Brassica plants, which include a wide range of important vegetable crops such as Chinese cabbage, leafy greens, cabbage, broccoli, and cauliflower, play a vital role in daily human life and are significant in agricultural production. While traditional breeding methods have achieved considerable success in improving these crops, they are limited in their ability to utilize genetic resources from completely different species. These valuable genes are often difficult to access even within closely related plants, making it challenging to achieve certain key breeding goals. The development of plant genetic engineering has provided a breakthrough by enabling the transfer of exogenous genes across species, offering new possibilities for crop improvement.
Research on transgenic plants began in the late 1970s and early 1980s, initially focusing on a few plant species where cells could be regenerated after being 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. introduced the leaf disc method to infect tobacco explants with At, leading to the first transgenic tobacco plants. Since then, over hundreds of genes have been identified globally, more than 120 transgenic plants have been developed, over 3,000 have undergone field trials, and by 1998, 30 had received commercial approval. This progress has drawn global attention to the importance of plant genetic engineering in agriculture.
A variety 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 approximately 80% of all transgenic plants produced so far.
**Research Progress on Transgenic Technology in Brassica Vegetables**
Recent years have seen significant advancements in the genetic transformation of Brassica vegetables, although challenges still remain. Below is an overview of the current state of research on the plant regeneration and gene transformation systems in Brassica vegetables.
**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, along with differing requirements for hormone types and ratios. For example, cabbage and cauliflower show relatively high regenerability, often requiring auxins like IAA, NAA, or IBA combined with cytokinins such as 6-BA or ZT. However, the induction of adventitious buds in cabbage and Chinese cabbage is more challenging, often linked to genetic factors associated with chromosome group A. Recent studies have shown that the use of phenylurea cytokinins, such as CPPU and TDZ, significantly enhances the regeneration frequency in these species.
(2) **Role of AgNO3 in Enhancing Adventitious Bud Induction**
Ethylene, an important plant hormone, plays a crucial role in regulating adventitious bud formation. Studies have demonstrated that reducing ethylene levels through genetic manipulation or the addition of silver nitrate (AgNO3) can significantly improve regeneration efficiency. For instance, adding AgNO3 to the culture medium has led to increased adventitious shoot formation in previously recalcitrant genotypes of cabbage and Chinese cabbage.
(3) **Impact of Explant Type and Seedling Age**
The type of explant and the age of the seedling also influence the success of in vitro regeneration. Leaves, hypocotyls, cotyledons, and cotyledonary petioles are commonly used explants. Research indicates that cotyledon explants generally show higher regeneration rates compared to hypocotyls. Additionally, seedlings aged between 4–8 days tend to yield better results.
**2. Genetic Transformation System in Brassica Vegetables**
(1) **Antibiotic Selection**
Co-cultivation with Agrobacterium is the primary method for transforming Brassica vegetables. After co-cultivation, antibiotics such as carbenicillin are typically used to eliminate Agrobacterium, preventing its overgrowth and subsequent damage to the explant tissue. Carbenicillin has been found to promote adventitious bud formation without inhibiting regeneration, unlike cephalosporin, which may suppress it.
(2) **Transformation Screening**
The neomycin phosphotransferase gene (NPT II) is widely used as a selectable marker. However, kanamycin, which is commonly used for selection, can inhibit adventitious bud formation. To overcome this, researchers have adopted strategies such as using lower concentrations of kanamycin during initial regeneration and gradually increasing the concentration later. This approach not only improves transformation efficiency but also ensures effective screening of transgenic plants.
(3) **Soaking of Explants in Agrobacterium Solution**
Exposure time to Agrobacterium is critical. Prolonged exposure can lead to browning and reduced regeneration. Therefore, soaking explants for only 1–5 minutes is standard practice. Pre-culturing explants before transformation has also been shown to reduce browning and enhance regeneration success.
**Transgenic Plants of Brassica Vegetables**
Several transgenes have been successfully introduced into Brassica vegetables, including CpTI, Bt, TI, MTII, auxin synthase, anti-black rot, capsid, CaMV, NPTII, and GUS genes. These modifications have improved resistance to pests, diseases, and environmental stresses, demonstrating the potential of genetic engineering in crop improvement. For example, transgenic lines resistant to thiram and insect pests have been developed, and some have shown promising performance in both greenhouse and field trials. Ongoing studies continue to explore the expression and stability of these transgenes across generations.
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