Breeding method of bletilla triplod plant and application thereof

Abstract

The present application belongs to the technical field of new plant variety breeding, and particularly relates to a breeding method of a bletilla triplod and application thereof. The breeding method of the bletilla triplod comprises the following steps: screening a bletilla diploid; artificially inducing and identifying a bletilla tetraploid; culturing the identified bletilla tetraploid to flowering; and hybridizing the bletilla tetraploid and the bletilla diploid to obtain a bletilla triplod fruit; wherein a mutagen used for the induction is a mixed aqueous solution of 10 ppm p-dichlorobenzene and 5 ppm alpha-bromonaphthalene. The present application first creates a new bletilla triplod variety, and establishes a complete set of bletilla triplod advantage utilization and breeding method. The present application first obtains a new bletilla triplod germplasm, and lays a scientific foundation for bletilla triplod breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant new variety breeding technology, and in particular relates to a method for propagating triploid Bletilla striata plants and its application. Background Technology

[0002] Bletilla striata ( Bletilla striata (Thunb) Rchb.f.) belongs to the genus Bletilla in the family Orchidaceae. Bletilla Bletilla striata is a perennial herbaceous plant, mainly distributed from the Yangtze River basin to the south and southwest. It is widely distributed in Anhui, Jiangxi, Hubei, Sichuan, Guizhou, Yunnan, Shaanxi, and Gansu provinces of my country, and is cultivated throughout these regions. There are approximately six species in the genus *Bletilla* worldwide, all native to East Asia. China has *Bletilla striata* (Chinese Bletilla). B. sinensis ),noob( B. formosana ), Bletilla striata ( B. striata ) and yellow-flowered white basil ( B. ochracea There are 4 types. Bletilla striata varieties can be broadly divided into two categories: one is purple-flowered Bletilla striata, with purplish-red flowers, including both large and small varieties; the other is yellow-flowered Bletilla striata, with yellowish-white flowers. There are also a few variants of Bletilla striata with flowers in white, blue, yellow, and pink, but mostly purple and red. Due to its rich and vibrant flower colors, elegant flower shape, and beautiful leaves, Bletilla striata also has extremely high ornamental value.

[0003] Bletilla striata is one of the commonly used traditional Chinese medicinal herbs, with its dried tubers used medicinally. As a traditional Chinese medicine, it has a wide range of medicinal applications. The tubers contain hundreds of active compounds, including steroids, terpenoids, esters, ethers, biphenes, dihydrophenanthrenes, and bibenzyl groups, as well as trace elements such as boron, molybdenum, manganese, iron, zinc, and copper. They are also rich in starch, glucose, volatile oils, and mucilage. Bletilla striata possesses hemostatic, tissue-regenerating, anti-inflammatory, antibacterial, gastric mucosal-protective, lung-tonifying, skin-nutrition-improving, and wrinkle-preventing effects. It is widely used in the pharmaceutical, food, and daily chemical industries, possessing extremely high medicinal and economic value.

[0004] Due to long-term exploitative harvesting and lack of protection, Bletilla striata resources are dwindling, leading to a severe shortage of medicinal raw materials. It is now listed as one of China's 30 rare and endangered natural medicinal herbs. Hybrid breeding and new variety development of Bletilla striata in my country are still lagging behind, relying primarily on artificial cultivation, which has resulted in germplasm degradation, reduced effective component content, and stagnant yield. Strengthening the protection of Bletilla striata germplasm resources, genetic improvement, and new variety development is urgently needed.

[0005] Polyploidy and hybridization play an important role in the evolution of higher plants. Genome triplication is not common in nature, but it plays an important role in evolution. Among them, triploid (3x) is the lowest multiple of polyploidy, which is produced by hybridization of tetraploid (4x) and diploid (2x), and has all the properties of polyploidy. Whether it is a naturally occurring triploid or an artificially produced triploid, it has hybrid vigor produced by hybridization. Therefore, triploid has the double advantages of polyploidy and hybridization, and its advantages are huge and irreplaceable. The huge nature of triploid vegetative organs is fully utilized, and the use of triploid hybrid vigor is favored. Triploid advantage utilization has great potential in medicinal plants, especially in Chinese medicine with roots and tubers as medicine. However, there are few reports at present. There is no report on the study of triploid of Bletilla striata. Although there are reports on the study of polyploidy of Bletilla striata, they are all about tetraploid (Chinese patent: CN202311182684.7; Lu Guangyao, master's thesis).

