Pepper distant hybrid breeding method using embryo rescue and application thereof

Abstract

The application relates to the field of plant breeding, and provides a pepper distant hybridization breeding method adopting embryo rescue, which comprises the following steps: respectively planting a female parent line pepper, a male parent Yunnan shan pepper and a bridge parent pepper ZY24; collecting Yunnan shan pepper pollen and pepper ZY24 pollen, mixing and preparing to obtain mixed pollen; pollinating the mixed pollen to pistil stigma of the female parent line pepper; taking the pollinated young embryo, inoculating the young embryo in an embryo induction medium for in-vitro culture, transferring the young embryo to a subculture medium after cotyledon growth, transferring the seedling to a rooting culture medium after the seedling grows healthily, and transplanting after acclimatization. The above pepper distant hybridization breeding method adopting embryo rescue significantly improves the fruit setting rate, embryo acquisition rate and seedling rate of pepper distant hybridization by controlling the planting environment of the parents, adopting mixed pollen pollination of the bridge parent, optimizing the formula of the embryo induction medium and cooperating with gibberellin spraying treatment, and provides an efficient, stable and repeatable technical means for pepper germplasm resource innovation.

Description

Technical Field

[0001] This application relates to the field of plant breeding technology, specifically to a method for distant hybridization breeding of peppers using embryo rescue and its application. Background Technology

[0002] chili( Capsicum spp . Long-term artificial breeding has led to a narrowing of the genetic base of cultivated chili peppers, resulting in slow progress in improving important traits such as disease resistance and stress tolerance. Wild chili pepper resources contain abundant excellent genes for disease resistance, insect resistance, and stress tolerance; introducing these genes into cultivated varieties through distant hybridization is an important direction for chili pepper breeding. (Capsicum genus) Capsicum It contains approximately 41 species, of which only 5 have been domesticated as cultivated varieties, including the annual pepper (Pepper arguta var. spp.). Capsicum annuum L., abbreviated as L. CA ), Chinese chili peppers ( Capsicum chinenseJacq. abbreviation CC ), Berry Pepper ( Capsicum baccatum L . abbreviation CB ), hairy pepper ( Capsicum pubescensRuiz.&Pav. abbreviation CP ) and drooping peppers ( Capsicum frutescens L., abbreviated as L. CF ).in, CA It is the most widely cultivated variety in my country, and CC (Chinese chili peppers) are known for their unique spicy flavor and resilience, and are mainly distributed in tropical and subtropical regions. Studies have shown that... CC Germplasm resources contain abundant superior genes for disease resistance, insect resistance, heat tolerance, and waterlogging tolerance. Introducing these genes... CA Cultivated varieties are of great significance for broadening the genetic base of cultivated peppers in my country and creating breakthrough breeding materials. However, CA and CC These are different chili pepper species, and there are serious post-fertilization barriers between them, which manifest as slow development of hybrid embryos, premature degeneration or abortion of the endosperm, and the immature embryos stop growing and turn brown and die in the early stages of development, making it difficult to obtain mature hybrid seeds.

[0003] Embryo rescue technology involves separating early-developing embryos from the mother plant and culturing them in vitro on an artificial culture medium, allowing them to overcome the abortion stage and continue developing into complete plants. It is currently the most effective method to overcome post-fertilization obstacles. However, existing technologies still have the following shortcomings: (1) unclear parental planting environment conditions, leading to mismatched flowering periods or low pollen viability; (2) low pollination efficiency and strong incompatibility in distant hybridization. Although there are reports of bridge parental lines or mixed pollen pollination strategies, [the text abruptly ends here]. CA × CC(3) The timing of embryo sampling often depends on experience and lacks clear morphological indicators, resulting in unstable embryo survival rate; (4) The culture medium formula often follows the conventional species scheme and lacks a special formula for pepper embryo development characteristics. In addition, during the culture process, the callus tissue proliferates excessively and competes with the embryo for nutrients, inhibiting the normal differentiation and seedling formation of the embryo; (5) There is a lack of rapid and reliable early authenticity identification methods for hybrid plants. Traditional morphological or chromosome identification has a long cycle and a large workload. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a method for distant hybridization breeding of peppers using embryo rescue.

