Method for cultivating unfertilized ovary of allium macrostemon bunge in vitro and application thereof

Abstract

The application provides an in-vitro culture method for cultivating Allium fistulosum unpollinated ovary and application thereof, and belongs to the technical field of plant tissue culture.The method comprises the following steps: taking the intact ovary of Allium fistulosum which is not pollinated as an explant, and performing induction culture, proliferation culture and rooting culture.Through the culture of the intact ovary and the one-step induction differentiation method, the method is simple, the induction rate is high, the operability is strong, a large number of Allium fistulosum unpollinated ovary culture regenerated plants can be obtained, and breeding materials which can be applied to the selection and breeding of different Allium fistulosum types are screened out, compared with the traditional breeding method, the breeding period is greatly shortened.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and in particular relates to a method for in vitro culture of unpollinated ovaries of cultivated leeks and its application. Background Technology

[0002] Leeks (Allium tuberrosum Rottl. ex Spr.) are one of the oldest cultivated edible vegetables. They are not only rich in nutrients and possess antibacterial and antioxidant activities, but also have other biological characteristics, earning them the reputation of being a vegetable with both edible and medicinal uses. They are highly adaptable, heat-resistant, and cold-resistant, and are cultivated worldwide. In China, the main cultivated and consumed leeks are of three species: wild leek (A. ramosum syn. odorum), broadleaf leek (A. hookeri Thwaiter. Enum.), and leek (A. tuberrosum Rottl. ex Spr.). Leeks have complex genetic traits, a large genome, and a unique seed reproduction method, exhibiting parthenogenesis and apomixis, making them cross-pollinated plants. Although leeks are self-compatible, they often show a high tendency for cross-pollination during self-pollination and exhibit severe inbreeding inhibition. Furthermore, due to the asynchronous development of male and female gametes, they are highly heterozygous. In production, the first generation of hybrids is mainly used. When carrying out hybridization breeding, it is necessary to cultivate high-purity male sterile parent lines, inbred lines, or self-incompatible lines. Therefore, the process of cultivating chive varieties with excellent traits through traditional breeding techniques is very tortuous.

[0003] Unpollinated ovary culture is a method for artificially inducing female gametophytes to develop into complete plants, and it is an important technique in haploid breeding. Research on ovary culture in chives is relatively limited. Chinese scholars Tian Huiqiao et al. (1989) explored the embryo development process and ovary culture conditions in chive ovary culture, laying the foundation for subsequent research. International research on obtaining superior chive breeding materials mainly utilizes cell engineering culture, not only using asexual organs of chives (leaves, root tips, flowers, inflorescences, stems, etc.) as explants to induce the acquisition of regenerated plants, but also conducting in-depth research on the rescue of distant hybrid embryos and haploid culture. In subsequent studies, some scholars successfully obtained regenerated plants through ovary culture. Wang Mingyao et al. (2005) selected flower buds as explants for the tissue culture of chive flowers. The results showed that flower buds were greatly affected by the addition of 6-BA and NAA in the culture medium. The optimal culture conditions were MS + 6-BA 1.0 mg / L + KT 1.0 mg / L + NAA 1.0 mg / L. Wang Liya et al. (2017) cultured chive ovaries and screened MS as the suitable basic culture medium. MS + 2,4-D 0.1 mg / L + ZT 0.5 mg / L had the best induction effect, with an average budding time of 42 days after inoculation. The differentiation medium was MS + NAA 0.1 mg / L + 6-BA 0.5 mg / L, and the rooting medium was MS + NAA 0.2 mg / L. Generally, 97% of chive seedlings completed rooting after 30 days. Yang et al. (2021) showed that the optimal induction medium for culturing unpollinated ovaries of chives was 0.1 mg / L 2,4-D, 0.5 mg / L ZT, and 9% sucrose. The culture methods used in these studies were all relatively complex, and no further identification or screening of haploid or double-haploid plants obtained from the culture of unpollinated ovaries of chives was conducted. Furthermore, patent CN107047311A discloses a "method for in vitro culture of unfertilized ovaries of chives," which involves culturing slices of unfertilized ovaries. Because unfertilized ovaries of chives are small, this method is not only cumbersome but also makes it difficult to distinguish whether the regenerated plants developed from the ovary wall or the ovules. Therefore, providing a simpler method for culturing unpollinated ovaries of chives with a high induction rate is of great significance to the development of chive breeding. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for in vitro culture of unpollinated ovaries of cultivated leeks, which has a high induction rate and can quickly obtain a large number of regenerated plants from unpollinated ovaries of leeks.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] A method for in vitro culture of unpollinated ovaries of cultivated leeks, the method comprising the following steps: using unpollinated ovaries of leeks as intact explants, induction culture, proliferation culture, and rooting culture are performed; the induction culture medium is: MS medium + 30-100 g / L sucrose + 0.1-0.4 mg / L 2,4D + 0.5-2 mg / L 6-BA + 8-10 g / L agar; the proliferation culture medium is: MS medium + 25-35 g / L sucrose + 0.1-0.3 mg / L NAA + 0.3-1 mg / L 6-BA + 8-10 g / L agar; the rooting culture medium is: MS medium + 25-35 g / L sucrose + 8-10 g / L agar.

