What research and innovation do people have on pumpkin germplasm resources?

Study on distant hybridization of 1. pumpkin.

Since German Drude (19 17) started the experiment of interspecific hybridization of pumpkin, scholars at home and abroad have been constantly exploring methods and techniques, trying to break the barriers of interspecific hybridization and realize the transfer of excellent quality, insect resistance and stress resistance among different species through conventional interspecific hybridization, somatic hybridization and molecular biology. Although some progress has been made, no ideal or consistent results have been achieved. The hybridization study of five cultivated pumpkins showed that the interspecific relationship of Cucurbita was close and the interspecific hybridization was complicated. See table 17-4 for the test results of some studies.

Table 17-4 results of interspecific hybridization of Cucurbita

A study by Yong Cheng An et al. (200 1) showed that Chinese pumpkins have a high affinity with Indian pumpkins, but an average affinity with gray-seeded pumpkins. F 1 generation is almost sterile, but the affinity with zucchini is the worst. Gray-seeded pumpkin has a certain genetic relationship with Chinese pumpkin, Indian pumpkin and zucchini, among which Chinese pumpkin has the highest genetic relationship. Summing up previous studies, Whitaker T.W.( 1962), an American cucurbitaceae crop expert, thinks that among the annual pumpkin species, Indian pumpkin species are closely related to American pumpkin species, while Indian pumpkin species are far away from American pumpkin species. Chinese pumpkin and Indian pumpkin have high compatibility, and interspecific hybrids can be obtained by crossing them, especially when Indian pumpkin is the female parent. The compatibility between Chinese pumpkin and gray-seed pumpkin is average, and F 1 generation is almost sterile, but the compatibility with American pumpkin is the worst. Gray-seeded pumpkin is closely related to American pumpkin; Perennial pumpkin and black seed pumpkin are far from annual pumpkin, but black seed pumpkin is close to Indian pumpkin and American pumpkin. From the analysis of genetic relationship, the evolution time of five pumpkin varieties may exist in a certain order, and Chinese pumpkin is in the middle stage of interspecific hybridization among annual pumpkins. However, it should be noted that in interspecific hybridization experiments, different varieties of the same species will get different test results, which is one of the reasons for the complicated genetic relationship between different species of pumpkin. 1983 FAO described the genetic relationship among pumpkin varieties in the global report "Cucurbitaceae Plant Genetic Resources", as shown in figure 17-3.

Fig. 17-3 schematic diagram of interspecific hybridization of five cultivated pumpkins.

At present, researchers are interested in combining the excellent quality traits of Indian pumpkin with the insect-resistant traits of Chinese pumpkin through interspecific hybridization technology to cultivate high-quality interspecific hybrids. At present, there are many successful examples, such as Pearson, O.H. et al. (195 1) and the new Tosa series pumpkin bred by Nagaoka Horticultural Seedling Factory in Japan. In addition, there are not many pumpkin germplasm resources with special resistance to diseases and insect pests, and only a few wild pumpkin germplasm resources have disease resistance, such as >: C. Ecuador [high antiviral disease-zucchini yellow mosaic virus, etc. ], > Clostridium Qiao Ben Oki [[High resistance to powdery mildew (>: powdery mildew)],> High resistance to powdery mildew and virus diseases, high resistance to powdery mildew, is a good disease-resistant germplasm resource. Some disease-resistant genes have been successfully transferred into cultivated species by distant hybridization. For example, American researcher Whitaker( 1959) found that the wild species >: C.lundelliana pumpkin can cross with any of the five cultivated species, so it is a good bridge variety of pumpkin. Zucchini and wild disease-resistant varieties >: Zucchini is not easy to cross. In order to transfer disease-resistant genes into pumpkin cultivars, researchers at Cornell University (Thomas W.Whitaker, 1986) have used Chinese pumpkin variety Butternut as a bridge variety. First, they used >: C.martinezii×C.moschata for hybridization, and then crossed the obtained F 1 generation with zucchini, and successfully obtained zucchini materials resistant to powdery mildew and cucumber mosaic virus disease. At the same time, through this method, the high-quality traits and insect-resistant traits of Chinese pumpkin have also been transferred to pumpkin varieties. Australian scholar (Herrington, Master, 2002. ) was successfully transformed into > C. Ecuador and > C. The antiviral gene of' Nigerian' pumpkin germplasm was transformed into pumpkin, and Indian pumpkin varieties Redlands Traiblazer and Dulong QHI and Chinese pumpkin varieties Sunset QHI were cultivated, which were resistant to zucchini yellow mosaic virus (ZYMV), papaya ringspot virus-watermelon strain (PRSV-W) and watermelon mosaic virus (WMV).

