Biotechnology Bulletin | Establishment and application of efficient hairy root genetic transformation system for broccoli

Genome editing technology can achieve precise genetic modification at specific loci, and as a new generation of breeding technology, it has been widely applied in crop breeding. Among them, the CRISPR/Cas system has become the most widely used system in the field of plant genome editing due to its low cost, easy operation, and high efficiency, and has been preliminarily applied in broccoli variety improvement^［10-13］However, stable genetic transformation relies on tissue culture processes, which have long cycles and limited efficiency, limiting the rapid screening of editing materials. Therefore, establishing an efficient editing efficiency evaluation system before stable transformation is of great significance for accelerating broccoli gene editing breeding.

The protoplast transient transformation system is commonly used for the validation of gene editing vector activity and iterative testing of gene editing tools. However, this method has problems such as complex operation, strong empiricism, and difficulty in reflecting the actual editing efficiency in vivo. And by Agrobacterium tumefaciens（Agrobacterium rhizogenes）The mediated hairy root transformation method has the advantages of easy operation and high efficiency. The T-DNA on its Ri plasmid can be integrated into the plant genome and induce the production of hairy roots, thereby obtaining composite plants in a short period of time^［16］This system has been successfully applied in gene function analysis and target site activity verification in various plants. However, there are significant differences in the response of different species to transformation conditions, and optimization is needed for key factors. At present, there is a lack of research on hairy root transformation in broccoli.

Based on this, Professor Li Jun's team from Hebei Agricultural University established an efficient broccoli hairy root transformation system mediated by Agrobacterium tumefaciens using RUBY as the reporter gene. By systematically optimizing the transformation conditions of Agrobacterium tumefaciens, the expression efficiency of exogenous genes was significantly improved, and the system was validated to have good applicability in cabbage, mustard, and cauliflower. This system can be used for rapid evaluation of gene editing target site activity and has achieved efficiency validation of CRISPR/Cas12a gene editing vectors at multiple target sites. This study provides an efficient and reliable technical platform for the functional research and precise genetic improvement of broccoli genes.

Research results

Cut broccoli rootless seedling explants 0.5-1 cm below the growth point in a sterile environment, immerse them in Agrobacterium tumefaciens solution for 15 minutes, and incubate them in the dark for 2 days before transferring them to MS solid medium to induce hairy roots. After about 25 days of cultivation, a large number of hairy roots appear at the wound site, and the efficiency of hairy root induction can reach 100%. Through system optimization exploration, it was found that K599 is the optimal Agrobacterium strain for broccoli hairy root transformation, with the best infection concentration OD₆₀₀=0.3； The optimal infection period is 3 days of seedling age; when the AS concentration is 200 μ mol/L, the average positive rate of hairy roots is the highest.

A: Disinfection and sowing of broccoli seeds; B: Broccoli cultivation; C: Acquisition of rootless seedlings; D: Agrobacterium infection in rootless seedlings; E: Remove excess bacterial liquid; F: Co cultivation; G: De bacteria; H: Absorb surface moisture; I: Rooting cultivation; J: Positive rooted plants. The ruler in the picture is 2 cm

The positive rates of hairy roots in cabbage, mustard greens, and cauliflower were 42.2%, 41.1%, and 61.1%, respectively. This indicates that the system has good applicability in various Brassica vegetables.

Using laboratory optimized CRISPR/Cas12a knockout vector for targeted editing of broccoliPDS（BolC3t19948H）Genes. Among the 33 tested plants, 22 were found to have integrated gene editing vectors, with an integration efficiency of 66.7%. Further target site amplification and enzyme digestion validation were performed on the integrated plants, and expected sized bands appeared in some samples. Sanger sequencing confirmed delta at the target site, indicating gene editing occurred in hairy roots with an editing efficiency of 36.4%.

Further validate the applicability of the system in other Brassica crops. The results showed that among the 35 tested plants, 20 plants detected vector integration, with an integration efficiency of 57.1%. Target site amplification and enzyme digestion analysis showed that some samples had uncut bands, and further Sanger sequencing confirmed multiple base deletions at the target site, indicating gene editing occurred in hairy roots with an editing efficiency of 25.0%.

A: PB-BpttCas12a vector map; B: CRISPR/Cas12a mediated BoPDS gene editing; C: CRISPR/Cas12a mediated BoARF16 gene editing; D: CRISPR/Cas12a mediated BoBADH2 gene editing. Blue letters represent target sites, red letters represent PAM sequences

In summary, the established hairy root transformation system can be used for rapid evaluation of gene editing vector activity and is applicable to various Brassica crops, providing an efficient and convenient technical platform for gene function research and gene editing.

Title: Establishment and Application of an Efficient Hairy Root Genetic Transformation System in Broccoli

Journal Name: Biotechnology Bulletin

DOI: 10.13560/j.cnki.biotech.bull.1985.2026-0460

URL: https://scholarcast.cast.org.cn/top-news/2102958706797555712