Ehretia asperula Zoll. et Mor. (“Xạ đen”) is a valuable medicinal plant widely used in traditional Vietnamese medicine due to its rich content of bioactive compounds. This study investigated the phytochemical composition, antioxidant activity, and genetic diversity of samples collected from three regions in Vietnam. ITS marker and methods for quantifying secondary metabolites were used to assess the genetic diversity and chemical composition of E. asperula across different collection sites. Although ITS1 sequence analysis showed no significant genetic variation among accessions, there were notable differences in secondary metabolite content. Plants from Hoa Binh province contained the highest levels of total phenolic, flavonoids, and rosmarinic acid, followed by those from Dong Nai and Vinh Phuc provinces. Among the tested solvents, an ethanol-water (30 : 70, v/v) proved most effective for extracting the targeted compounds. In addition, a micropropagation protocol was successfully established using nodal explants from the plants collected from Hoa Binh province. Optimal surface sterilization was achieved with 0.1% HgCl2 for 16 min. Shoots were most efficiently regenerated from nodal explants on MS medium supplemented with 3.0 mg l−1 kinetin. Shoot proliferation was most effective on MS medium supplemented with 1.5 mg l−1 2-isopentenyladenine, while root induction reached 100% efficiency on MS medium containing 1.0 mg l−1 indole-3-butyric acid. These findings highlight the potential of E. asperula as a sustainable natural antioxidant source and support its continued use in traditional medicine.

In plants, the functional characterization of essential genes is often hindered by the lethality associated with complete loss-of-function alleles. Here, we present a genome editing-based strategy to generate viable hypomorphic alleles through selective removal of the translation start codon. Using Arabidopsis thalianaNON-SMC ELEMENT 1, which encodes a conserved component of the Structural Maintenance of Chromosomes (SMC) 5/6 complex involved in DNA repair and genome stability, as a model, we generated CRISPR-Cas9-edited alleles lacking the start codon. These homozygous mutants exhibited severe developmental defects, including stunted growth and failure to form true leaves. Moreover, they displayed molecular hallmarks of genome instability, such as increased DNA fragmentation, upregulation of DNA repair and cell cycle checkpoint genes, and root meristem cell death. Complementation assays using wild-type and mutated NSE1 genomic constructs confirmed that these alleles retained partial gene function. Overall, the use of start codon removal as an editing strategy is a robust and broadly applicable approach for generating hypomorphic alleles without relying on transcript-level manipulations, such as RNAi. This work therefore provides a practical demonstration of a novel editing strategy for dissecting the functions of essential genes that are otherwise genetically intractable. Consequently, this approach expands the functional genomics toolkit and opens new avenues for basic plant biology and advanced biotechnological applications.

Sugars in root exudates play a pivotal role in shaping plant-microbe interactions in the rhizosphere, serving as carbon sources and signaling molecules that orchestrate microbial behavior, community structure, and plant resilience. Recent research has shed light on the dynamics of sugar levels in root exudates, the factors that influence their secretion, and the mechanisms by which these sugars drive microbial colonization and community assembly in the rhizosphere. Microbial communities, in turn, contribute to plant physiological changes that enhance growth and stress tolerance. While well-studied sugars such as glucose, sucrose, and fructose are known to promote chemotaxis, motility, and biofilm formation, emerging evidence suggests that less-studied sugars like arabinose and trehalose may also play significant roles in microbial interactions and stress resilience. Key challenges remain, including the accurate measurement of labile sugars that are rapidly metabolized by microbes, and the elucidation of genetic mechanisms underlying rhizosphere metabolic interactions in both host plants and microbes. Addressing these challenges will advance our understanding of sugar-mediated interactions and inform the development of sustainable agricultural innovations.

Biotechnology, also referred to as genetic engineering or recombinant DNA (rDNA), is an industrial methodology that utilizes scientific research on DNA to enable pragmatic implementations. Recombinant DNA (rDNA) pertains to an artificially generated form of DNA resulting from the fusion or insertion of one or more DNA molecules. This methodology has introduced innovative prospects for progress in medical genetics and biomedicine, as it facilitates the alteration of microbes, animals, and plants to generate medically advantageous chemicals with expeditious effectiveness. The application of recombinant DNA technology plays a crucial part in refining health outcomes by facilitating the development of innovative vaccinations and treatments. This chapter provides a succinct summary of recombinant DNA (rDNA) and its wide-ranging applications in several academic fields.

Medicinal herbs are essential to many conventional and contemporary health-care systems around the world. However, the increased demand for these plants has also meant a rise in adulteration, which puts the public’s health and the effectiveness of herbal medicines at grave risk. In response to this difficulty, DNA-based methods have become effective means of validating therapeutic plants and separating them from adulterants. Several research investigations have indicated the potential prevalence of substitution and/or adulteration, collectively stated as substitution, within the raw herbal market for medicinal plants. Such practices could pose risks to the well-being and safety of consumers. The substitution of species could have detrimental effects on consumer health, potentially leading to severe allergies and failing to produce the intended therapeutic effects. Identifying species adulteration visually in the raw herbal trade proves challenging due to the typically dry state of the plants, resulting in the loss of their original characteristics. In recent times, various methods have emerged for the identification of medicinal plants in trade, such as morpho-taxonomical keys, histological techniques, chemical fingerprinting, and DNA-based approaches, each with its own set of advantages and drawbacks. However, a notable limitation of morphological techniques is their inability to identify powdered market samples, while chemical and histological approaches are susceptible to variations based on the age and season of collection. The accuracy, dependability, and cost of DNA barcoding have made it a popular technology for identifying medicinal plant materials in commercial settings. To verify the legitimacy of medicinal plants, this chapter offers a thorough overview of DNA-based methods, such as DNA barcoding, SNP analysis, HRM analysis, digital PCR, next-generation sequencing (NGS), and DNA fingerprinting. It also highlights the methods, benefits, drawbacks, and applications of these methods.

