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.

Biotechnological Perspectives to Combat the COVID-19 Pandemic: Precise Diagnostics and Inevitable Vaccine Paradigms.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause for the ongoing global public health emergency. It is more commonly known as coronavirus disease 2019 (COVID-19); the pandemic threat continues to spread aroundthe world with the fluctuating emergence of its new variants. The severity of COVID-19 ranges from asymptomatic to serious acute respiratory distress syndrome (ARDS), which has led to a high human mortality rate and disruption of socioeconomic well-being. For the restoration of pre-pandemic normalcy, the international scientific community has been conducting research on a war footing to limit extremely pathogenic COVID-19 through diagnosis, treatment, and immunization. Since the first report of COVID-19 viral infection, an array of laboratory-based and point-of-care (POC) approaches have emerged for diagnosing and understanding its status of outbreak. The RT-PCR-based viral nucleic acid test (NAT) is one of the rapidly developed and most used COVID-19 detection approaches. Notably, the current forbidding status of COVID-19 requires the development of safe, targeted vaccines/vaccine injections (shots) that can reduce its associated morbidity and mortality. Massive and accelerated vaccination campaigns would be the most effective and ultimate hope to end the COVID-19 pandemic. Since the SARS-CoV-2 virus outbreak, emerging biotechnologies and their multidisciplinary approaches have accelerated the understanding of molecular details as well as the development of a wide range of diagnostics and potential vaccine candidates, which are indispensable to combating the highly contagious COVID-19. Several vaccine candidates have completed phase III clinical studies and are reported to be effective in immunizing against COVID-19 after their rollout via emergency use authorization (EUA). However, optimizing the type of vaccine candidates and its route of delivery that works best to control viral spread is crucial to face the threatening variants expected to emerge over time. In conclusion, the insights of this review would facilitate the development of more likely diagnostics and ideal vaccines for the global control of COVID-19.

The present and potential future methods for delivering CRISPR/Cas9 components in plants.

BACKGROUND: CRISPR/Cas9 genome editing technology is a DNA manipulation tool for trait improvement. This technology has been demonstrated and successfully applied to edit the genome in various species of plants. The delivery of CRISPR/Cas9 components within rigid plant cells is very crucial for high editing efficiency. Here, we insight the strengths and weaknesses of each method of delivery. MAIN TEXT: The mutation efficiency of genome editing may vary and affected by different factors. Out of various factors, the delivery of CRISPR/Cas9 components into cells and genome is vital. The way of delivery defines whether the edited plant is transgenic or transgene-free. In many countries, the transgenic approach of improvement is a significant limitation in the regulatory approval of genetically modified crops. Gene editing provides an opportunity for generating transgene-free edited genome of the plant. Nevertheless, the mode of delivery of the CRISPR/Cas9 component is of crucial importance for genome modification in plants. Different delivery methods such as Agrobacterium-mediated, bombardment or biolistic method, floral-dip, and PEG-mediated protoplast are frequently applied to crops for efficient genome editing. CONCLUSION: We have reviewed different delivery methods with prons and cons for genome editing in plants. A novel nanoparticle and pollen magnetofection-mediated delivery systems which would be very useful in the near future. Further, the factors affecting editing efficiency, such as the promoter, transformation method, and selection pressure, are discussed in the present review.

The Rise of the CRISPR/Cpf1 System for Efficient Genome Editing in Plants.

Cpf1, an endonuclease of the class 2 CRISPR family, fills the gaps that were previously faced in the world of genome engineering tools, which include the TALEN, ZFN, and CRISPR/Cas9. Other simultaneously discovered nucleases were not able to carry out re-engineering at the same region due to the loss of a target site after first-time engineering. Cpf1 acts as a dual nuclease, functioning as an endoribonuclease to process crRNA and endodeoxyribonuclease to cleave target sequences and generate double-stranded breaks. Additionally, Cpf1 allows for multiplexed genome editing, as a single crRNA array transcript can target multiple loci in the genome. The CRISPR/Cpf1 system enables gene deletion, insertion, base editing, and locus tagging in monocot as well as in dicot plants with fewer off-target effects. This tool has been efficiently demonstrated into tobacco, rice, soybean, wheat, etc. This review covers the development and applications of Cpf1 mediated genome editing technology in plants.

Indirect regeneration and genetic fidelity analysis of acclimated plantlets through SCoT and ISSR markers in Morus alba L. cv. Chinese white.

A reliable protocol was developed for in vitro micro propagation of Morus alba L.cv. Chinese white. Initially, friable callus was induced (242.8 and 128.5 mg) from in vivo leaf and nodal explants on Murashige and Skoog's (MS) medium amended with 4.0 µM/L of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3.0 µM/L of Naphthalene acetic acid (NAA) respectively within 3 weeks. Shoot regeneration (12.2 and 8.6) was obtained from leaf and node derived callus on 6-benzylaminopurine (BAP) + Thidiazuron (TDZ) at 2.5 + 2.0 and 7.5 + 2.0 µM/L concentrations respectively, after 4 weeks of incubation. In vitro shoots were rooted (90 %) on half strength MS medium with 7.5 µM/L indole-3 butyric acid (IBA) and plantlets were hardened in plastic pots contained farmyard manure, sand and garden soil in 1:1:2 ratio. The genetic stability of plantlets were confirmed by start codon targeted (SCoT) and inter simple sequence repeats (ISSR) primers based molecular analysis.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant.

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.