T1 Plants Research Articles

Marker-Assisted Selection of Jacalin-Related Lectin Genes OsJRL45 and OsJRL40 Derived from Sea Rice 86 Enhances Salt Tolerance in Rice.

Soil salinization limits rice growth and is an important restriction on grain yield. Jacalin-related lectins are involved in multiple stress responses, but their role in salt stress responses and use as molecular markers for salt tolerance remain poorly understood. Salt stress treatments and RT-qPCR analyses of Sea Rice 86 (SR86), 9311, and Nipponbare (Nip) showed that OsJRL45 and OsJRL40 enhanced tolerance of salt stress in SR86. Molecular markers based on sequence differences in SR86 and the salt-sensitive variety, 9311, in the intergenic region between OsJRL45 and OsJRL40 were validated in recombinant inbred lines derived from SR86 and 9311, hybrid populations, and common rice varieties. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that OsJRL45 and OsJRL40 interacted. Co-transformation of Nip with OsJRL45 and OsJRL40 derived from SR86 had no effect on the mature phenotype in T2 plants; however, salt stress at the three-leaf stage led to significant increases in CAT, POD, SOD, and Pro contents, but reduced MDA content in transgenic plants. Transcriptomic analysis identified 834 differentially expressed genes in transgenic plants under salt stress. GO and KEGG enrichment analyses indicated that metabolic pathways related to antioxidant responses and osmotic balance were crucial for salt-stress tolerance. Thus, molecular markers based on nucleotide differences in OsJRL45 and OsJRL40 provide a novel method for identifying salt-tolerant rice varieties.

The transcription factor TaNF-YB4 overexpression in wheat increases plant vigor and yield

Ensuring food security is a priority in developing countries. This study aimed to improve wheat yield by overexpressing the TaNF-YB4 transcription factor, which is involved in carbon assimilation and stress tolerance. An expression cassette for TaNF-YB4 was developed in a modified wheat transformation vector (pSB219) and examined through transient expression in Nicotiana tabacum, followed by Agrobacterium-mediated transformation of wheat variety FSD-2008. T0 transgenic plants were propagated to obtain T3 generation PCR-positive plants. Transgene expression was assessed in PCR-verified T2 plants using RT-PCR and qRT-PCR at six weeks post-germination. qRT-PCR analysis using the ΔΔCT method indicated higher TaNF-YB4 expression in transgenic lines than in the wild-type control plants. Improved agronomic and phenotypic traits were observed with a 6-36% increase in 1000-grain weight in the selected transgenic lines. Root architecture assessments demonstrated enhanced root length, surface area, and projected area in transgenic lines compared with wild-type plants. Additionally, notable variances in total chlorophyll, protein, and sugar content levels were observed between the transgenic lines and control plants, demonstrating statistical significance with a p-value ≤ 0.05. This study indicates that low-level constitutive expression of TaNF-YB4 can enhance wheat yield, presenting a viable strategy for improving wheat productivity.

Research of salt tolerance of genetically modified wheat plants with an additional copy of the ornithine-Δ-aminotransferase gene

Aim. To investigate the level of resistance to salt stress of T3 and T4 seed generation plants of genetically modified wheat (Triticum aestivum L.) with an additional copy of the ornithine-δ-aminotransferase (oat) gene and their original genotypes. Methods. Determination of the content of free L-proline (Pro) and physiological and morphometric parameters. Results. The level of Pro was studied and the morphometric and growth parameters of the offspring of transgenic plants and their original forms under normal / stress conditions were analyzed. Conclusions. T3 and T4 wheat plants under salinity conditions had a higher percentage and higher rate of seed germination compared to the original genotypes. During in vitro cultivation of seedlings, a stress state was observed at doses of 250 and 300 mM NaCl, at which the percentage of survival of transgenic variants was 83.3, non-transgenic only 33.3. Under conditions of in vivo salt stress, T3 and T4 plants had taller shoots and longer roots compared to the original forms. The survival rate of genetically modified plants was ~ 90 %, non-transgenic plants about 60 %. There was no significant difference in the accumulation of free L-proline between the investigated plant variants. It increased in transgenic seedlings on the 21st day of stress under conditions of artificially simulated salinity.