[0007] Preferably, the ovary is taken from the early flowering stage or the peak flowering stage, and the ovary development stage is from 2 days to 1 day before flowering.

[0008] Preferably, when the number of roots of the chive regenerated plant obtained from the rooting culture is ≥6 and the root length is ≥6cm, the chive regenerated plant is transplanted to a substrate for acclimatization culture.

[0009] Preferably, the matrix is ​​a mixture of peat, coconut coir, cow dung, and perlite in a volume ratio of 0–4:2–6:1:1.

[0010] Preferably, the culture conditions for the induction culture, proliferation culture and rooting culture are: culture temperature 22-27℃, light intensity 3200-3700 lux, 16 hours of light and 8 hours of darkness per day.

[0011] Another object of the present invention is to provide an induction medium for in vitro culture of unpollinated ovaries of chives, wherein the induction medium is: MS medium + 30-100 g / L sucrose + 0.1-0.4 mg / L 2,4D + 0.5-2 mg / L 6-BA + 8-10 g / L agar.

[0012] Another object of the present invention is to provide a proliferation medium for in vitro culture of unpollinated ovaries of chives, wherein the proliferation medium is: MS medium + 25-35 g / L sucrose + 0.1-0.3 mg / L NAA + 0.3-1 mg / L 6-BA + 8-10 g / L agar.

[0013] Another object of the present invention is to provide a rooting medium for in vitro culture of unpollinated ovaries of chives, wherein the rooting medium is: MS medium + 25-35 g / L sucrose + 8-10 g / L agar.

[0014] Another object of the present invention is to provide the application of the in vitro culture method, the induction medium, the proliferation medium, or the rooting medium in the propagation and / or maintenance of superior traits of leeks.

[0015] Another object of the present invention is to provide the application of the in vitro culture method, the induction medium, the proliferation medium, or the rooting medium in the improvement of leek varieties.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides a method for in vitro culture of unpollinated ovaries of cultivated chives. Using unpollinated, intact ovaries of chive varieties with superior traits as material, this invention employs a simple method with a high induction rate and strong operability through intact ovary culture and a one-step induction differentiation method. It can obtain a large number of regenerated plants from unpollinated chive ovaries and can be applied to breeding materials for different chive varieties. Compared with traditional breeding methods, it significantly shortens the breeding cycle. Attached Figure Description

[0018] Figure 1 : Process diagram of obtaining regenerated plants from unpollinated, intact ovaries of chives. A: Collected flower buds; B: Ovary after treatment; C: Ovary swelling (induction culture for 14 days); D: Regenerated plant (induction culture for 40 days); E: Proliferation culture; F: Rooting culture;

[0019] Figure 2 The effect of different genotypes on embryogenesis; different lowercase letters in the figure indicate significant differences (P < 0.05).

[0020] Figure 3 Ovary morphology at different developmental stages: A: Open flower bud morphology; B: Flower bud morphology 1 day before flowering; C: Flower bud morphology 2 days before flowering; D: Flower bud morphology 3 days before flowering; E: Flower bud morphology 4 days before flowering; F: Anther morphology of open flower bud; G: Anther morphology of flower bud 1 day before flowering; H: Anther morphology of flower bud 2 days before flowering; I: Anther morphology of flower bud 3 days before flowering; J: Anther morphology of flower bud 4 days before flowering; K: Open ovary morphology; L: Ovary morphology of flower bud 1 day before flowering; M: Ovary morphology of flower bud 2 days before flowering; N: Ovary morphology of flower bud 3 days before flowering; O: Ovary morphology of flower bud 4 days before flowering; The material in the figure is 21-CJ46.

