Towards developing resistance to chickpea chlorotic dwarf virus through CRISPR/Cas9-mediated gene editing using multiplexed gRNAs

Journal of Plant Diseases and Protection - Mastreviruses are an emerging group of viruses transmitted by leafhoppers and infect both monocot and dicot plants. Chickpea chlorotic dwarf virus...     

Effects of simulated acid rain and root-knot nematode on tomato

Effects were examined of simulated acid rain and of root-knot nematode, Meloidogyne incognita race 1, on plant growth, yield, photosynthetic pigments and leaf epidermal characters of tomato ( Lycopersicon esculentum cv. Pusa Ruby). Sequential inoculation exposures (pre-, post-, and concomitant with nematode inoculation) were carried out in a greenhouse. Intermittent treatments of simulated acid rain at pH 3.2 caused white-to-tan irregular lesions on both the upper and lower surfaces of tomato leaves. The foliar symptoms were more pronounced on nematode-infected plants. Simulated acid rain (pH 3.2) and/or nematode infection suppressed plant growth, yield and pigment synthesis, the effects being greatest in post-inoculation treatments compared with simulated acid rain at pH 6.8. The total weight of fruits per plant was greatly suppressed owing to simulated acid rain or nematode infection. Chlorophyll a was found to be more sensitive to simulated acid rain or nematode infection than other leaf pigments. Root penetration, galling, egg mass production, and fecundity (number of eggs per egg mass) of M. incognita were enhanced at pH 5.6 and suppressed at pH 3.2 compared with pH 6.8. Nematode infection or simulated acid rain at pH 3.2 suppressed stomata and trichome development (number and size). Simulated acid rain treatments at pH 5.6 had a positive effect on number and size of trichomes, but a negative effect on stomata. The apertures of stomata were wider on tomato leaves exposed to simulated acid rain, especially at pH 3.2, than at pH 6.8.     

Wheat Seed Presoaking for Improved Germination

Seed presoaking improves wheat germination under marginal moisture conditions. The duration of seed presoaking was studied at The University of Arid Agriculture, Rawalpindi, using 10 wheat varieties. Seed presoaking beyond 12 h does not improve germination further, and beyond 21 h, germination rate is drastically reduced.     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Expression profiling of transgenes (Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the Cry1Ac gene has revolutionized insect pest control since its adoption, although the development of resistance in insect pests has reduced its efficacy.  After 10 years of cultivating Bacillus thuringiensis (Bt) cotton with a single Cry1Ac gene, growers are on the verge of adopting Bt cotton that carries the double gene (Cry1Ac+Cry2A) due to its better effectiveness against insect pests.  Thus, the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.  The expression levels of the Cry1Ac and Cry2A genes were evaluated in the leaves of 10 genotypes (2 parents and 8 F₁ hybrids) at 30 days after sowing (DAS), while samples of leaves, bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.  The F₁ hybrids were developed through reciprocal crosses between two Bt (CKC-1, CKC-2) and two non-Bt (MNH-786, FH-942) parents.  The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay (ELISA).  The results showed that the MNH786×CKC-1 hybrid had the highest concentrations of Cry1Ac gene at 30 DAS (3.08 µg g^–1) and 110 DAS (1.01 µg g^–1) in leaves.  In contrast, the CKC-2×MNH-786 hybrid showed the lowest concentrations of Cry1Ac gene at 30 DAS (2.30 µg g^–1) and 110 DAS (0.86 µg g^–1).  The F₁ hybrid FH-942×CKC-2 showed the highest concentrations of Cry2A gene at 30 DAS (8.39 µg g^–1) and 110 DAS (7.74 µg g^–1) in leaves, while the CKC-1×MNH-786 hybrid expressed the lowest concentrations of Cry2A gene at 30 DAS (7.10 µg g^–1) and 110 DAS (8.31 µg g^–1).  A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS, whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.  When the expression pattern was compared between various plant parts in genotype CKC-2, it was found that leaves had higher concentrations of Cry1Ac (3.12 µg g^–1) and Cry2A (8.31 µg g^–1) at 70 DAS, followed by bolls (Cry1Ac (1.66 µg g^–1) and Cry2A (8.15 µg g^–1)) and flowers (Cry1Ac (1.07 µg g^–1) and Cry2A (7.99 µg g^–1)).  The genotype CKC-2 had higher concentrations of Cry1Ac (3.12 µg g^–1) and Cry2A (8.31 µg g^–1) in the upper canopy but less accumulation (2.66 µg g^–1 of Cry1Ac, 8.09 µg g^–1 of Cry2A) in the lower canopy at 70 DAS.  Similarly, at 110 DAS, the expression levels of Cry1Ac and Cry2A in upper and lower canopy leaves were 1.52 and 7.92 µg g^–1, and 0.99 and 7.54 µg g^–1, respectively.  Hence, the current study demonstrates that different genotypes showed variable expression for both of the Cry1Ac and Cry2A genes during plant growth due to different genetic backgrounds.  The Cry2A gene had three-fold higher expression than Cry1Ac with significant differences in expression in different plant parts.  The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1Ac and Cry2A for sustainable cotton production worldwide.     

