New Advances in Transgenic Research of Brassica Vegetables

Brassica plants include many important vegetable crops, such as Chinese cabbage, green vegetables, cabbage, broccoli, broccoli, etc. These vegetables are closely related to people's daily lives and occupy a certain position in agricultural production. Conventional breeding methods have made great achievements in vegetable breeding, but because of their limitations, they cannot use genetic resources that exist in completely different other species. These resources are difficult to find in close relatives, making it difficult for some breeding goals that people desperately hope. achieve. The application of plant genetic engineering technology can break through the boundaries between species and transfer exogenous useful genes to provide new breeding methods for further improvement of varieties.

Research on transgenic plants began in the late 1970s and early 1980s, when plants were only regenerated from cells transformed with wild-type Ri and Ti plasmids on a few plant species. In 1983 Zambryski et al., De Block et al. (1984) reported that gene transfer of Agrobacterium tumefaciens (At) and Agrobacterium rhizogenes (Ar) with the excision genes were performed to obtain transgenic plants of normal morphology. In 1985, Horsch et al. pioneered the leaf disc method to infect tobacco explants with At to obtain transgenic tobacco. To date, hundreds of genes have been isolated worldwide, more than 120 transgenic plants have been obtained, more than 3,000 transgenic plants have been approved for field trials, and 30 plants have been approved for commercial use by 1998. The importance of plant genetic engineering to agricultural development has drawn worldwide attention.

A variety of gene transformation techniques have been developed such as Agrobacterium-mediated method, gene gun transformation method, PEG-mediated transformation method, electroporation perforation transformation method, microinjection transformation method, laser microbeam transformation method, plant germ cell transformation method. Ultrasound transformation, liposome-mediated transformation, virus-mediated transformation, etc. Among them, Agrobacterium-mediated gene transformation systems are the most widely used, and 80% of the transgenic plants that have been obtained use this transformation technique.

I. Research progress on transgenic technology of Brassica vegetables

In recent years, remarkable progresses have been made in the research of gene transformation of Brassica vegetables, but there are also some problems. The following is the current research status of the plant regeneration receptor system and gene transformation system of Brassica vegetables.
1. Plant regeneration system for in vitro culture of Brassica vegetables

(1) Effect of hormone on adventitious bud induction of explants

In vitro culture of plants, exogenous cytokinin and auxin play an important role in the induction and growth of adventitious buds. With different genotypes, the degree of difficulty in inducing adventitious buds is different, and the requirements for hormone types and ratios are also different. Adventitious bud induction of cabbage, cauliflower is relatively easy, usually in the medium by adding IAA, NAA, IBA and other auxin hormones and with cytokinins 6-BA, ZT, you can get a higher frequency of plant regeneration [1,3 , 5, 6]. The induction of adventitious buds in cabbage and Chinese cabbage is more difficult. Studies have shown that this is determined by their genetic group A chromosomes. Previous studies have used more traditional adenine cytokinins and progressed slowly. Our group chose the phenylurea cytokinin CPPU which has attracted people’s attention in recent years, and the frequency of adventitious bud induction of cabbage and Chinese cabbage was significantly increased [2,8]. Recently, Wang Lingjian et al. used another phenylurea cytokinin TDZ [7] in tissue culture of Chinese cabbage and achieved similar results. The application of phenylurea cytokinin is one of the important reasons for the breakthrough in the gene transformation of vegetables in recent years.

(2) The promotion of adventitious bud induction by AgNO3

As an important plant hormone, ethylene, like other hormones, has an important influence on the induction of adventitious buds in tissue culture. Pau et al. successfully introduced antisense ACC oxidase gene into Brassica juncea by Agrobacterium-mediated method, which resulted in a decrease in ACC oxidase activity in transgenic plants and their offspring explants. Ethylene release in vitro was significantly decreased during the in vitro culture. The frequency of adventitious shoot induction increased dramatically, demonstrating that morphogenesis of adventitious buds is controlled by regulatory genes involved in ethylene synthesis. Studies have reported that it is difficult to obtain regenerated plants from in vitro culture of Chinese cabbage, which is associated with the production of large amounts of ethylene during the explant culture. Silver ions can competitively bind ethylene receptors to eliminate ethylene inhibition of adventitious bud morphogenesis. In turn, the frequency of inducing adventitious buds in vitro was increased. Therefore, in recent years, many researchers in the in vitro culture of Brassica vegetables began to add AgNO3 to the culture medium, which led to a large increase in the frequency of adventitious bud induction of some genotypes of cabbage, Chinese cabbage, and cabbage that were previously difficult to regenerate.[2] ,4,5,7,8].

