T1 Plants Research Articles

Effect of Different Spacing and Time of Planting on Growth and Yield of Chinese Cabbage (Brassica rapa) under Prayagraj Agro Climatic Condition

A field experiment was conducted during the Rabi season in 2022-23 at vegetable research farm, Department of Horticulture, Naini Agricultural Institute, Sam Higginbottom University of Agriculture, Technology & Sciences, Prayagraj (UP). The experiment consisted of nine treatments combinations with three plants spacing viz. (i) 30 x 45cm, (ii) 45 x 60cm and (iii) 45 x 45cm and three levels of sowing date viz. (i) November 5th 2022, (ii) November 12th 2022, (iii) November 19th 2022, were included in the study in Randomized Block Design with three replications. The growth, yield and quality contributing all characters were significantly influenced by the treatments. The highest plant height, number of leaves, weight of head per plant, chlorophyll content and length of outer leaves (cm) as recorded in T6 (D2S3) (plants sowing November 12th 2022 with spacing 45 x 45cm) followed by T3 (D1S3) and lowest in T7 (D3S1). The maximum weight of head per m2, yield per plot (79.6kg) and yield per hectare (89.1t ha) were recorded -1 in T4 (D2S1) (plants sowing November 12th 2022 with spacing 30 x 45cm) followed by T1 (D1S1) and minimum weight of head per m2, yield per plot (35.6kg) and yield per hectare (199.0 t ha-1) were recorded in T8 (D3S2) of Chinese cabbage.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy

BackgroundPlant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease.ResultsInitially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1–3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, ‘transgene-free’ T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants.ConclusionOur results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

An efficient and accurate droplet digital PCR method for rapid transgene copy number detection and homozygous identification in cotton (Gossypium hirsutum)

Transgene copy number characterization and homozygous identification are important steps for both the scientific research and transgenic breeding in plant. Traditionally, Southern blotting, quantitative PCR, genome sequencing and genetic analysis are the major methods to achieve these objectives. These methods are laborious, time-consuming and cost-expensive. Digital PCR is a breakthrough technology that is able to provide sensitive and absolute nucleic acid quantification. However, its application is rarely studied in transgene copy number detection and homozygous identification, especially in cotton, an important industrial crop worldwide. Here, we developed a droplet digital PCR (ddPCR) system to rapidly and efficiently detect the transgene copy number in cotton. The GhTPS, a single gene in per subgenome of Gossypium hirsutum, and NPT II, a marker gene in the transgenic T-DNA, were selected as the endogenous reference gene and exogenous insertion sequences, respectively. Results showed that probe concentration at 150 nM and DNA concentration at 5.0 ng/µL were the best conditions to perform ddPCR in tetraploid cotton. The detected T-DNA copy numbers obtained from the developed ddPCR system were in consistent with the results from Southern blotting analysis and genome resequencing results, respectively, demonstrating that the developed ddPCR system is accurate and efficient in identifying the T-DNA copy number. Moreover, the homozygous and heterozygous T1 offspring plants from a T0 plant were efficiently distinguished using this approach, greatly shortening the selection cycles of homozygous plants. The ddPCR system was further applied to detect the transgenic Bt copy numbers in commercialized cotton varieties, indicating the suitability and convenience in identifying transgenic cotton with different origins. This study provides a novel, rapid and efficient method for copy number and homozygous determination in transgenic cotton.

Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis.

Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T1 plants. This system was used to confer Z-abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z-abienol were detected in the leaves of homozygous T1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.

CRISPR/Cas9 editing characteristics of multiple transgenic generations in Fortunella hindsii, an early flowering mini-citrus

