In view of the lack of domestic cauliflower germplasm resources, it is the most economical and effective way to obtain new germplasm by directly introducing excellent first-generation hybrids from abroad and screening excellent germplasm resources through self-purification. At present, domestic cauliflower breeding units have used this method to separate a large number of new germplasm resources and inbred lines, and used these germplasm resources to breed many excellent new cauliflower varieties, such as Baifeng, Jin Xue 88, Yunshan 1, Fenghua 60 and Xiahua 6.
(b) intraspecific hybridization creates new types.
Different subspecies or varieties of cabbage are easy to cross, and excellent germplasm resources and even new varieties can be created through intraspecific hybridization. Sun Deling et al. (2002) used the hybridization of broccoli, broccoli and purple broccoli, and purple broccoli and broccoli. The single bulb of its offspring is greatly improved, close to broccoli, with a color between the male parent and the female parent, while the vitamin C content is close to broccoli, which is 22% ~ 60.6% higher than broccoli, and the total sugar content is 9.8% ~ 33. 1% (table1-/kloc-0
Table 1 1- 1 Contents of Total Sugar and Vitamin C in New Broccoli
(C) Biotechnology and innovation of cauliflower germplasm resources
Conventional breeding has played a great role in the innovation of cauliflower germplasm resources, but usually the beneficial genes that can stabilize inheritance are narrow or lacking; Most beneficial genes are controlled by many minor genes, so it is difficult to select these genes and determine the genotypic differences. With the development of biotechnology, we can improve the pertinence of breeding on the basis of traditional breeding, overcome some problems in conventional breeding, and further broaden the innovation and utilization of beneficial germplasm resources. At present, more and more researchers begin to pay attention to the innovation of germplasm resources by biotechnology.
1. Cell Engineering and Broccoli Germplasm Resources Innovation
(1) Techniques of anther culture and isolated microspore culture In the early 1960s, Guha and Maheshwari pioneered the method of inducing haploid by anther culture. Since then, anther culture has become one of the important ways to induce haploid and has been applied in crop breeding. In cauliflower, Wang Huaiming (1992) studied the embryogenesis in cauliflower anther and pollen culture, and observed the process of pollen grains developing into embryoids in anthers and the chromosome ploidy of regenerated plants. Zhang Xiaoling (2002) and others think that magnetic field pretreatment can obviously improve the induction rate of anther culture callus. Chen (2004) used five cauliflower varieties as materials for anther culture to obtain regenerated plants and seeds.
Because the method of anther culture can't rule out the possibility that regenerated plants come from somatic cells, the research on obtaining regenerated plants from anther culture has been slow for many years. The method of isolated microspore culture can solve this problem well, so the method of isolated microspore culture to obtain regenerated plants has been paid more and more attention. At present, this technology has been successfully applied to Chinese cabbage, non-heading Chinese cabbage, heading Chinese cabbage, kale, Brussels sprouts, kale, kohlrabi, leaf mustard, turnip cabbage and cauliflower. Vegetable Research and Engineering Center of Beijing Academy of Agriculture and Forestry Sciences, Horticulture Research Institute of Henan Academy of Agricultural Sciences, Tianjin Kerun Vegetable Research Institute and other units have successively carried out isolated microspore culture of cauliflower, initially established a technical system of isolated microspore culture of cauliflower, obtained DH strains of some varieties of cauliflower, and cultivated excellent new varieties of cauliflower.
DH pure lines can be obtained quickly and effectively by isolated microspore culture. DH strain has stable genetic characteristics and can obtain randomly arranged gametes from its parents. Because isolated microspore culture can quickly homozygous heterozygous parents, it can screen specific traits controlled by multiple genes in one step, obviously improve the selection probability and accelerate the breeding process. Geng Jianfeng et al. (2002) bred a cauliflower DH hybrid "Yuxue 60" with early maturity, heat tolerance, white flower head, good quality and strong disease resistance by using two self-incompatibility lines produced by isolated microspore culture. Sun Deling (2002) combined isolated microspore culture with conventional techniques to innovate germplasm resources and DH strain materials.
Isolated microspore culture technology has also been applied to distant and interspecific hybridization breeding of Brassica plants. Shi et al. (1993) obtained isolated microspore embryos and regenerated plants from interspecific hybrids between Brassica napus and Orychophragmus violaceus, Brassica napus and Brassica juncea, respectively, which opened up an innovative germplasm resource way for distant and interspecific hybrid breeding of Brassica plants.