[0005] To achieve the above objectives, this application provides a method for distant hybridization breeding of peppers using embryo rescue, comprising the following steps:

[0006] (1) The male parent Yunnan Shuanla, the parental parent chili ZY24, and the female parent chili pepper were planted respectively; pollen from Yunnan Shuanla and chili pepper ZY24 were collected and mixed to prepare mixed pollen; (2) The mixed pollen is applied to the stigma of the female parent, pepper; (3) Take the pollinated embryos and inoculate them into the embryo induction medium for in vitro culture. After the embryos grow cotyledons, transfer them to the subculture medium. After the seedlings grow vigorously, transfer them to the rooting medium. After hardening and acclimatization, transplant them.

[0007] In one embodiment, in step (1), chili peppers, Yunnan hot pot peppers, and chili pepper ZY24 are planted in an artificial climate chamber; The artificial climate chamber has the following environmental conditions: daytime temperature 25-28℃, nighttime temperature 18-20℃, light intensity 6000 Lux, photoperiod of 16 hours of light / 8 hours of darkness, and indoor relative humidity ≥80%.

[0008] In one embodiment, in step (1), the mass ratio of Yunnan hot pot pollen and chili ZY24 pollen in the mixed pollen is 1:1 to 4:1.

[0009] In one embodiment, in step (2), 0.5h-1h after pollination with mixed pollen, gibberellin solution is sprayed onto the stigma and ovary surface by atomization; the concentration of the gibberellin solution is 50-100mg / L.

[0010] In one embodiment, in step (3), the embryos are taken 15-25 days after pollination, and the longitudinal diameter of the embryos is 1.0-3.0 mm. When the seedlings grow to 3-4 cm in height and are vigorous, they are transferred to the rooting culture medium. After 7-10 days of hardening and acclimatization, the seedlings are transplanted.

[0011] In one embodiment, in step (3), the embryo induction medium is formulated as MS basal medium + 0.5-2.0 mg / L GA3 + 0.25-1.0 mg / L KT, and the pH of the medium is 5.8.

[0012] In one embodiment, in step (3), the embryo induction medium is formulated as MS basal medium + 0.5-2.0 mg / L GA3 + 0.25-1.0 mg / L KT + 10-50 mg / L spermidine, and the pH of the medium is 5.8.

[0013] In one embodiment, in step (3), the subculture medium is formulated as MS basal medium + 0.5-1.0 mg / L 6-BA + 0.1-0.5 mg / L IAA, and the pH of the medium is 5.8.

[0014] In one embodiment, in step (3), the rooting medium is formulated as 1 / 2 MS basal medium + 0.1-0.5 mg / L IBA + 0.05-0.2 mg / L NAA, and the pH of the medium is 5.8.

[0015] In one embodiment, during step (3), if callus tissue appears around the embryo during in vitro culture in the embryo induction medium, the callus tissue is removed using a dissecting needle, leaving only the embryo body.

[0016] Based on a general inventive concept, the present invention also provides the application of the above-mentioned chili pepper distant hybridization breeding method using embryo rescue in the creation of chili pepper germplasm resources.

[0017] Compared with the prior art, this application has the following beneficial effects: (1) The present invention improves pollination efficiency by using a mixture of Yunnan hot pot pollen and chili ZY24 pollen to pollinate the stigma of chili pepper, overcoming the incompatibility barrier between species and significantly improving the fertilization rate.

[0018] (2) The timing of embryo removal in this invention is precise. The timing of embryo removal is determined by pollination time and longitudinal diameter of the embryo, so as to avoid embryo death caused by removing the embryo too early or too late.

[0019] (3) The environmental conditions of this invention are clearly defined. By precisely controlling the temperature, light and humidity of the artificial climate chamber, the parent plants are guaranteed to grow uniformly and have high pollen viability, providing a repeatable environmental basis for distant hybridization.