Impact of predictive selection of LbCas12a CRISPR RNAs upon on- and off-target editing rates in soybean.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized creating targeted genetic variation in crops. Although CRISPR enzymes have been reported to have high sequence-specificity, careful design of the editing reagents can also reduce unintended edits at highly homologous sites. This work details the first large-scale study of the heritability of on-target edits and the rate of edits at off-target sites in soybean (Glycine max), assaying ~700 T1 plants each resulting from transformation with LbCas12a constructs containing CRISPR RNAs (crRNAs) predicted to be either "unique" with no off-target sites or "promiscuous" with >10 potential off-targets in the soybean genome. Around 80% of the on-target edits observed in T0 plants were inherited in the T1 generation, and ~49% of the total observed on-target edits in T1 were not observed at T0, indicating continued activity of LbCas12a throughout the life cycle of the plant. In planta editing at off-target sites was observed for the Promiscuous but not the Unique crRNA. Examination of the edited off-target sites revealed that LbCas12a was highly tolerant to mismatches between the crRNA and target site in bases 21-23 relative to the start of the protospacer, but even a single mismatch in the first 20 nt drastically reduced the editing rate. In addition, edits at off-target sites have lower inheritance rates than on-target edits, suggesting that they occur later in the plant's lifecycle. Plants with a desired on-target edit and no off-target edits could be identified in the T1 generation for 100% of the T0 plants edited with the Unique crRNA compared with the 65% of T0 plants edited with the Promiscuous crRNA. This confirms that proper crRNA selection can reduce or eliminate off-target editing. Even when potential off-target sites are predicted, plants containing only the intended edits can still be identified and propagated.

OsIPK1 frameshift mutations disturb phosphorus homeostasis and impair starch synthesis during grain filling in rice.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) catalyzes the final step in phytic acid (InsP6) synthesis. In this study, the effects of OsIPK1 mutations on InsP6 synthesis, grain filling and their underlying mechanisms were investigated. Seven gRNAs were designed to disrupt the OsIPK1 gene via CRISPR/CAS9 system. Only 4 of them generated 29 individual insertion or deletion T0 plants, in which nine biallelic or heterozygous genotypes were identified. Segregation analysis revealed that OsIPK1 frameshift mutants are homozygous lethality. The biallelic and heterozygous frameshift mutants exhibited significant reduction in yield-related traits, particularly in the seed-setting rate and yield per plant. Despite a notable decline in pollen viability, the male and female gametes had comparable transmission rates to their progenies in the mutants. A significant number of the filling-aborted (FA) grains was observed in mature grains of these heterozygous frameshift mutants. These grains exhibited a nearly complete blockage of InsP6 synthesis, resulting in a pronounced increase in Pi content. In contrast, a slight decline in InsP6 content was observed in the plump grains. During the filling stage, owing to the excessive accumulation of Pi, starch synthesis was significantly impaired, and the endosperm development-specific gene expression was nearly abolished. Consistently, the activity of whereas AGPase, a key enzyme in starch synthesis, was significantly decreased and Pi transporter gene expression was upregulated in the FA grains. Taken together, these results demonstrate that OsIPK1 frameshift mutations result in excessive Pi accumulation, decreased starch synthesis, and ultimately leading to lower yields in rice.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat

BackgroundCRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.ResultsWe identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.ConclusionBased on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Creating large-scale genetic diversity in Arabidopsis via base editing-mediated deep artificial evolution

BackgroundBase editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants.ResultsIn this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding.ConclusionsHere, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.

Improvement of Flowering Stage in Japonica Rice Variety Jiahe212 by Using CRISPR/Cas9 System.

