Brassica plants, including important vegetable crops like Chinese cabbage, leafy greens, cabbage, broccoli, and cauliflower, play a significant role in daily human life and agricultural production. Traditional breeding methods have contributed greatly to the development of these vegetables, but they are limited in their ability to utilize genetic resources from distantly related species. This limitation has made it challenging to achieve certain breeding goals that are highly desired. The emergence of plant genetic engineering technology has provided new possibilities by breaking down species barriers and enabling the transfer of exogenous useful genes, thus opening up new avenues for crop improvement.
Research into transgenic plants began in the late 1970s and early 1980s. Initially, only a few plant species could be regenerated from cells transformed with wild-type Ri and Ti plasmids. In 1983, Zambryski et al. and De Block et al. (1984) reported successful gene transfer using *Agrobacterium tumefaciens* (At) and *Agrobacterium rhizogenes* (Ar), resulting in transgenic plants with normal morphology. By 1985, Horsch et al. pioneered the leaf disc method, which allowed the transformation of tobacco explants and led to the first transgenic tobacco plants. Since then, hundreds of genes have been identified worldwide, more than 120 transgenic plants have been developed, over 3,000 have undergone field trials, and by 1998, 30 had received commercial approval. The potential of plant genetic engineering to revolutionize agriculture has attracted global attention.
A wide range of gene transformation techniques have since been developed, including Agrobacterium-mediated transformation, gene gun, PEG-mediated, electroporation, microinjection, laser microbeam, plant germ cell transformation, ultrasound, liposome-mediated, and virus-mediated methods. Among these, the Agrobacterium-mediated system is the most widely used, accounting for about 80% of all transgenic plants produced.
### Research Progress on Transgenic Technology of Brassica Vegetables
Recent years have seen significant progress in the genetic transformation of Brassica vegetables, although challenges remain. This section outlines the current status of plant regeneration systems and gene transformation techniques in Brassica crops.
#### 1. Plant Regeneration System for In Vitro Culture of Brassica Vegetables
**(1) Effect of Hormones on Adventitious Bud Induction**
In vitro culture relies heavily on exogenous cytokinins and auxins to induce and promote adventitious bud formation. Different genotypes exhibit varying degrees of difficulty in this process, as well as differing requirements for hormone types and ratios. For example, inducing adventitious buds in cabbage and cauliflower is relatively straightforward when media contain auxins such as IAA, NAA, or IBA combined with cytokinins like 6-BA or ZT. However, cabbage and Chinese cabbage are more challenging due to genetic factors related to their group A chromosomes. Recent studies have shown that phenylurea cytokinins, such as CPPU and TDZ, significantly enhance the frequency of adventitious bud induction in these species, marking a major breakthrough in vegetable gene transformation.
**(2) Promotion of Adventitious Bud Induction by AgNO3**
Ethylene, an important plant hormone, plays a key role in adventitious bud formation. Studies have demonstrated that reducing ethylene levels through the use of silver nitrate (AgNO3) can enhance shoot regeneration. For instance, Pau et al. introduced an antisense ACC oxidase gene into *Brassica juncea*, leading to reduced ethylene release and increased adventitious shoot formation. Similarly, adding AgNO3 to the culture medium has proven effective in improving regeneration rates in difficult-to-regenerate Brassica varieties such as Chinese cabbage and kale.
**(3) Explant Type and Seedling Age**
The choice of explant material and seedling age significantly affects the success of in vitro regeneration. Leaves, hypocotyls, cotyledons, and cotyledonary petioles are commonly used explants. Cotyledon explants generally show higher regeneration frequencies, while hypocotyls tend to perform less well. Seedlings aged between 4 to 8 days are typically most suitable for transformation experiments.
#### 2. Brassica Vegetable Genetic Transformation System
**(1) Selection of Antibiotics**
Co-cultivation of explants with *Agrobacterium* is a common method for obtaining transgenic Brassica plants. After co-cultivation, antibiotics are used to eliminate *Agrobacterium*, preventing overgrowth that could damage the explant. Cephalosporin is toxic to some explants, while carbenicillin promotes adventitious bud formation without inhibiting growth. Therefore, carbenicillin is preferred during transformation, though it may hinder root development, so it is often omitted or reduced in rooting media.
**(2) Transformation Screening**
The neomycin phosphotransferase gene (NPT II) is widely used as a selection marker for transgenic Brassica plants. However, kanamycin can inhibit adventitious bud formation, making it necessary to adjust its concentration during the selection process. Techniques such as low-concentration screening followed by gradual increase or delayed addition of kanamycin after initial regeneration have improved transformation efficiency and ensured the survival of transgenic plants.
**(3) Soaking Explants in Agrobacterium Solution**
Brassica explants are highly sensitive to *Agrobacterium*, and prolonged exposure can cause browning and reduce transformation success. Short soaking times (1–5 minutes) are typically used, and pre-culturing explants for 2–3 days before infection helps reduce browning and improve regeneration rates.
### Transgenic Plants of Brassica Vegetables
Several transgenes have been successfully introduced into Brassica vegetables, including insect-resistant genes such as CpTI, Bt, TI, MTII, and others. These transgenic plants have shown promising results in terms of pest resistance and improved traits.
For example, the CpTI gene was introduced into non-heading Chinese cabbage and other Brassica varieties, resulting in transgenic plants with enhanced resistance to pests. Similarly, the Bt gene was integrated into cabbage, and the TI gene from sweet potato was introduced into broccoli, showing good performance against local pests. The MTII gene, responsible for cadmium binding, was also successfully transferred, demonstrating improved tolerance under stress conditions.
Overall, the development of transgenic Brassica vegetables represents a major advancement in agricultural biotechnology, offering sustainable solutions to improve yield, quality, and resistance to environmental stresses. Continued research and refinement of transformation techniques will further enhance the potential of these genetically modified crops.
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