A method for creating a four-ploid germplasm of asparagus

Abstract

The application belongs to the technical field of plant breeding, and discloses a method for creating asparagus tetraploid germplasm, which comprises the following steps: selecting a single female plant of a tetraploid variety with excellent traits as a female parent, selecting a single male plant of a diploid variety with excellent traits as a male parent, and performing hybridization to obtain a triploid plant; then, performing hybridization between a triploid male plant material and a tetraploid female plant material which is different from the previous variety; and finally, selecting an asparagus tetraploid germplasm which takes into account the advantages of tetraploidy and the growth of diploidy. The method is simple and easy to implement, and can achieve directional screening. The asparagus tetraploid germplasm obtained by the method takes into account the advantages of tetraploidy and the growth of diploidy, and provides excellent germplasm for breeding tetraploid varieties with short time from sowing to harvesting, fast growth and high yield.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, and specifically relates to a method for creating tetraploid asparagus germplasm. Background Technology

[0002] Asparagus (Asparagus officinalis L.) is a perennial vegetable belonging to the genus Asparagus in the family Asparagaceae. It is dioecious, and the tender stems, which are the edible part, have extremely high nutritional and medicinal value. They are rich in vitamins, amino acids, proteins, and minerals, and are therefore known as the "King of Vegetables".

[0003] Currently, the asparagus varieties circulating in the market are generally diploid (green and white asparagus) and tetraploid (purple asparagus). Triploid asparagus is usually obtained by crossing tetraploid plants with diploid plants, but the success rate is very low, and there are very few reports of triploid asparagus to date. There are generally two ways to obtain tetraploid asparagus. One is to select superior lines from existing tetraploid varieties as male and female parents, and breed tetraploids using conventional hybridization breeding and tissue culture techniques. This is the main method for breeding tetraploid varieties at present. The other method is to use chemical mutagens such as colchicine to double the diploid plants to obtain tetraploids. However, this method has problems such as low doubling rate and survival rate of asparagus polyploid induction, inconsistent doubling ratios of plants, the presence of a large number of chimeras, and a significant reduction in the fruit set rate of individual tetraploid plants.

[0004] Compared to diploids, tetraploids often exhibit advantages such as taller plants, thicker stems, higher biomass, and stronger disease resistance. However, their growth cycle (from seedling to harvest, not the entire growth process from planting to senescence and death) is longer and their growth is slower. Therefore, to shorten the cultivation time and bring the product to market as soon as possible, it is necessary to select and breed tetraploid varieties with fast growth and good germplasm. However, since asparagus is a perennial vegetable, the conventional breeding method involves selecting superior parent lines, creating hybrid combinations, screening high-yield combinations through comparative trials, and then using tissue culture to propagate the parent lines and establish seed production fields. This breeding process is very lengthy. If we can first create tetraploid germplasm with a short growth period as breeding material, and then quickly breed fast-growing, high-yielding, and high-quality tetraploid varieties for production, we can effectively solve the problem of the long asparagus breeding process. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for creating tetraploid asparagus germplasm that is simple to operate and can achieve targeted screening. Through this method, tetraploid materials that combine the advantages of tetraploids and the growth vigor of diploids can be obtained, providing excellent germplasm for breeding tetraploid varieties with short time from sowing to harvest, fast growth and high yield.

[0006] To solve the above technical problems, the technical scheme of the present application is:

[0007] A method for creating a four-ploid germplasm of asparagus, comprising the following steps:

[0008] a. Select high-quality diploid and tetraploid asparagus seeds, respectively, for seedling and planting, and after flowering, select a female plant of the tetraploid variety with excellent traits as the female parent, and a male plant of the diploid variety with excellent traits as the male parent for hybridization, and after the fruit is mature, select seeds with full grains to retain;

[0009] b. The seeds obtained in step a are subjected to seedling, and through chromosome ploidy identification, a triploid plant is selected for planting, and within 1-2 years after planting, phenotypic identification (plant growth state, appearance of shoots, etc.) is performed, and a male plant with excellent traits is selected for preservation;

[0010] c. The triploid male plant material selected in step b and the female plant material of other excellent (non-step a variety) tetraploid with excellent traits are hybridized after flowering, and after the fruit is mature, seeds with full grains are selected for preservation;

[0011] d. The full seeds obtained in step c are subjected to seedling and chromosome ploidy identification, and a tetraploid material is selected for planting;

[0012] e. Within 1-2 years after planting the tetraploid material obtained in step d, phenotypic identification is performed, and an asparagus four-ploid germplasm that takes into account the advantages of tetraploid and the growth of diploid is selected.

