Method for obtaining pepper interspecific hybrid seedlings by embryo rescue

Abstract

The application discloses a method for obtaining pepper interspecific hybrid seedlings through embryo rescue, and comprises the following steps: (1) obtaining pepper hybrid embryos, and transferring the pepper hybrid embryos into a germination culture medium to perform germination culture; (2) transferring the pepper hybrid embryos subjected to the germination culture into a subculture medium to perform subculture for 3-4 times, and culturing until the pepper hybrid embryos appear into embryoid; and (3) transferring the pepper hybrid embryos appearing into embryoid into a growth and development culture medium to perform growth and development and rooting culture, and obtaining pepper interspecific hybrid seedlings. The method for obtaining pepper interspecific hybrid seedlings through embryo rescue can rescue the pepper hybrid embryos with fertilization defects through the technical means of embryo rescue, and obtain pepper hybrid seedlings, thereby providing great help for interspecific hybrid culture of peppers.

Description

Technical Field

[0001] This invention belongs to the field of plant in vitro culture technology, and particularly relates to a method for obtaining interspecific hybrid seedlings of pepper by embryo rescue. Background Technology

[0002] Chili peppers (Capsicum) originated in tropical America and belong to the Solanaceae family. They are a widely cultivated vegetable crop with significant economic value worldwide. In recent years, high spiciness and high resistance have become important directions in chili pepper breeding. With the promotion of modern facility agriculture, the cultivation area of ​​chili peppers in China has gradually increased. Among them, the most widely planted chili pepper variety in my country is *C. annuum*, which is characterized by high yield and strong adaptability, but its resistance varies considerably. *C. frutescens* and *C. chinense* varieties combine high spiciness and strong resistance, but their cultivation conditions are greatly limited by region and their yields are very low. Furthermore, research shows that interbreeding among various chili pepper cultivars presents varying degrees of reproductive barriers.

[0003] Interspecific hybridization is a type of distant hybridization that involves the artificial transfer of exogenous genes into cultivated varieties to cultivate superior varieties. It is an effective way to broaden the genetic base of chili pepper cultivars and create germplasm resources. In chili pepper interspecific hybridization, incompatibility exists. This manifests in two main ways: first, the inability to form normal seeds; second, the death or sterility of hybrid offspring, as well as large segregation and slow stabilization. Incompatibility in chili pepper interspecific hybridization is mainly divided into pre-fertilization barriers and post-fertilization barriers. In most distant hybridizations, shriveled or degenerated seeds are usually harvested, a phenomenon considered to be caused by post-fertilization developmental barriers. Embryo rescue technology is the fundamental method to solve the problem of seed degeneration and the inability to successfully obtain hybrid varieties from distant hybrids.

[0004] Embryo rescue technology can effectively create new chili germplasm resources, aggregate more beneficial traits, and broaden the genetic background of major cultivated varieties. However, chili embryo rescue technology is not yet perfect, and the following problems exist: (1) Chili embryo rescue technology has certain reliability issues and cannot guarantee that every embryo can be successfully rescued. (2) Current chili embryo rescue technology is not ideal for some specific varieties or variants of chili and needs further improvement and optimization. (3) Current chili embryo rescue technology still needs to be improved in terms of cost and efficiency, and more economical and efficient methods and approaches need to be found. (4) The lack of promotion and application of chili embryo rescue technology has prevented it from being widely used and recognized. More practice and cases are needed to prove its effectiveness and feasibility. Therefore, it is very important to establish an efficient embryo rescue technology system and improve the success rate of distant hybridization. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the current technology for rescuing chili embryos is not yet perfect. This invention overcomes the shortcomings and defects mentioned in the background section and provides a solution.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A method for obtaining interspecific hybrid seedlings of pepper through embryo rescue includes the following steps:

[0008] (1) Obtain chili hybrid embryos, transfer the embryos into germination medium, and germinate them at 25±2℃ with a photoperiod of 12h.

[0009] (2) Transfer the germinated pepper hybrid embryos to a subculture medium and subculture them three times until embryoids appear in the embryos;

[0010] (3) The embryonic embryos that have appeared are transferred to the growth and development culture medium for growth, development and rooting culture to obtain interspecific hybrid seedlings of pepper.

[0011] This application describes a three-stage culture process for embryos: germination culture, subculture in a subculture medium, and rooting growth in a growth and development medium. Each stage effectively utilizes its culturing properties.

[0012] Germination culture: Overcomes low seed viability and natural sterility, and improves seed germination rate;

[0013] Subculture: Continuous subculturing maintains the growth and reproduction of plants, further expanding the cultivation scale;

[0014] Rooting culture: promotes the growth and development of young plant roots;

[0015] Combining these three steps effectively overcomes the problem of hybrid embryo abortion, yields rare hybrids, breaks seed dormancy, promotes embryo germination, rapidly propagates superior varieties, and shortens the breeding cycle.

