Plant Biotechnology Reports最新文献

Six popular and widely cultivated arabica coffee (Coffea arabica L.) varieties of commercial importance namely Selection 5B, Selection 13, Selection 11, Selection 8, Selection 7.3 and Selection 3 were tested for their genetic identity with ISSR markers. Fifteen ISSR primers were tested using genomic DNA of selected coffee varieties. Pooled genomic DNA of all the six varieties was amplified with each ISSR primer with an average of four loci per primer. The size range of locus amplified by all the fifteen primers was ranging from 100 to 1200 bp depending upon on the ISSR primers. Only three out of fifteen primers, namely ISSR4, ISSR6 and ISSR8, were screened based on the number of amplified locus and size range from low to high. The selective ISSR primers distinguished all the six varieties of coffee with unique markers. ISSR 4 amplified two unique markers with a locus size 1300 bp and 950 bp for Sln.5B and 180 bp and 150 bp for Sln.13. ISSR6 had produced five varietal-specific markers with a locus size of 180 bp in Sln.5B, 1250 bp in Sln.11, 350 bp in Sln.3. ISSR8 had amplified seven unique loci across the coffee varieties with 700 bp and 800 bp in Sln.5B, 200 bp and 500 bp in Sln.11 and one locus each in Sln.7.3 and Sln.3 with 300 bp and 150 bp respectively. Repeated amplification of genomic DNA of all the six varieties of coffee with selective ISSR primers produced consistent ISSR genetic fingerprints. Selective ISSR primers were validated with marker parameters resolving power (RP), effective multiplex ratio (EMR), marker index (MI) and polymorphic information content (PIC). Utilisation of these markers in arabica coffee genetic improvement is discussed.

Multiple myeloma (MM) is an incurable disease characterized by malignant plasma cells within the bone marrow, and its increasing occurrence has highlighted the need for innovative strategies to address relapse and treatment resistance. Given the substantial expression of programmed death ligand 1 (PD-L1) in the human multiple myeloma cell line RPMI8226, we propose PD-L1 as a promising target for multiple myeloma therapy. Here, we successfully engineered an anti-PD-L1 monoclonal antibody (mAb) within a plant-based system. Building upon our previous findings, we germinated seeds derived from transgenic plants under in vitro conditions. Afterward, we screened the resulting seedlings for expression of the anti-PD-L1 mAb using polymerase chain reaction (PCR) and western blot analyses. Anti-PD-L1 mAbs were successfully purified from plant leaves and characterized through SDS-PAGE analysis. Our findings, which were confirmed via indirect enzyme-linked immunosorbent assay (ELISA), validate the binding affinity of the anti-PD-L1 mAb to recombinant PD-L1 protein. Furthermore, we investigated the interaction between the plant-derived anti-PD-L1 mAb and Fc gamma receptor I (FcγRI) as well as Fc gamma receptor IIIa (FcγRIIIa) molecules, confirming robust affinity. Additionally, the antibody’s binding affinity to the human multiple myeloma cancer cell line RPMI8226 was confirmed via cell ELISA. Our findings demonstrated that, unlike existing therapeutics, the plant-derived anti-PD-L1 antibody not only effectively binds to human recombinant PD-L1 protein but also to FcγRI and FcγRIIIa. These findings suggest the potential of plant-derived anti-PD-L1 mAb for the development of innovative therapies against multiple myeloma, emphasizing the need for further research and preclinical evaluation.

Brassica is an essential genus in agriculture, and gene flow from living modified (LM) organisms to native relatives or non-modified cultivars of the crucifer family is a critical issue in Asia. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, and accurate method for identifying LM crops. This study aimed to develop a LAMP assay for the laboratory diagnosis and field analysis of three novel LM canola events: T45, 73496, and MON88302. The genomic DNA of each LM canola was amplified by incubating at 63 °C for 30 min with a newly developed 2 × LAMP premix containing Bst DNA polymerase and event-specific primer sets. The sensitivity of the LAMP assay for the three canola events in our condition ranged from 100 pg·μL⁻¹ to 1 ng·μL⁻¹. Genetic elements of the three LM events were identified through a combination of the LAMP assay, a syringe-type DNA extraction kit, and a portable isothermal amplifier. This study proposes a novel, simple system based on isothermal amplification for the DNA detection of the three LM canola events in a survey field.

