aBIOTECH最新文献

Tobacco (Nicotiana tabacum) plants synthesize the psychoactive pyridine alkaloid nicotine, which has sparked growing interest in reducing nicotine levels through genome editing aiming at inactivating key biosynthetic genes. Although stable transformation-mediated genome editing is effective in tobacco, its polyploid nature complicates the complete knockout of genes and the segregation of transgenes from edited plants. In this study, we developed a non-transgenic genome editing method in tobacco by delivering the CRISPR/Cas machinery via an engineered negative-strand RNA rhabdovirus vector, followed by the regeneration of mutant plants through tissue culture. Using this method, we targeted six berberine bridge enzyme-like protein (BBL) family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis, in the commercial tobacco cultivar Hongda. We generated a panel of 16 mutant lines that were homozygous for mutations in various combinations of BBL genes. Alkaloid profiling revealed that lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels, while other BBL members played a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine. Preliminary agronomic evaluation identified two low-nicotine lines with growth phenotypes comparable to those of wild-type plants under greenhouse and field conditions. Our work provides potentially valuable genetic materials for breeding low-nicotine tobacco and enhances our understanding of alkaloid biosynthesis.

Crop breeding requires a balance of tradeoffs among key agronomic traits caused by gene pleiotropy. The molecular manipulation of genes can effectively improve target traits, but this may not reduce gene pleiotropy, potentially leading to undesirable traits or even lethal conditions. However, molecular editing of cis-regulatory elements (CREs) of target genes may facilitate the dissection of gene pleiotropy to fine-tune gene expression. In this study, we developed a pipeline, in potato, which employs open chromatin to predict candidate CREs, along with both transient and genetic assays to validate the function of CREs and CRISPR/Cas9 to edit candidate CREs. We used StCDF1 as an example, a key gene for potato tuberization and identified a 288 bp-core promoter region, which showed photoperiodic inducibility. A homozygous CRISPR/Cas9-editing line was established, with two deletions in the core promoter, which displayed a reduced expression level, resulting in late tuberization under both long-day and short-day conditions. This pipeline provides an alternative pathway to improve a specific trait with limited downside on other phenotypes.

Tomato (Solanum lycopersicum) and potato (Solanum tuberosum), two integral crops within the nightshade family, are crucial sources of nutrients and serve as staple foods worldwide. Molecular genetic studies have significantly advanced our understanding of their domestication, evolution, and the establishment of key agronomic traits. Recent studies have revealed that epigenetic modifications act as “molecular switches”, crucially regulating phenotypic variations essential for traits such as fruit ripening in tomatoes and tuberization in potatoes. This review summarizes the latest findings on the regulatory mechanisms of epigenetic modifications in these crops and discusses the integration of biotechnology and epigenomics to enhance breeding strategies. By highlighting the role of epigenetic control in augmenting crop yield and adaptation, we underscores its potential to address the challenges posed by a growing global population as well as changing climate.

The effect of fungicides on the plant-rhizosphere microbiome is a subject of ongoing debate, but whether any alteration in the rhizosphere microbiome could affect plant health is an issue that has not been thoroughly investigated. To address this deficiency, we analyzed the rhizosphere microbiome of wilt disease—resistant and disease-susceptible cucumber cultivars to determine whether (and which) plant-associated microorganisms have a role in disease resistance. We further assessed whether the fungicides thiophanate-methyl and carbendazim affect the rhizosphere microbiome, which may contribute to the plant’s immune response. Based on results acquired with both radicle-inoculation and soil-inoculation methods, cultivars Longyuanxiuchun (LYXC) and Shuyan2 (SY2) were identified as being disease resistant, whereas Zhongnong6 (ZN6) and Zhongnong38 (ZN38) were susceptible. The microbiome structure differed substantially between the resistant and susceptible plants, with LYXC and SY2 each having a significantly greater Shannon index than Zhongnong38. These results revealed that the disease-resistant cucumber cultivars recruited more beneficial bacteria, i.e., Bacillus, in their rhizosphere soil; as such, Bacillus was identified as a keystone genus in the microbial co-occurrence network. Thus, the presence of Bacillus may help cucumbers defend against fungal pathogens within the rhizosphere. Bacillus subtilis strain LD15, which was isolated from LYXC rhizosphere soil, could suppress pathogen growth, in vitro, and reduce disease severity in pot assays. Moreover, evidence also confirmed the accumulation of LD1 in the rhizosphere soil of resistant cucumber cultivars. For LYXC, application of thiophanate-methyl or carbendazim altered the microbiome structure, decreased bacterial diversity, and reduced the abundance of Bacillus species. Finally, pot assays verified that fungicide application decreased the proportion of LD15 in rhizosphere soil. From a microbial perspective, thiophanate-methyl and carbendazim may weaken the rhizobacteria-mediated defense response of cucumbers against cucumber Fusarium wilt disease. Our findings reveal a role for the rhizosphere microbiome in protecting plants from pathogens and constitute a reference for assessing the ecotoxicological risk of pesticides to non-target soil microorganisms.