[0016] Preferably, the method for obtaining the chili hybrid embryo includes:

[0017] Chili peppers were used as the parents for interspecific hybridization. The two materials were cross-pollinated after entering the full flowering period. Five weeks after pollination, the young fruits were harvested weekly, and the surface of the young fruits was disinfected. The disinfected young fruits were cut open to obtain the chili pepper hybrid embryos.

[0018] Preferably, the hybridization pollination method includes:

[0019] One afternoon before hybridization, select healthy plant buds and bag them; during hybridization, collect fresh pollen from the bagged buds, perform artificial emasculation and hybridization, and re-bag and label them after hybridization.

[0020] Preferably, the surface disinfection method includes: disinfecting the surface of the young fruit with a 75% ethanol solution for 30 seconds, and cutting open the disinfected young fruit with a sterile scalpel.

[0021] Preferably, the parent varieties of the chili pepper are the annual long chili pepper C. annuum 2022M17 as the female parent and the shrub chili pepper C. frutescens XML as the male parent.

[0022] Hybridization can produce more genetic variations, resulting in more mutants and new varieties.

[0023] The paternal parent, C. frutescens, may provide more disease resistance or adaptive genes, enhancing the stress resistance of the hybrid offspring.

[0024] The male parent, C. frutescens, may have higher yield or agronomic traits, and the traits of C. annuum can be improved through hybridization.

[0025] At the same time, genetic differences between the father and mother may lead to instability in hybrid offspring, and some offspring may exhibit undesirable traits.

[0026] C. annuum and C. frutescens may have different reproductive and growth habits, leading to abnormal growth and development in hybrid offspring. These offspring may be infertile, making seed propagation and targeted selection impossible. Therefore, embryo rescue technology is needed to assist in obtaining interspecific hybrid seedlings.

[0027] Preferably, the germination medium is formulated as MS + 25-35 g / L sucrose + 0.4-0.6 mg / L GA3 + 0.04-0.06 mg / L NAA + 5-8 g / L agar, with a pH of 5.8-6.0.

[0028] Preferably, the growth and development medium is formulated as MS + 25-35 g / L sucrose + 0.4-0.6 mg / L GA3 + 0.15-0.25 mg / L TDZ + 5-8 g / L agar, with a pH of 5.8-6.0.

[0029] Preferably, the subculture medium is formulated as MS + 25-35 g / L sucrose + 5-8 g / L agar, and the pH of the medium is 5.8-6.0.

[0030] The above three culture media are optimized and selected specifically for pepper embryos.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) This invention provides a method for obtaining chili interspecific hybrid seedlings by embryo rescue, which can rescue chili hybrid embryos with fertilization defects through embryo rescue technology and obtain chili hybrid seedlings, which greatly helps the interspecific hybridization culture of chili;

[0033] (2) This invention uses an annual chili pepper (C. annuum) as the female parent and a shrub chili pepper (C. frutescens) with high capsaicin content as the male parent for distant hybridization. Combined with embryo rescue technology, high-spiciness germplasm resources are created. Currently, the scheme of this invention has successfully bred hybrid varieties with high capsaicin content, with a spiciness of 175,000 SHU. The hybrid vigor is obvious, and the yield and disease resistance are greatly improved. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The HDLs of annual chili pepper C. annuum 2022M17 and shrub-like chili pepper C. frutescens in Example 1;

[0036] Figure 2 These are photos of embryo rescue cultured embryos and seedlings from Example 1;

[0037] Figure 3 This is a photo of the seedlings of the high-spiciness germplasm that was finally successfully bred in Example 1. Detailed Implementation

[0038] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0039] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0040] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0041] Example 1

[0042] A method for obtaining interspecific hybrid seedlings of pepper through embryo rescue includes the following steps:

[0043] I. Interspecific hybridization

[0044] The annual pepper C. annuum 2022M17 was used as the female parent, and the shrub-like pepper C. frutescens HDL was used as the male parent (images of the female and male parents are shown below). Figure 1 As shown in the figure, both the maternal and paternal parents in this embodiment were provided by the Hunan Provincial Vegetable Research Institute. Interspecific hybridization was conducted using *C. annuum* 2022M17 and *C. frutescens* XML as parents: hybridization and pollination were performed after both materials entered their full flowering period. One day before hybridization, large flower buds from robust plants were selected, bagged with tracing paper, and fresh pollen was collected during hybridization. Artificial emasculation and hybridization were then performed, followed by bagging and labeling.