Dynamics of organic carbon and nitrogen in deep soil profile and crop yields under long-term fertilization in wheat-maize cropping system

Soil organic carbon (SOC) and nitrogen (N) are two of the most important indicators for agricultural productivity. The primary objective of this study was to investigate the changes in SOC and N in the deep soil profile (up to 100 cm) and their relationships with crop productivity under the influence of long-term (since 1990) fertilization in the wheat-maize cropping system. Treatments included CK (control), NP (inorganic N and phosphorus (P) fertilizers), NPK (inorganic N, P and potassium fertilizers), NPKM (NPK plus manure), and M (manure). Crop yield and the properties of topsoil were measured yearly from 2001 to 2009. C and N contents were measured at five different depths in 2001 and 2009. The results showed that wheat and maize yields decreased between 2001 and 2009 under the inorganic fertilizer (NP and NPK) treatments. The average yield between 2001 and 2009 under the NP, NPK, NPKM, and M treatments (compared with the CK treatment) increased by 38, 115, 383, and 381%, respectively, for wheat and 348, 891, 2 738, and 1 845%, respectively, for maize. Different long-term fertilization treatments significantly changed coarse free particulate (cfPOC), fine free particulate (ffPOC), intramicroaggregate particulate (iPOC), and mineral-associated (mSOC) organic carbon fractions. In the experimental years of 2001 and 2009, soil fractions occurred in the following order for all treatments: mSOC>cfPOC>iPOC>ffPOC. All fractions were higher under the manure application treatments than under the inorganic fertilization treatments. Compared to the inorganic fertilization treatments, manure input enhanced the stocks of SOC and total N in the surface layer (0–20 cm) but decreased SOC and N in the deep soil layer (80–100 cm). This reveals the efficiency of manure in increasing yield productivity and decreasing risk of vertical loss of nutrients, especially N, compared to inorganic fertilization treatments. The findings provide opportunities for understanding deep soil C and N dynamics, which could help mitigate climate change impact on agricultural production and maintain soil health.     

Phenotypic variations of wheat cultivars from the North China Plain in response to cadmium stress and associated single nucleotide polymorphisms identified by a genome-wide association study

Understanding the genetic mechanisms for cadmium (Cd) uptake and translocation in common wheat (Triticum aestivum) is of significance in food Cd contamination control. In this study, a diverse panel of 132 wheat cultivars was collected from the North China Plain. The cultivars were evaluated in terms of their phenotypic variations in response to Cd stress and subjected to a genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with the phenotypic variations at the seedling stage. Significant phenotypic variations with high heritability were observed among the wheat cultivars exposed to 40 μmol L^-1 Cd for the studied traits, including root length (RL), shoot length (SL), root and shoot dry biomasses (RDW and SDW, respectively), root and shoot Cd concentrations (RCD and SCD, respectively), and Cd translocation factor (TF). Mean RCD, SCD, and TF ranged from 1.0 to 33.8, 0.125 to 2.022, and 0.009 to 0.321 mg g^-1, respectively. Cluster analysis showed that wheat cultivars with higher RL, SL, RDW, and SDW under Cd stress were able to accumulate more Cd in root, leading to a lower Cd TF. Mixed linear model-based association analysis detected 17 novel significant marker-trait associations (MTAs), four of which were significant at a genome-wide scale. Most of the significant MTAs controlled Cd TF and explained 17.17%-26.47% of the phenotypic variations. Some of the SNP loci were physically close to a reported Cd-related quantitative trait locus or gene on wheat chromosomes. Results of this study provided a list of wheat cultivars with the potential of low Cd accumulation and enriched our knowledge on the genetic basis of Cd uptake and translocation in wheat. Pyramiding breeding of superior alleles detected in this study may additionally reduce Cd accumulation of improved wheat cultivars with excellent agronomic traits.     

Bound water characteristics and microstructure evolution during uniaxial compression of mucky silty clay

Journal of Soils and Sediments - Compression studies can provide insights into evaluating the engineering potential and environmental impact of clay. The objective of this study was to...     

Production of aromatic amino acids and related compounds from p-hydroxyphenylacetic acid by rumen microorganisms in vitro

Suspensions of mixed rumen bacteria (B), protozoa (P), and mixed rumen microorganisms (BP) prepared from rumen contents of fistulated goats were anaerobically incubated with 1 mM p‐hydroxyphenylacetic acid (HPA) at 39°C for 24 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of microbial cells in all incubations were analyzed by HPLC. Large amounts of Tyr (32.1, 42.7 and 36.1% of disappeared HPA in B, P and BP, respectively) were produced from HPA during a 12 h incubation period. The formation of Tyr in P (178.6 µmol/g MN) was 1.5 and 2 times higher than in B and BP, respectively. Phe (7–11% of the disappeared HPA) and Trp (3–6% of the disappeared HPA) were also synthesized from HPA in B, P, and BP. Phe synthesis in P (46.3 µmol/g MN) was 1.7 times higher than in B but, in contrast, Trp synthesis in B, was 1.6 times higher than in P. The metabolites p‐hydroxyphenylpyruvic acid (in the range of 5–14% of disappeared HPA), phenylacetic acid (1–11%), p‐hydroxybenzoic acid (3–7%) and benzoic acid (1–6%) were produced from HPA in B, P and BP. Phenylpropionic acid (6% of the disappeared HPA) was produced only in B and BP.