The CRISPR/Cas9 editing system is presently regarded as one of the most efficient tools for genome editing and has been implemented in perennial fruit crops. However, the characteristics of transgenic plants and their subsequent generation have rarely been reported in fruit trees. In this study, 15 distinct preserved germplasm resources of Hongkong kumquat (Fortunella hindsii) were utilized to selecting suitable genotypes for genetic transformation. The shoot regeneration efficiency, seed number per fruit, and epicotyl diameter displaid a moderate positive correlation with transformation efficiency, with correlation coefficients of 0.6267, 0.5429, and 0.5388, respectively. The transcription factor RKD family gene FhRWP was knocked out in kumquat, resulting in the production of seven T0 and 34 T1 plants. Editing efficiency of T0 generation ranged from 1.62% to 68.11%, with a significant difference in editing activity among cases. Up to 23 editing types were detected in a single positive transformant, demonstrating that the high degree of citrus editing chimerism. Not all of T1 lines exhibited an increase in editing efficiency relative to T0. The descendants of T0-A2/A3 were all negative, while the other group of T1 lines showed both higher chimera and editing efficiencies of up to 92% than the others. Finally, the CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. This study provides novel insights into the application of the CRISPR/Cas9 system in asexual plants and serves as a reference for gene function verification in citrus and other species.

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.).

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Wheat Transformation with ScTPS1-TPS2 Bifunctional Enzyme for Trehalose Biosynthesis Protects Photosynthesis during Drought Stress

Wheat cultivation makes an important contribution to human nutrition. Trehalose synthesis plays a role in the tolerance to drought stress. A bifunctional TPS-TPP enzyme gene from yeast was used to obtain transgenic wheat plants to increase trehalose synthesis. Mature wheat embryos were transformed using pGreen rd29A::TPS1-TPS2 or pGreen 35S::TPS1-TPS2 constructs. The transgene presence in mature leaves of T3 plants was confirmed by sequencing a PCR fragment of the inserted transgene. Transgenic and NT plants were submitted to drought stress for eight days. Transformed wheat lines retained a higher relative water content than NT plants during drought stress, and the Rubisco activity was unaffected. Plants transformed with the 35S construct showed a lower photosynthetic rate and lower fructose 1–6-bisphosphatase (FBPase) activity during drought, suggesting that constitutive trehalose and sucrose synthesis caused a reduced ribulose 1,5-bisphosphate (RuBP) regeneration. Lines transformed with the rd29A promoter showed a higher photosynthetic rate after eight days of drought, as the RuBP regeneration was unaffected. Transgenic wheat plants had higher biomass and grain weight than NT plants after drought. These results suggest that trehalose synthesis improves photosynthesis during stress and induces changes in the activity of some Calvin-cycle enzymes, reflected in plant metabolism and growth.

Development of Agrobacterium-mediated in planta transformation protocol through coleoptile in rice.

Genetic modification of rice is mainly carried out by Agrobacterium-mediated transformation of callus accompanied by tissue culture. It is time consuming, laborious and unapplicable for cultivars unable to induce callus. In this study, we have reported a novel gene transfer protocol that involves pulling out primary leaf from coleoptile and injection of Agrobacterium culture into the empty channel. Out of 25 plants survived after injection of Agrobacterium tumefaciens EHA105 culture harboring pCAMBIA1301-RD29A-AtDREB1A, 8 T0 plants revealed the expected size of around 811bp corresponding to AtDREB1A gene and Southern blotting analysis on 18 T1 plants suggested introgression of AtDREB1A. 3 T2 lines (7-9, 12-3, 18-6) exhibited accumulation of free proline and soluble sugars, yet increase of chlorophyll content, but decrease of electrolyte leakage and methane dicarboxylic aldehyde under cold stress condition at the vegetative growth stage. Yield components investigation on T2 lines showed earlier heading date and no yield loss compared to wild type plants grown under normal condition. GUS expression analysis and integrated transgene detection in T0 and T1 plants followed by evaluation of cold stress tolerance in T2 lines suggest the advantage of this in planta transformation protocol to obtain transgenic rice.

Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity.

Transcriptional corepressors of the Topless (TPL) family regulate plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of the TPL family roles in transcriptional regulation. Chromatin immunoprecipitation with sequencing (ChIP-Seq) was performed on Arabidopsis thaliana lines expressing GFP-tagged Topless-related 1 (TPR1-GFP) with and without constitutive immunity via Enhanced Disease Susceptibility 1 (EDS1). RNA-Seq profiling of the TPR1-GFP lines and pathogen-infected tpl/tpr mutants, combined with measuring immunity, growth, and physiological parameters was employed to investigate TPL/TPR roles in immunity and defense homeostasis. TPR1 was enriched at promoter regions of c. 1400 genes and c. 10% of the detected binding required EDS1 immunity signaling. In a tpr1 tpl tpr4 (t3) mutant, resistance to bacteria was slightly compromised, and defense-related transcriptional reprogramming was weakly reduced or enhanced, respectively, at early (< 1 h) and late 24 h stages of bacterial infection. The t3 plants challenged with bacteria or pathogen-associated molecular pattern nlp24 displayed photosystem II dysfunctions. Also, t3 plants were hypersensitive to phytocytokine pep1 at the level of root growth inhibition. Transgenic expression of TPR1 rescued these t3 physiological defects. We propose that TPR1 and TPL family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity.