[0021] Figure 4 The effect of different sampling periods on embryo yield;

[0022] Figure 5 The effect of different culture conditions on embryo emergence rate;

[0023] Figure 6 The effect of different exogenous hormones added to the induction medium on the embryo emergence rate;

[0024] Figure 7 The effect of different induction media on embryo emergence rate;

[0025] Figure 8 : Rooting of chives in different rooting media: A: Rooting of chives in medium M; B: Rooting of chives in medium M1; C: Rooting of chives in medium M2;

[0026] Figure 9 : Regenerated superior strains, A: Regenerated plants obtained from 20-CJ24; B: Regenerated plants obtained from 21-CJ46. Detailed Implementation

[0027] This invention provides a method for in vitro culture of unpollinated ovaries of cultivated chives. The method includes the following steps: using intact, unpollinated ovaries of chives as explants, and performing induction culture, proliferation culture, and rooting culture. The process of obtaining regenerated plants from unpollinated ovaries of cultivated chives according to this invention is as follows: Figure 1 As shown.

[0028] The cultivated leek described in this invention is leek (A. tuberosum Rottler ex Spr.), which is a leek variety with excellent varietal characteristics and easy embryo formation in ovary culture, and uses an unpollinated, intact ovary of the leek as an explant.

[0029] In this invention, the ovary is selected during the initial flowering or full bloom stage, and the ovary development period is from 2 days to 1 day before flowering. From 8:00 AM to 10:00 AM, unopened flower buds are selected from healthy, disease-free flowering plants to dissect and extract the complete ovary. As one embodiment, the flower bud diameter is 3.85–4.06 mm. After collection with tweezers, the buds are placed in a resealable bag, then placed in an ice box and brought back to the plant. They are stored at 0–4°C, preferably for 20–25 hours, before being removed and processed. The flower bud processing method includes: soaking in 75% medical alcohol for 18–22 seconds in a sterile laminar flow hood, rinsing 2–3 times with sterile water, then disinfecting with 1% sodium hypochlorite for 13–17 minutes, rinsing 2–3 times with sterile water, and then placing the disinfected flower buds on dry sterile filter paper to remove excess moisture from the surface. The petals, filaments, and anthers are removed from the flower buds using pointed tweezers to extract the complete ovary.

[0030] In this invention, one end of the complete ovary connected to the receptacle is inoculated onto an induction medium. The induction medium is: MS medium + 30-100 g / L sucrose + 0.1-0.4 mg / L 2,4D + 0.5-2 mg / L 6-BA + 8-10 g / L agar, preferably MS medium + 60-95 g / L sucrose + 0.15-0.3 mg / L 2,4D + 0.8-1.5 mg / L 6-BA + 9 g / L agar, more preferably MS medium + 90 g / L sucrose + 0.2 mg / L 2,4D + 1 mg / L 6-BA + 9 g / L agar; the pH is adjusted to 5.8-5.9.

[0031] The induction culture conditions of this invention are: culture temperature 22–27℃, light intensity 3200–3700 lux, 16 hours of light and 8 hours of darkness per day; preferably, the culture temperature is 23–26℃ and the light intensity is 3400–3600 lux, more preferably, the culture temperature is 25℃ and the light intensity is 3500 lux. Regenerated plants can be directly obtained by culturing in the induction medium for 35–42 days.

[0032] In this invention, the regenerated plants obtained through induced culture are removed, the roots are cut off, and the buds and leaves of 0.4–0.5 cm are retained. These are then inoculated into a proliferation medium for further proliferation culture. The proliferation medium is: MS medium + 25–35 g / L sucrose + 0.1–0.3 mg / L NAA + 0.3–1 mg / L 6-BA + 8–10 g / L agar, preferably MS medium + 27–32 g / L sucrose + 0.15–0.25 mg / L NAA + 0.4–0.6 mg / L 6-BA + 9 g / L agar, more preferably MS medium + 30 g / L sucrose + 0.2 mg / L NAA + 0.5 mg / L 6-BA + 9 g / L agar; the pH is adjusted to 5.8–5.9. The plants are cultured on the proliferation medium for 30–35 days.

[0033] The propagation and culture conditions of the present invention are as follows: culture temperature 22-27℃, light intensity 3200-3700 lux, 16 hours of light and 8 hours of darkness per day; preferably, the culture temperature is 23-26℃ and the light intensity is 3400-3600 lux, more preferably, the culture temperature is 25℃ and the light intensity is 3500 lux.

[0034] This invention involves transferring the proliferating plants to a rooting medium for rooting culture. The rooting medium is: MS medium + 25-35 g / L sucrose + 8-10 g / L agar, preferably MS medium + 30 g / L sucrose + 9 g / L agar; the pH is adjusted to 5.8-5.9.