[0020] (4) The culture medium and management system of this invention are matched, the embryo induction culture medium formula is optimized, and callus tissue is removed during the culture process to promote embryo development and improve the seedling rate. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The illustration shows cutaway photographs of young fruits 15 days, 20 days, and 25 days after pollination in Example 1 of the present invention. Figure 2 A schematic photograph of embryo culture in Example 1 of the present invention is shown; Figure 3 A schematic photograph of seedling cultivation in Embodiment 1 of the present invention is shown; Figure 4 A photograph of chili pepper SJ008-5-2-1 in Embodiment 1 of the present invention is shown schematically; Figure 5 A photograph of Yunnan-style hot pot is shown schematically in Embodiment 1 of the present invention; Figure 6 A photograph of chili pepper ZY24 is shown schematically in Embodiment 1 of the present invention; Figure 7 The illustration shows the F1 plants obtained by distant hybridization in Example 1 of the present invention. Detailed Implementation

[0023] To facilitate understanding of this application, the following description will be more comprehensive and detailed in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of this application is not limited to the following specific embodiments.

[0024] 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 this application.

[0025] 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.

[0026] The MS basal culture medium package used in the following examples contains: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate heptahydrate, 85 mg / L potassium dihydrogen phosphate, 0.5 mg / L potassium iodide, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate tetrahydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 27.8 mg / L ferrous sulfate, 37.8 mg / L disodium EDTA, 100 mg / L inositol, 0.5 mg / L sodium EDTA. mg / L niacin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L thiamine hydrochloride, 2 mg / L glycine, 0.5 mg / L folic acid, 0.05 mg / L biotin, 0.05 mg / L La-naphthaleneacetic acid, 0.25 mg / L kinetin, 0.2 mg / L gibberellin, 6 g / L agar powder, 30 g / L sucrose.

[0027] Example 1: A method for distant hybridization breeding of peppers using embryo rescue includes the following steps: (1) Parental planting Using chili pepper SJ008-5-2-1 as the female parent, Yunnan Shuanla (a local variety from Hongde, Yunnan) as the male parent, and chili pepper ZY24 as the bridging parent; among which chili pepper SJ008-5-2-1 is... CA , Figure 4 Photo of chili pepper SJ008-5-2-1; Yunnan hot pot chili pepper. CC , Figure 5 Photos of Yunnan-style hot pot; chili pepper ZY24 is... CC and CA Lines obtained by self-pollination of high-generation offspring after distant hybridization Figure 6 Photograph of chili pepper ZY24. Chili pepper SJ008-5-2-1 was published in Zhou Shudong, Zhang Zhuqing, Chen Wenchao, et al. Breeding of new chili pepper variety Bolai Xinhongxiu [J]. Hunan Agricultural Sciences, 2019, (3): 11-13. DOI: 10.16498 / j.cnki.hnnykx.2019.003.004.; Chili pepper ZY24 was published in Pei Zhuoqiang, Yang Hang, Song Yu, et al. Effects of different plant growth regulators on in vitro culture of chili pepper cotyledon explants [J]. Hunan Agricultural Sciences, 2026, (1): 72-76. DOI: 10.16498 / j.cnki.hnnykx.2026.001.013.

[0028] Chili peppers SJ008-5-2-1, Yunnan Shuanla, and ZY24 were planted in an artificial climate chamber. The environmental conditions in the artificial climate chamber were controlled as follows: daytime temperature 25-28℃, nighttime temperature 18-20℃; light intensity 6000 Lux; photoperiod of 16 hours light / 8 hours darkness; relative humidity ≥80%. The chili pepper plants (SJ008-5-2-1, Yunnan Shuanla, and ZY24) were regularly watered and fertilized to ensure robust plant growth.

[0029] (2) Pollen collection One day before flowering, select large buds about to open from Yunnan Shuanla and pepper ZY24, carefully peel off the petals with tweezers to expose the anthers. Collect the anthers in clean petri dishes and place them at room temperature under natural ventilation for 24 hours to allow for natural pollen dispersal. Collect the pollen from Yunnan Shuanla and pepper ZY24 separately from the clean petri dishes, mix them evenly at a 1:1 mass ratio to obtain mixed pollen, and store it in a refrigerator at 4℃ for short-term use.