The barley genome encodes a complete set of MADS-box proteins sharing homology with components of the ABCDE model, which explains the molecular basis of floral organ identity in angiosperm flowers. Although the E-class members are universally expressed across floral whorls and crucial for flower development in Arabidopsis and rice, the functional role of the barley E-class LOFSEP subfamily (comprising MADS1, MADS5, and MADS34) remains elusive, particularly during spikelet formation. Here, we characterize the single, double and triple lofsep mutants in barley in an attempt to overcome the anticipated genetic redundancy. Surprisingly, loss of function of all LOFSEP members only disturbs lemma development, either converting this hull organ into a leaf-like structure or reducing its size. The inner organs, including lodicules, anthers and pistil remain unaffected. A systematic interrogation of how ABCDE class genes are affected in all whorls of the mutants was undertaken. Generally, in the lemma and palea of the lofsep mutants, A- and E-class genes are hyperactivated, B- and C- classes are slightly repressed, and D-class genes show unchanged expression in these inner organs. Intriguingly, loss of function of MADS6, an AGL6 member closely related to the E-class genes, leads to most organs being transformed into lemma-like organs with new spikelets generated from the center of the flower. Contrasting with rice, these findings suggest barley LOFSEPs may have regressed in determining floral organ identity, and this could be partially compensated by HvMADS6.

Viable pollen is crucial for fertilization, but pollen is generally highly susceptible to heat stress. A quick, reliable method for testing the heat-stress tolerance of pollen is needed to improve the heat-stress tolerance in plants, but current methods require considerable space and labor. In addition, many such methods only test tolerance to a single constant temperature, making it time-consuming to screen heat tolerance over a wide temperature range and to examine the dynamics of pollen viability at different temperatures. To address this issue, we aimed to: (1) develop an easy, reliable method for measuring pollen viability at different temperatures; and (2) identify the best temperature range for screening pollen with high heat-stress tolerance. We harvested mature pollen from wheat (Triticum aestivum) plants and transferred it to a 96-well plate filled with liquid medium containing sucrose. We placed the plate in a PCR machine operating under a gradient PCR program to simultaneously test a range of temperatures. After incubating the pollen for 4 h, at temperatures ranging from 21.9 to 47 °C, we examined the pollen grains under a light microscope and employed a specific image analysis pipeline to assess the effects of temperature on pollen morphology, germination, and tube growth. This method facilitated the high-throughput screening of many pollen samples, enabling rapid, reliable, and precise analysis of pollen viability in response to temperature. Our approach should be applicable to other plant species and could be used to identify quantitative trait loci or genes influencing heat stress tolerance in pollen for breeding programs.

Sorghum, the fifth largest global cereal crop, comprises various types, such as grain, sweet, forage, and biomass sorghum, delineated by their designated end uses. Among these, sweet sorghum (Sorghum bicolor (L.) Moench) stands out for its unique versatility, exceptional abiotic stress tolerance and large biomass serving the multi-purpose of high-sugar forage, syrup, and biofuel production. Despite its significance, functional genomic research and biotechnological breeding in sweet sorghum are still in nascent stages, necessitating more efficient genetic transformation and genome-editing techniques. This study unveils Gaoliangzhe (GZ), an elite sweet sorghum variety for heightened resistance to salinity and drought. Through the establishment of an Agrobacterium tumefaciens‐mediated genetic transformation and CRISPR/Cas9-based genome-editing system in GZ, a breakthrough is achieved. Using genome-editing technology, we first produced a fragrant sweet sorghum line by targeting the BETAINE ALDEHYDE DEHYDROGENASE 2 (SbBADH2) gene. Our results establish a strong foundation for further functional genomic research and biotechnological breeding of sweet-sorghum varieties.

CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees, yet precise editing remains challenging in these species. Prime editing (PE), a revolutionary technology for precise editing, allows for arbitrary base substitutions and the insertion/deletion of small fragments. In this study, we focused on the model tree poplar 84K (Populus alba × P. glandulosa). We used the 2 × 35S promoter to express a fusion protein of spCas9 nickase (nCas9) and engineered Moloney murine leukemia virus (MMLV), and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA (epegRNA) and Nick gRNA, pioneering the establishment of the Prime Editor 3 (PE3) system in dicot poplar. Single-base substitutions, multiple-base substitutions, and small-fragment insertions/deletions were edited into three endogenous target genes. The desired edits were identified in hygromycin-resistant (transformed) calli at seven out of nine target sites, with an average editing efficiency ranging from 0.1 to 3.6%. Furthermore, stable T₀ plants contained the desired edits at four out of nine targets, with editing efficiencies ranging from 3.6 to 22.2%. Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.