Phytoene Desaturase Gene Research Articles

Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon.

CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (PDS) gene in melon. The constructed vectors were delivered to host plants using Agrobacterium-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a PDS editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.

Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter.

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species.

Construction and optimization of the TRV-mediated VIGS system in Areca catechu embryoids

Areca catechu, a member of the palm family Arecaceae native to tropical regions, holds significant economic importance as a tropical crop. This study explores the application of virus-induced gene silencing (VIGS) in A. catechu embryogenic callus tissues, utilizing the phytoene desaturase gene (AcPDS) of A. catechu as a reporter gene. Results demonstrate that employing EHA105 strain, an Agrobacterium infection liquid concentration of OD600 = 0.5, an infection duration of 5 min, acetosyringone (AS) concentration of 21.5 mg/L, co-cultivation at 19 °C for 2 days followed by 28 °C for 3 days, and a subsequent 14-day post-co-cultivation period, the experimental group (pTRV1+pTRV2-AcPDS) exhibited significantly reduced gene expression compared to the control group CK (pTRV1+pTRV2), manifesting the anticipated photobleaching phenotype by the 21st day. RT-PCR analysis revealed that AcPDS gene expression in TRV-mediated photobleached A. catechu embryos decreased to 0.227 times that of the control group CK. This established VIGS system in A. catechu embryoids establishes an effective experimental platform for studying functional genes in A. catechu.

An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Generation and characterization of a depigmented variant of Chlorella pyrenoidosa for application in food products

Microalgae are promising natural resources for an array of essential nutrients and health-promoting compounds. The past decade has witnessed their increasing popularity as a value-added ingredient in the food industry. However, some of their intrinsic properties, especially their typical green color and off-flavor, may compromise consumer acceptability and remain a key challenge for food scientists. This study aimed to generate and characterize a chemically induced pigment-deficient mutant (C49) of Chlorella pyrenoidosa. In contrast to the wild type (WT), C49 could not grow photoautotrophically or mixotrophically. However, when grown heterotrophically, it exhibited a growth curve similar to that of WT. Pigment analysis verified the presence of only trace amounts of chlorophylls (0.27 ± 0.02 mg/g) and carotenoids (0.036 ± 0.003 mg/g) in the C49 which were only ∼1.7% and ∼1% of those in the WT, respectively. C49 also had higher carbohydrate (62.75% ± 2.42%) and lower protein (26.54% ± 1.00%) content than the WT (37.41% ± 2.25% and 36.21% ± 1.97%, respectively). Genetic assays revealed inactivation of the phytoene desaturase gene as a key mechanism underlying the reduced xanthophyll-synthesizing capability of C49, thus causing its failure to accumulate chlorophyll and develop chloroplasts. Transcriptomic and RT-PCR analyses indicated that the high carbohydrate content was likely due to reduced starch degradation. Furthermore, C49 demonstrated an emulsifying capacity comparable to that of WT over a wide pH range (3–8) in oil-in-water emulsions consisting of 5% cell-disrupted homogenate in deionized water and rapeseed oil (1:1, v/v). The results of this study support that depigmented Chlorella pyrenoidosa possesses promising potential for application in the food industry.

Identification of genus Deinococcus strains by PCR detection using the gyrB gene and its extension to Bacteria domain

In radiation-resistant bacteria belonging to the genus Deinococcus, transposition events of insertion sequences (IS elements) leading to phenotypic changes from a reddish color to white were detected following exposure to gamma irradiation and hydrogen peroxide treatment. This change resulted from the integration of IS elements into the phytoene desaturase gene, a key enzyme in the carotenoid biosynthesis pathway. To facilitate species identification and distinguish among Deinococcus strains, the gyrB gene encoding the B subunit of DNA gyrase was utilized. The s gnificance of the gyrB gene is well recognized not only in genome replication through the regulation of supercoiling but also in phylogenetic analysis providing support for 16S rRNA-based identification. Its mutation rate surpasses that of the 16S rRNA gene, offering greater resolution between closely related species, particularly those exhibiting >99% similarity. In this study, phylogenetic analysis was conducted comparing the 16S rRNA and gyrB gene sequences of Deinococcus species. Species-specific and genus-specific primers targeting Deinococcus species were designed and experimentally validated for selective amplification and rapid identification of the targeted species. This approach allows for the omission of 16S rRNA sequencing in the targeted Deinococcus species. Therefore, the gyrB gene is useful for identifying bacterial species and genus-level detection from individual microbes or microbial consortia using specialized primer sets for PCR amplification.

CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.)