[0030] (3) Pollination and hormone treatment On the day the pepper plant SJ008-5-2-1 blooms, between 8:00 and 10:00 AM, take the fresh mixed pollen obtained in step (2), dip a clean fine brush into the mixed pollen, and gently apply it to the surface of the stigma of the pistil of the pepper plant SJ008-5-2-1 to ensure that the stigma is fully pollinated. After pollination, mark the plant with a tag and record the pollination date. After pollination, let the plant stand for 45 minutes to allow the pollen to complete its initial attachment and germination. Then, use a handheld sprayer to evenly spray an 80 g / L gibberellin solution in a very fine mist onto the stigma and ovary of the pistil to promote pollen germination and fertilization.

[0031] (4) Embryo rescue After pollination, regularly observe the development of the young fruit. Eighteen days after pollination, when the young fruit has noticeably enlarged, harvest it. In a clean bench, immerse the young fruit in 75% ethanol for 30 seconds for disinfection, then rinse three times with sterile water. Carefully cut open the fruit with a sterile scalpel, and under a stereomicroscope, use a dissecting needle to remove the embryo with a longitudinal diameter of 1.5 mm, taking care to avoid damaging the embryo. Figure 1 Photos of the young fruit cut open 15 days after pollination, 20 days after pollination, and 25 days after pollination.

[0032] The extracted embryos were inoculated into embryo induction medium, with 10 embryos inoculated into each bottle, and then placed in a tissue culture room for culture. Figure 2This is a photograph of the embryo culture. The embryo induction medium was formulated as MS basal medium + 0.5 mg / L GA3 (gibberellin) + 0.25 mg / L KT (kinetin) + 30 mg / L spermidine, with a pH of 5.8. Culture conditions were: temperature 25 ± 2℃, relative humidity 70%-80%, light intensity 2000 Lux, and a photoperiod of 16 hours light / 8 hours dark. During culture, embryo development was observed every 3-5 days. If callus tissue appeared around the embryo, it was immediately removed with a sterile dissecting needle, preserving only the embryo itself to ensure normal embryonic development.

[0033] Once the embryo has developed two cotyledons, the cotyledons are removed using a sterile scalpel in a clean bench and transferred to a subculture medium for further culture. The subculture medium is formulated as MS basal medium + 0.5 mg / L 6-BA (6-benzylaminopurine) + 0.1 mg / L IAA (3-indoleacetic acid), with a pH of 5.8. Subculture conditions are the same as those for isoembryo induction culture.

[0034] When the subcultured plantlets grow to a height of 3-4cm ( Figure 3 (Photo of seedling culture) The plants were transferred to rooting medium for rooting culture. The rooting medium formula was MS basal medium + 0.1 mg / L IBA (3-indolebutyric acid) + 0.05 mg / L NAA (α-naphthaleneacetic acid), and the pH of the medium was 5.8. Rooting culture conditions were based on homoembryonic induction culture conditions.

[0035] (5) Hardening off seedlings and transplanting Once the rooted plants have developed 3-5 healthy white roots, open the culture bottle cap and place them under natural light for 7-10 days to harden them off, allowing them to gradually adapt to the external environment. Keep the substrate moist during hardening off. After hardening off, remove the plants from the culture medium, wash the roots to remove any remaining medium, and transplant them into nutrient pots filled with sterilized substrate. Cover with plastic film to retain moisture, and gradually ventilate to reduce humidity. Once the plants have established themselves, transplant them to the field for normal cultivation and management, and harvest F1 seeds. Figure 7 These are F1 plants obtained through distant hybridization.

[0036] Example 2: The method of distant hybridization breeding of peppers using embryo rescue in this embodiment is the same as that in Example 1, except that the embryos in the young fruit are taken 15 days after pollination.

[0037] Example 3: The method of distant hybridization breeding of peppers using embryo rescue in this embodiment is the same as that in Example 1, except that the embryos in the young fruit are taken 20 days after pollination.

[0038] Example 4: The method of distant hybridization breeding of peppers using embryo rescue in this embodiment is the same as that in Example 1, except that the embryos in the young fruit are taken 25 days after pollination.