[0006] Chinese patent CN202311182684.7 reports a method for inducing and cultivating Bletilla striata polyploidy, which is characterized by liquid culture of Bletilla striata seeds, and obtaining Bletilla striata polyploidy by ① placing protocorms in colchicine for doubling, and ② culturing doubled protocorms in different culture media at different stages. The Bletilla striata polyploid plant is a tetraploid.

[0007] In 2015, Lu Guangyao of Zhejiang University of Chinese Medicine reported the induction of Bletilla striata polyploidy in his master's thesis "Study on New Rapid Propagation Technology of Bletilla striata", which is characterized by: morphological preliminary identification of the induced diploid Bletilla striata as a part of the polyploid Bletilla striata variant plant, and chromosome counting analysis, with a chromosome number of 2n=64, proving that the variant Bletilla striata plant is a polyploid Bletilla striata.

[0008] Currently, the cultivation of Bletilla striata triploid in China is still in the blank stage, and there is no report. SUMMARY

[0009] The present application first creates a new strain of Bletilla striata triploid and establishes a complete set of advantage utilization and breeding methods for Bletilla striata triploid. The present application first obtains a new germplasm of Bletilla striata triploid, which lays a scientific foundation for Bletilla striata triploid breeding.

[0010] The present application provides a method for cultivating Bletilla striata triploid, comprising the following steps:

[0011] (1) Screening Bletilla striata diploid;

[0012] (2) Artificial induction, identification and screening of Bletilla striata tetraploid;

[0013] (3) Cultivating the identified tetraploid obtained in step (2) to flowering;

[0014] (4) crossing the tetraploid obtained in step (3) with the diploid of step (1) to obtain a white-bark magnolia triploid;

[0015] Further, the mutagen used for induction in step (2) is a mixed aqueous solution of 10 ppm p-dichlorobenzene and 5 ppm alpha-bromonaphthalene.

[0016] Further, the specific method for artificial induction of the white-bark magnolia tetraploid in step (2) is as follows: selecting white-bark magnolia as the mutagenic material for tetraploid, sowing the seeds on a seedbed to obtain round corms, and before the green bud points of the corms emerge, using a mutagen for artificial induction: 20-22 DEG C, intermittent treatment for 2-3 times, 24-48 hours for each time.

[0017] Further, the process of the screening is as follows: taking a new root of white-bark magnolia, cutting the root part of the living tissue, treating the cut part with 0.002 M 8-hydroxyquinoline at 20-22 DEG C for 3.5-4 hours, fixing the treated part with a fixing solution, preparing a chromosome specimen by smear method, and then staining the specimen with a staining solution for 40 minutes, washing with tap water, and air-drying.

[0018] Further, the fixing solution is methanol: glacial acetic acid = 3:1.

[0019] Further, the staining solution is a phosphate buffer solution: Giemsa mother liquor = 40:1.

[0020] The application provides an application of a method in preparation of a white-bark magnolia triploid.

[0021] The application provides a seedling breeding method of the white-bark magnolia triploid, wherein the seeds in the fruit pods of the obtained triploid are uniformly mixed with activated carbon, the seeds are uniformly sowed on a seedbed by using a 20-mesh sieve, misty water is sprayed twice a day to keep the seedbed substrate moist, after the seeds germinate into seedlings, water is sprayed once a day, water-soluble fertilizer is sprayed once a week for 2 weeks, and ventilation is kept to obtain triploid seedlings.

[0022] Further, the ratio of the seeds in the fruit pods of the triploid to the activated carbon is 1:20-30.

