Plant Science

Gene stacking, the process of introducing multiple genes into a single plant to enhance desired traits, is essential for plant genetic improvement through both conventional breeding and genetic transformation. In general, transformation-based gene stacking can be achieved through either co-transformation to simultaneously introduce multiple genes or sequential multi-round transformation. While co-transformation is generally faster and more efficient than sequential multi-round transformation, it often requires two selectable marker genes, which confer resistance to antibiotics, for selecting transgenic events. However, in most cases, there is only one best selectable marker gene for a specific plant species or genotype. Also, it is harder to optimize the concentrations of two antibiotics for co-transformation than using one antibiotic for selecting transgenic events. To overcome this challenge, we recently developed an innovative split selectable marker system for plant co-transformation, allowing the use of one selectable marker gene to select transgenic events. This method involves constructing two binary vectors, each carrying a subset of genes of interest and a partial fragment of the selectable marker gene, which is connected to a partial intein fragment. Following Agrobacterium-mediated co-transformation, plants harboring both binary vectors are selected using a single antibiotic, such as kanamycin. This split-marker system can be used to co-transform multiple genes into both herbaceous and woody plants, accelerating genetic improvement of polygenic traits or integrative improvement of multiple traits to simultaneously increase crop yield and quality.

Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement, or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method’s robustness was validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from Escherichia coli K12 strain, followed by a target-specific transfer into A. tumefaciens EHA105 strain. The optimized method minimizes DNA degradation and maximizes uptake by Agrobacterium cells, making it a reproducible and accessible protocol for various genetic engineering applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability, requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of plant biotechnology.

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens–mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering A. tumefaciens to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer A. tumefaciens to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered A. tumefaciens.

Agrobacterium rhizogenes is a soil bacteria with extensive infectivity, which can infect almost all dicotyledonous plants and a few monocotyledonous plants to induce root nodules. This is caused by the root-inducing plasmid, which contains genes responsible for the autonomous growth of root nodules and crown gall base synthesis. Structurally, it is similar to the tumor-inducing plasmid in that it mainly contains the Vir region, the T-DNA region, and the functional region of crown gall base synthesis. Its T-DNA is integrated into the nuclear genome of the plant with the assistance of Vir genes, causing hairy root disease in the host plant and the formation of hairy roots. The roots produced by Agrobacterium rhizogenes–infested plants are characterized by a fast growth rate, high degree of differentiation, physiological, biochemical, and genetic stability, and ease of manipulation and control. In particular, the hairy root system is an efficient and rapid research tool for plants that have no affinity for transformation by Agrobacterium rhizogenes and low transformation efficiency. The establishment of germinating root culture system for the production of secondary metabolites in the original plants through the genetic transformation of natural plants mediated by root-inducing plasmid in Agrobacterium rhizogenes has become a new technology combining plant genetic engineering and cell engineering. It has been widely used in a variety of plants for different molecular purposes, such as pathological analysis, gene function verification, and secondary metabolite research. Chimeric plants obtained by induction of Agrobacterium rhizogenes that can be expressed instantaneously and contemporarily are more rapidly obtained, compared to tissue culture and stably inheritable transgenic strains. In general, transgenic plants can be obtained in approximately one month.

Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide.

Identification of novel genes and their functions in rice is a critical step to improve economic traits. Agrobacterium tumefaciens-mediated transformation is a proven method in many laboratories and widely adopted for genetic engineering in rice. However, the efficiency of gene transfer by Agrobacterium in rice is low, particularly among japonica and indica varieties. In this protocol, we elucidate a rapid and highly efficient protocol to transform and regenerate transgenic rice plants through important key features of Agrobacterium transformation and standard regeneration media, especially enhancing culture conditions, timing, and growth hormones. With this protocol, transformed plantlets from the embryogenetic callus of the japonica cultivar ‘Taichung 65’ may be obtained within 90 days. This protocol may be used with other japonica rice varieties.

Sweet basil (Ocimum basilicum) is a popular herb with high economic value and is currently threatened by a severe oomycete disease. An efficient transformation method is a prerequisite for gene functional analysis to accelerate molecular breeding and deploy effective disease management strategies, and breeding through genetic engineering. Here we present a detailed protocol for a highly efficient Agrobacterium tumefaciens-mediated transformation method for sweet basil, which was established based on a previously reported method by other researchers, with modifications on several aspects, including growth of sweet basil, age of plants used for explants, preparation and concentration of Agrobacteria. This protocol allows researchers in academia and agroindustry to generate transgenic sweet basil plants in an easy, quick and highly reproducible manner. In addition, this protocol may be applicable to transform other species within the genus Ocimum.