[0035] The rooting culture conditions of this invention are as follows: culture temperature 22-27℃, light intensity 3200-3700 lux, 16 hours of light and 8 hours of darkness per day; preferably, the culture temperature is 23-26℃ and the light intensity is 3400-3600 lux, more preferably, the culture temperature is 25℃ and the light intensity is 3500 lux.

[0036] In this invention, when the number of roots of the regenerated chives obtained from rooting culture is ≥6 and the root length is ≥6cm, the regenerated chives are transplanted into a potting substrate for acclimatization cultivation. The potting substrate is a mixture of peat moss, coconut coir, cow manure, and perlite in a volume ratio of 0–4:2–6:1:1, preferably 4:2:1:1. Preferably, 3–5 kg / m³ of perlite can be added to the potting substrate. 3 Slow-release Osmocote No. 5, preferably with an added amount of 4kg / m³ 3 Slow-release Osmocote No. 5.

[0037] As one feasible approach, the present invention involves removing the caps from tissue culture seedling bottles, placing them in a culture room for 2 days, washing away the culture medium from the roots, and then transplanting them into a potting substrate for acclimatization culture.

[0038] After transplanting, conventional water and fertilizer management is carried out. One year later, the agronomic traits of the plants, such as plant height, plant width, leaf length, leaf width, pseudostem length, pseudostem thickness, root length, root thickness, and number of tillers, are investigated. Based on the analysis of the agronomic trait survey data, compared with the original leek donor, plants with traits such as long and thick pseudostems, large and thick leaves, and resistance to bolting can be selected as leek breeding materials for yellow leek. Plants with strong tillering ability, early bolting, and high bolting quantity are selected as breeding materials for leek with bolting.

[0039] The present invention also provides the application of the in vitro culture method, the induction medium, the proliferation medium, or the rooting medium in the propagation and / or maintenance of superior traits of leeks.

[0040] The present invention also provides the application of the in vitro culture method, the induction medium, the proliferation medium, or the rooting medium in the improvement of leek varieties.

[0041] In a specific embodiment of the present invention, the MS medium consisted of: NH4NO3 1650 mg / L, KNO3 1900 mg / L, CaCl2·2H2O 440 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, FeSO4·7H2O 27.8 mg / L, and Na2-EDTA·2H2O. 37.3 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride (vitamin B6) 0.5 mg / L, thiamine hydrochloride (vitamin B1) 0.1 mg / L, glycine 2.0 mg / L.

[0042] In specific embodiments, the plant growth regulators used in this invention—6-benzylaminopurine (6-BA), kinetin (KT), 2,4-dichlorophenoxyacetic acid (2,4-D), zeatin (ZT), thiabendazole (TDZ), and 1-naphthaleneacetic acid (NAA)—are all conventional commercially available products.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] A method for in vitro culture of unpollinated ovaries of chives includes the following steps:

[0046] 1. Flower bud collection: Select chive varieties with excellent varietal characteristics and easy embryo formation in ovary culture. One day before the peak flowering period, between 8:00 and 10:00 am, select unopened flower buds from healthy flowering plants free from pests and diseases. Collect them with tweezers, put them in a resealable bag, place them in an ice box, and bring them back.

[0047] 2. Flower Bud Treatment: Place the harvested flower buds in a 4℃ refrigerator for 24 hours. After 24 hours, remove them and select buds with a diameter of 3.85–4.06 mm for treatment. In a sterile laminar flow hood, first soak the buds in 75% medical alcohol for 20 seconds, rinse twice with sterile water, then disinfect with 1% sodium hypochlorite for 15 minutes, and rinse three times with sterile water. Finally, place the disinfected buds on dry sterile filter paper to remove excess moisture from the surface.

[0048] 3. Ovary Retrieval: Using sharp tweezers, remove the petals, filaments, and anthers to extract the intact ovary (see...). Figure 1 (B) in the middle;

[0049] 4. Inoculation and Induction: The end of the ovary connected to the receptacle was inoculated onto the induction medium, which consisted of MS medium containing 0.2 mg / L 2,4-D, 1 mg / L 6-BA, 90 g / L sucrose, and 9 g / L agar, with the pH adjusted to 5.8. The medium was then placed in a light-controlled culture chamber under the following conditions: 25℃, 3500 lx light intensity, 16 h light and 8 h dark per day. Regenerated plants were obtained directly after 40 days of culture.

