Plant Biotechnology Reports最新文献

Stomata are specialized pores that play a vital role in gas exchange and photosynthesis. Microscopic images are often used to assess stomatal characteristics in plants; however, this can be a challenging task. By utilizing Matterport’s Mask R-CNN implementation as the foundational model, fine-tuning was conducted on a dataset of 810 microscopic images of Hedyotis corymbosa leaves’ surfaces for automated stomatal pores detection. The outcomes were promising, with the model achieving a convergence of 98% mean average precision (mAP) for both detection and segmentation. The training loss and validation loss values converged around 0.18 and 0.37, respectively. Regression analyses demonstrated the statistical significance (p values ≤ 0.05) of predictor parameters. Notably, the tightest cluster of data points was observed in stomata pore area measurements, followed by width and length. This highlights the precision of the stomatal pore area in characterizing stomatal traits. Despite challenges posed by the original dataset’s low-resolution images and artifacts like dust, bubbles, and blurriness, our innovative utilization of the Mask R-CNN algorithm yielded commendable outcomes. This research introduces a robust approach for stomatal phenotyping with broad applications in plant biology and environmental studies.

Throughout the globe morphological, biochemical and genetic variability exists in chilli and is harnessed to achieve specific breeding objectives. In this study, chilli germplasm was characterized based on horticultural traits, biochemical quantification and simple sequence repeat (SSR) polymorphism for diversity estimation. A total of 36 SSR primers were utilised to study the genetic divergence among 48 genotypes of chilli collected from nine states of India. Among the 36 primers, sixteen amplified null alleles. A total of 41 alleles were detected with average 2.05 alleles per locus. The largest number of alleles (5) were obtained with marker CAMS-234. The polymorphic information content ranged from 0.06 to 0.72 with an average of 0.50. On the basis of SSR analysis, the UPGMA cluster classified 48 genotypes into three groups. There was significant variability in germplasm for all morpho-biochemical traits. Kashi Anmol (100.50 q/ha) expressed the highest yield. Highest vitamin C content at green stage was recorded in IC-561635 (187 mg/100 g) and the greatest capsaicin content (9547.90 µg/g) equivalent to pungency of 171,862.2 Scoville heat units (SHU) was recorded in Bhut Jolokia. Principal component analysis indicates that the first five principal components explain 74.63% per cent of the total variation. Additionally, analysis of molecular variance (AMOVA) showed that 1% of the total genetic variation occurred among the population and 99% genetic variation within the populations, whereas the pairwise F_st specified the moderate genetic variation ranging from 0.002 to 0.020. The present investigation has strengthened the knowledge of genetic worth of this germplasm for application in various genetic improvement programmes.

This study aims to establish an efficient protocol for protoplast isolation, cell division, and callus induction in two carnation cultivars, Chabaud and Giant Chabaud, by examining the influence of different types of plant tissue, enzyme concentrations, incubation times, cotyledon ages, and medium compositions. Our results indicate that protoplast yield varies significantly between different plant tissues, with true leaves offering the highest yield and viability, especially under a 0.1% driselase concentration and a 6-h incubation time. We observed that increasing the driselase concentration to 1.0% significantly reduced protoplast yields in all tissues tested. In addition, the age of the cotyledons notably affected protoplast yield, with younger cotyledons providing higher yields. The Murashige and Skoog medium supplemented with 1 mg/L zeatin and 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) proved to be the most effective for promoting cell division and colony formation from protoplasts derived from cotyledons and true leaves. The study also found that plant growth regulators (PGRs) significantly influence callus proliferation, with differences observed between protoplast sources.

The CRISPR/Cas9-based gene editing system for the direct delivery of pre-assembled Cas9 ribonucleoproteins (RNPs), consisting of a Cas9 nuclease and a single guide RNA (sgRNA), into plant protoplasts enables DNA-free gene editing without introducing foreign gene into plants. Here, we described the optimization of CRISPR/Cas9 RNPs delivery into cabbage protoplasts for efficient DNA-free gene editing. We determined the insertion and deletion (indel) frequency of BoMYBL2-1, a negative regulatory gene for anthocyanin biosynthesis in cabbage (Brassica oleracea var. capitata). We optimized the molar ratio of Cas9 to sgRNA and the incubation time of RNP–protoplast transfection to enhance the indel frequency under various conditions. Based on the BoMYBL2-1 nucleotide sequences, we designed nine sgRNAs to target BoMYBL2-1. Our in vitro digestion assay showed that all sgRNAs were able to cleave the targeted fragment. When the sgRNA and Cas9 proteins were subsequently transfected into protoplasts isolated from cabbage cotyledons, the deep sequencing results showed that the indel frequency of sgRNAs in BoMYBL2-1 was the highest (7.4%) with sgRNA3. We compared various molar ratios of Cas9 and sgRNA and incubation times of RNP–protoplast transfection to optimize transfection and ensure high indel frequency. The highest frequency was observed when the Cas9:sgRNA ratio was 1:10. Furthermore, when the incubation time for RNP–protoplast transfection was 1 min and 3 min, the indel frequency was higher than 25%. Altogether, these results provide valuable information on the optimized conditions for high-efficiency gene editing using CRISPR/Cas9 RNP delivery into cabbage protoplasts.