Photosynthesis is responsible for sustained plant productivity and ensures food supply. The change in global climatic patterns affects photosynthesis that subsequently reduces plant yield and poses threat to food security. Photosynthesis relies on a dual nature enzyme ribulose 1, 5 bisphosphate carboxylase oxygenase (Rubisco), which can fix CO₂ as well as O₂. The fixation rate of CO₂ to O₂ depends upon the relative concentration of CO₂ inside chloroplast. Higher level of CO₂ results in improved photosynthesis, however, its concentration depends upon environmental conditions. Under adverse climate conditions, the CO₂ level drops down that leads to increased oxygenation which impedes the photosynthesis and reduces plant productivity. The impact is more significant and apparent specifically in C₃ plants. Attempts have been made to address the loss in photosynthesis and multiple strategies have been adapted to date that focus on improvement of photosynthesis in C₃ plants. In this review, we have discussed the multiple strategies being employed by different researchers to date for improvement of photosynthesis. The strategies discussed in this review include: improving the performance of Rubisco, engineering CO₂-concentrating mechanism of C₄ photosynthesis into C₃ species, transformation of bicarbonate transporters from cyanobacteria into chloroplasts of C₃ plants, and establishment of photorespiratory bypasses to catabolise toxic glycolate in shortest possible pathway.

Plant triterpenoids are secondary metabolites with high chemical diversity and interesting biological properties. Chestnuts are deciduous trees in the genus Castanea, and their nuts have been used as an important food. In this work, we identified various types of triterpenes, such as α-amyrin, β-amyrin, lupeol, and friedelin, in the leaves and/or stem bark of Korean chestnut (Castanea crenata). Triterpene biosynthesis occurs by the cyclization of 2,3-oxidosqualene to triterpenes, catalyzed by oxidosqualene cyclases (OSCs). A total of 65 putative OSC sequences were obtained from the leaf transcriptome data of C. crenata plants using PacBio sequencing. We selected 5 putative OSC unigenes, named CcOSC1-5, for functional characterization of genes involved in triterpene biosynthesis. Functional characterization of the CcOSC1-5 genes by heterologous expression in erg7 mutant yeast revealed that both CcOSC1 and CcOSC2 had a similar function, encoding multifunctional triterpene synthases producing mainly β-amyrin and a small amount of α-amyrin and lupeol. CcOSC3 encodes mixed amyrin synthase, which mainly produces β-amyrin and a small amount of α-amyrin. CcOSC4 produced mainly α-amyrin and lupeol and a small amount of β-amyrin. CcOSC5 encodes an enzyme for lupeol production as a single product. In conclusion, we identified various triterpenes and functionally characterized the triterpene synthase genes that participate in β-amyrin, α-amyrin, and lupeol biosynthesis in C. crenata.

Citrus spp. are recalcitrant to in vitro shoot regeneration and we report an improved in planta protocol for genetic transformation of rough lemon that bypasses shoot regeneration in tissue culture. The features of the protocol were the use of an Agrobacterium suspension with an OD_{600 nm} = 0.6–1.0 supplemented with 100 μg acetosyringone, gentle shaking of embryo axes pricked at shoot apical meristems (from 2-day-old germinating seeds) at 70 rpm during agro-infection, followed by growth and development of plantlets at 30 °C. PCR screening of 2-month-old T₀ plants revealed the presence of an amplicon corresponding to the β-1,3-glucanase gene in the primary branches of 25 plants with a transformation efficiency of 7.74%. PCR analysis of the secondary branches of these plants after 18 months showed chimerism, i.e., the coexistence of transformed and untransformed branches in all 25 plants. Quantification of β-1,3-glucanase expression in the transformed secondary branches by qRT-PCR showed that plant number 32 had maximum (3.71-fold) relative transgene expression. The qRT-PCR analysis of all four tertiary branches arising from the transformed secondary branch of plant number 32 showed no significant differences in expression among themselves and from the transformed secondary branch, suggesting restoration of the transformed branches with uniform expression and dissociation of chimerism. Scanning electron microscopy examination of leaves from secondary and tertiary branches that uniformly expressed the transgene showed a smooth, waxy surface with non-significant variation in stomata, which had a narrow opening and a mean pore length of 4.22 ± 0.25–5.09 ± 0.36 µm. In contrast, the leaves of untransformed branch had a rough surface and a significantly large stomatal opening with a mean pore length of 7.82 ± 0.67 µm. The micro-morphological characteristics of the leaves confirmed the dissociation of chimerism in the transformed tertiary branches of plant number 32. The study demonstrates identification of chimerism after in planta transformation using PCR technique, and the novelty relates to monitoring dissociation of chimerism in transformed tertiary branches of T₀ generation using qRT-PCR analysis and its corroboration by electron microscopy. The protocol for genetic transformation in plants described in the present study can be used for trait improvement by transgenesis.