In the distant hybridization experiment, pollen sterility or poor seed development often occurs in F 1 generation or early hybridization 2 ~ 3 generations. In order to preserve offspring, backcross method and embryo rescue technology are often combined. Wall J.R.( 1954) obtained the hybrid of Chinese pumpkin and zucchini by embryo culture technology. Wasek R. (1982) also obtained the hybrid offspring of zucchini and >: C. pumpkin. Pearson O.H et al. (195 1) put forward that the problem of pollen sterility can be solved by doubling the progeny of distant hybridization with colchicine. With regard to the difficulty and variability of interspecific hybridization in pumpkin, the research of Wall and York( 1960) thinks that the diversity of gametes contributes to the success of interspecific hybridization, that is, heterozygous genotypes are easier to hybridize than homozygous genotypes. Cucumber in zucchini is usually easier to cross with Chinese pumpkin than other types of zucchini.

Cytological and Molecular Biology of Pumpkin

In the aspect of construction and application of pumpkin plant regeneration system, from Schoroeder( 1968), the pumpkin plant regeneration system (Biserka J.,191; Carol G., 1995) and organogenesis pathway (Ananthakrishnan G., 2003; Krishnan K., 2006), and conducted a lot of research on various conditions affecting the regeneration rate. In vitro megaspore culture; Fujita K.( 1988) and others used Chinese pumpkin (> pumpkin seed house in vitro) as explants, and took materials during and after flowering. Results The frequency of ovule regeneration in flowering stage was significantly higher than that after flowering, which indicated that explants had strong differentiation ability and high regeneration frequency in the active stage of plant growth and development. At the same time, the study also found that pretreatment of ovary at 5℃ for 2 days can promote the occurrence of embryoid. Sheng Yuping et al. (2002) studied the tissue culture and rapid propagation of 1 pumpkin. By comparing the effects of disinfection time, explant source and hormone combination, the tissue culture and rapid propagation technology of pumpkin 1 suitable for production was established. The results show that the best disinfection method is to disinfect the surface with 70% ethanol for 30s, and then disinfect it with mercuric chloride15 min; The bud induction rate of terminal buds is higher than that of cotyledons. The medium suitable for the growth of test-tube seedlings is 2/3 ms+ba 0.5 mg/L, and 1/2MS has the best effect on the formation of adventitious roots.