[0050] 5. Propagation: Remove the young regenerated plants, cut off the roots that have grown out, retain the buds and 0.5 cm leaves, and inoculate them into the proliferation medium, which is: MS + 0.2 mg / L NAA + 0.5 mg / L 6-BA + 30 g / L sucrose + 9 g / L agar, and adjust the pH to 5.8; place them in a light incubator for further cultivation under the following conditions: temperature 25℃, light intensity 3500 lx, 16 h light and 8 h dark per day; proliferation culture for 30 days;

[0051] 6. Rooting: Transfer the plants after propagation culture to the rooting medium for rooting culture. The rooting medium is: MS + 30 g / L sucrose + 9 g / L agar powder; the culture conditions are: temperature 25℃, light intensity 3500 lx, 16 h light and 8 h dark per day, until roots grow.

[0052] 7. Acclimation and Transplanting: When the regenerated leek plants have at least 6 roots and a length of at least 6cm, they can be acclimated and transplanted. After placing the tissue culture seedlings in the culture room for 2 days with the caps off, wash off the culture medium from the roots and transplant them into a potting substrate. The potting substrate is prepared by mixing peat moss, coconut coir, cow manure, and perlite in a volume ratio of 4:2:1:1.

[0053] Example 2

[0054] The effect of different plant material sources on embryo emergence rate.

[0055] The plant materials were collected by the Guizhou Provincial Institute of Horticulture and introduced and screened from the experimental base of the Guizhou Provincial Institute of Horticulture. The experimental materials and their characteristics are shown in Table 1.

[0056] Table 1. Materials used in the unpollinated ovary culture of chives

[0057] 1 20-CJ1 The broad-leaved leek with a triangular cross-section of the pseudostem has strong tillering ability and is cold-resistant and disease-resistant. Danzhai County Danzhai broad-leaf chives 2 20-CJ2 Broad-leaved chives with a triangular pseudostem cross-section are relatively cold-resistant and disease-resistant. Yang Ai in Gui'an New District broadleaf chives 3 20-CJ17 The broad-leaved leek has a triangular pseudostem cross-section, strong tillering ability, and is relatively cold-resistant and disease-resistant. Liping County Liping broad-leaf chives 4 20-CJ26 The broad-leaved leek with a triangular pseudostem cross-section has strong tillering ability and is relatively cold-resistant and disease-resistant. Shiqian County Shiqian broadleaf chives 5 20-CJ27 The broad-leaved leek with a triangular cross-section of the pseudostem has strong tillering ability and is cold-resistant and disease-resistant. Danzhai County Danzhai Broadleaf Leek No. 2 6 20-CJ9 Cultivated chives with a circular pseudostem cross-section have strong tillering ability. Zunyi City Zunyi leeks 7 20-CJ10 Cultivated chives with a circular pseudostem cross-section have strong tillering ability. Jinsha County Mukong chives 8 20-CJ24 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant. Xifeng County Xifeng Leeks 9 20-CJ28 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant and disease-resistant. Duyun City Duyun chives 10 20-CJ29 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are cold-resistant and disease-resistant. Xingyi City Xingyi Leeks 11 20-CJ31 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant and disease-resistant. Zhijin County Leeks 12 20-CJ35 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant and disease-resistant. Bozhou District, Zunyi City Water chestnut and chives 13 20-CJ38 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are cold-resistant and disease-resistant. Puding County Puding Leeks 14 21-CJ1 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are cold-resistant. Fusheng Company Green Leek Treasure 15 21-CJ24 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and strong disease resistance. Fusheng Company 791 Superior Series 16 21-CJ26 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are cold-resistant and disease-resistant. Shandong Snow Chives 17 21-CJ32 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant and disease-resistant. Fusheng Company Rich Leek Tai No. 1 18 21-CJ38 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are cold-resistant and disease-resistant. Fugou County, Henan Province North and South Green Leeks 19 21-C43 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant and disease-resistant. Pinglong Leek Industry Chinese Divine Sword 20 21-CJ46 Cultivated chives with a circular pseudostem cross-section have strong tillering ability and are relatively cold-resistant and disease-resistant. Pinglong Leek Industry Prince of Leeks

[0058] For the 20 different genotypes in Table 1, unpollinated ovary culture was performed. Flower buds were collected, treated, and ovaries were obtained using the method described in Example 1. The obtained ovaries were then induced in MS medium containing 0.1 mg / L 2,4-D, 0.5 mg / L 6-BA, 30 g / L sucrose, and 9 g / L agar at pH 5.8. Five bottles were inoculated for each variety, with 10 ovaries per bottle, and three replicates were performed for each genotype. After inoculation, the ovaries were placed in a 4°C refrigerator for 1 day, then transferred to a 25°C thermostatic incubator for 14 days of dark culture, followed by a light culture chamber with the following conditions: 25°C, 3500 lux light intensity, 16 h light, 8 h dark. Embryo emergence rate was calculated after 35 days. The effects of different genotypes on embryo emergence are as follows: Figure 2 As shown.

