future pest management means targeting various classes of insects including dipterans, coleopterans, hemipterans, lepidopterans, hymenopterans and isopterans. However, the extent of RNAi in individual class varies due to underlying mechanisms. The present review focuses on three major insect classes viz hemipterans, lepidopterans and coleopterans and the rationale behind this lies in the fact that studies pertaining to RNAi has been extensively performed in these groups. Additionally, these classes harbour major agriculturally important pest species which require due attention. Interestingly, all the three classes exhibit varying levels of RNAi efficiencies with the coleopterans exhibiting maximum response, while hemipterans are relatively inefficient. Lepidopterans on the other hand, show minimum response to RNAi. This has been attributed to many facts and few important being endosomal escape, high activity dsRNA-specific nucleases, and highly alkaline gut environment which renders the dsRNA unstable. Various methods have been established to ensure safe delivery of dsRNA into the biological system of the insect. The most common method for dsRNA administration is supplementing the diet of insects via spraying onto leaves and other commonly eaten parts of the plant. This method is environment-friendly and superior to the hazardous effects of pesticides. Another method involves submergence of root systems in dsRNA solutions and subsequent uptake by the phloem. Additionally, more recent techniques are nanoparticle- and Agrobacterium-mediated delivery systems. However, due to the novelty of these biotechnological methods and recalcitrant nature of certain crops, further optimization is required. This review emphasizes on RNAi developments in agriculturally important insect species and the major hurdles for efficient RNAi in these groups. The review also discusses in detail the development of new techniques to enhance RNAi efficiency using liposomes and nanoparticles, transplastomics, microbial-mediated delivery and chemical methods.

Genetic estimation of grain yield and its attributes in three wheat (Triticum aestivum L.) crosses using six parameter model.

Grain yield is a complex polygenic trait representing a multiplicative end product of contributing yield attributes governed by simple to complex gene interactions. Deciphering the genetics and inheritance of traits/genes influencing yield is a prerequisite to harness the yield potential in any crop species. The objective of the present investigation was to estimate genetic variance components and type of gene action controlling yield and its component traits using six populations (P1, P2, F1, F2, BC1 and BC2) of the three bread wheat crosses. Cross I (25th HRWSN 2105 × WH 1080), cross II (22ndSAWYT323 × RSP 561) and cross III (22ndSAWYT333 × WH 1080) involving elite stripe rust resistant wheat genetic stocks in combination with commercial check varieties were used for analysis. A combination of morpho-physiological, biochemical and disease influencing traits were evaluated, thus exploring the possibility of multi-trait integration in future. Results revealed that the estimated mean effects (m) were highly significant for all the traits in all crosses, indicating that selected traits were quantitatively inherited. The estimate of dominant gene effect was highly significant for plant height, number of tillers per plant in all the three crosses. Grain yield per plant was highly significant in the cross II while total protein content was highly significant in both crosses II and III. Glycine betaine content showed significant additive genes effect. Duplicate epistasis was the most significant for traits like plant height, total protein content and grain yield per plant. Dominance gene effect was more important than additive gene effects in the inheritance of grain yield and most other traits studied. The magnitude of additive X additive gene effects was high and positively significant whereas dominance × dominance was negatively significant for most of the traits studied in the three crosses. Additive × dominance gene effects was of minor significance, thus indicating that selection for grain yield and its components should be delayed to later generations of breeding.

Comparative studies and identification of camptothecin produced by an endophyte at shake flask and bioreactor.

The fungus showing homology with Nodulisporium by 28S ribosomal gene sequencing, which has been discovered as an endophyte on medicinal plant Nothapodytes foetida, was found to produce 45 and 5.5 microg of camptothecin (CPT) per gram of mycelia at bioreactor and at shake flask, respectively, which was further quantified and characterised by various spectroscopic analyses.