Lead (Pb) is one of the most toxic heavy metals (HMs) for plants and the environment. Sweetpotato [Ipomoea batatas (L.) Lam], the sixth most important food crop in the world, is tolerant to various environmental stresses, owing to its high antioxidant capacity. In this study, we selected sweetpotato cultivars showing high tolerance to lead (Pb) for phytoremediation-related applications. Young seedlings of 20 sweetpotato cultivars were treated with 30 mM Pb. Daeyumi (KO-12) and Dahomi (KO-5) were selected as Pb-tolerant and -sensitive cultivars, respectively, based on their photosynthetic activity and growth inhibition index (I₅₀). In the Pb treatment, hydrogen peroxide and malondialdehyde contents of KO-12 were 1.5-fold less than those of KO-5. In addition, KO-12 showed a higher ability to accumulate Pb in roots and leaves than KO-5. Expression levels of four Pb-responsive genes, including the metallothionein gene IbMT1, were higher in the roots and leaves of KO-12 than in those of KO-5. Interestingly, KO-12 showed greater tolerance to high Pb concentrations than sunflower and rapeseed, which have been well-studied for phytoremediation. Our results suggest that sweetpotato is a suitable biomaterial for the phytoremediation of soils contaminated with HMs, including lead, for sustainable agriculture.

Cesium (Cs) toxicity has deleterious effects on plant growth and development. However, the molecular mechanism of the toxic effect of Cs on plants has been poorly understood. To obtain insights into the molecular events occurring in plants under Cs stress, we performed a comparative transcriptomic analysis between control and Cs-treated plants via RNA-seq. We identified 183 differentially expressed genes (141 upregulated and 42 downregulated) under Cs stress (1.5 mM CsCl). Gene ontology (GO) analysis using differentially expressed genes in Cs stress indicated that Cs triggered plant stress signaling pathways like reactive oxygen species (i.e., hydrogen peroxide). Further KEGG and MapMan metabolic pathway analyses revealed that many abiotic/biotic stress signaling pathways were highly induced. In particular, heat shock protein family genes were substantially induced upon exposure to Cs stress. We investigated the root growth of several knockout mutants of heat shock protein family genes and found that heat stress response was compromised in these mutants compared to wild type plants. It suggested that heat shock protein genes including HSP17s, HSP23s, HSP101, and HSFA2 proteins are deployed upon exposure to Cs for plant stress tolerance. Our study provided novel insights into the molecular events occurring in Cs-stressed plants.

Zinc-finger homeodomain transcription factors (ZF-HD TFs) are relatively a small gene family in Arabidopsis involved in plant development and stress response. However, the biological functions of ZF-HD TFs remain largely undiscovered. Here, we aimed to elucidate the evolutionary history and functional role of ZF-HD TFs in other species, by performing phylogenic analysis and domain and motif identification studies in Arabidopsis, sorghum (Sorghum bicolor), and moss (Physcomitrella patens). Forty-two ZF-HD TF proteins were classified into two distinct subfamilies based on the conserved ZF Cys/His-rich dimerization and homeodomain (HD) domains. The phylogenetic tree of proteins was further divided into five groups based on the similarity of sequences, and three distinct motifs were defined in the amino acid sequences. Genetic analysis revealed that the moss PpZF-HD1, Pp3c1_15290, gene partially rescued the amiR zf-HD-79 mutant lines at phenotypic and molecular levels. Subcellular localization studies revealed that moss PpZF-HD1 was localized in the cytosol and nuclei. Phylogenetic analysis and genetic complementation revealed that ZF-HD TFs play functional roles in regulating plant architecture, which is conserved in Arabidopsis, sorghum, and moss. Although our study is only a preliminary exploration into ZF-HD TFs, it provides a novel perspective that will help future researchers better understand the biological role of ZF-HD proteins in plants.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is a threat to soybean cultivation, leading to substantial economic losses. Here, an RNA sequencing analysis was conducted to identify genes associated with the response of wild soybean (Glycine soja) to C. sojina. Differentially expressed genes (DEGs) were identified by comparing the gene expression of C. sojina-inoculated plants with that of non-inoculated plants. A total of 1642 DEGs (790 up-regulated and 852 down-regulated) were identified in C. sojina-inoculated wounded leaves compared with non-inoculated wounded leaves. The DEGs were analyzed for gene ontology and the KEGG pathway to identify the key genes responsible for the response to C. sojina and the corresponding pathways. In GO analysis, ‘Defense response’ was highlighted, while in KEGG analysis, ‘Metabolic pathways’ and ‘Flavonoid biosynthesis’ were emphasized. A total of 67 DEGs were categorized within the 'biotic stress' MapMan category, with ‘Redox state,’ ‘Cell wall,’ and ‘Secondary metabolites’ showing the highest abundance of assigned DEGs. DEGs associated with the phenylpropanoid pathway (GsALDH and GsAOMT-like), cell wall remodeling (GsPME12), and reactive oxygen species (GsGSTUs), were identified in plants inoculated with C. sojina compared to non-inoculated plants. Additionally, Gs2MF3OR-like (encodes an enone oxidoreductase) and Gsα-DOX1-like (involved in oxidative stress) also participated in the response of wild soybean to the disease. Our results suggest potential C. sojina-resistant genes that could serve as targets for further functional characterization, as well as for soybean molecular breeding programs aimed at improving FLS resistance.