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Efficient and stable plant genetic transformation systems are pivotal for gene function analysis and molecular breeding. Traditional hybrid breeding faces inherent challenges such as reproductive isolation and lengthy breeding cycles. In contrast, gene editing technologies based on genetic transformation enable precise manipulation of target genes and directed improvement of agronomic traits, significantly accelerating the breeding process. However, existing transformation techniques heavily rely on tissue culture, which is complex and constrained by plant genotype preferences, incomplete regeneration systems, and low transformation efficiencies. Statistics indicate that only about 0.1% of the approximately 370,000 plant species worldwide have established stable transformation systems, severely limiting gene function studies and crop improvement efforts. To overcome this bottleneck, researchers have recently focused on developing non-tissue culture genetic transformation technologies that leverage plants' innate regenerative capabilities to efficiently introduce exogenous genes under natural growth conditions.
Agrobacterium-mediated non-tissue culture transformation systems have gained attention due to their simplicity, short cycles, and high efficiency.
Figure 1. (A) Floral dipping method; (B) Cut-dip-budding (CDB) delivery method; (C) Extremely simplified cut-dip-budding delivery method; (D) Cut-dip-bud-in situ delivery method; (E) Regenerative activity-dependent in planta injection delivery method; (F) Seed inoculation method. Red indicates a positive regenerating organ or plant. (Feng, M., et al., 2025)
The floral dip method, initially applied to Arabidopsis thaliana, involves immersing early-stage floral tissues in an Agrobacterium suspension, often with the aid of vacuum infiltration, to facilitate T-DNA insertion into the ovule genome, resulting in the production of transgenic seeds through sexual reproduction. This technique bypasses tissue culture steps, offering a straightforward and time-efficient process. Over 20,000 T-DNA insertion lines have been successfully generated in Arabidopsis using this method. Its application has been extended to other crops, including Brassica napus, cabbage, wheat, and soybeans. However, transformation efficiencies are generally low (0.01%-4%) and highly dependent on the flowering stage, necessitating multiple generations of selfing to obtain homozygous lines. Therefore, it is more suitable for self-pollinating species with short life cycles.
The CDB method utilizes the asexual propagation potential of plant vegetative organs. By wounding explants such as root segments and exposing them to Agrobacterium rhizogenes, transgenic roots are induced, which can then regenerate into shoots through a "cut-dip-budding" process. Initially established in rubber dandelion, this method achieved positive root regeneration efficiencies of 40%-50% and shoot regeneration rates of 15%-25%, approximately ten times higher than traditional floral dip methods. To further reduce the cycle time, an extreme simplification of the CDB system (ES-CDB) was developed, omitting the hairy root induction step and shortening the transformation cycle from 14 weeks to 2 weeks. This system has shown excellent performance in species like Coronilla varia, sweet potato, medicinal plants such as Schizonepeta tenuifolia, and succulents. Notably, it holds significant potential for Asteraceae and woody plants with root suckering abilities, such as poplar and jujube. However, in woody fruit trees like citrus and peach, while positive roots can be induced, the difficulty in regenerating shoots from roots limits the acquisition of complete transgenic plants. To address this, in situ transformation methods involve removing the shoot apical meristem and inoculating the wound with Agrobacterium to directly induce positive shoot regeneration, providing a solution for plants where only above-ground traits need to be observed.
The RAPID method focuses on the strong regenerative capacity of meristematic tissues. By microinjecting Agrobacterium into the vicinity of the vascular cambium, transgenic positive organs are induced. In sweet potato, this method achieved transformation efficiencies ranging from 12.5% to 37.5% across six different genotypes, with a transformation cycle of only 3 to 10 weeks, reducing the duration by over 75% and increasing efficiency by more than 100-fold compared to traditional tissue culture methods. Successful applications have also been reported in potato, Panax notoginseng, and lily. Compared to the CDB method, RAPID ensures more reliable infection by delivering Agrobacterium directly into meristematic tissues through pressure injection. In species with weaker regenerative abilities, such as tobacco and tomato, co-expression of developmental regulatory genes like WUSCHEL (WUS) or PLETHORA5 (PLT5) can significantly enhance the acquisition rate of positive shoots.