[0059] Depend on Figure 2 It can be seen that the embryo emergence rate of unpollinated ovary materials of different genotypes of chives is different. Cultivated chives with circular pseudostem cross-sections can all produce embryos, but the embryo emergence rate varies significantly. The highest is 21-CJ46 at 80%, and the lowest is Guijiu No. 1 at 13.33%. Broad-leaved chives cannot produce embryos. Genotype is the key factor affecting the embryo emergence rate of chive ovary culture.

[0060] Example 3

[0061] The effect of different sampling periods on embryo yield.

[0062] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that the flowering periods of the samples were: initial flowering, full bloom, and late full bloom. Samples were taken at different ovary development stages during each flowering period: the day of flowering (0), 1 day before flowering (-1), 2 days before flowering (-2), 3 days before flowering (-3), and 4 days before flowering (-4). Flowers on the day of flowering needed to be bagged in advance. All other conditions were the same as in Example 1. The embryo percentage was calculated after 35 days. Ovary morphology at different developmental stages is shown in [Figure 1]. Figure 3 The effect of different sampling periods on embryo yield is shown in [reference needed]. Figure 4 .

[0063] It is evident that the embryo emergence rate of chives varies depending on the flowering stage and ovary development stage. The ovary development stage has a certain impact on gynogenetic development, with stronger floral organ development vitality observed during the initial flowering and full bloom stages. In this invention, the highest embryoid induction rate was achieved by sampling 2 days to 1 day before flowering during the initial and full bloom stages. The initial flowering stage and 1 day before flowering are the optimal times for sampling unpollinated chive ovaries. The highest embryo emergence rate occurs 1 day before flowering because this is the period of maturation of the female gametophytes within the chive ovary.

[0064] Example 4

[0065] The effect of different culture conditions on embryo emergence rate.

[0066] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that after the ovaries were inoculated into the induction medium, the culture conditions were as follows: the culture conditions of Example 1, high temperature (33℃) stress for 1, 2, 3, and 4 days followed by culture at 25℃, low temperature (4℃) stress for 1, 2, 3, and 4 days followed by culture at 25℃, and dark culture at 25℃ for 14 days followed by culture in a light-controlled culture room. These were recorded as: 25℃, 33℃ 1d, 33℃ 2d, 33℃ 3d, 33℃ 4d, 4℃ 1d, 4℃ 2d, 4℃ 3d, 4℃ 4d, and dark 14d + 25℃. All other conditions were the same as in Example 1. Ten ovaries were collected per bottle, and five bottles were inoculated for each treatment, with three replicates. The effect of different culture conditions on the embryo emergence rate is shown in [reference needed]. Figure 5 .

[0067] Different culture conditions have varying effects on embryo emergence rate. Directly placing the inoculated embryos in a 25°C light-cured culture room (Example 1) yielded the best results and was also simple and convenient. The next best option was cultured at 4°C for 1 day followed by transfer to a light-cured culture room. However, cultured after heat shock treatment at 33°C did not produce ideal results, with a low embryo emergence rate and ovary drying. This may be because high or low temperature treatments do not stimulate embryo-like formation in unpollinated leek ovaries; a regular light-dark culture condition is more effective in promoting embryo-like formation in unpollinated leek ovaries.

[0068] Example 5

[0069] The effect of adding different exogenous hormones to the induction medium on the embryo emergence rate.

[0070] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that MS medium was used as the basal medium with a sucrose concentration of 30 g / L. The types and amounts of exogenous hormones added to induction media A, B, and C are shown in Table 2. MS without sucrose was used as the control (CK). Other conditions were the same as in Example 1. Ovaries were inoculated into different induction media for induction culture, and the embryo emergence rate was calculated after 35 days. The effect of different induction media on the embryo emergence rate is shown in [Table 2]. Figure 6 .