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica-japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica-japonica interspecific hybrid rice breeding with wider adaptability.

CRISPR–Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes

Calcium-dependent protein kinases (CDPKs) act as key signal transduction enzymes in plants, especially in response to diverse stresses, including herbivory. In this study, a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses. We developed a strategy for screening insect-resistance genes from a CRISPR–Cas9 mutant library of GhCPKs. The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants. The average target-gene coverage was 86.18%, the genome editing rate was 89.49%, and the editing heritability was 82%. An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects. The mutant that showed the clearest insect resistance, cpk33/74 (in which the homologous genes GhCPK33 and GhCPK74 were knocked out), was selected for further study. Oral secretions from Spodoptera litura induced a rapid influx of Ca2+ in cpk33/74 leaves, resulting in a significant increase in jasmonic acid content. S-adenosylmethionine synthase is an important protein involved in plant stress response, and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2. In addition, virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S. litura. This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

CRISPR/CasRx-mediated resistance to Soybean mosaic virus in soybean

Soybean mosaic virus (SMV), an RNA virus, is the most common and destructive pathogenic virus in soybean fields. The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to viruses; hence, this study aimed to engineer SMV resistance in soybean using this system. Specifically, multiple sgRNAs were designed to target positive- and/or negative-sense strands of the SMV HC-Pro gene. Subsequently, the corresponding CRISPR/CasRx vectors were constructed and transformed into soybeans. After inoculation with SMV, 39.02%, 35.77%, and 18.70% of T1 plants were confirmed to be highly resistant (HR), resistant (R), and mildly resistant (MR) to SMV, respectively, whereas only 6.50% were identified as susceptible (S). Additionally, qRT-PCR and DAS-ELISA showed that, both at 15 and 30 d post-inoculation (dpi), SMV accumulation significantly decreased or was even undetectable in HR and R plants, followed by MR and S plants. Additionally, the expression level of the CasRx gene varied in almost all T1 plants with different resistance level, both at 15 and 30 dpi. Furthermore, when SMV resistance was evaluated in the T2 generation, the results were similar to those recorded for the T1 generation. These findings provide new insights into the application of the CRISPR/CasRx system for soybean improvement and offer a promising alternative strategy for breeding for resistance to biotic stress that will contribute to the development of SMV-immune soybean germplasm to accelerate progress towards greater soybean crop productivity.

OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

Transgenic Arabidopsis thaliana plants expressing bacterial γ-hexachlorocyclohexane dehydrochlorinase LinA

Backgroundγ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation.ResultsWe generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h.ConclusionThe transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.

Callus-specific CRISPR/Cas9 system to increase heritable gene mutations in maize.

A callus-specific CRISPR/Cas9 (CSC) system with Cas9 gene driven by the promoters of ZmCTA1 and ZmPLTP reduces somatic mutations and improves the production of heritable mutations in maize. The CRISPR/Cas9 system, due to its editing accuracy, provides an excellent tool for crop genetic breeding. Nevertheless, the traditional design utilizing CRISPR/Cas9 with ubiquitous expression leads to an abundance of somatic mutations, thereby complicating the detection of heritable mutations. We constructed a callus-specific CRISPR/Cas9 (CSC) system using callus-specific promoters of maize Chitinase A1 and Phospholipid transferase protein (pZmCTA1 and pZmPLTP) to drive Cas9 expression, and the target gene chosen for this study was the bZIP transcription factor Opaque2 (O2). The CRISPR/Cas9 system driven by the maize Ubiquitin promoter (pZmUbi) was employed as a comparative control. Editing efficiency analysis based on high-throughput tracking of mutations (Hi-TOM) showed that the CSC systems generated more target gene mutations than the ubiquitously expressed CRISPR/Cas9 (UC) system in calli. Transgenic plants were generated for the CSC and UC systems. We found that the CSC systems generated fewer target gene mutations than the UC system in the T0 seedlings but reduced the influence of somatic mutations. Nearly 100% of mutations in the T1 generation generated by the CSC systems were derived from the T0 plants. Only 6.3-16.7% of T1 mutations generated by the UC system were from the T0 generation. Our results demonstrated that the CSC system consistently produced more stable, heritable mutants in the subsequent generation, suggesting its potential application across various crops to facilitate the genetic breeding of desired mutations.