A Fast and Cost-Effective Genotyping Method for CRISPR-Cas9-Generated Mutant Rice Lines.

With increasing throughput in both the generation and phenotyping of mutant lines in plants, it is important to have an efficient and reliable genotyping method. Traditional workflows, still commonly used in many labs, have time-consuming and expensive steps, such as DNA purification, cloning and growing E. coli cultures. We propose an alternative workflow where these steps are bypassed, using Phire polymerase on fresh plant tissue, and ExoProStar treatment as preparation for sequencing. We generated CRISPR-Cas9 mutants for ZAS (ZAXINONE SYNTHASE) in rice with two guide RNAs. Using both a traditional workflow and our proposed workflow, we genotyped nine T1 plants. To interpret the sequencing output, which is often complex in CRISPR-generated mutants, we used free online automatic analysis systems and compared the results. Our proposed workflow produces results of the same quality as the old workflow, but in 1 day instead of 3 days and about 35 times cheaper. This workflow also consists of fewer steps and reduces the risk of cross contamination and mistakes. Furthermore, the automated sequence analysis packages are mostly accurate and could easily be used for bulk analysis. Based on these advantages, we encourage academic and commercial labs conducting genotyping to consider switching over to our proposed workflow.

Mesh Crop Cover Optimizes the Microenvironment in a Tropical Region and Modifies the Physiology and Metabolome in Tomato

In tropical regions, high light levels can lead to increased photooxidative damage in plants. Thus, reducing solar radiation could have a substantial impact on crop performance. This study aimed to evaluate the physiological responses and metabolic profile of two tomato varieties grown in microenvironments modified with cover meshes under a high light level and a warm climate. The experiment was achieved under high solar irradiance and an unfavorably high temperature. The varieties “Moneymaker” (MM) and “Campeche 40” (C40) were grown from 45 to 130 days after sowing at four solar irradiance levels: 100% (T1), 80% (T2), 75% (T3), and 50% (T4). In both varieties, the plants grown under the lowest irradiances (T3 and T4) were the tallest, with larger leaf areas, and accumulated more aerial and root biomass. Under moderate shading (T2), plants took better advantage of the light and had the highest photochemical quenching coefficient (qP) (C40 = 0.60 and MM = 0.48) and the highest electron transport rate (ETR). However, T3 and T4 plants had the highest net assimilation rate (23.6 and 23.9 µmol m−2 s−1 in C40, and 22.7 and 22.6 µmol m−2 s−1 in MM, respectively) and the highest A/Ci coefficients. Although both tomato varieties accumulate similar metabolites, MM leaves accumulate more glucose and C40 leaves accumulate more proline and valine. Furthermore, MM leaves accumulate more glycine and GABA under high radiation, and C40 leaves accumulate more proline and valine than leaves under 50% shade (T4). We conclude that using meshes in areas with high irradiance could be an alternative to reduce abiotic stress factors in plants.

Biotechnology of α-linolenic acid in oilseed rape (Brassica napus) using FAD2 and FAD3 from chia (Salvia hispanica)