[0071] Table 2 Induction medium formulation

[0072]

[0073] Depend on Figure 6 It is known that embryos cannot emerge when MS medium without sucrose is used as the induction medium. The highest embryo emergence rate of unpollinated leek ovaries was observed when 0.5 mg / L 6-BA + 0.1 mg / L 2,4-D were added to the induction medium, followed by TDZ and 2,4-D, while the lowest embryo emergence rate was observed when only TDZ was added. Exogenous hormones promote embryo emergence in plant ovary culture. Commonly used exogenous hormones are mainly divided into two categories: cytokinins and auxins. Different exogenous hormones have different effects on the embryo emergence rate of unpollinated leek ovaries in vitro. The induction medium provided by this invention can effectively improve the embryo emergence rate.

[0074] Example 6

[0075] The effect of different induction media on embryo emergence rate.

[0076] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that the composition of the induction medium is shown in Table 3, while other conditions are the same as in Example 1. Ovaries were inoculated into different induction media for induction culture, and the different media were labeled as shown in Table 3. The embryo emergence rate was calculated after 35 days. The effect of different induction media on the embryo emergence rate is shown in [Table 3]. Figure 7 .

[0077] Table 3 Induction medium formulation

[0078]

[0079] Depend on Figure 7 It was found that the induction medium containing 90 g / L sucrose + 1 mg / L 6-BA + 0.2 mg / L 2,4-D had the best induction effect, with an induction rate approaching 90%. Since unpollinated ovaries cannot synthesize carbohydrates when cultured in vitro, a carbon source is an essential component of the culture medium. Sucrose is commonly used as a carbon source in in vitro gynogenetic culture, but the concentration varies depending on the species. In this invention, sucrose concentrations of 30-90 g / L can provide a carbon source for in vitro culture. This invention uses different concentrations of sucrose and different ratios of 2,4-D and 6-BA as exogenous hormones for ovary culture, which can improve the embryo emergence rate.

[0080] Example 7

[0081] The effect of different proliferation media on the proliferation coefficient of adventitious shoots.

[0082] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that the hormone composition in the proliferation medium is as shown in Table 4, while other conditions are the same as in Example 1. The induced adventitious shoots were inoculated into different proliferation media for proliferation culture. The different media are labeled as shown in Table 4. The formulations of different proliferation media and the effects of different proliferation media on the proliferation coefficient of leek regenerated plants are shown in Table 4.

[0083] Table 4. Effects of different proliferation media on the proliferation coefficient of regenerated chive plants.

[0084]

[0085] Table 4 shows that the proliferation media using 6-BA and NAA as exogenous hormones in this invention have high proliferation coefficients. The highest proliferation coefficient (3.8) was achieved when MS + 30 g / L sucrose + 0.5 mg / L 6-BA + 0.2 mg / L NAA was used as the proliferation medium for regenerated seedlings, resulting in robust plant growth. After obtaining regenerated plants through induction culture, these plants need to be propagated to obtain more regenerated plants.

[0086] Example 8

[0087] The effects of different rooting media on the root growth of regenerated chive plants.

[0088] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that excessively long leaves of the chive seedlings were trimmed, and individual tissue culture seedlings were transferred to three rooting media: M (MS + 30 g / L sucrose + 9 g / L agar powder), M1 (MS + 0.2 mg / L NAA + 30 g / L sucrose + 9 g / L agar powder), and M2 (1 / 2 MS + 0.2 mg / L NAA + 30 g / L sucrose + 9 g / L agar powder). All other treatments were the same as in Example 1. Five bottles were inoculated for each treatment, with five individual seedlings per bottle, and three replicates were performed. Rooting time and conditions were recorded to screen for the optimal induction medium. See below for chive rooting conditions in different rooting media. Figure 8 The effects of different rooting media on the survival rate of regenerated plants after transplanting are shown in Table 5.

[0089] Table 5. Effects of different rooting media on the survival rate of regenerated plantlets after transplanting.

[0090]

[0091]

[0092] From Table 5 and Figure 8 It can be seen that, when inducing root growth in healthy plants, compared with conventional MS and 1 / 2 MS media with 0.2 mg / L NAA, the best rooting effect is achieved by using MS medium without any hormones + 30 g / L sucrose + 9 g / L agar powder, resulting in more roots, stronger root growth, and higher survival rate after transplanting.

[0093] Example 9

[0094] The effects of different potting substrates on the growth of regenerated chive plants.