(3) Explant type and seedling age

Different explant materials, due to the difference in the balance of their endogenous plant hormones, make the frequency of adventitious buds different. For example, Ondre et al.'s test results in tobacco showed that when tobacco plant explants were introduced with the regulatory gene ipt related to cytokinin synthesis, the transformed tissue became a tumory expression tumor; and the regulatory gene iaaM involved in auxin synthesis was introduced. With and iaaH, the transformed tissue shows a root-expressing tumor. So choose

Appropriate explant material is very necessary. Explants of Brassica vegetables in vitro typically include leaves, hypocotyls, cotyledons, and cotyledonary pedicels. According to studies, adventitious buds in cotyledon explants have a high frequency of induction [2,4,7,8] for Chinese cabbage and Chinese cabbage, and hypocotyls for cabbage and cauliflower are generally lower [1,5,6,9]. Seedlings of sterile seedlings are usually suitable for 4-8 days [1-9].

2. Brassica vegetable genetic transformation system

(1) Selection of antibiotics

Co-cultivation of explants with Agrobacterium is the main approach for obtaining transformed plants of Brassica vegetables in recent years (Table 1). After co-cultivation, it is necessary to use antibiotics to kill Agrobacterium, otherwise the over-reproduction of Agrobacterium causes the death of transformed cells at the incision of the explant and the difficulty of adventitious shoot induction. Commonly used antibiotics are cephalosporins and carbenicillin. In the co-cultivation of the explants of Brassica napus with Agrobacterium tumefaciens, Cherest et al. and De Blook et al. observed that cephalosporin has toxic effects on explant materials and inhibits plant differentiation. Our research shows that cephalosporin inhibited the adventitious bud induction of cabbage, cabbage and Chinese cabbage [1,2,7,8] and carbenicillin promoted adventitious bud induction of cabbage and Chinese cabbage[2,8] ], No inhibition of adventitious buds induced on the cabbage hypocotyl [1], therefore in the gene transformation of Brassica vegetables Carbenicillin should be used to kill Agrobacterium. However, carbenicillin is not conducive to rooting [3], so no carbenicillin is added to the rooting medium, or the amount is reduced.

(2) Transformer Screening

The most commonly used screening marker gene in the transformation of Brassica vegetables is the neomycin phosphotransferase gene (NPT II), which confers resistance to kanamycin in transformed cells. Pua et al. used Agrobacterium tumefaciens to transform the longitudinal sections of Brassica napus stalks, and no stem buds were differentiated on the kanamycin selective medium. Therefore, kanamycin may be considered to affect the acquisition of transgenic plants. The results of this research show that kanamycin inhibited the adventitious bud induction of cabbage [2,7], Chinese cabbage [8], and cabbage [1], which affected the development of genetic transformation work. For this purpose, we adopted the first to induce adventitious shoots on a medium containing a lower concentration of kanamycin, and then to increase the concentration of kanamycin during the propagation of adventitious shoots; or after the infection of Agrobacterium In the adventitious bud induction medium without kanamycin, the adventitious buds or seedlings were transferred to the growth medium containing kanamycin after the induction of adventitious buds, and the non-transformed plants were eliminated. This technical measure not only overcomes the obstacles of plant regeneration, but also plays a screening role for marker genes. Using this method, a group of Cana has been obtained from the vegetables Dwarf yellow, Suzhou blue, 973, the black leaves of the middle foot and dwarf black leaves, cabbage Xiafeng 42 and 426, the cabbage heart 23 and the black leaf head. Resistance to transformed plants.

(3) Explants soaked in Agrobacterium solution

The explants of Brassica vegetables are more sensitive to Agrobacterium, and the cells at the wound after infection with Agrobacterium are likely to cause browning due to allergic reactions, which seriously affect the frequency of adventitious bud induction and gene transformation. Wei Zhiming et al. [5] studied cabbage and found that when the hypocotyl segment and the Agrobacterium infection time exceed 5-10 minutes, the hypocotyl segments are brownish to death after transformation, and the frequency of adventitious bud induction is greatly reduced. . Therefore, the 1-5 minute soaking time is commonly used in genetic transformation of Brassica vegetables [1,2,3,5,7,8]. In addition, Wei Zhiming et al. pre-incubated cabbage hypocotyl for 2-3 days before gene transformation, significantly reduced the browning phenomenon, and greatly increased the frequency of adventitious bud induction [5], indicating that the appropriate time of pre-culture is conducive to gene transformation. Increased efficiency.