In pumpkin genetic engineering breeding, it has been reported that antiviral genes were transferred into pumpkin. The effective genes used in pumpkin antiviral genetic engineering mainly come from viruses, and coat protein gene strategy is widely used in pumpkin. It has been reported that the coat protein genes of watermelon mosaic virus (WMV), cucumber mosaic virus (CMV), pumpkin mosaic virus (SqMV) and zucchini macular mosaic virus (ZYMV) of cucurbitaceae crops were transferred into cucurbitaceae crops. In the United States, the coat protein (CP) gene of CMV pathogenic strain has been introduced into commercial varieties of pumpkin and melon, and this resistance is controlled by a dominant single gene. At Cornell University, the CP gene of SqMV- 1 has been introduced into pumpkin, showing high resistance, especially the transformation of WMV coat protein gene into pumpkin strain, which is controlled by a single gene and is not affected by temperature and virus concentration. In addition, the CP gene of ZYMV virus has been transferred to cucurbitaceae crops in the United States. Brent Lowell (1999) and others compared the disease-resistant materials bred by genetic engineering methods and the disease-resistant or disease-tolerant varieties bred by traditional breeding methods with the common susceptible hybrid varieties, and identified the effects of virus diseases through field disease symptom investigation and enzyme-linked immunosorbent assay (ELISA). The results showed that Watermelon Mosaic Virus (WMV) was the most common virus and caused the most diseases. Transgenic varieties show strong disease resistance and high yield, and most transgenic varieties have higher disease resistance and yield than control varieties. The results of enzyme-linked immunosorbent assay (ELISA) showed that coat protein could be detected in some transgenic plants, which was related to plant resistance. Transgenic zucchini plants showed slight symptoms of virus infection after harvesting commercial melons. Pang Shizhong (2000) and others studied the pathogen-mediated resistance pathway and obtained the pumpkin strain carrying the coat protein gene of pumpkin mosaic virus. Three independent strains of R 1 (sensitive, recoverable and disease-resistant) were inoculated in greenhouse, net room and field with SqMV. Almost all resistant lines (SqMV- 127) showed disease resistance in greenhouse and field. The sensitive strain (SqMV-22) showed symptoms and spread to the whole plant. The transcription of foreign genes in CP transgenic plants was analyzed. The results showed that the resistant strain SqMV- 127 showed post-transcriptional silence of CP gene. The evidence is that the transcription level of CP gene in resistant plants is very high, but the accumulation is very small, and CP protein can not be detected. This is the first report of transgenic pumpkin resistance to SqMV.

In recent years, it has been reported that two or more CP genes have been transformed into cucurbitaceae crops abroad. Tricolj D.M and M Fuchs (1995, 1998) studied the resistance of transgenic pumpkins ZW-20 and ZW-20B expressing ZYMV and WMV coat protein genes in Geneva. The results showed that the above two varieties showed a high level of resistance to the mixed inoculation of these two viruses. Five transgenic pumpkin strains expressing CMV, ZYMV and WMV CP genes were tested in the field. The results showed that the transgenic strain C2W-3 expressing three CP genes, CMV, ZYMV and WMV, showed the highest resistance and no systemic infection. ZW-20, a strain expressing ZMV and WMV CP genes, showed a high level of resistance to ZMV and WMV. Three strains expressing CMV single CP gene, ZMV and WMV, were C- 14, Z-33 and W- 164, respectively, showing the same symptoms as the control, but the onset was delayed for 2-4 weeks. It can be seen that the more CP genes expressed in the same plant, the stronger the resistance.

In China, the CP gene of WMV was also transferred to melon, watermelon and cucumber. Transfer CP gene into pumpkin, melon, tomato and pepper; The CP gene of cucumber mosaic virus was transferred into tobacco. But at present, all the domestic reports are the experimental results of CP gene being transferred into a single virus.

In the application of molecular markers in pumpkin, Stachel( 1998) and others used 40 random primers to analyze 20 pumpkin lines (self-bred for 6 generations), among which 34 primers amplified 1 16 polymorphic bands. Through cluster analysis, 20 materials were divided into three categories, which were basically consistent with the traditional classification system. Gwanama et al. (2000) used 16 random primers to conduct RAPD analysis on 3 1 Chinese pumpkin materials collected from Zambia and Malawi, and amplified 39 polymorphic bands. Cluster analysis classified 3 1 germplasm into 4 categories, Malawi into 3 categories and Zambia into 1 categories. The genetic distance of Malawi samples was 0.32 ~ 0.04, and that of Zambia samples was 0.26 ~ 0.04. Li et al. (2000, 2007) analyzed the genetic relationship of 23 domestic and foreign breeding materials and varieties of three kinds of pumpkins by technology, and found that the genome analysis results of three kinds of pumpkins were completely consistent with the traditional taxonomy results, and at the same time, the technology revealed that the genetic relationship of different pumpkin varieties (lines) was basically consistent with their geographical origin and morphological characteristics. At the same time, the research group used BC6 near-isogenic line established by (dwarf Chinese pumpkin× Indian pumpkin )× Indian pumpkin as population, and obtained molecular markers closely linked with dwarf genes of Chinese pumpkin by RAPD technique, with linkage distance of 2.29cM. Li Junli et al. (2005) analyzed the genetic diversity of 70 pumpkin germplasm by RAPD technique. 70 pumpkin germplasm were classified into three categories by systematic cluster analysis: Chinese pumpkin, American pumpkin and Indian pumpkin, which was consistent with the traditional classification results. At the same time, the germplasm was subdivided into three groups, the first group was divided into three groups, the second group was divided into six groups, and the third group was divided into five groups. The results show that there is a certain correlation with geographical origin and morphological characteristics. The results of principal component analysis and systematic cluster analysis are basically the same, but systematic cluster analysis can provide more information in revealing the relationship between closely related individuals. Ferriol et al. (200 1) conducted random primer analysis on 8 pumpkin germplasm, and found that the interspecific genetic distance was obviously greater than the intraspecific genetic distance, which was consistent with the results of classification according to fruit characters.