[0013] Preferably, the diploid asparagus seed variety in step a is "Champion", "Lu Asparagus No. 7", and the tetraploid asparagus seed variety is "Purple Passion" and "Jingzilu No. 2".

[0014] Further, the diploid asparagus seed variety in step a is "Lu Asparagus No. 7", and the tetraploid asparagus seed variety is "Purple Passion"; the tetraploid asparagus seed variety in step c is "Jingzilu No. 2".

[0015] Preferably, the identification in step b is performed by a method combining flow cytometry and chromosome identification to select triploid material; leaf tissue (flow cytometry) and root tip tissue (chromosome identification) at 60-80 days after seedling are selected for identification.

[0016] Preferably, the identification in step d is identified by flow cytometry combined with chromosome identification method, and tetraploid materials are selected; leaf tissue (flow cytometry) and root tip tissue (chromosome identification) at 60-80 days of seedling are selected respectively for identification. Preferably, the observation method in step e is that the seeds obtained in step c are simultaneously seedling with the diploid and tetraploid seeds in step a, and the traits are compared, and the single plant with fast growth and excellent appearance of shoot is selected from the tetraploid materials selected in step d.

[0017] Due to the adoption of the above technical solutions, the application has the following beneficial effects:

[0018] The application utilizes the tetraploid female plant with excellent traits as the female parent and the diploid male plant as the male parent for hybridization, and then utilizes the tetraploid female plant with excellent traits as the female parent and the triploid male plant as the male parent for hybridization, and then the tetraploid germplasm that takes into account the advantages of tetraploid and the growth of diploid is screened; the method is simple and easy to implement, and can achieve directional screening, and provides an excellent germplasm resource bank for screening the tetraploid variety that overcomes the defects of the prior art, has a short time from sowing to harvesting, fast growth of plants, and excellent asparagus traits. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 for different periods and different growth conditions of materials; wherein

[0020] A (champion), B (purple passion), and C (triploid male plant obtained by hybridization of the female parent "purple passion" and the male parent "champion" in 2021) were all seedling in a greenhouse on March 8, 2022, planted in a greenhouse on June 19, 2022, and photographed on April 10, 2023;

[0021] D (champion), E (Jingzilu No. 2), and F (tetraploid germplasm created by the application) were all seedling in a winter warm greenhouse on September 21, 2024, planted in a greenhouse on March 12, 2025, and photographed on July 29, 2025;

[0022] G (champion), H (triploid male plant), and I (tetraploid germplasm created by the application) were all photographed on the same period (April 5, 2025); G and I were seedling in a winter warm greenhouse on September 28, 2023, planted in a greenhouse on March 19, 2024, and H was seedling in a greenhouse on March 8, 2022, planted in a greenhouse on June 19, 2022;

[0023] Figure 2 The photos are for seed selection at different fullness; wherein A is full seed; B is underfull seed; and C is empty seed;

[0024] Figure 3The growth situation diagram of the four-ploid asparagus created by the application after the second year of planting; wherein the left is the whole plant growth situation diagram; the right is the above-ground stem growth situation detail diagram, the number of sprouting branches is large, the growth is strong, and the sprout package head is tight. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with the examples.

[0026] Seedling raising method: (1) seedling tray preparation: 32-hole hole tray seedling is adopted, and prepared seedling substrate is directly purchased and loaded into the tray for standby use; (2) seed treatment: the seeds are soaked in clean water at room temperature for 2-3 days, and the water is changed 1-2 times every day during the soaking period; (3) sowing: the treated seeds are sown one by one in the seedling tray, and sufficient water is poured after covering the soil; (4) seedling management: control the temperature and humidity, keep the temperature and humidity of the seedbed before the seed germination, control the temperature between 25-30 degrees, and keep it humid; timely topdressing (20 days once, 30 kg / acre of N, P, K, Ca fertilizer), pour water after topdressing, and ventilate to prevent fertilizer damage.

[0027] Planting: rain-sheltered cultivation, planting in the greenhouse, the specific method is as follows: (1) the land is fully ploughed to 40-50 cm, the planting ditch with a depth of 30-40 cm in the north-south direction is opened, the row spacing is 150 cm, the organic fertilizer and the compound fertilizer are applied 3-5 days before planting, mixed with the soil, finally pour water thoroughly, the soil is sunken, the depth of the planting ditch is about 25 cm at this time, and the planting is carried out when the soil humidity is appropriate; (2) the seedlings with an age of 60-80 days are planted, the hole is dug according to the plant spacing of 50 cm, the size of the hole is appropriate to put the root group, the soil is covered, the water is poured thoroughly, the planting depth is 12-15 cm below the ground level, the seedling is planted, the soil is covered shallowly, about 4-5 cm, and the soil is covered in several times, finally the planting ditch position becomes the planting ridge.

