Ilex cornuta propagation method with stable character based on apomixis phenomenon

Abstract

The invention discloses a character-stable Chinese holly propagation method based on an apomixis phenomenon, and relates to the technical field of Chinese holly breeding and propagation. According to the method, fine flower thinning is carried out on screened female ilex cornuta plants with excellent characters before flowering of the female ilex cornuta plants; bagging and isolating immediately after flower thinning so as to completely eradicate external pollen pollution; after fruit setting, the bagging type is changed, and a protective agent is sprayed, so that physiological fruit falling is prevented; and finally, harvesting seeds and sowing to obtain a clonal offspring with the genetic character highly consistent with that of the female parent. According to the method, bagging isolation and physiological regulation are combined, the apomixis characteristic of the ilex cornuta is effectively utilized, and breeding with stable characters of excellent single plants is achieved. The method has the remarkable advantages of simplicity and convenience in operation, low cost, high propagation efficiency and stable progeny characters, and is suitable for new Chinese holly variety breeding, rapid propagation and new plant variety protection.

Description

TECHNICAL FIELD

[0001] The present application relates to the breeding and propagation technology field of Ilex cornuta, more particularly to a trait-stable Ilex cornuta propagation method based on apomixis. BACKGROUND

[0002] Ilex cornuta is an important ornamental and medicinal plant, with peculiar leaf shape, bright red fruit, high ornamental value, strong stress resistance, and pruning tolerance. The trait fixation of its excellent single plant (such as specific leaf shape, compact plant type, and dense fruit) is the core of breeding work.

[0003] The traditional propagation method has the following limitations: Ilex cornuta is a dioecious plant, and seeds are produced through cross-pollination. Firstly, the seedling offspring produced by seed propagation is extremely unstable in traits, and cannot maintain the single trait of the excellent mother plant, which is not suitable for commercial variety production. Although conventional cutting and grafting can maintain the traits of the mother plant, the propagation coefficient is low, the rooting speed is slow, and it is limited by seasons, especially for some selected strains, rooting is difficult, the root system is weak, and the resistance is not as good as seedlings. The cost of tissue culture and rapid propagation is high, and the technology is complex, and the system establishment is time-consuming and laborious.

[0004] Fortunately, Ilex cornuta has apomixis of nucellar embryo. This means that the embryo in its seeds can develop directly from the nucellar tissue of the mother plant, bypassing meiosis and fertilization, thereby producing clonal seedlings that are identical to the mother plant in genetic background. This provides the possibility of asexual reproduction through seeds, and can obtain seedling offspring with developed root system and strong resistance. However, bagging isolation can cause severe physiological fruit drop, resulting in a sharp decrease in fruit setting rate, which is the main technical bottleneck for utilizing this characteristic to propagate and fix the excellent traits of Ilex cornuta. So far, no efficient technology system has been established to utilize this apomixis characteristic to achieve stable propagation of excellent single plants of Ilex cornuta.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a trait-stable Ilex cornuta propagation method. SUMMARY

[0006] Therefore, the present application provides a trait-stable Ilex cornuta propagation method based on apomixis. The method is simple and effective, and by fine thinning of flowers before flowering, stage bagging isolation, and physiological regulation, the apomixis characteristic of Ilex cornuta is fully utilized to achieve efficient and stable propagation of its excellent traits, aiming to solve the problem in the prior art that there is no method to effectively utilize the apomixis characteristic to achieve stable propagation of excellent single plants.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A kind of based on the stable trait of apomixis phenomenon Propagation method of Sabina, comprising the following steps:

[0009] S1 thinning: before the flowering of Sabina female plants, thinning treatment is carried out;

[0010] S2 bag isolation: after thinning, the flower branch of the remaining flower bud is covered with a pollen isolation bag, the bag opening is tightened to prevent foreign pollen;

[0011] S3: after the stigma of the flower in the bag withers and the ovary swells, the bag is removed, or the pollen isolation bag is replaced with a net bag that prevents insects and birds;

[0012] S4 spray protective agent: after replacing the bag, a mixed solution containing 5-15 mg / L gibberellic acid and 0.01-0.015 mg / L brassinolide is sprayed onto the leaves and young fruits on the same day;

[0013] S5 seed collection and sowing: after the fruits ripen, the seeds are harvested and sown to obtain Sabina seedlings consistent with the traits of the female parent.