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon–intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 μg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

A novel and efficient Apple latent spherical virus-based gene silencing method for functional genomic studies in Chenopodium quinoa

Quinoa (Chenopodium quinoa Willd.), with its resilience in harsh environments and excellent nutritional value, has become crucial for global food security. Despite recent progress in genomic research, the inability to perform functional studies in quinoa due to the absence of transformation techniques remains a significant obstacle. In this work, we present the development of a novel Apple Latent Spherical Virus (ALSV)-mediated virus-induced gene silencing (VIGS) protocol that will allow to perform functional genomics studies in quinoa in a fast and simple way. The method was fine-tuned using ALSV plasmids that carry partial gene sequences of phytoene desaturase (PDS) from Nicotiana benthamiana and quinoa. The developed technique involves an initial inoculation in Nicotiana plants through agroinfiltration with Agrobacterium tumefaciens cultures carrying the different viral constructs. Viral extracts were prepared using local or systemic leaves, which were then used as inoculum to infect quinoa leaves through mechanical damage. The method was successfully tested in two contrasting quinoa varieties, although some differences were observed in infection phenotype and viral susceptibility. The effect of insertion sequence size in the viral vectors was also analyzed, resulting in differences in bleaching or chlorosis phenotype and impact on plant growth. The presence of the virus in inoculated plants was confirmed, and the reduction in PDS gene expression in silenced plants was verified. Because quinoa lacks stable transformation protocols, limiting heterologous expression assays, our ALSV-based VIGS protocol is very attractive for loss-of-function gene studies.

Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotypeused to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Phytoene Desaturase (PDS) Gene-Derived Markers Identify “A” and “B” Genomes in Banana (Musa spp.)

Phytoene desaturase (PDS) is a plant enzyme involved in carotenoid biosynthesis. The PDS gene has been used as a selective marker for genome editing in several plant species, including banana (Musa spp.). Its knockout promotes dwarfism and albinism, characteristics that are easily recognizable and highly favorable. In Musa spp., the A genome increases fruit production and quality, whereas the B genome is associated with tolerance to biotic and abiotic stresses. The objective of this study was to identify a molecular marker in the PDS gene to easily discriminate the A and B genomes of banana. A 2166 bp fragment for the “PDSMa” marker was identified as polymorphic for the A genome (identification accuracy of 99.33%), whereas ~332 and ~225 bp fragments were detected for the “PDSMb” marker with 100% accuracy using MedCalc software. In this study, we used genotypes with A and B genomes that are used in the genetic improvement of bananas and an accession with the BT genome. It was not possible to differentiate the accession with the BT genome from the others, suggesting that the markers do not have the capacity to separate the T genome from the A and B genomes. To the best of our knowledge, this is the first study to use the PDS gene to determine doses of the A genome and identify the B genome in Musa spp., which will aid in evaluating the genomic constitution of banana hybrids and accessions at the seedling stage and accelerating their classification in crop genetic improvement programs.

In silico design of CRISPR/Cas9 guide RNA for the knockout of the phytoene desaturase gene in sweet potato (Ipomoea batatas L.)

This study aimed to design in silico guide RNA (sgRNA) for CRISPR/Cas9-mediated knockout of the phytoene desaturase (PDS) gene in sweet potato (Ipomoea batatas L.). The sequence of the coding region of the IbPDS gene is 1791 base pairs (bp) long, and these, in turn, are equivalent to 572 amino acids. The amino acid sequence of the IbPDS gene was compared with the homologous sequences of other nearby plant species, showing that it presents a close similarity with PDS of Ipomoea triloba and Ipomoea nil with 98.60% and 97.73%, respectively. CRISPR RGEN Tools provided 113 results for the IbPDS gene, filtering to 24 and selecting three sgRNA sequences for the design of the gene editing vector, which were sgRNA 1 (5'-ACCTCATCAGTCACCCTGTCNGG-3'), sgRNA 2 (5'- CCTCCAGCAGCAGTATTGGTTGGTTTGNGG -3') and sgRNA 3 (5'- CTGAACTCTCCTGGTTGGTTGTTNGG -3'). The predicted secondary structures of the selected sgRNAs present efficient sgRNA structures for gene editing of the target gene. The PMH-Cas9- 3xsgRNA vector for CRISPR/Cas9-mediated knockout of the IbPDS gene was designed in silico with three sgRNA sequences and one Hygromycin resistance marker. Keywords: Gene editing, sgRNA, IbPDS, gene editing vector, Hygromycin.

CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

Improvements in the Appearance and Nutritional Quality of Tomato Fruits Resulting from Foliar Spraying with Silicon.