α-Linolenic acid (ALA, 18:3Δ9,12,15) is an essential fatty acid for humans since it is the precursor for the biosynthesis of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). Modern people generally suffer from deficiency of ALA because most staple food oils are low or lack ALA content. Biotechnological enrichment of ALA in staple oil crops is a promising strategy. Chia (Salvia hispanica) has the highest ALA content in its seed oil among known oil crops. In this study, the FAD2 and FAD3 genes from chia were engineered into a staple oil crop, oilseed rape (Brassica napus), via Agrobaterium tumefaciens-mediated transformation of their LP4-2A fusion gene construct driven by the seed-specific promoter PNapA. In seeds of T0, T1, and T2 lines, the average ALA contents were 20.86, 23.54, and 24.92%, respectively, which were 2.21, 2.68, and 3.03 folds of the non-transformed controls (9.42, 8.78, and 8.22%), respectively. The highest seed ALA levels of T0, T1, and T2 plants were 38.41, 35.98, and 39.19% respectively, which were 4.10–4.77 folds of the respective controls. FA-pathway enzyme genes (BnACCD, BnFATA, BnSAD, BnSCD, BnDGAT1, BnDGAT2, and BnDGAT3) and positive regulatory genes (BnWRI1, BnLEC1, BnL1L, BnLEC2, BnABI3, BnbZIP67, and BnMYB96) were all significantly up-regulated. In contrast, BnTT1, BnTT2, BnTT8, BnTT16, BnTTG1, and BnTTG2, encoding negative oil accumulation regulators but positive secondary metabolism regulators, were all significantly down-regulated. This means the foreign ShFAD2-ShFAD3 fusion gene, directly and indirectly, remodeled both positive and negative loci of the whole FA-related network in transgenic B. napus seeds.

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants

BackgroundCas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.ResultsTo improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.ConclusionsOur results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

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

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. 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. 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.

CRISPR/Cas9-mediated gene editing to confer turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa).

Genome editing approaches, particularly the CRISPR/Cas9 technology, are becoming state-of-the-art for trait development in numerous breeding programs. Significant advances in improving plant traits are enabled by this influential tool, especially for disease resistance, compared to traditional breeding. One of the potyviruses, the turnip mosaic virus (TuMV), is the most widespread and damaging virus that infects Brassica spp. worldwide. We generated the targeted mutation at the eIF(iso)4E gene in the TuMV-susceptible cultivar "Seoul" using CRISPR/Cas9 to develop TuMV-resistant Chinese cabbage. We detected several heritable indel mutations in the edited T0 plants and developed T1 through generational progression. It was indicated in the sequence analysis of the eIF(iso)4E-edited T1 plants that the mutations were transferred to succeeding generations. These edited T1 plants conferred resistance to TuMV. It was shown with ELISA analysis the lack of accumulation of viral particles. Furthermore, we found a strong negative correlation (r = -0.938) between TuMV resistance and the genome editing frequency of eIF(iso)4E. Consequently, it was revealed in this study that CRISPR/Cas9 technique can expedite the breeding process to improve traits in Chinese cabbage plants.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Shelf-life extension of Fragaria × ananassa Duch. using selenium nanoparticles synthesized from Cassia fistula Linn. leaves.

Fragaria × ananassa Duch. (Strawberry) fruit is susceptible to postharvest diseases, thus decrease in quality attributes, such as physiological and biochemical properties leads to decrease in shelf life. The objective of the present study was to check the effect of Selenium NP's and packaging conditions on the shelf life of strawberry (Fragaria × ananassa Duch) fruits. The shelf life was observed with 4 days intervals and examined for characteristics such as physiological weight loss, moisture content, percentage decay loss, peroxidase, catalase, and DPPH radical scavenging. The quality change of postharvest Fragaria × ananassa Duch. was monitored by the application of selenium nanoparticles (T1 plant extract in 10 mM salt solution, T2 plant extract in 30 mM salt solution, T3 plant extract in 40 mM salt solution, T4 distilled water; control) in different packaging materials (plastic bags, cardboard, and brown paper) at different storage conditions (6°C and 25°C). 10 mM, 20 mM, and 30 mM solution of sodium selenite salt, prepared from 1 M stock solution. Selenium nanoparticles were synthesized using Cassia fistula L. extract and sodium selenite salt solution. Polyvinyl alcohol (PVA) was used as a stabilizer. The nanoparticles were characterized through UV-visible spectroscopy and X-Ray diffractometer (XRD). It was observed that the strawberry Fragaria × ananassa Duch. Treated with T1 (CFE and 10 mM salt solution) stored in plastic packaging at ±6°C showed the best physiological parameters and hence the treatment is recommended for storage without affecting the quality of strawberry fruit up to 16 days.