[0039] Example 5: The method of distant hybridization breeding of peppers using embryo rescue in this embodiment is the same as that in Example 1, except that the embryo induction medium is formulated as MS basal medium + 1.0 mg / L GA3 + 0.5 mg / L KT + 30 mg / L spermidine.

[0040] Example 6: The method of distant hybridization breeding of peppers using embryo rescue in this embodiment is the same as that in Example 1, except that the embryo induction medium is formulated as MS basal medium + 1.0 mg / L GA3 + 0.5 mg / L KT + 50 mg / L spermidine.

[0041] Example 7: The method of distant hybridization breeding of peppers using embryo rescue in this embodiment is the same as that in Example 1, except that the embryo induction medium is formulated as MS basal medium + 1.0 mg / L GA3 + 0.5 mg / L KT.

[0042] Comparative Example 1: A method for distant hybridization breeding of peppers using embryo rescue includes the following steps: (1) Parental planting Using chili pepper SJ008-5-2-1 as the female parent and Yunnan scallion pepper as the male parent, both chili peppers were planted in an artificial climate chamber. The environmental conditions in the artificial climate chamber were controlled as follows: daytime temperature 25-28℃, nighttime temperature 18-20℃; light intensity 6000 Lux; photoperiod of 16 hours light / 8 hours darkness; relative humidity ≥80%. The chili pepper SJ008-5-2-1 and Yunnan scallion pepper plants were regularly watered and fertilized to ensure robust plant growth.

[0043] (2) Pollen collection One day before flowering, select large, nearly open buds from the Yunnan shrub orchid, carefully peel off the petals with tweezers to expose the anthers. Collect the anthers in clean petri dishes and place them in a well-ventilated area at room temperature for 24 hours to allow for natural pollination. Collect the pollen from the clean petri dishes separately and store them in a 4°C refrigerator for short-term use.

[0044] (3) Pollination and hormone treatment On the day the chili pepper SJ008-5-2-1 blooms, between 8:00 and 10:00 AM, take the fresh Yunnan chili pepper pollen obtained in step (2), dip a clean fine brush into the mixed pollen, and gently apply it to the surface of the stigma of the chili pepper SJ008-5-2-1 plant to ensure full pollination. After pollination, mark the plant with a tag and record the pollination date. After pollination, let the plant stand for 45 minutes to allow the pollen to complete initial attachment and germination. Then, use a handheld sprayer to evenly spray the gibberellin solution in a very fine mist onto the stigma and ovary of the pistil to promote pollen germination and fertilization.

[0045] (4) Embryo rescue After pollination, regularly observe the development of the young fruit. Eighteen days after pollination, when the young fruit has noticeably enlarged, harvest it. In a clean bench, immerse the young fruit in 75% ethanol for 30 seconds for disinfection, then rinse three times with sterile water. Carefully cut open the fruit with a sterile scalpel, and under a stereomicroscope, use a dissecting needle to remove the embryo with a longitudinal diameter of 1.5 mm, taking care to avoid damaging the embryo.

[0046] The extracted embryos were inoculated into embryo induction medium, with 10 embryos per bottle, and placed in a tissue culture room for culture. The embryo induction medium was formulated as MS basal medium + 0.5 mg / L GA3 + 0.25 mg / L KT + 30 mg / L spermidine, with a pH of 5.8. The culture conditions were: temperature 25 ± 2℃, relative humidity 70%-80%, light intensity 2000 Lux, and a photoperiod of 16 hours light / 8 hours dark. During culture, the embryo development was observed every 3-5 days. If callus tissue appeared around the embryo, it was immediately removed with a sterile dissecting needle, leaving only the embryo itself to ensure normal embryonic development.

[0047] Once the embryo has developed two cotyledons, the cotyledons are removed using a sterile scalpel in a clean bench and transferred to a subculture medium for further culture. The subculture medium is formulated as MS basal medium + 0.5 mg / L 6-BA + 0.1 mg / L IAA, with a pH of 5.8. Subculture conditions are the same as those for homoembryo induction culture.