[0095] Using 21-CJ46 from Example 2 as the test material, the difference between this example and Example 1 is that the potting substrate formula is as shown in Table 6, while other conditions are the same as in Example 1. After rooting, acclimatization was carried out. The acclimatization method was as follows: after rooting, the bottle cap was removed, and the plant was placed in a greenhouse for 2 days. The culture medium was then cleaned and the plant was ready for transplanting. The plant was transplanted into five different potting substrate formulas (see Table 7) and cultured in an artificial climate chamber. The culture conditions were: 12 hours of light, 3500 lux of light intensity, and day / night temperature of 25℃ / 18℃. During this period, routine water, fertilizer, and pest and disease management were carried out. After 14 days of culture, the survival rate and growth and development of the chives were recorded, and the results are shown in Table 7.

[0096] Table 6 Formulas for Different Potting Substrates

[0097] T1 Peat:coconut coir:cow dung:perlite volume ratio 6:0:1:1 T2 Peat:coconut coir:cow dung:perlite volume ratio 4:2:1:1 T3 Peat:coconut coir:cow dung:perlite volume ratio 3:3:1:1 T4 Peat:coconut coir:cow dung:perlite volume ratio 2:4:1:1 T5 peat:coconut coir:cow dung:perlite volume ratio 0:6:1:1

[0098] Table 7. Survival rate and growth of chives under different substrate cultivation conditions.

[0099]

[0100] As shown in Table 7, the suitable cultivation substrate for the growth of chive plants grown from unpollinated ovaries is peat moss: coconut coir: cow manure: perlite in a volume ratio of 4:2:1:1.

[0101] Example 10

[0102] Identification, evaluation, and screening.

[0103] Using the method described in Example 1, 500 regenerated plants were obtained from 15 leek experimental varieties numbered 6-20 in Table 1. Agronomical traits of the regenerated plants, including plant height, plant width, leaf length, leaf width, pseudostem length, pseudostem diameter, root length, root diameter, and number of tillers, were investigated one year after transplanting. The results are shown in Table 8. Figure 9 .

[0104] Table 8 Results of the survey on agronomic traits of superior regenerated lines

[0105]

[0106] From Table 8 and Figure 9 It was found that, compared with the original donor plants, six lines superior to the donor were obtained through culture of the unpollinated ovaries of 20-CJ24. These lines exhibited long and robust pseudostems, long, broad, and thick leaves, and tolerance to bolting, but weaker tillering ability. These traits align with the breeding objectives for leek yellow varieties. Five lines were obtained from the unpollinated ovaries of 21-CJ46, demonstrating strong tillering ability, early bolting, and abundant bolting. These characteristics align with the breeding objectives for bolting leeks. Unpollinated ovary culture of leeks can obtain double haploid materials, which is an important method for monoploid breeding of leeks. The regenerated plants obtained are directly developed from the female gametophytes of the donor material, thus allowing for the emergence of super-parental lines.

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for in vitro culture of unpollinated ovaries of cultivated leeks, characterized in that, The method includes the following steps: using an unpollinated, intact ovary of leek as an explant, induction culture, proliferation culture, and rooting culture are carried out; The culture medium for induction culture is: MS medium + 30~100g / L sucrose + 0.1~0.4mg / L 2,4D + 0.5~2mg / L 6-BA + 8~10g / L agar; The culture medium for proliferation culture is: MS medium + 25~35 g / L sucrose + 0.1~0.3 mg / L NAA + 0.3~1 mg / L 6-BA + 8~10 g / L agar; The rooting culture medium is: MS medium + 25~35g / L sucrose + 8~10g / L agar.

2. The in vitro culture method according to claim 1, characterized in that, The ovary is selected during the initial flowering period or the peak flowering period, and the ovary development period is from 2 days to 1 day before flowering.

3. The in vitro culture method according to claim 1, characterized in that, When the number of roots of the regenerated chives obtained from the rooting culture is ≥6 and the root length is ≥6cm, the regenerated chives are transplanted into a substrate for acclimatization culture.

4. The in vitro culture method according to claim 3, characterized in that, The substrate is a mixture of peat, coconut coir, cow dung and perlite in a volume ratio of 0~4:2~6:1:

1.

5. The in vitro culture method according to claim 1, characterized in that, The culture conditions for the induction culture, proliferation culture and rooting culture are as follows: culture temperature 22~27℃, light intensity 3200~3700 lux, 16 hours of light and 8 hours of darkness per day.

6. The application of the in vitro culture method according to any one of claims 1 to 5 in the propagation and / or preservation of superior traits of leeks.

7. The application of the in vitro culture method according to any one of claims 1 to 5 in the improvement of leek varieties.

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