The seed inoculation method targets the embryo of germinating seeds, enhancing Agrobacterium infection efficiency through ultrasonic and vacuum infiltration treatments. Early applications in Arabidopsis achieved transformation efficiencies of only 0.32%. However, optimized systems have reached 31.3%-38.6% in peanut, stabilized around 30% in soybean, and even exceeded 90% in citrus seeds. This genotype-independent method, termed "Genotype-Independent Transformation Technology" (GIFT), allows for the processing of thousands to tens of thousands of seeds simultaneously, facilitating the large-scale creation of mutant libraries and elite germplasm. Nevertheless, challenges remain in monocotyledonous plants like maize and sorghum, as well as in the grapevine, and the high heterozygosity in some plants may affect the accuracy of phenotypic analyses.
For species with weak regenerative abilities, DR-assisted transformation offers a breakthrough. Overexpression of key developmental genes such as BABY BOOM (BBM), WUSCHEL (WUS), and GRF-GIF chimeras can significantly enhance cell regeneration potential. For instance, expressing ZmBBM/ZmWUS in maize inbred lines increased transformation efficiency to over 40%. GRF-GIF chimeras, which do not cause developmental abnormalities, have been widely applied in wheat, citrus, and hemp. Under non-tissue culture conditions, the expression of the WUS2-ipt combination in tobacco leaf meristems significantly improved the editing efficiency of albino shoots, demonstrating compatibility with gene editing technologies.
VIGE leverages virus-induced gene silencing (VIGS) mechanisms by incorporating target genes into viral vectors for transient expression. This approach has been widely used for gene function validation in difficult-to-transform plants like pepper and peach. Although most viruses do not integrate stably into the genome, tobacco rattle virus (TRV) fused with the FT gene can direct vectors to meristems, enabling the development of VIGS technology in conjunction with CRISPR-Cas systems. Using high-capacity vectors like potato virus X (PVX) and tomato spotted wilt virus (TSWV), heritable mutations have been achieved in tobacco and pepper with efficiencies up to 77.9%, paving the way for the creation of non-transgenic edited germplasm.
Key factors affecting transformation efficiency include explant selection, strain compatibility, and optimization of operational parameters. Explant types should align with plant reproductive characteristics: floral organs or seeds for sexually reproducing crops, and vegetative organs like roots and stems for asexually reproducing species. Young meristematic tissues, such as rubber dandelion seedlings or basal stems of 15-day-old sweet potato plants, are preferred for their strong regenerative abilities. Strain selection is species-specific—Agrobacterium rhizogenes K599 achieves over 90% efficiency in sweet potato CDB transformations, while Agrobacterium tumefaciens AGL1 performs better in the RAPID system.
Precise control of bacterial concentration (OD600 0.6-1.0) and infection duration (10-30 minutes) is crucial, as excessive levels can cause tissue necrosis. Enhanced infection methods like vacuum infiltration, injection, and ultrasonic treatment significantly improve efficiency; combining ultrasound and vacuum treatment in peanut seeds increased transformation rates by 100-fold. Visual markers such as GFP and Ruby are gradually replacing traditional antibiotic selection due to their non-destructive detection capabilities, with Ruby's red coloration enabling rapid visual identification in sweet potato and snapdragon.
Alternative transformation techniques have also made progress. The pollen tube pathway introduces exogenous DNA into the embryo sac via the pollen tube and has been applied in over 40 plant species, including cotton, wheat, and rose, though it suffers from low efficiency and poor reproducibility. Particle bombardment uses gene guns to deliver DNA-coated metal particles into cells, overcoming genotype limitations and being particularly suitable for monocotyledonous crops, albeit with high equipment costs and randomness. Nanoparticle delivery employs materials like carbon dots to penetrate cell walls and deliver nucleic acids, achieving transient silencing in tobacco and cotton. Combining this approach with CRISPR-Cas ribonucleoproteins holds promise for stable genetic editing without foreign DNA integration
At Creative Biogene, we specialize in plant genetic transformation services, including both traditional tissue culture and advanced non-tissue culture platforms. Whether you are working on staple crops, woody species, medicinal plants, or recalcitrant genotypes, we offer customized, efficient, and scalable transformation systems tailored to your species and project needs. Our services encompass Agrobacterium-mediated transformation, genome editing, and virus-induced gene editing.
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