[0048] When the subcultured plantlets reach a height of 3-4 cm, they are transferred to rooting medium for rooting culture. The rooting medium formula is MS basal medium + 0.1 mg / L IBA + 0.05 mg / L NAA, with a pH of 5.8. Rooting culture conditions are based on homoembryonic induction culture conditions.

[0049] (5) Hardening off seedlings and transplanting Once the rooted plants have developed 3-5 healthy white roots, open the culture bottle cap and place them under natural light for 7-10 days to harden them off, allowing them to gradually adapt to the external environment. Keep the substrate moist during hardening off. After hardening off, remove the plants from the culture medium, wash the roots to remove any remaining medium, and transplant them into nutrient pots filled with sterilized substrate. Cover with plastic film to retain moisture, and gradually ventilate to reduce humidity. Once the plants have established themselves, transplant them to the field for normal cultivation and management, and harvest F1 seeds.

[0050] In Comparative Example 1, Yunnan spicy pollen was used to replace the mixed pollen in Example 1.

[0051] Comparative Example 2: The method of distant hybridization breeding of peppers using embryo rescue in this comparative example is the same as that in Example 1, except that the embryos in the young fruit are taken 5 days after pollination.

[0052] Comparative Example 3: The method of distant hybridization breeding of peppers using embryo rescue in this comparative example is the same as that in Example 1, except that the embryos in the young fruits are taken 10 days after pollination.

[0053] Comparative Example 4: The method of distant hybridization breeding of peppers using embryo rescue in this comparative example is the same as that in Example 1, except that the embryo induction medium is formulated as MS basal medium.

[0054] Comparative Example 6: This comparative example does not involve embryo rescue steps; the remaining methods are the same as in Example 1.

[0055] Pollen from Examples 1-7 and Comparative Examples 1-6 was used to pollinate 80 female flowers in each group. The fruit set rate, embryo germination rate, and seedling survival rate were recorded for each group, and the results are shown in Table 1. The experiment was repeated three times, and the results are expressed as mean ± standard deviation. SPSS 27.0 was used for statistical analysis, and Duncan's method was used for multiple comparisons. Specifically, fruit set rate = (number of fruits set / number of pollinated flowers) × 100%; embryo germination rate = (number of germinated embryos / number of inoculated embryos) × 100%; seedling survival rate = (number of germinated embryos / seedling survival rate) × 100%.

[0056] As shown in Table 1, regarding fruit set rate, except for Comparative Examples 1 and 5, the fruit set rates of all other examples and Comparative Examples 2, 3, and 4 were in the high range of 44.34% to 49.12%, with no significant differences among them. Regarding embryo germination rate and seedling rate, Example 5 performed best (53.6%, 94.34%), while Examples 3, 4, 5, 6, and Example 7 also performed well; Comparative Examples 1, 2, and 5 were completely ineffective, and Comparative Examples 3 and 4 had extremely low embryo germination rates or poor seedling rates.

[0057] Table 1: Embryo rescue efficiency of Examples 1-7 and Comparative Examples 1-6

[0058] Note: Different lowercase letters in the same column of the table indicate significant differences. P <0.05).

[0059] Example 8: The efficiency of distant hybridization and embryo rescue was compared between different chili peppers (H21-1, M2012-3 and XJ34-1) and Yunnan hot pot chili pepper and chili pepper ZY24.

[0060] H21-1, M2012-3 and XJ34-1 respectively CA Using a cultivated variety as the female parent, Yunnan Shuanla as the male parent, and chili pepper ZY24 as the bridging parent, distant hybridization and embryo rescue were carried out in the same manner as in Example 1 to obtain the hybrid F1 generation.

[0061] Each female parent plant had 30 flowers pollinated. Fruit set rate (number of fruits / number of pollinated flowers × 100%), embryo germination rate (number of germinated embryos / number of inoculated embryos × 100%), and seedling survival rate (number of tissue culture seedlings / number of germinated embryos × 100%) were recorded. The experiment was repeated three times, and the results are expressed as mean ± standard deviation. Duncan's new multiple range method was used for multiple comparisons, and the results are shown in Table 2.