(2) Protoplast fusion technology Protoplast fusion, also called somatic cell fusion, is a hybrid of two protoplasts. It is not the combination of male and female gametes, but the fusion of somatic cells and complete genetic material. It can break the sexual isolation and cross incompatibility between species, thus widely polymerizing various excellent genes, significantly increasing the variation range and creating new germplasm resources. Therefore, this technology has been paid more and more attention by genetic breeders. Since Carlson et al. obtained the first tobacco somatic hybrid plant in 1972, the technical system has been continuously improved and developed, and cell fusion has been successful in many species. In the mid-1980s, it was reported that somatic hybrid plants were obtained from 65,438+05 intraspecific combinations, 38 interspecific combinations and 65,438+03 intergeneric combinations. By the 1990s, regenerated plants were obtained by adding 65,438+04 intraspecific hybrids, 62 interspecific hybrids and 47 intergeneric hybrids, and two cytoplasmic hybrids were differentiated. The hybrid plants obtained in Brassica of Cruciferae are Arabidopsis thaliana, Brassica napus, Brassica napus+Brassica napus and Brassica napus+cauliflower.
The difference between protoplast fusion technology and conventional sexual hybridization is that somatic hybridization has no meiosis, and protoplast fusion of two diploid cells produces tetraploid hybrid plants, while sexual hybridization with the same parent only produces diploid hybrids.
Cytoplasmic hybrids can be obtained by protoplast fusion, which provides a new breeding way for cultivating cauliflower varieties such as cytoplasmic male sterility and herbicide resistance. At present, the main types of cytoplasmic male sterility in cauliflower transferred by sexual hybridization and backcross are Ogura cytoplasmic male sterility and Polima cytoplasmic male sterility. However, it usually takes 6 ~ 1 1 year to transfer cytoplasmic male sterile genes by conventional breeding. Protoplast fusion technology can overcome the problems of age-old or cross incompatibility caused by sexual backcross transfer, and open up a new way for the effective utilization of cauliflower heterosis. Hui Zhiming (2005) studied the transfer of cytoplasmic male sterility of Xiaocang radish to cauliflower by protoplast fusion technology, and obtained interspecific somatic hybrid plants of cauliflower and Xiaocang radish cytoplasmic Brassica napus. Therefore, protoplast fusion technology has been applied to the research field of cauliflower sterility, and a large number of male sterile plants have been obtained, which is an effective way to cultivate new male sterile germplasm.
Protoplast fusion can overcome the incompatibility of distant hybridization and transfer the stress resistance of wild varieties. Cauliflower production is often threatened by pests and diseases, but due to long-term artificial cultivation and directional selection, the disease-resistant and stress-resistant genes in existing breeding materials have become narrower and narrower, which is far from meeting the needs of further improving the multi-resistance of varieties to diseases and adversity. Strengthening the utilization of excellent resistance genes in wild materials is an effective way to further innovate basic breeding materials. Wild vegetables have formed a high degree of disease resistance under long-term natural selection. Through distant hybridization with wild type, the disease resistance and stress resistance of existing varieties can be greatly improved, but distant hybrids usually show incompatibility, which seriously limits their application in variety improvement. Asymmetric hybrids obtained by protoplast fusion technology can overcome the incompatibility of distant hybridization and transfer the resistance of wild species. Yao Xingwei (2005) used asymmetric protoplast fusion technology to transfer the stress resistance of wild species to cauliflower (the donor kale has excellent characteristics such as high photosynthetic efficiency, resistance to white rust, aphid, black spot and salt tolerance), and obtained 17 hybrid through * * experiment, and its stress resistance is being further identified. It can be seen that breeders pay more and more attention to the exploration and utilization of wild resources, and the combination of cell engineering and molecular biology in biotechnology is an effective way to utilize wild resources at this stage.