II. Transgenic Plants of Brassica Vegetables

(1) CpTI gene

The research group carried out the study of transgenic CpTI genes in cabbage [1], Chinese cabbage [8], and green cabbage [2]. In the past three years, it has been obtained from non-heading Chinese cabbage dwarf yellow, Suzhou blue, 973, middle black leaves, dwarf black leaves, Chinese cabbage Xiafeng 42, 426, black cabbage head 23, and black leaf head. Transgenic plants that are resistant to thiram. PCR and Southern blot molecular tests demonstrated that the insect-resistant gene, CpTI, had been introduced into transformed plants. Non-heading Chinese cabbage dwarf yellow, Chinese cabbage cabbage Xiafeng 42, cabbage cabbage heart 23 and black leaf head transgenic contemporary plants showed different levels of insect resistance in the greenhouse insects. The segregation of insect resistance traits was observed in the natural progeny of the field and in the artificial larvae test of the transgenic plants of the Safflower Core 23 transgenic plants (T1 generation), and the strains with the same insect resistance performance were selected from the T2 generation. system. In addition, individual plants with resistance to insect resistance were selected from the progeny (T1 generation) of the progeny of the black leaf, dwarf black leaf, and black cabbage head of transgenic plants of non-heading Chinese cabbage.

Fang Hongxuan et al. (1997)[6] introduced the CpTI gene into the cabbage cultivars Jingfeng and Yingchun. The experiments showed that the kanamycin resistance screening, ELISA detection, NPTII detection, and Southern blot detection results were highly consistent. The results of insect resistance test showed that there were significant differences in the anti-insect effects of the three transgenic clones. Different plants in the same clone were also significantly different. This indicates that the expression regulation mechanism of foreign genes in transgenic plants is complex and needs further study.

(2) Bt gene

Wei Zhiming et al. (1998)[5] introduced the Bt gene into the female parent and the male parent of the cabbage cultivar Xiafeng Cabbage. Southern blot hybridization results showed that most of the genetic transformation of Cabbage was single copy number integration. A few are 2 and 3 copy number integrations.

(3) TI gene

Ding et al. (1998) [9] introduced the TI gene cloned from sweet potato into broccoli and PCR-amplified the DNA of contemporary transgenic plants to obtain a 0.66 kb DNA fragment. Hybridization with a TI cDNA probe demonstrated that these fragments have a TI gene sequence. TI gene expression products with a molecular weight of 24 KD were detected in all 6 transgenic plants. The field resistance test showed that the transgenic plants had better insecticidal effects on the two local pests Spodoptera litura and Plutella xylostella. Genetic analysis of the R1 and R2 generations is ongoing.

(4) MTII gene

Chen Shuhui et al. (1998)[10] isolated the cadmium binding protein gene (MTII) from guinea pigs and transformed it into broccoli using Agrobacterium-mediated method. Southern blot analysis showed that the MTII gene had been introduced into broccoli. The experiment also showed that the promoter was For rbcS, the expression level and cadmium tolerance were higher when the promoter was CaMV35S.

(5) Auxin synthase gene

Jiang Yan et al. (1998)[11] introduced the gene into Brassica oleracea. The number of lateral roots was increased in M2 plants. Adventitious roots occurred earlier and more when axillary buds were cut. A large number of adventitious roots naturally occurred when hypocotyls were cultured in vitro. .

(6) Anti-Black Rot Gene

Liao Fangxin et al. (1998) [12] introduced the gene into cabbage through pollen tube introduction. According to the test, 56 out of 237 offspring showed disease resistance.

(7) Capsid and CaMV genes

Passelegue et al. (1996)[13] introduced the capsid gene and CaMV gene into cauliflower through Agrobacterium-mediated method. The integration of T-DNA in the plant genome has multiple copies, and no virus coat protein was detected in the capsid-transformed plants. .

(8) NPTII gene and GUS gene

These two genes are reporter genes, and some studies have reported the results of transferring the above reporter gene alone. Christey et al. (1997) [14] introduced the NPTII gene into Brassica oleracea, and Takasaki et al. (1997) [15] introduced NPTII and GUS gene into non-heading Chinese cabbage.

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