Thirdly, the innovation of pumpkin germplasm resources.

According to the introduction of (Encyclopedia of Wild Vegetable Horticulture), Japan began the research on the innovation of pumpkin germplasm resources as early as the 1940s, and bred many Indian pumpkin varieties with good quality and early maturity, and Chinese pumpkin varieties with good quality in the middle and late maturity, and also took the lead in the field of rootstock pumpkin research in the world. European and American countries pay more attention to the study of ornamental pumpkins and carved pumpkins (mostly zucchini species), while the study of Chinese pumpkin species (Wesel-Beaver L.,1994; Maynard. N, 1994, 1996) are mostly concentrated on Butternut varieties, and varieties with short vines, semi-short vines, antiviral diseases and powdery mildew have been selected. Before 1980s, the research on pumpkin germplasm innovation in China only stayed at the level of conventional breeding of local varieties. After 1980s, many fresh pumpkin varieties (Zheng Hanpan,1998; Liu Yisheng, 20065438+0; Li, 2006; Jia, 2007; Luo Fuqing, 20065438+0; Qianyi, 200 1), such as honey pumpkin, auspicious 1 pumpkin, Jinghong chestnut pumpkin, short-vine Lv Jing chestnut pumpkin, Jingmi chestnut pumpkin, Li Hong, Jinxing, sweet chestnut and many other high-quality and high-yield hybrids; Large-scale multi-seed pumpkin varieties have been cultivated, such as Meiya Syracuse 1 and Heilongjiang Wuxia (Ji Xinwen, 2002).

In recent years, vegetable breeders at home and abroad have been carrying out research on how to improve pumpkin quality, yield, adaptability, disease resistance and insect resistance, and have made some new progress in germplasm innovation research.

(1) Innovate pumpkin germplasm by conventional cross-selection technology.

In the late 1980s, the innovation of pumpkin germplasm resources made great progress. The Vegetable Research Institute of Shanxi Academy of Agricultural Sciences has successively bred a new pumpkin variety Wuman 1 ~ 4 series. Because of its unique dwarfing characteristics, it is suitable for close planting, easy to manage, good in fruit and high in yield, especially suitable for young pumpkins in some southern provinces and regions, and soon accepted by the market. At the end of 1990s, the vegetable research institute of Hengyang City, Hunan Province successively cultivated four series of early-maturing vegetable pumpkins, namely, Linghao 1, Linghao 2 and Linghao 4. This series of varieties has strong stress resistance, high yield, strong continuous fruit setting, sweet taste, good quality and good storage and transportation resistance. It entered the market in the mid-1970s and developed rapidly in China's domestic market in the 1990s. According to incomplete statistics, the planting area of this variety is as high as 60,000 km^2 per year, and the seed sales and planting area are increasing at the rate of 10% per year. Even Myanmar, Vietnam, the United States and other countries have introduced planting. This variety was approved by Guangdong Crop Variety Approval Committee on 1997. In addition to the above fresh pumpkin varieties, there are also naked pumpkins selected by the Vegetable Research Institute of Shanxi Academy of Agricultural Sciences. Because its seeds have no exocarp, it has become an excellent new Chinese pumpkin variety with both seeds and melon meat. In recent years, China's scientific research level in the innovation research of Indian pumpkin germplasm has also been significantly improved. Most of the innovative germplasm resources are bred by using high-quality germplasm resources introduced from abroad or at home, and many stable, excellent and distinctive inbred lines have been bred through self-crossing, hybridization, backcrossing and multi-generation self-crossing. Then parents are selected according to the breeding objectives, and the varieties needed in production are bred, such as Lu Jing chestnut pumpkin, Ginkgo chestnut, Jixiang 65433 and so on. These studies have played a positive role in improving the level of pumpkin breeding and production in China.