[0028] Flow cytometry identification method: leaf tissue is taken for determination, and the specific method is referred to the literature (Dolezel, J. et al. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protoc ols, 2233-2244 (2007). https: / / doi.org / 10.1038 / nprot.2007.310) operation.

[0029] Chromosome identification: the root tip chromosome observation method is adopted, the root tip tissue is taken for determination, and the specific method is referred to the literature operation (Li Xia, et al. Cytological study on chromosome of male and female asparagus [J]. Seed, 2022, 41 (07): 88-91).

[0030] Example 1

[0031] a. Select diploid variety "champion", tetraploid variety "purple passion" asparagus seed, respectively, seedling, planting, after flowering, select the tetraploid female plant (purple passion) with excellent traits as the female parent (the number of hybrid flowers is 139), and the diploid male plant (champion) with excellent traits as the male parent to carry out hybridization. After the fruit matures, 48 seeds are obtained (8 of which are full seeds);

[0032] (Asparagus is a perennial plant, a single asparagus has several thousand flowers, one flower hybridization produces one fruit, and a fruit contains a maximum of 6 seeds; therefore, only one female plant and one male plant are selected for hybridization breeding experiment)

[0033] b. The seeds obtained in step a are seedling, and the ploidy identification is carried out by flow cytometry combined with chromosome identification when the seedling is 60-80d, 17 triploid plants are obtained, of which 9 are male plants, named 23-1-1 to 23-1-9, and cultivation and screening are carried out (see table 1);

[0034] c. The triploid male plant material (23-1-2) with excellent traits selected in step b and the tetraploid female plant material (different from "purple passion" in step a) "Jingzilu No. 2" with excellent traits are hybridized after flowering (a total of 120 flowers are hybridized), and 37 seeds are obtained after the fruit matures (36 of which are full seeds) (see table 3);

[0035] d. The seeds obtained in step c are seedling to obtain 35 seedlings, and the ploidy identification is carried out by flow cytometry combined with chromosome identification when the seedling is 60-80d, 31 tetraploid plants are obtained;

[0036] e. The tetraploid material obtained in step d is planted for 1-2 years, and the traits are investigated, from which 25 tetraploid germplasm with the advantages of tetraploid and the growth of diploid are obtained.

[0037] Example 2

[0038] Referring to the method of example 1, the tetraploid "purple passion" female plant is selected as the female parent and the diploid "Lu Asparagus No. 7" male plant is selected as the male parent; the tetraploid "Jingzilu No. 2" female plant is selected as the female parent and the diploid "champion" and "Lu Asparagus No. 7" male plants are selected as the male parent to carry out hybridization, and the breeding process is the same as example 1. The triploid male plant materials obtained are named 23-2-1 to 23-2-17, 23-3-1 to 23-3-7, and 23-4-1 to 23-4-12 (see table 1).

[0039] 23-1-2, 23-2-7, 23-3-4, and 23-4-6 were hybridized with "Purple Passion," and 23-2-7 was hybridized with "Jingzi Lu No. 2." The breeding process was the same as in Example 1, and the number of seeds obtained were 9, 20, 32, 56, and 112 respectively (see Table 3). After planting and screening, 2, 9, 11, 30, and 56 tetraploid germplasm plants were obtained that combined the advantages of tetraploids with the growth vigor of diploids.

[0040] Comparative Example 1

[0041] Using the same method as in Example 1, diploid female plants of "Champion" and "Lu Asparagus No. 7" were used as female parents and tetraploid male plants of "Purple Passion" and "Jing Zi Lu No. 2" were used as male parents for hybridization. Among them, the triploids obtained from the hybridization of "Champion" and "Purple Passion" yielded a total of 3 male plants, named 23-5-1 to 23-5-3 respectively; the triploids obtained from the hybridization of "Champion" and "Jing Zi Lu No. 2" yielded a total of 3 male plants, named 23-6-1 to 23-6-3 respectively; the triploids obtained from the hybridization of "Lu Asparagus No. 7" and "Purple Passion" yielded a total of 6 male plants, named 23-7-1 to 23-7-6 respectively; the triploids obtained from the hybridization of "Lu Asparagus No. 7" and "Jing Zi Lu No. 2" yielded a total of 2 male plants, named 23-8-1 and 23-8-2 respectively (see Table 2).