In order to address a specific agronomic challenge, plant biotechnology can be used in combination with conventional plant breeding techniques, transgenic approaches, integrated crop management techniques, or a combination of all these approaches. Biotechnologies such as cellular and tissue culture and molecular genome analysis can be used to cope with genetic erosion and strengthen ex situ collections and in situ conservation. As a result of recent advances in genetics and molecular biology, improved and novel agricultural products have been produced in many countries around the world. The transfer of genetic information into plant cells has been facilitated through the utilization of tissue culture techniques. Tissue culture is not a distinct field of plant science but, rather, a collection of experimental techniques for growing a large number of isolated plants under sterile and controlled conditions. Tissue culture techniques have become increasingly popular among research laboratories in recent years in exploring a wide range of basic and applied topics concerning higher plants. However, tissue culture methods are not restricted to research. There are lots of different techniques used in tissue culture, like micropropagation and embryo rescue, callus and cell suspension plant regeneration, microspore culture, etc., all of which are used to facilitate large-scale plant propagation. Micropropagation has been proven to be very successful in producing high-quality planting material for a wide range of crops. The DNA-based approach includes, but is not limited to, the use of isolates, amplifiers, modifications, and recombines of DNA; genetic engineering to create GMOs; marker and probe technologies for gene mapping; functional and structural genetics; and DNA fingerprinting for precise genotyping. A key part of this approach is the development of transgenic individuals using recombinant DNA techniques. This approach involves the isolation, cloning, combination, and reinsertion of genetic material through a range of techniques. This research will help elucidate the fundamental principles of regenerating plants from precursor cells, providing a solid foundation for the implementation of plant micropropagation and genetic modifications.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for genome editing in various fruit trees, including apple (Malus × domestica). In previous studies, transfer DNA (T-DNA) expressing genome editing tools, Streptococcus pyogenes Cas9 (SpCas9) and single guide RNA (sgRNA), was stably integrated into the apple genome via Agrobacterium-mediated transformation. However, due to self-incompatibility, long generation period, and the high heterozygosity of apple, removing only the integrated T-DNA from the apple genome by crossbreeding while maintaining the introduced varietal trait is difficult. Therefore, an efficient SpCas9-sgRNA delivery system without transgene insertion is required for genome editing of apple. In this study, we used geminivirus-derived replicons (GVRs) for the transient expression of genome editing tools. Small DNA vectors were deconstructed by splitting the elements necessary for the production of GVRs from bean yellow dwarf virus into two vectors. Production of GVRs using these vectors was demonstrated in Arabidopsis and apple cells. Genome editing was improved by using the GVR-producing vectors with genome editing tools in Arabidopsis protoplasts. The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA, enabling the detection of targeted mutations introduced in the endogenous apple genome. These findings demonstrate the utility of GVRs in genome editing via transient gene expression in apple. It can be expected that our GVR-based genome editing technology has potential utility for transgene-free genome editing in apple.

The long 5' untranslated region (5'UTR) exhibits enhancer activity in translation of rice OsMac3 mRNA. In this report, we describe elements of OsMac3 5'UTR that may be responsible for its enhancer activity, including a long uORF and several secondary structure elements. OsMac3 5'UTR can be dissected into three stem-loop structures SL1, small SL and SL2, where the uORF starts within SL1 and ends within SL2. As expected, uORF inhibits translation of downstream ORF since deletion of the uORF AUG or the SL1 stem-loop increases translation by approximately two-fold. Thus, the 158 nt 3' region of the 5'UTR lacking SL1 together with the AUG uORF, which has significant enhancer activity, was named dMac3. We investigated two critical regions within dMac3 mRNA that influence its translation: SL2, which destabilization potentially decreases translation activity, and another 13 nt located downstream of SL2. We further confirmed that dMac3 promotes mRNA translation initiation in an in vitro translation system and during transient expression in either cultured cells or Nicotiana benthamiana leaves. Thus, the dMac3 5'UTR is a useful tool for efficient protein production in various in vitro and in vivo translation systems.

Quinoa, a pseudocereal and leafy vegetable native to South America, is highly nutritious and can grow in harsh environments. One of the most prominent morphological features of quinoa is that the above-ground portion is covered with a layer of epidermal bladder cells (EBCs), and the role of EBCs in quinoa's high stress tolerance is of interest. Recent studies have shown that two WD40-repeat proteins, Reduced number of EBC (REBC) and REBC-like1, are required for EBC formation and that EBCs contribute defense mechanisms against biotic stress rather than abiotic stress. However, the role of EBCs in drought stress tolerance remains controversial due to the pleiotropic effects of these genes, including their impact on plant growth. Here, we show that REBC and REBC-like1 mediate water retention in detached quinoa leaves. Using a virus-induced gene silencing (VIGS) system, we found that downregulation of both REBC and REBC-like1 had no apparent effect on plant growth, but reduced the number of EBCs in both lowland and highland quinoa lines. Further, downregulation of both genes increased water loss in detached leaves of quinoa plants, supporting the notion that EBCs mediate water retention in quinoa leaves. Interestingly, we found higher EBC density in the southern highland lines grown in drier areas. Thus, we demonstrate that the effective use of VIGS in the analysis of genes with pleiotropic effects allows analyses that were difficult to perform using mutants alone, and that unlike mutants, functional genomics studies of quinoa can be easily performed in various lines using VIGS.