[0023] The application provides an application of a method in preparation of white-bark magnolia triploid seedlings.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The present application provides a "Baihe triploid breeding method", which effectively overcomes the shortcomings of the prior art, and achieves the increase of Baihe medicinal material yield, quality and drug effective component content by using Baihe triploid heterosis. The present application uses "p-dichlorobenzene + alpha-bromonaphthalene" mixed mutagen to artificially induce tetraploid of Baihe white flower, successfully obtains Baihe white flower homologous tetraploid, and the genome chromosome is 2n=4x=72. Then, the tetraploid Baihe white flower is used as a female parent (4x♀), and the diploid Baihe purple flower is used as a male parent (2x♂) to carry out cross breeding, that is, 4x♀x2x♂, and the triploid Baihe is obtained.

[0026] Since the triploid is spatially isolated from other species and cannot be sexually crossed, the first generation of cross breeding is a permanent F1 hybrid, which has the double advantages of hybrid and polyploid. Therefore, the triploid Baihe solves the problems of Baihe variety confusion, germplasm degradation, medicinal material yield reduction and drug content instability. The heterosis of the triploid Baihe new variety (line) can ensure that the yield, quality and drug component content of Baihe medicinal material remain stable and increase. The method of the present application is simple, the input cost is low, and from tetraploid induction, triploid hybridization and transformation, triploid seedling propagation to field cultivation, no sterile operation process is needed, it is easy to popularize, and it is deeply loved by the majority of farmers. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a diploid Baihe parent diagram in Example 1, wherein Figure a is a diploid Baihe white flower, Figure b is a diploid Baihe white flower genome chromosome, 2n=2x=36; Figure c is a diploid Baihe purple flower, and Figure d is a diploid Baihe purple flower genome chromosome, 2n=2x=36.

[0028] Figure 2 It is a white flower protocorm cluster bud and cluster seedling diagram of intermittent mutagenesis treatment in Example 1, wherein Figure a is a protocorm to be treated for tetraploid, Figure b is a protocorm differentiated into cluster seedlings after 30 days of treatment, Figure c is a protocorm cluster bud after 2 weeks of treatment, and Figure d is a white flower seedling to be identified after 60 days of treatment.

[0029] Figure 3 It is a comparison of diploid, triploid and tetraploid Baihe (1-year-old seedlings) diagram in Example 1, wherein Figure a is a diploid plant, Figure b is a diploid genome chromosome, 2n=2x=36; Figure c is a triploid plant, Figure d is a triploid genome chromosome, 2n=3x=54; Figure e is a tetraploid plant, and Figure f is a tetraploid genome chromosome, 2n=4x=72.

[0030] Figure 4 It is a triploid Baihe seedling breeding diagram in Example 2.

[0031] Figure 5For Example 2, the roots and corms of diploid, triploid, and tetraploid white bamboo are compared (1 year old). In the figures, Fig. a and Fig. c are the roots and corms of diploid white bamboo seedlings; Fig. b and Fig. d are the roots and corms of triploid white bamboo seedlings; and Fig. e is a comparison of the leaves of diploid, triploid, and tetraploid white bamboo seedlings. DETAILED DESCRIPTION

[0032] Example 1

[0033] Investigation, collection, morphological analysis, and chromosome number identification of white bamboo germplasm resources.

[0034] Three main cultivars in Yunnan were collected, ① yellow-flowered white bamboo (Bambusa multiplex Bletilla ochracea Schltr.), ② white-flowered white bamboo (Bambusa multiplex Bletilla striata war. alba ), and ③ purple-flowered three-pronged white bamboo (Bambusa multiplex Bletilla striata (Thunb.) Reichb. f). The results of chromosome identification are as follows: ①-③ are all diploid, 2n = 2x = 36 Figure 1 a-b).

[0035] Tetraploid artificial induction: According to the results of morphological analysis and chromosome identification, white-flowered white bamboo was selected as the tetraploid mutation material. First, the seeds of white-flowered white bamboo were directly sown in the seedbed. When the seeds turned green and became green nodules, i.e., protocorms, before the protocorms sprouted green bud points, a mixed chemical mutagen was used to induce tetraploidy Figure 2 a).