Using protoplast fusion technology can transfer the excellent quality traits of some varieties, which provides a new way to improve the nutritional quality of cauliflower. High yield and high quality of vegetables have always been the goal pursued by people. P.S.Jourdan( 1989) used cauliflower and herbicide-resistant Brassica napus for protoplast fusion test, and obtained hybrid plants with high herbicide-resistant characteristics. B.Navratilove et al. (1997) used cauliflower and rhizobia-resistant horseradish as experimental materials, and obtained somatic hybrid plants of cauliflower and horseradish by protoplast fusion technology. Hu (2002) used Brassica napus and Orychophragmus violaceus with high contents of linoleic acid and palmitic acid to carry out somatic cell fusion test. The hybrid plants obtained by protoplast fusion test not only increased linoleic acid and palmitic acid content, but also significantly decreased erucic acid content, which significantly improved the variety quality.
2. Molecular marker technology and innovation of cauliflower germplasm resources
Using easily identifiable genetic markers to assist selection is an important means to improve selection efficiency and reduce breeding blindness. The rapid development of molecular marker technology in recent 20 years provides a new way for crop breeding. Using DNA molecular markers can make early selection, improve the accuracy of selection and breeding efficiency, and help shorten the breeding cycle.
Self-incompatibility of self-incompatibility lines screened by (1) molecular markers is an important genetic characteristic of cross-pollination fertilization and genetic recombination in higher plants. Scholars at home and abroad have done a lot of research on the genetic mechanism of self-incompatibility According to Lewis( 1979), self-incompatibility was found in 74 families of angiosperms. The study of screening self-incompatibility lines by molecular markers has also been reported in China. Huang (200 1) obtained the differential fragments related to cauliflower self-incompatibility by analysis. Song Lina (2005) used RAPD and ISSR molecular markers to separate linkage markers to determine self-incompatibility.
(2) Identification of disease-resistant germplasm resource Zhang Feng (1999) Using AFLP technology, four markers closely linked with black rot resistance genes were screened from a pair of near-isogenic lines of cauliflower with black rot resistance and susceptibility. Liu Song (2002) used the near allelic lines C7 12 and C73 1 of Tianjin Kerun Vegetable Research Institute as materials to screen out the RAPD marker OP224/ 1600 linked with cauliflower black rot resistance gene RXC, and transformed it into a stable SCAR marker, which can quickly and accurately screen resistant materials. Grain Rain (2007) used the disease-resistant and susceptible near isogenic lines of cauliflower as experimental materials, and obtained three molecular markers related to the disease-resistant genes by ISSR: ISSR 1 1000, ISSR2 1500 and ISSR1870. In addition, the differential expression of cauliflower strains resistant to black rot was analyzed by cDNA-AFLP technique, and a gene fragment related to black rot resistance was preliminarily obtained, which was confirmed to be an inducible gene fragment related to signal transduction of induced systemic resistance (ISR). In addition, two homologous sequences RGA330-7 and NBS5-100 were obtained from the resistant strains by homologous sequence candidate gene method and nbs map method. Sequence analysis showed that these two fragments may be related to the black rot resistance gene of cauliflower. In addition, protein sequences predicted by two RGAs were compared with protein sequences of seven known plant disease resistance genes, and a phylogenetic tree was constructed. The clustering results show that the two RGA fragments obtained in this study should belong to non-TIR-NBS-LRR type. Finally, the changes of cytosine methylation level and methylation pattern in the genome before and after pathogen stress were analyzed by epigenetics, and the molecular mechanism of resistance to black rot was discussed from the perspective of gene expression regulation.
(3) Using molecular marker technology to detect genetic variation can occur not only in the whole genome, but also in specific genes or gene clusters, structural genes, regulatory genes and single nucleotides. Mutation can be induced spontaneously or artificially in cultured plant cells without mutagen treatment. The mutation frequency is generally 10-5 ~ 10-8. When treated with mutagen, it can be increased to 10-3, but mutagen often causes side effects such as decreased fertility. Leroy (2000,2001) and others used cauliflower hypocotyl for tissue culture, and ISSR method was used to detect the interplant polymorphism of callus formation, cell proliferation and regenerated plants after seedling formation. It is considered that the regenerated plants induced by tissue culture have genetic polymorphism, which proves that tissue culture can induce and screen genetic variation.
(4) Screening cauliflower male sterile germplasm resources by molecular marker technology. Plant male sterility is a genetic phenomenon that can not produce viable pollen, which exists widely in the plant kingdom. Male sterility has been found in 6 17 varieties or interspecific hybrids of 320 species in 43 families, which is of great value in the utilization of crop heterosis.