[0062] Table 2: Comparison of embryo rescue efficiency between different maternal parents and distant relatives of *Shuanla* (a type of hybrid rice).

[0063] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0064] Table 2 shows that there were significant differences in embryo rescue efficiency when different chili pepper maternal parents were crossbred with Yunnan Sichuan pepper. Regarding fruit setting rate, H21-1 was significantly higher than M2012-3 and XJ34-1, while there was no significant difference between the latter two. Regarding embryo germination rate, there were significant differences among the three maternal parents, with H21-1 being the highest, followed by M2012-3, and XJ34-1 the lowest. Regarding seedling rate, there were no significant differences among the three maternal parents, with H21-1, M2012-3, and XJ34-1 all having seedling rates greater than 88%. This indicates that the hybridization compatibility between different chili pepper maternal parents and Yunnan Sichuan pepper is genotype-dependent, but all can yield distant hybrid plants, meeting breeding needs.

[0065] Those skilled in the art will understand that, in addition to the specific female chili varieties listed in this embodiment, other conventional chili varieties are also applicable to the distant hybridization and embryo rescue method of the present invention. The technical effect of the present invention does not depend on a specific female variety and has universality.

[0066] The above are merely preferred embodiments of this application. It should be noted that this application is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principles of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should also be considered within the scope of protection of this application.

Claims

1. A method for distant hybridization breeding of chili peppers using embryo rescue, characterized in that, Includes the following steps: (1) The male parent Yunnan Shuanla, the parental parent chili ZY24, and the female parent chili pepper were planted respectively; pollen from Yunnan Shuanla and chili pepper ZY24 were collected and mixed to prepare mixed pollen; (2) The mixed pollen is applied to the stigma of the female parent, pepper; (3) Take the pollinated embryos and inoculate them into the embryo induction medium for in vitro culture. After the embryos grow cotyledons, they are transferred to the subculture medium. After the seedlings grow to 3-4 cm in height, they are transferred to the rooting medium and transplanted after hardening and acclimatization.

2. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (1), chili peppers, Yunnan hot pot peppers, and chili pepper ZY24 were planted in an artificial climate chamber. The artificial climate chamber has the following environmental conditions: daytime temperature 25-28℃, nighttime temperature 18-20℃, light intensity 6000 Lux, photoperiod of 16 hours of light / 8 hours of darkness, and indoor relative humidity ≥80%.

3. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (1), the mass ratio of Yunnan hot pot pollen and chili ZY24 pollen in the mixed pollen is 1:1-4:

1.

4. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (2), 0.5-1 hour after pollination with mixed pollen, the gibberellin solution is sprayed onto the stigma and ovary surface by atomization; the concentration of the gibberellin solution is 50-100 mg / L.

5. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (3), the embryos are taken 15-25 days after pollination, and the longitudinal diameter of the embryos is 1.0-3.0 mm; the seedlings are transplanted after 7-10 days of hardening and acclimatization.

6. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (3), the embryo induction medium is formulated as MS basal medium + 0.5-2.0 mg / L GA3 + 0.25-1.0 mg / L KT, with a pH of 5.8; the subculture medium is formulated as MS basal medium + 0.5-1.0 mg / L 6-BA + 0.1-0.5 mg / L IAA, with a pH of 5.

8.

7. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 6, characterized in that, In step (3), the embryo induction medium is formulated as MS basal medium + 0.5-2.0 mg / L GA3 + 0.25-1.0 mg / L KT + 10-50 mg / L spermidine, and the pH of the medium is 5.

8.

8. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (3), the rooting medium is formulated as 1 / 2 MS basal medium + 0.1-0.5 mg / L IBA + 0.05-0.2 mg / L NAA, and the pH of the medium is 5.

8.

9. The method for distant hybridization breeding of peppers using embryo rescue as described in claim 1, characterized in that, In step (3), if callus tissue appears around the embryo during the in vitro culture of the embryo induction medium, the callus tissue is removed by dissecting needle, leaving only the embryo body.

10. The application of the chili pepper distant hybridization breeding method using embryo rescue as described in any one of claims 1-9 in the creation of chili pepper germplasm resources.

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