[0036] The mixed chemical mutagen is a mixed aqueous solution of 10 ppm p-dichlorobenzene and 5 ppm α-bromonaphthalene, and the treatment is intermittent for 3 times at 20-22 °C, with each treatment lasting for 24-48 hours.

[0037] Tetraploid identification and screening of white bamboo: After the protocorms differentiated into seedlings and the root systems grew to 1-2 cm, chromosome ploidy identification was performed Figure 2 d). A part of the root system of the white bamboo seedling treated by the mutagen was treated with 0.002 M 8-hydroxyquinoline for 3.5-4 hours, then fixed with 3:1 (methanol: glacial acetic acid) fixing solution, and chromosome specimens were prepared by the smearing method. After natural air drying, the specimens were dyed with Giemsa staining solution, and observed and analyzed under a microscope. A pure tetraploid white bamboo with 2n = 4x = 72 was screened out Figure 3 e-f).

[0038] The Giemsa staining solution is: Giemsa stock solution: phosphate buffer solution (1:40), room temperature staining for 40 minutes, and then washed with running water until the staining solution is completely washed off, and then naturally air dried.

[0039] Example 2

[0040] 1. Hybrid breeding of triploid Bletilla striata

[0041] A triploid Bletilla striata hybrid was obtained by crossing a tetraploid Bletilla striata (4x ♀) with a diploid Bletilla striata (2x ♂) when the tetraploid and diploid Bletilla striata were in bloom for 3-5 days, i.e. 4x ♀ x 2x ♂. Figure 3 c-d).

[0042] 2. Seedling breeding of triploid Bletilla striata

[0043] The triploid Bletilla striata hybrid was first mixed with activated carbon at a ratio of 1:20, and the seeds were evenly spread on the seedbed using a 20-mesh sieve. The seedbed substrate was composed of undergrowth humus, vermiculite, and nutrient soil at a ratio of 1:1:1. The temperature was maintained at 20-25 °C, the humidity was 60%-80%, and the light transmittance was about 25%. The seeds were sprayed with water in a misting manner twice a day. After the seeds germinated and the seedlings grew, the humidity was maintained at 50%-60% and the seeds were sprayed with water once a day. The results, as shown in Figure 4 , showed that the germination rate of the seeds reached more than 80%. Comparisons of the roots, corms, and leaves of one-year-old diploid, triploid, and tetraploid Bletilla striata showed that the triploid Bletilla striata exhibited obvious polyploidy and hybrid vigor. Figure 3 a-f, Figure 5 a-e).

[0044] Planting and management of triploid Bletilla striata: When the triploid Bletilla striata roots were lush and corms appeared at the root, they could be transplanted in the field. The planting and field management were performed according to the conventional methods for Bletilla striata.

[0045] Test Example 1

[0046] 1. From May to June 2020, an investigation of Bletilla striata germplasm resources was conducted, and the main cultivars distributed in Yunnan were collected, morphologically analyzed, and identified for chromosome number. New roots of Bletilla striata were taken, and the root meristems were cut and treated with 0.002 M 8-hydroxyquinoline at 20-22 °C for 3.5-4 hours. The samples were fixed with 3:1 (methanol: glacial acetic acid) fixing solution, prepared into chromosome specimens by smearing, dyed with a 40:1 (phosphate buffer solution: Giemsa mother solution) dyeing solution for 40 minutes, rinsed with tap water, air-dried, and observed and analyzed under a microscope. The results, as shown in Figure 1 , showed that Bletilla striata var. alba ( Bletilla striata ) and Bletilla striata var. purpurea ( alba ) were both diploid, with 2n = 2x = 36 ( Bletilla striata ). A pure diploid Bletilla striata var. alba was selected as the tetraploid mutagenesis material. Figure 1

[0047] ​2. From March to May 2021, artificial pollination was performed on the diploid Baihua Baiji (artificial pollination was performed 3-5 days after Baihua Baiji bloomed, and homogametic pollination was adopted).