CRISPR-Cas9-mediated editing of GmARM improves resistance to multiple stresses in soybean

The growth and development of soybean plants can be affected by both abiotic and biotic stressors, such as saline-alkali stress and Phytophthora root rot. In this study, we identified a stress-related gene—GmARM—whose promoter contained several hormone-response and stress-regulatory elements, including ABRE, TCA element, STRE, and MBS. qRT-PCR analysis showed that the expression of GmARM was the highest in seeds at 55 days after flowering. Furthermore, this gene was upregulated after exposure to saline-alkali stress and Phytophthora root rot infection at the seedling stage. Thus, we generated GmARM mutants using the CRISPR-Cas9 system to understand the role of this gene in stress response. T3 plants showed significantly improved salt tolerance, alkali resistance, and disease resistance, with a significantly higher survival rate than the wildtype plants. Moreover, mutations in GmARM affected the expression of related stress-resistance genes, indicating that GmARM mutants achieved multiple stress tolerance. Therefore, this study provides a foundation for further exploration of the genes involved in resistance to multiple stresses in soybean that can be used for breeding multiple stress-resistance soybean varieties.

Synergistic impact of Serendipita indica and Zhihengliuella sp. ISTPL4 on the mitigation of arsenic stress in rice.

Arsenic (As) is a highly toxic metal that interferes with plant growth and disrupts various biochemical and molecular processes in plants. In this study, the harmful effects of As on rice were mitigated using combined inoculation of a root endophyte Serendipita indica and an actinobacterium Zhihengliuella sp. ISTPL4. A randomized experiment was conducted, in which rice plants were grown under controlled conditions and As-stressed conditions. The control and treatment groups consisted of untreated and non-stressed plants (C1), treated and non-stressed plants (C2), stressed and untreated plants (T1), and stressed and treated plants (T2). Various phenotypic characteristics such as shoot length (SL), root length (RL), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW), and root dry weight (RDW) and biochemical parameters such as chlorophyll content, protein content, and antioxidant enzymatic activities were evaluated. The activity of various antioxidant enzymes was increased in T2 followed by T1 plants. Furthermore, high concentrations of phytohormones such as ethylene (ET), gibberellic acid (GA), and cytokinin (CK) were found at 4.11 μmol mg-1, 2.53 μmol mg-1, and 3.62 μmol mg-1 of FW of plant, respectively. The results of AAS indicated an increased As accumulation in roots of T2 plants (131.5 mg kg-1) than in roots of T1 plants (120 mg kg-1). It showed that there was an increased As accumulation and sequestration in roots of microbial-treated plants (T2) than in uninoculated plants (T1). Our data suggest that this microbial combination can be used to reduce the toxic effects of As in plants by increasing the activity of antioxidant enzymes such as SOD, CAT, PAL, PPO and POD. Furthermore, rice plants can withstand As stress owing to the active synthesis of phytohormones in the presence of microbial combinations.