[0042] The tetraploid female plants of 23-7-1 and "Jingzi Lu No. 2" were hybridized after flowering (a total of 149 flowers were hybridized). After the fruit matured, 51 seeds were obtained (42 of which were plump seeds) (see Table 3).

[0043] Comparative Example 2

[0044] Referring to the method in Example 1, tetraploid female plants of "Purple Passion" were selected as the female parent and diploid male plants of "Lu Asparagus No. 7" as the male parent; tetraploid female plants of "Jing Zi Lu No. 2" were selected as the female parent and diploid male plants of "Lu Asparagus No. 7" as the male parent for hybridization. The breeding process was the same as in Example 1. Twenty and fourteen triploid female plants were obtained, respectively named 23-2-18 to 23-2-37 and 23-4-13 to 23-4-26.

[0045] Two cultivars, 23-2-21 and 23-4-13, were crossed with tetraploid male plants of 'Jingzi Lu No. 2'. After flowering, the crosses were performed (96 and 104 flowers respectively). Upon fruit ripening, no fully developed seeds were obtained; the number of underdeveloped seeds was 6 and 9 respectively. Due to the insufficient number of fully developed seeds, effective planting and selection were not feasible, and subsequent experiments were abandoned.

[0046] Results and Analysis

[0047] The hybridization results of different paternal and maternal parents in Examples 1, 2 and Comparative Example 1 are statistically summarized and listed in Tables 1, 2 and 3 respectively:

[0048] Table 1. Results of hybridization of different diploid (paternal) and tetraploid (maternal) parents in Examples 1 and 2.

[0049]

[0050] Among these, "plump" seeds refer to seeds that are approximately hemispherical or short-oval in shape, with a black seed coat that is smooth and glossy, and abundant endosperm (see...). Figure 2 A); "Unsatisfied" refers to seeds with irregular shapes, numerous seed coat wrinkles, and insufficient endosperm (see...). Figure 2 B); "Silty seed" refers to a seed with only one seed coat and no endosperm (see...). Figure 2 C).

[0051] Table 2 shows the hybridization results of different diploid (paternal) and tetraploid (maternal) lines in Comparative Example 1.

[0052]

[0053] Tables 1 and 2 show the results of reciprocal crosses using diploid and tetraploid plants as the male and female parents, respectively. The tables show that using a tetraploid plant as the female parent and a diploid plant as the male parent resulted in a high proportion of plump seeds and a high overall seed count. The best combination was using the tetraploid "Purple Passion" as the female parent and the diploid "Lu Asparagus No. 7" as the male parent, producing a total of 176 seeds, with 17 plump neutrons. The seed production rates relative to the number of hybrid flowers (150) were 117.3% (total seed count) and 11.3% (plump seed count), respectively. The second best combination was using the tetraploid "Jing Zi Lu No. 2" as the female parent and the diploid "Lu Asparagus No. 7" as the male parent, producing a total of 91 seeds, with 9 plump seeds. The seed production rates relative to the number of hybrid flowers (132) were 68.9% and 6.82%, respectively.

[0054] Using a diploid as the female parent and a tetraploid as the male parent, the number of plump seeds and the proportion of total seeds obtained by hybridization were low. Using the tetraploid "Purple Passion" as the male parent and the diploid "Lu Asparagus No. 7" as the female parent, the highest number of total seeds obtained was 52, but no plump seeds were obtained. The seed production rate was 31.4% (total seed count) relative to the number of hybrid flowers (169). Using the tetraploid "Jing Zi Lu No. 2" as the male parent and the diploid "Champion" as the female parent, the lowest number of total seeds obtained was 20, with 5 plump neutrons. The seed production rates were 15.5% and 3.88% respectively relative to the number of hybrid flowers (129).

[0055] Table 3: Results of hybridization in Examples 1 and 2, where triploid male plants were the father and tetraploid female plants were the mother.

[0056]

[0057] Table 3 shows the statistical results of hybridization between triploid male plants as the male parent and tetraploid female plants as the female parent. Using "Purple Passion" as the female parent, hybridization was performed with 23-1-2, 23-2-7, 23-3-4, and 23-4-6 as the male parent, yielding total seed numbers of 9, 20, 32, and 56 respectively. The number of plump seeds was 9, 15, 15, and 11 respectively. Overall, 23-3-4 and 23-4-6 showed better results, indicating that using different varieties as the two tetraploid female parent materials is preferable.