Living modified organisms (LMOs) in South Korea are managed under the Transboundary Movement, Etc. of LMOs Act, the domestic implementation law of the Cartagena Protocol on Biosafety. The Ministry of Environment (MOE) oversees the risk review and safety management of LMOs for environmental remediation, and consultative review on the risk posed to the natural ecosystem by LMOs for other purposes. In particular, the MOE operates a risk assessment institute to promote scientific and systematic safety management by standardizing an evaluation criterion for the relevant LMOs. These must be established according to the standards of the LMOs Act. In this review, a new technique for reliable management of the LMO facility, including a confined field, was proposed to comply with the safety standards of the MOE. Based on the analysis of the LMOs Act and the practices of established LMO facilities, potential facility management directions under the MOE were discussed from the perspectives of the facility, cultivation, waste, record, and education. These suggestions can help physically isolate LMO facilities and prevent the unintentional release of LMOs, thus helping avoid hybridization between LMOs and non-LMOs. In addition, a monitoring system was proposed to prevent transgene escape and subsequent hybridization with the wild relatives of LMOs. These proposals can be widely used for safety management when researching the development and commercialization of LMOs at facilities under the MOE.

The Lamiaceae family is included in the angiosperms and comprises over 7000 species, many of which are of considerable ecological, economic, and cultural importance. We seek to establish a taxonomic basis by examining the speciation timeline in Lamiaceae using phylogenetics and publicly available transcriptome data. Since Ks is steadily accumulated over time in plants for environmental adaptation until speciation occurs, the timing of speciation can be estimated from examination of Ks values. A total of 24 species included in the Lamiaceae family used in our analysis belongs to four subfamilies. We performed transcriptome assembly for each of the 24 species using trimmed data collected from public databases. We compiled groups of gene families in which at least one copy of the gene is present in each species from orthologous groups among unigenes. From these groups, we obtained a total of 450,014 single nucleotide polymorphisms (SNPs) across 27 species, incorporating three additional outgroup species. Subsequently, a tree was created using these SNPs. In our tree, the outgroup species were clearly located externally, confirming the proximity of species within the same subfamily. The Ks peak corroborated the outcomes observed in the phylogenetic tree. We estimated the rate of sequence evolution and divergence time for each species on the phylogenetic tree by referencing the time of divergence among the Lamiaceae family. In particular, Clinopodium serpyllifolium, Lavandula × intermedia, Phlomis fruticosa, and Volkameria inermis were analyzed for the first time. Our study helps with the understanding of the function of plants included in the Lamiaceae family and is expected to provide a fundamental resource that can be used to pinpoint the molecular and genomic evolution of the Lamiaceae family.

β-Conglycinin (7S) and glycinin (11S) are the two major storage proteins in soybean seeds. Storage proteins occupy a large portion of soybean seeds, which is the main obstacle to accumulating additional foreign proteins. Thus, we produced 7S-edited soybean plants using CRISPR/Cas9 via Agrobacterium-mediated transformation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed that the α′-subunit (76 kDa) of β-conglycinin protein disappeared from the 7S-edited T₂ line (#19–8). The removal of the band was caused by the early termination of the CG-1 gene by insertion/deletion (in/del) mutations with an efficiency of 78.5% generated by genome editing. Soybeans with low-storage proteins could be used for improved foreign protein production in seed.

Chalcone synthase (CHS), a key enzyme in plant flavonoid synthesis, is essential for plant tolerance to abiotic stress. However, little research on CHS from the earliest terrestrial plants, such as mosses, has been reported. Here, the biological function of a CHS gene from Antarctic moss Pohlia nutans (PnCHS1) was studied. PnCHS1 had a 32.8–53.7% similarity to CHS from other species, however it still had highly conserved motifs of CHS such as Catalytic site (Asn366, His333) and Co-A binding site (Ser146). Subcellular localization analysis showed that PnCHS1 was distributed in the cell membrane and in the membranes of endothelial organelles. Heterologous expression of PnCHS1 increased flavonoid content in 5-day-old Arabidopsis grown with 24 h light and 17-day-old Arabidopsis cultured with sucrose, as well as anthocyanin content in the latter. PnCHS1 heterologous expression in Arabidopsis increased plant tolerance to salt stress, including a high germination rate and a long taproot. Heterologous expression of PnCHS1 boosted tolerance to oxidative stress while decreasing the sensitivity to ABA. Under H₂O₂ or ABA stress, the expression pattern of PnCHS1, ROS scavenging enzyme gene (FeSOD1, FeSOD2, Cu-Zn-SOD2, and Cu-Zn-SOD3) and three genes of ABA signal pathway (RAB18, RD29B, and NCED3) were considerably up-regulated by real-time quantitative analysis. It indicates that PnCHS1 could enhance plant tolerance to NaCl and oxidative stresses, and may play a role in the adaptation of Antarctic moss to extreme environments.