Inheritance of signs of resistance to osmotic stresses in genetically modified wheat

Aim. To investigate the inheritance of transgenes and the preservation of increased resistance to drought in the seed generations of genetically modified wheat plants with a double-stranded RNA (dlRNA) suppressor of the proline dehydrogenase (pdh) gene. Methods. PCR, determination of free L-proline (Pro) content and yield structure indicators. Results. Under the influence of osmotic stress, T4 wheat plants were selected with elements that form dlRNA suppressor of the pdh gene of wheat, which retain the sign of increased resistance to water deficit. It was found that T4 plants had a higher content of Pro than their original forms under both normal cultivation conditions and water deficit. It is shown that during droughts, biotechnological plants were characterized by higher grain productivity compared to the original genotypes, while under conditions of sufficient moisture provision, the differences in the elements of the yield structure were insignificant. Conclusions. The analysis of seed generations of transgenic wheat showed variability in the manifestation of signs of increased resistance to osmotic stress, which is due to the instability of recombinant DNA in generations. Testing of T4 wheat under the effects of osmotic stress showed the fact of successful selection of plants with introduced elements that form dlRNA suppressor of the pdh gene and cause the sign of increased resistance to water deficit in seed generations.

Continual improvement of CRISPR-induced multiplex mutagenesis in Arabidopsis.

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We foundthat the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.

Inoculants of Arbuscular Mycorrhizal Fungi Influence Growth and Biomass of Terminalia arjuna under Amendment and Anamendment Entisol

Entisol soil is hard and compact in nature, rendering it high in bulk density, which influences root penetration adversely and thereby poor plant growth. In this experiment, used seven treatments in different combination in normal soil, were used as growth media for the Terminalia arjuna seedling. T3 (60% entisol) found the best as it gave the highest biomass in the species regardless of arbuscular mycorrhizal fungi (AMF) treatment. AMF treatment enhanced the growth and biomass of plants significantly in all the given treatments. AMF colonization observed a maximum in tertiary roots. T1 (100% entisol soil) exhibited the highest degree of AMF colonization in tertiary roots, resulting in the highest mycorrhiza dependency of plants for this soil. The addition of normal soil to entisol soil was found to decrease the bulk density, resulting in increased root diameter, and T3 plants exhibited the highest biomass and AMF compatibility for T. arjuna species. The T. arjuna plant’s growth and biomass responded positively to AMF in all types of treatments. The plant’s growth and biomass were highest in the T3 treatment, which had a bulk density of 1.50 g/cm3. In this study, we combined the entisol with mycorrhizal inoculation of the nursery growing medium to promote plant growth and biomass, improve the plant’s ability to hold water and absorb nutrients, and lower the entisol’s bulk density. The T. arjuna (Roxb) plant responds very favorably to mycorrhiza inoculation in nursery conditions with the entisol growth medium.

Transcriptional and physio-chemical responses of Tectona grandis L. triggered by teak defoliator

Tectona grandis (L) is a popular timber species cultivated in the Indian sub-continent and other countries because of its economic significance. However, the lack of quality planting material with special reference to defoliator herbivory is a major constraint because it negatively influences the establishment of thriving plantations. To address this, the so-called pest response in teak seedlings was investigated at the biochemical and molecular levels. The stress was levied by allowing defoliator infestation on 1-year-old seedlings for nine days at 3-day intervals, whereas control plants were maintained as uninfected. A considerable difference was observed for the considered traits among the infested plants at different intervals compared with the control. Total soluble protein, soluble sugars, tannin, and lignin accumulation increased with increasing stress intensity. Furthermore, catalase, polyphenol oxidase, and phenol content increased after defoliator feeding, whereas peroxidase activity decreased compared with control plants. In addition, six herbivory stress-responsive genes revealed the molecular response of teak. Among them, four genes viz. zinc finger protein (ZFP2), ethylene-responsive factor (ERF-1), catalase-1 (Cat1) and peroxidase super family protein (Psfp) were highly expressed in the infected plants as compared with the control plants. In contrast, the MYB-DNA binding domain gene was downregulated in infected plants compared to uninfected plants. Cytochrome P450 was up-regulated in T1 and T2 plants, while it was down-regulated in T3. Genes were highly expressed as the stress frequency increased. This study offers an improved understanding of biochemical and transcriptional defense responses against the teak defoliator, which could further support teak improvement for quality planting material production.

Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin’s mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.