[0058] A comparison of hybridization results using 23-1-2, 23-2-7, and 23-7-1 as male parents and "Jing Zi Lu No. 2" as female parent versus "Purple Passion" as female parent and 23-3-4 and 23-4-6 as male parents showed that the number of plump seeds and the total number of seeds obtained with "Jing Zi Lu No. 2" as female parent and 23-1-2 and 23-2-7 as male parents were higher than the number of hybrid flowers. Among them, the combination with 23-2-7 as male parent had the best effect, with a total of 112 seeds and 96 plump seeds. The seed production rates were 81.8% and 70.1% respectively relative to the number of hybrid flowers (137).

[0059] Example 3 Planting Experiment

[0060] The seeds obtained in steps b and d of Examples 1, 2, and Comparative Example 1 (the planting experiment in Comparative Example 2 was abandoned due to insufficient experimental data) were simultaneously raised as seedlings with their parent varieties. Growth was observed in different materials, and superior germplasm was selected. Specific growth conditions are shown in the attached figures. Figure 1 A, B, and C are a comparison chart of the growth of diploid male parent variety "Champion" (green bamboo shoot), tetraploid female parent variety "Purple Passion" (purple bamboo shoot), and triploid male plant (seed of offspring obtained by crossing A and B) when they were raised at the same time. It can be seen from the chart that the tetraploid variety grows slowly, has fewer branches, and is relatively robust, while the diploid variety grows quickly and has more branches. The triploid offspring obtained by the cross grows quickly, has more branches, and is purple in color. Figure 1 The chart compares the seedling growth of D, E, and F, representing the diploid male parent variety "Champion" (green shoot), the tetraploid female parent variety "Jingzi Lu No. 2" (purple shoot), and the tetraploid germplasm created in this invention (obtained by crossing triploid seeds from D and "Purple Passion" with E). It shows that the tetraploid female parent variety "Jingzi Lu No. 2" (…) is… Figure 1 E) Slow growth and fewer branches, while diploid varieties grow quickly and have more branches. The tetraploid germplasm created in this invention ( Figure 1 F) Rapid growth, numerous branches, and purple color, combining the advantages of tetraploid and diploid growth (see morphology of the whole plant and above-ground stems). Figure 3 ). Figure 1G, H, and I are respectively the "champion" and triploid male plants (and) Figure 1 (same as material B), the tetraploid germplasm created in this invention (and...) Figure 1 (F material same) Picture of the shoot emergence period. At this period, the tetraploid varieties 'Purple Passion' and 'Jingzi Lu No. 2' had not yet produced shoots.

[0061] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for creating a tetraploid germplasm of Asparagus officinalis, characterized in that: The method comprises the following steps: a. Select high-quality "Lu Asparagus No. 7" diploid and "Purple Passion" tetraploid asparagus seeds, and carry out seedling and planting, respectively. After flowering, select the "Purple Passion" tetraploid female plant as the female parent and the "Lu Asparagus No. 7" diploid male plant as the male parent for hybridization. After the fruit matures, select the seeds with full grains to reserve; b. The seeds obtained in step a are subjected to seedling, and through chromosome ploidy identification, the triploid plants are selected for planting, and after planting for 1-2 years, the male plants with excellent traits are selected and reserved; c. The triploid male plant material obtained in step b and the female plant material of "Jingzilu No. 2" tetraploid are hybridized after flowering, and after the fruit matures, the seeds with full grains are selected and reserved; d. The full seeds obtained in step c are subjected to seedling and chromosome ploidy identification, and the tetraploid material is selected for planting; e. The tetraploid material obtained in step d is observed for 1-2 years after planting, and the asparagus tetraploid germplasm is screened.

2. The method of creating a tetraploid stock of Asparagus officinalis according to claim 1, wherein: The identification in step b is carried out by combining flow cytometry with chromosome identification, and the triploid material is selected; the leaf tissue and root tip tissue at 60-80 days after seedling are selected for identification.

3. The method of creating tetraploid stock of Asparagus officinalis as claimed in claim 1, wherein: The identification in step d is carried out by combining flow cytometry with chromosome identification, and the tetraploid material is selected; the leaf tissue and root tip tissue at 60-80 days after seedling are selected for identification.

4. The method of creating tetraploid stock of Asparagus officinalis as claimed in claim 1, wherein: The observation method in step e is that the seeds obtained in step c and the "Lu Asparagus No. 7" diploid and "Purple Passion" tetraploid seeds in step a are simultaneously subjected to seedling and trait comparison, and the single plants with fast growth and excellent asparagus appearance traits are selected from the tetraploid material selected in step d.

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