Wang (2006) used cauliflower male sterile variety A and restorer line B as materials, amplified by PCR with primer +/P6-, and found a 300bp differential fragment, which can be used as a molecular marker to identify male sterile lines. Wang (2005) obtained the cauliflower cytoplasmic male sterility related gene kndx6 12 by searching the nucleic acid and protein database. The preliminary results showed that the cytoplasm of sterile cauliflower used in the experiment may also be Ogura type, which provided conditions for further research and utilization of cauliflower male sterility gene at molecular level.
(5) Construction of genetic linkage map of cauliflower and its application in breeding. Genetic linkage map refers to a chromosome linear linkage map with chromosome recombination exchange rate as the relative length unit and genetic markers as the main body. Molecular marker genetic linkage map indicates the relative position of DNA fragment corresponding to each marker on chromosome, which is the basis of the application of molecular markers in crop genetics and breeding. The theoretical basis of constructing molecular marker linkage map is chromosome exchange and recombination. Since 1986, molecular genetic maps, namely molecular marker linkage maps, have been established for major crops, providing a technical platform for gene location, gene cloning and assisted selection for crop design and breeding. It has played a very important role in the fields of genetic theory, functional genomics and genetic breeding.
Li (200 1) and others used SRAP and AFLP techniques to locate the RI of 86 cabbage× cauliflower. The map consists of 130 SRAP markers and 120 AFLP techniques. These markers were evenly distributed in 9 linkage groups, covering 2 165cM. Grain Rain (2007) used AFLP and NBS mapping methods to construct the first genetic linkage map of cauliflower with F2 as the mapping population. The map includes 9 linkage groups, the total length of linkage groups is 668.4cM, and the average distance between adjacent markers is 2.9cM. Among 234 AFLP markers and 265,438+0 NBS markers, NBS markers are distributed in 8 linkage groups and arranged in clusters in the genome. This map is helpful to further obtain resistance genes by providing possible resistance gene sites. At the same time, the distribution and composition of RGA in the whole cauliflower genome were studied, which also provided reference for understanding the distribution and evolution of resistance genes. It can be further used in molecular marker-assisted breeding.
(6) Innovation of cauliflower germplasm resources with different colors In recent years, many new varieties that are difficult to obtain by traditional gardening techniques have been obtained by using genetic engineering technology, such as purple, white and three different colors of Petunia embedded in purple and white. However, these technologies usually need to know the related genes to obtain the cDNA of the target gene, and then introduce these foreign genes into the target plant to change the color and pattern. Crisp et al. put forward a model from the genetic performance of the hybrid offspring of the white-flowered variety and the green variety: Wiwi gene controls white to be dominant to yellow, and relies on the dominant gene gr 1gr2 to be green. Dickson reported that the cauliflower variety PI 1832 14 introduced from Egypt is pure white even if it is completely exposed to sunlight. It is thought to be controlled by 2 or 3 pairs of dominant genes. Singh and others reported that two pairs of genes can cover the leaves of the flower ball and prevent the flower ball from changing color due to sunlight. Li Ling et al. (2000) studied the near-isogenic lines of cauliflower and yellow flower, and screened a specific band of white flower strain, which was preliminarily identified as unique to white flower strain by Northern dot blot hybridization. At the same time, the mRNA of near isogenic lines of cauliflower and yellow flower was analyzed by Smart cDNA-AFLP silver staining technique, in which two pairs of primers had three polymorphic bands between the two expression gene libraries, one of which was isolated from the white flower strain * * * *; The differential fragment of a white flower strain was screened out, which laid the foundation for cloning color-related genes.
3. Transgenic technology and innovation of cauliflower germplasm resources
The development of transgenic technology is of great significance to accelerate the innovation of cauliflower germplasm resources. At present, a large number of new cauliflower varieties with male sterility, disease resistance, insect resistance and good quality have been bred, which has produced great social and economic benefits.
(1) Germplasm resource innovation of cauliflower male sterility In recent years, the research on cauliflower male sterility has made progress, and some genes or chimeras related to infertility have been found. This laid a foundation for further clarifying the molecular mechanism of cauliflower male sterility and guiding the cultivation of new sterile lines. Bhalla( 1998) integrated the pollen-related gene Bcp 1 into the plasmid PBI1kloc-0/,and transformed it into the cotyledons of cauliflower by Agrobacterium tumefaciens, thus obtaining a new cauliflower germplasm with 50% pollen sterility.