[0045] Five weeks after pollination, young fruits are harvested once a week. The surface of the young fruit is disinfected with 75% ethanol for 30 seconds, and the young fruit is cut open with a sterile scalpel to remove the embryo.

[0046] II. Obtaining interspecific hybrid seedlings of chili peppers through embryo rescue

[0047] This application selected 100 chili pepper seeds for embryo rescue experiments, and conducted three groups, with 20 seeds cultured in each culture dish.

[0048] (1) Obtain chili hybrid embryos and transfer the embryos into conical flasks containing germination medium. The germination medium is MS + 30 g / L sucrose + 0.5 mg / L GA3 + 0.05 mg / L NAA + 6 g / L agar, pH 5.8. Germination culture is carried out at 25 ± 2℃ and a photoperiod of 12 h.

[0049] (2) The germinated pepper hybrid embryos were transferred to a subculture medium and subcultured 3 times. The subculture medium was MS + 30 g / L sucrose + 6 g / L agar powder, and the pH of the medium was 5.8. The embryos were cultured until embryoids appeared.

[0050] (3) The embryonic embryos that have appeared are transferred to the growth and development medium, which is MS + 30 g / L sucrose + 6 g / L agar powder, and the pH of the medium is 5.8. Growth, development and rooting culture are carried out to obtain interspecific hybrid seedlings of pepper.

[0051] The hybrid combination C. annuum 2022M17 × C. frutescens HDL produces seeds in green fruits, but the seeds are not fully developed. The embryo rescue technique described in this embodiment can be used to obtain seeds that are not fully developed. Figure 2 The healthy interspecific hybrid seedlings shown have a survival rate of 60%.

[0052] III. Creation of High-Spiciness Germplasm Resources

[0053] The cultivation of interspecific hybrid seedlings includes the following steps:

[0054] 1. Select high-spiciness chili germplasm resources as the female parent for hybridization breeding.

[0055] 2. Screen and identify chili germplasm resources, and select individuals with high spiciness as hybrid parents.

[0056] 3. Conduct hybridization combinations of chili germplasm resources to cultivate a large number of hybrid offspring.

[0057] 4. Perform embryo rescue treatment on hybrid offspring, using appropriate hormone combinations to rescue embryos that cannot grow normally and allow them to continue growing.

[0058] 5. Select and breed hybrid offspring that have undergone embryo rescue to identify individuals with high spiciness.

[0059] 6. Conduct offspring trials on the selected high-spiciness individuals, and carry out continuous hybridization and selection to ultimately fix the high-spiciness quality.

[0060] 7. Ultimately, a series of high-spiciness chili germplasm resources were obtained. These high-spiciness germplasm resources include... Figure 3 As shown.

Claims

1. A method for obtaining interspecific hybrid seedlings of chili peppers through embryo rescue, characterized in that, Includes the following steps: (1) Interspecific hybridization was carried out using pepper as the parent. The two materials were cross-pollinated after entering the full flowering period. After 5-6 weeks of pollination, the young fruits were harvested weekly, the surface of the young fruits was disinfected, the disinfected young fruits were cut open to obtain pepper hybrid embryos, and the embryos were transferred to germination medium and cultured under the conditions of 23-27℃ and photoperiod of 10-14h. The formula of the germination medium was MS + 25-35g / L sucrose + 0.4-0.6 mg / L GA3 + 0.04-0.06 mg / L NAA + 5-8 g / L agar, and the pH of the medium was 5.8-6.

0. The parent of pepper was an annual chili pepper as the female parent and a shrub pepper as the male parent. (2) Transfer the germinated pepper hybrid embryos to a subculture medium and subculture 3-4 times until the embryos develop embryoids; the subculture medium is formulated as MS + 25-35 g / L sucrose + 5-8 g / L agar, and the pH of the medium is 5.8-6.

0. (3) The embryonic embryos that have appeared are transferred to the growth and development medium for growth, development and rooting culture to obtain interspecific hybrid seedlings of pepper. The formula of the growth and development medium is MS + 25-35 g / L sucrose + 0.4-0.6 mg / L GA3 + 0.15-0.25 mg / L TDZ + 5-8 g / L agar, and the pH of the medium is 5.8-6.

0.

2. The method as described in claim 1, characterized in that, The hybridization pollination method includes: 10-12 hours before hybridization, select healthy plant buds and bag them; during hybridization, collect fresh pollen from the bagged buds, perform artificial emasculation and hybridization, and re-bag and label them after hybridization.

3. The method as described in claim 1, characterized in that, The surface disinfection method includes: disinfecting the surface of the young fruit with a 75% ethanol solution for 30-45 seconds, and cutting open the disinfected young fruit with a sterile scalpel.

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