Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus.

BACKGROUND: Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. METHODS AND RESULTS: The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CONCLUSIONS: CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Expression of radish defensin (RsAFP2) gene in chickpea (Cicer arietinum L.) confers resistance to Fusarium wilt disease.

BACKGROUND: Chickpea (Cicer arietinum L.), a major nutritional source cultivated worldwide, is vulnerable to several abiotic and biotic stresses, including different types of soil-borne pathogens like Fusarium oxysporum f. sp. ciceri, which causes root rot disease and severely affects productivity. METHODS AND RESULTS: In this study, putative transgenic plants were obtained with the Radish defensin (Rs-AFP2) gene through Agrobacterium tumefaciens mediated transformation using the embryo axis explants. Transgenes were confirmed in 18 putative transgenic plants with PCR-specific primers for nptII and Rs-AFP2 genes. Twelve transgenic plants were established successfully under greenhouse conditions. The T0 plants were allowed for self-pollination to obtain T1 seeds. The T1 plants, selected for Fusarium wilt assay using Fusarium oxysporum f. sp. Cicero, showed different resistance levels, from moderate to high levels in comparison to control plants (wild-type) which exhibited severe wilt symptoms. CONCLUSION: Our results suggest the application of Radish defensins (RsAFP1/RsAFP2 genes) for improving pathogen resistance in chickpea.

A review on CRISPR/Cas-based epigenetic regulation in plants.

Epigenetic changes are the heritable modifications in genes without altering DNA sequences. The epigenetic changes occur in the plant genomes to regulate gene expression patterns, which were used to regulate different biological processes, including coping various environmental stresses. These changes, including DNA methylation, non-coding RNA regulation, and histone modification, play a vital role in the transcription and translation processes to regulate gene expression. Gene engineering for the development of stress-tolerant crops via the DNA methylation pathway initially needs a proper selection of genes and its promoter. Manipulating epigenetics requires genetic engineering tools such as Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas). However, CRISPR/Cas9 mediated epigenetic editing refers to transcriptional reprogramming at the targeted sites using epigenetic enzymes fused with decatalytical Cas9 (dCas9). This review focused on the different epigenetic mechanisms in plants and their potential contribution to developing epigenetic tools. The dCas9 endonuclease tethered with transcriptional repressor or activator domain leads to CRISPR inhibitor (CRISPRi) or activator (CRISPRa) for regulating gene expression. The dCas9 has been successfully fused with other various effector domains for constructing epigenetic tools, including the DNA methyltransferase 3A (DNMT3A), or the DNA demethylase TET. Multiple efforts have been made to improve epigenome editing in plants. Initially, incorporating SunTag into the dCas9-EpiEffector complex was used as an epigenetic tool; demethylation of target loci with dCas9-SunTag-TET1 futher increased its efficiency. Additionally, SunTag could also be fused with the dCas9-DNMT3A complex to augment CpG methylation at a targeted loci.

A grobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC.

A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg L-1 and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg L-1. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), ß-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03251-x.

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato.

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l-1 in combination with 0.1 mg l-1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA3) (1.0 mg l-1), IAA (0.5 mg l-1), and ZEA (0.5 mg l-1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l-1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes.