What innovative research have scientists conducted using Chinese cabbage germplasm resources?

(1) hybridization and distant hybridization

1. Embryo, ovule and ovary culture in vitro

In vitro culture of embryo, ovule and ovary is a common method to overcome the obstacle of distant hybridization of Brassica plants. You Zhu (1978) successfully applied ovary culture to embryo rescue of interspecific hybrids between Chinese cabbage and cabbage for the first time. The interspecific hybrid between Chinese cabbage and white mustard was successfully obtained by ovary culture in Gong (1995).

2. Protoplast fusion

Cell fusion avoids the process of sexual intercourse, so there is no problem of fertilization incompatibility In recent years, great progress has been made in somatic hybridization of higher plants. When sexual hybridization cannot be carried out, interspecific and intergeneric hybrids can be obtained by somatic hybridization. Takeshita et al. (1980) artificially synthesized Brassica napus by protoplast fusion of cabbage and Chinese cabbage. Somatic cell fusion opens up a new way to obtain new materials and varieties from hybrid combinations with distant genetic relationship or extremely low fertilization compatibility.

3. Multi-generation continuous backcrossing

Multi-generation continuous backcross method has certain effect on overcoming hybrid sterility in interspecific or intergeneric distant hybridization. In backcrossing, as for which original parent to backcross, it depends on which parent will retain the excellent genetic traits in the offspring to be backcrossed. If the first backcross is not enough, the second or third backcross can be continued. According to the related research of Chinese cabbage, it is worth explaining that the backcross seed setting strength is related to the hybrid generation and parent type.

4. Induced diploid

Because some distant hybrids do not have homologous genomes or complete genomes, hybrids are completely sterile. Artificial treatment to induce diploid can successfully overcome sterility. If the hybrid seedlings are treated with colchicine, they can produce diploids and successfully overcome sterility. It should be pointed out that not all the sterility of distant hybridization F can be overcome by chromosome doubling. Only when the meiosis of F cannot be paired because the chromosomes from parents are not homologous, and only a few can be paired, doubling the number of hybrid chromosomes will improve the fertility of hybrids.

Chinese cabbage has been widely hybridized between species and genera. Warwick, SI and A.Francis( 1994) listed a large number of examples of distant hybridization in the Guide to Wild German Germplasm and Related Crops in Brasilia.

(2) Cell engineering technology

Haploid has been the goal pursued by plant breeders since the beginning of this century. Haploid can be obtained by isolated microspore culture and anther culture, and then DH plants can be formed. However, compared with anther culture, the isolated microspore population is used for isolated microspore culture, and both embryos and regenerated plants come from microspore cells, which eliminates the interference of anther wall and tapetum tissue. On the other hand, isolated microspore culture technology can obtain microspore embryos and regenerated plants with high embryoid incidence in a wide range of genotypes, and microspore plants have the characteristics of naturally doubling into diploids, so isolated microspore culture has a very attractive application prospect in genetic and breeding research.

In the early 1970s, Nitsch et al. (1973) established the isolated microspore culture technology when studying the anther culture of Datura stramonium. Lichter( 1982) was first obtained in the isolated microspore culture of Brassica napus, and its application in Chinese cabbage breeding has become more and more mature in the past 20 years. At the end of 1980s, Sato et al. (1989) first conducted isolated microspore culture of Chinese cabbage. In the early 1990s, Cao Mingqing and others (1992) took the lead in microspore culture of Chinese cabbage in China. At present, several units in China have carried out research work in this field and successfully applied it to breeding practice. Li Genyi et al (1999; In 2000, excellent new varieties Baiyu 1 1 and Baiyu 7 were bred by isolated microspore culture technology. Cao Mingqing et al. (1993) used isolated microspore culture technology to obtain herbicide-resistant Chinese cabbage plants. At present, the microspore culture technology of Chinese cabbage has become a conventional technology to innovate germplasm resources and plays an increasingly important role.

Isolated microspore culture technology can also be used to screen various resistant mutants. Akmad et al. (199 1) treated early-maturing rapeseed microspores with ultraviolet radiation, and obtained a small number of offspring with enhanced resistance to Alternaria alternata and herbicide "CleanR". Cao Mingqing's research group once added Alternaria toxin to the culture medium. Therefore, microspore plants of Chinese cabbage with certain resistance to black spot disease were screened from the induced microspore embryos.

(3) Genetic engineering technology

With the establishment and continuous improvement of tissue culture and DNA recombination technology, modern biotechnology has been widely used in germplasm innovation and new variety breeding of many crops. However, due to the difficulty of tissue culture and regeneration system of Chinese cabbage, the application of transgenic technology in Chinese cabbage breeding has been limited to some extent. Since 1980s, the system of tissue culture and high-frequency plant regeneration of Chinese cabbage has been gradually established, and some breakthroughs have been made in transgenic research on this basis.

Yang Guangdong et al. (2002) introduced modified cowpea trypsin inhibitor gene (sck) into Chinese cabbage inbred line GP- 1 1 and hybrid No.4, and obtained kanamycin-resistant plants. PCR detection and Southern hybridization confirmed that sck gene had been integrated into Chinese cabbage genome. The activity detection of cowpea trypsin inhibitor showed that most transgenic plants had certain inhibitory activity on bovine trypsin, while the control non-transgenic plants had very low inhibitory activity. In vitro leaf feeding and field natural resistance identification further proved that transgenic plants had certain resistance to Pieris rapae.

Zhu Changxiang et al. (200 1) studied the factors affecting plant regeneration and gene transformation frequency of Chinese cabbage with the petiole of Fukuyama Bald. On this basis, the efficient regeneration system and efficient gene transformation system of Chinese cabbage were established, and the CP gene of turnip mosaic virus was introduced into Chinese cabbage to obtain transformed plants. PCR detection and Southern blot analysis proved that TuMV-CP gene had been integrated into Chinese cabbage genome. North blot analysis and ELISA showed that TuMV-CP was effectively expressed at transcription and translation levels. The genetic analysis of T generation of transgenic plants showed that the separation of foreign genes in the offspring of transgenic plants followed the law of 3: 1. The determination of disease resistance showed that transgenic plants had obvious anti-virus infection ability.

Liu Commune et al. (1998) obtained herbicide-resistant transgenic plants from microspore embryoids of Chinese cabbage. Cotyledon embryoids obtained from microspore culture of Chinese cabbage were rubbed with broken glass slag, then cultured with Agrobacterium tumefaciens, and several green seedlings were regenerated on the medium containing screening agent Basta. After self-pollination, the identification of Basta resistance of its offspring showed that there was an insertion point of bar gene in the genome of the resistant plant, and the microspores of the transformed plant were re-cultured. The segregation rate of Basta resistance in microspore plants of offspring showed that transgenic plants were hybrids.