[0014] As a preferred technical solution, S1 is performed before the flowering of Sabina, when the flower buds are swollen and the petals have not yet opened, and the thinning treatment specifically involves retaining 4 middle inflorescences per branch, and retaining 2 healthy flower buds per inflorescence.

[0015] As a preferred technical solution, the pollen isolation bag in S2 is a parchment paper bag.

[0016] As a preferred technical solution, the concentration of gibberellic acid in the mixed solution in S4 is 10 mg / L, and the concentration of brassinolide is 0.015 mg / L.

[0017] Another object of the present application is to provide the application of the above-mentioned Sabina propagation method in the selection of new Sabina varieties, the rapid propagation of Sabina, or the protection of new plant varieties.

[0018] Another object of the present application is to provide a protective agent for assisting the apomictic propagation of Sabina, which contains 5-15 mg / L gibberellic acid and 0.01-0.015 mg / L brassinolide.

[0019] As a preferred technical solution, the protective agent contains 10 mg / L gibberellic acid and 0.015 mg / L brassinolide.

[0020] Another object of the present application is to provide the application of the above-mentioned protective agent in improving the fruit setting rate of Sabina apomixis.

[0021] Advantages:

[0022] The method is simple and efficient in operation, the process is clear, the flower removing, bagging and pesticide spraying links are easy to operate, no complex instruments and laboratory techniques are needed, and the method is easy to be mastered by ordinary technicians and popularized and applied in the breeding of Chinese dwarf bayberry, so that the cost of breeding of excellent varieties is greatly reduced.

[0023] The strengthening nursing effect: spraying protective agents during the fruit setting period can effectively regulate the nutrient distribution in the body of Chinese dwarf bayberry, enhance the stress resistance, prevent physiological fruit drop, and ensure the stable development of precious seeds.

[0024] Highly stable traits: the varieties reproduced by the method have a clear and stable genetic background, and the population is uniform, which fundamentally ensures the high consistency of the offspring and the female parent traits, and is very beneficial to the application and protection of plant new variety rights. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0026] Figure 1 The female single plant of Chinese dwarf bayberry with excellent traits screened out; Figure 2 The photo of replacing the net bag after bagging; Figure 3 The seedling produced by bagging, showing consistency. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] Embodiment 1

[0031] 1. In February 2023, in the Chinese dwarf bayberry germplasm resource garden of Jiangsu Provincial Forestry Science Research Institute, according to the target breeding traits (such as leaf shape, plant type, branch density, cold and drought resistance, fruit size and density, etc.), a single female plant with excellent phenotype was selected, which had compact plant type and small and delicate leaves. The female gender was confirmed by observing the fruit setting condition, and the plate was marked.

[0032] 2. Thinning: On March 25, 2023, the flower buds of the plant were expanded, and the petals were not yet split. Fifteen flower branches were selected, and four middle inflorescences were retained on each branch. Only two of the strongest flower buds were retained on each inflorescence (a total of 120 flowers).

[0033] 3. Bagging isolation: Immediately after thinning, a sheepskin paper bag was put on, and the bag opening was tied tightly to prevent cross-pollination.

[0034] 4. Bag replacement: On April 10, the stigma had withered, and the ovary had expanded. The sheepskin paper bag was replaced with a white nylon mesh bag (the mesh bag was used to prevent birds from eating, so as to collect apomictic seeds).

[0035] 5. Spraying protective agents: On the same day after replacing the bag, a mixed solution containing 10 mg / L gibberellic acid and 0.015 mg / L brassinolide was sprayed on the leaves and young fruits until they were evenly wet.

[0036] 6. Seed collection and sowing: After the fruits in the bag developed normally to complete maturity (color turned red, and fruit flesh softened), the bag was carefully removed, and 62 mature fruits were collected, with a fruit setting rate of 51.67%. The fruit flesh was washed and dried to obtain 85 pure and full seeds. After sowing, a total of 38 seedling offspring were obtained, and the root system of the seedlings was developed, with a transplanting survival rate of 95%.