Research on silicon (Si), an element considered beneficial for plant growth, has focused on abiotic and biotic stress mitigation. However, the effect of Si on tomato fruit quality under normal growth conditions remains unclear. This study investigated the effects of applying different levels of Si (0 mmol·L-1 [CK], 0.6 mmol·L-1 [T1], 1.2 mmol·L-1 [T2], and 1.8 mmol·L-1 [T3]) in foliar sprays on tomato fruit quality cultivated in substrates, and the most beneficial Si level was found. Compared to CK, exogenous Si treatments had a positive influence on the appearance and nutritional quality of tomato fruits at the mature green, breaker, and red ripening stages. Of these, T2 treatment significantly increased peel firmness and single-fruit weight in tomato fruits. The contents of soluble sugars, soluble solids, soluble proteins, and vitamin C were significantly higher, and the nitrate content was significantly lower in the T2 treatment than in the CK treatment. Cluster analysis showed that T2 produced results that were significantly different from those of the CK, T1, and T3 treatments. During the red ripening stage, the a* values of fruits in the T2 treatment tomato were significantly higher than those in the other three treatments. Moreover, the lycopene and lutein contents of the T2 treatment increased by 12.90% and 17.14%, respectively, compared to CK. T2 treatment significantly upregulated the relative gene expression levels of the phytoene desaturase gene (PDS), the lycopene ε-cyclase gene (LCY-E), and the zeaxanthin cyclooxygenase gene (ZEP) in the carotenoid key genes. The total amino acid content in tomato fruits in the T2 treatment was also significantly higher than that of CK. In summary, foliar spraying of 1.2 mmol·L-1 exogenous Si was effective in improving the appearance and nutritional quality of tomato fruits under normal growth conditions. This study provides new approaches to further elucidate the application of exogenous silicon to improve tomato fruit quality under normal conditions.

Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N.alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

CRISPR/Cas9-mediated editing of the phytoene desaturase gene in Vanilla planifolia enabling targeted domestication

ABSTRACT Vanilla is an undomesticated species and the botanical source of vanilla extract. There has been increasing demand to expand vanilla cultivation in the Americas to strengthen the vanilla supply chain. Recent development in vanilla genomics resources has opened opportunities for interrogation of vanilla diversity and discovery of candidate genes responsible for key traits. The ability to precisely change genomic sequences could be a valuable tool for domesticating vanilla while enabling functional genomics. Here we report the successful biolistic-mediated transformation of seed-derived callus and CRISPR/Cas9 gene editing of the phytoene desaturase (PDS) gene in vanilla. Sequencing of leaves from regenerated plantlets displaying the expected albino phenotype showed short deletions from 1 to 163 base pairs in the phytoene desaturase gene at each of three target loci. The ability to apply gene editing to vanilla can now be used in combination with the vanilla genome to create targeted knockouts to support the domestication of vanilla.

Mesoporous Silica Nanoparticles Mediate SiRNA Delivery for Long-Term Multi-Gene Silencing in Intact Plants.

RNA interference (RNAi) is a powerful tool for understanding and manipulating signaling pathways in plant science, potentially facilitating the accelerated development of novel plant traits and crop yield improvement. The common strategy for delivering siRNA into intact plants using agrobacterium or viruses is complicated and time-consuming, limiting the application of RNAi in plant research. Here, a novel delivery method based on mesoporous silica nanoparticles (MSNs) is reported, which allows for the efficient delivery of siRNA into mature plant leaves via topical application without the aid of mechanical forces, achieving transient gene knockdown with up to 98% silencing efficiency at the molecular level. In addition, this method is nontoxic to plant leaves, enabling the repeated delivery of siRNA for long-term silencing. White spots and yellowing phenotypes are observed after spraying the MSN-siRNA complex targeted at phytoene desaturase and magnesium chelatase genes. After high light treatment, photobleaching phenotypes are also observed by spraying MSNs-siRNA targeted at genes into the Photosystem II repair cycle. Furthermore, the study demonstrated that MSNs can simultaneously silence multiple genes. The results suggest that MSN-mediated siRNA delivery is an effective tool for long-term multi-gene silencing, with great potential for application in plant functional genomic analyses and crop improvement.

RETRACTED ARTICLE: Agroinfiltration-based transient genome editing for targeting phytoene desaturase gene in kinnow mandarin (C. reticulata Blanco).

Citrus reticulata Blanco also known as kinnow mandarin is a widely grown horticultural crop in Punjab. CRISPR/Cas9 technology is being widely used for generation of varieties with increased resilience towards abiotic and biotic stresses as well as improved horticultural traits. Xanthomonas citri subsp. citri (Xcc)-mediated Agroinfiltration offers a fast and transgene-free method for the delivery of CRISPR/Cas9 constructs for systemic introduction into plants for functional genomics and expression studies. The technology is currently unexplored in kinnow mandarin. This study is aimed at establishing an efficient method of Cas9 delivery for transient knockout of PDS (phytoene desaturase) gene in kinnow mandarin. The construct pKO-119-PDS N-Cas9/sgRNA:PDS1 carrying sgRNA and Cas9 enzyme was delivered into the dorsal surface of young leaves of kinnow mandarin. The leaves showed albino patches at the point of injection within 60h. Two surfactants (Triton-X and Silwet™) were used to ease the Agroinfiltration process which resulted in variation in the expression of vector. The Sanger's analysis of the treated plants showed a substitution within the sgRNA region which resulted in change in amino acid from proline to serine. The protocol provides a feasible and an efficient method for genome editing in C. reticulata which could be helpful in future studies aimed at genome editing as well as genetic transformation.

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.