Foreign scholars (Whitaker T.W.,1959; Munger H.M.,1976; Provvidenti. and Robinson R.W.W., 1978) have done a lot of work in disease-resistant transfer and disease-resistant variety breeding of pumpkin. The main methods are multi-generation hybridization, backcross and selfing between cultivated species and disease-resistant wild species, and embryo rescue technology is used to transfer disease-resistant or insect-resistant genes into cultivated species, which greatly enriches and perfects the types of pumpkin germplasm resources and opens up a new way for pumpkin disease-resistant breeding. For example, Contin M.E.( 1978) and Andres T.C.(2000, 2002) reported that researchers at Cornell University in the United States obtained powdery mildew resistance by crossing and backcrossing C.lundelliana (related wild species), C.martinezii and C.moschata (cultivated species) respectively. Through interspecific hybridization, Australian scholars transferred genes of papaya against ringspot virus (PRSV), zucchini against yellow mosaic virus (ZYMV) and watermelon mosaic virus (WMV) from Nigerian local varieties of Ecuador and China to Indian pumpkin varieties and Chinese pumpkin varieties, and bred Redlands Pioneers, Dulong QHI and Sunset QHI. Lebeda A.( 1996) has introduced the cucumber mosaic virus (CMV) resistance gene of C.martinezii into zucchini, and the resistance is partially dominant. The results showed that Whitaker was resistant to ZYMV, cucumber mosaic virus, WMV and powdery mildew.

(two) the use of other technologies for pumpkin germplasm innovation

The combination of biotechnology and conventional selection technology can create new pumpkin specific germplasm, thus improving breeding efficiency and quality.

Obtaining new germplasm from pumpkin by haploid culture technology has not been reported, but Lee Y.K, Abrie A.L and Kwack S.N. (2003,2001,1988) reported that pumpkin seeds and ovules were induced to obtain callus, and then somatic embryos and regenerated plants were obtained. A lot of research has been done on various factors affecting regeneration, such as explants, hormones, genotypes and so on, and a relatively mature method has been formed. Zhao Jianping (1999) successfully obtained tissue culture seedlings of Aisi pumpkin by tissue culture technology. Liu Shuantao and others succeeded in rapid propagation of black-seeded pumpkin seedlings by tissue culture technology. The establishment of this technology laid a foundation for creating new germplasm through genetic transformation, rapid propagation, variety improvement, interspecific hybridization and embryo culture.

Zhang Xingguo et al. (1998) reported that somatic hybrid calli were obtained by fusing cucumber cotyledon protoplasts and Chinese pumpkin and pumpkin seed leaf protoplasts with polyethylene glycol and high calcium and high pH methods. The purpose is to expand the genetic background of the two genera and create new germplasm.

Radiation mutation technology plays an important role in artificially creating new germplasm, which can accelerate the process of artificial evolution, enrich the variation types of organisms and provide more selection opportunities for breeding. According to the report of Li Xiuzhen et al. (1996), the dried seeds of Xiao Ju pumpkin were treated with 60Co-γ rays, and three new excellent strains were selected after five generations.

Pumpkin is one of the important vegetable crops in China. Although some excellent varieties have been obtained by traditional breeding methods, the research depth and breadth of germplasm resources are far behind many other vegetable crops. In the future, we should strengthen the research on molecular marker technology, haploid culture technology and transgenic technology of pumpkin, and pay attention to the combination with traditional breeding technology, so that each pumpkin variety can establish various forms of transgenic system, optimize the molecular marker technology system, accelerate the labeling and cloning of important economic traits genes, and further improve the research level of pumpkin germplasm resources.