[0037] 7. Identification: During the seedling period, all plants showed the same leaf shape and dark green color as the mother plant, with a phenotypic consistency rate of 100%.

[0038] The results show that the present application successfully realizes the stable propagation of the excellent traits of the single plant of the plant.

[0039] Experimental Example 1

[0040] Effect of bagging isolation on the traits of the offspring after sowing

[0041] In this experimental example, 15 flower branches were selected, and natural pollination was allowed. The remaining operations were completely consistent with those of Example 1.

[0042] After sowing the collected fruits, it was observed and counted that 105 mature fruits were collected by natural pollination, with a fruit setting rate of 87.5%. The fruit flesh was washed and dried to obtain 367 pure and full seeds. Among the 90 seedlings produced by the seeds, only about 2 (2.22%) were similar to the mother plant, and the remaining plants showed significant trait separation (leaf shape became sharp, plant type became loose, etc.).

[0043] The results show that bagging isolation during the flowering period can effectively purify the apomictic offspring of the plant, and obtain propagation materials with highly stable traits.

[0044] Experimental Example 2

[0045] The effect of spraying different protective agents on the fruit setting rate is shown in Table 1.

[0046] In this experimental example, the concentrations of gibberellic acid and brassinolide are adjusted based on Example 1, and the rest of the operations are exactly the same as those in Example 1.

[0047] As shown in Table 1, after the fruits in the bags develop normally to full maturity, the bags are carefully removed, the mature fruits are harvested, the pulp is washed and dried, and the number of full seeds is counted. It is found that different protective agents and changes in the concentration ratio significantly affect the fruit setting rate of the Chinese plum yew. When no protective agent is sprayed, the fruit setting rate of the Chinese plum yew is only 5%, and 6 full seeds are obtained. The fruit setting rate of the Chinese plum yew in Example 1 is the highest, reaching 51.67%, and 85 full seeds are obtained, indicating that the protective agent with this concentration ratio has the best effect on improving the fruit setting rate and the number of seeds of the Chinese plum yew.

[0048] Table 1 Effect of different protective agents on the fruit setting rate

[0049]

[0050] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other.

[0051] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for propagating a plant of the genus Sabina based on a trait stable to apomixis, characterized in that, The method comprises the following steps: S1: flower thinning: before the female plant of the Chinese plumyew blooms, flower thinning is performed; S2: bagging and isolation: after the flower thinning, a bag that isolates pollen is used to cover the flower branch with the bud, the bag opening is tied tightly to prevent pollen from other flowers; S3: after the stigma of the flower in the bag withers and the ovary swells, the bag is removed or the bag that isolates pollen is replaced with a bag that prevents insects and birds; S4: after the bag is replaced, a mixed solution containing 5-15 mg / L gibberellic acid and 0.01-0.015 mg / L brassinolide is sprayed on the leaves and young fruits on the same day; S5: seed collection and sowing: after the fruits ripen, the seeds are collected and sown to obtain Chinese plumyew seedlings with the same traits as the female plant.

2. The method for propagating *Ilex cornuta* according to claim 1, characterized in that, S1: the Chinese plumyew blooms before the flower bud swells and the petals are not yet split, and the flower thinning is specifically to keep 4 middle inflorescences on each branch and 2 strong flower buds on each inflorescence.

3. The method for propagating *Ilex cornuta* according to claim 1, characterized in that, S2: the bag that isolates pollen is a sheepskin paper bag.

4. The method for propagating *Ilex cornuta* according to claim 1, characterized in that, S4: the concentration of the gibberellic acid in the mixed solution is 10 mg / L, and the concentration of the brassinolide is 0.015 mg / L.

5. The application of the Chinese plumyew propagation method in any of claims 1-4 in new variety breeding, rapid propagation or plant new variety protection.

6. A protective agent for assisting the propagation of pyracantha by apomixis, characterized by, The protective agent contains 5-15 mg / L gibberellic acid and 0.01-0.015 mg / L brassinolide.

7. The protective agent for assisted Tormentil apomixis propagation according to claim 6, characterized by that, The protective agent contains 10 mg / L gibberellic acid and 0.015 mg / L brassinolide.

8. The application of the protective agent in any of claims 6 or 7 in improving the rate of apomixis in Chinese plumyew.

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