(2) The innovation of cauliflower disease-resistant germplasm resources has also been reported in the cloning and transfer of cauliflower disease-resistant genes. Zhang Guihua et al. (200 1) took the cauliflower variety Chunqiu as the experimental material, and obtained the screened and transformed cauliflower under the mediation of Agrobacterium tumefaciens strain GV31kloc-0/carrying CaMV Bari- 1 gene VI.
There are two kinds of exogenous genes commonly used in transgenic insect-resistant research of cauliflower: endotoxin (Bt) gene and cowpea trypsin inhibitor (CpTI) gene, which have been successfully transformed into cauliflower, providing valuable experience for innovating cauliflower germplasm resources by transgenic technology.
Hua (1992), Cai Rongqi (2000), (2002) and Zhou Huanbin (2003) all used Agrobacterium-mediated method to transform Bt gene into cauliflower and successfully obtained transgenic plants.
CpTI gene belongs to Bowman-birk serine protease inhibitor, which can inhibit trypsin activity in midgut of many pests including Lepidoptera, Coleoptera and Orthoptera, and has broad-spectrum insect resistance. Lu Lingling (2004) integrated cowpea trypsin inhibitor (CpTI) gene into cauliflower genome by Agrobacterium tumefaciens to inhibit the growth and development of LEPIDOPTERA caterpillar. Xu Shuping (2002) introduced Bt gene and cowpea trypsin inhibitor gene (CpTI) into cauliflower through Agrobacterium-mediated genetic transformation to obtain transgenic cauliflower plants. Ding (1998) and others transformed the insect-resistant gene TI isolated from local sweet potato into cauliflower by Agrobacterium-mediated method. The results showed that the insect-resistant effect of transgenic plants was more obvious than that of control plants.
(3) Research Progress on Mutant Genes Related to Flowering Head Traits of Cauliflower Bowman( 1993) et al. first discovered cauliflower with flowering head mutation in Arabidopsis thaliana. Subsequently, Kempin Sa (1995) and others isolated two genes related to meristem activity of flowers from Arabidopsis thaliana, cauliflower and apetalous 1. Studies have shown that their functions are transcription factors. At the same time, the homologous gene of this gene in cauliflower varieties was studied, and it was found that the homologous gene in cauliflower was nonfunctional. This shows that the formation mechanism of cauliflower fleshy inflorescence morphology is closely related to this gene. Purugganan(2000) and others studied the polymorphism of CAL gene in wild-type and cultivated cauliflower, and found that there were differences between wild-type and cultivated cauliflower, and there was mutation in exon 5 of the gene in cultivated cauliflower. Lee B.Smith(2000) studied the origin and evolution of cauliflower bulbs by isolating two recessive alleles, BoCAL and BoAP 1, and obtained the genetic model of flower bulb development. It is considered that there is a strong correlation between BoCAL-a allele and the morphology of discrete inflorescences. The above results showed that the mutation of CAL gene inhibited the development of floral meristem, which was the genetic basis of cauliflower bulb formation. Zhao Sheng et al. (2003), Cao (2003) and (2000) transformed the BoCAL gene into cauliflower, and the transgenic cauliflower could not form a flower head, which confirmed that the foreign gene BoCAL could partially compensate for the loss of BoCAL gene function and partially restore the flower head phenotype of cauliflower. Therefore, by controlling the mutation degree and gene expression level of BoCAL gene, the appearance time and development speed of flower heads can be adjusted, thus providing a new way for cultivating new cauliflower varieties with high hardness.
In production practice, the changes of endogenous hormones and nutritional components after the flower ball is harvested lead to the gradual decline of internal quality, which seriously affects the commodity and edible nutritional value of the product. The senescence of bulbs is first manifested in the loss of chlorophyll in sepals. The synthesis of ethylene has a causal relationship with the loss of chlorophyll and subsequent yellowing. ACC oxidase (ACO) is the rate-limiting enzyme for ethylene synthesis, and the expression of ACO gene regulates the rate of ethylene production. Regulating the expression of ACO gene can delay the production of ethylene, which has been successful in many crops. Chen Yinhua (2005) designed a pair of degenerate primers according to the amino acid sequence of ACC oxidase in several closely related crops, and obtained a candidate fragment of 1202bp from cauliflower genome, thus obtaining a new cauliflower anti-aging material.