[0048] 3. From July to August 2021, the diploid Baihua Baiji fruits were harvested, and sowing was started in September. The sowing method was as follows: the seeds in the diploid Baihua Baiji fruit pods were taken, mixed with activated carbon, and the ratio of the seeds to the activated carbon was 1:20, and then the seeds were uniformly scattered on the seedbed using a 20-mesh sieve. The seedbed substrate was as follows: under-forest humus: vermiculite: nutrient soil (1:1:1). The temperature was maintained at 20-25°C, the humidity was 60%-80%, the light transmittance was about 25%, the seedbed substrate was kept moist by spraying water in a misty form twice a day, and after the seeds germinated and the seedlings grew, water was sprayed once a day, water-soluble fertilizer was sprayed once every two weeks, and ventilation was maintained.

[0049] 4. From October to November 2021, tetraploid artificial induction was performed. The diploid Baihua Baiji obtained in step 1 was selected as the tetraploid mutagenesis material, and the Baiji seed protocorms were taken. Before the green bud points appeared on the protocorms, a mixed chemical mutagen was used to induce tetraploidy. The mixed chemical mutagen was a mixed aqueous solution of 10 ppm p-dichlorobenzene + 5 ppm a-bromonaphthalene, and the protocorms were cultured in the dark at 20-22°C for 3 times of intermittent treatment, each time for 24-48 hours. The results are shown in Figure 2 b-d.

[0050] 5. From May to June 2022, tetraploid identification was performed. After the protocorms differentiated into seedlings and the root systems grew to 1-2 cm, the root parts of the seedlings were cut and the tissues were treated with 0.002 M 8-hydroxyquinoline for 3.5-4 hours, fixed with a fixing solution with a volume ratio of 3:1 (methanol: glacial acetic acid), and chromosome specimens were prepared by the smear method. After natural air drying, the specimens were dyed with Giemsa staining solution. Microscopic observation and analysis were performed. The results are shown in Figure 3 , and a homozygous tetraploid Baihua Baiji with 2n=4x=72 was screened out. The tetraploid mutagenesis rate reached 52.5%. Then the culture was continued until flowering.

[0051] First, different gradient treatment tests were performed (Table 1), 100-200 protocorms were treated in each group, and then the protocorms were transferred to normal culture. After 2 weeks, the survival rate of the protocorms was counted, and after 60 days, chromosome identification was performed, the number of tetraploid seedlings was counted, the tetraploid mutagenesis rate was analyzed, and non-aneuploids including octoploids (8x) were screened out. The results showed that the intermittent treatment method had the best tetraploid mutagenesis rate, which could reach more than 50% (Table 1).

[0052] Table 1. Effects of different treatment methods and times of mixed mutagens on Baihua Baiji

[0053]

[0054] 6. From March to April 2024, triploid hybridization was conducted. Using tetraploid white-flowered Bletilla striata as the female parent (4x♀) and diploid purple-flowered trifoliate Bletilla striata as the male parent (2x♂), i.e., 4x♀ × 2x♂, triploid hybridization was carried out to obtain triploid Bletilla striata hybrids. The results are as follows... Figure 3 cd, Figure 4 As shown.

[0055] 7. From July to August 2024, the triploid Bletilla striata fruits were harvested. Sowing began in September using direct seeding. Seeds from the triploid Bletilla striata pods were collected and first thoroughly mixed with activated carbon at a ratio of 1:20. The seeds were then evenly sown onto the seedbed using a 20-mesh sieve. The seedbed substrate consisted of forest humus, vermiculite, and nutrient soil (1:1:1). The temperature was maintained at 20–25 ℃, humidity at 60%–80%, and light transmittance at approximately 25%. The seedbed substrate was kept moist by misting twice daily. After seed germination and seedling emergence, water was sprayed once daily, and water-soluble fertilizer was applied every two weeks, while maintaining ventilation. Results showed that the germination rate of triploid Bletilla striata seeds was over 80%. Figure 4 ).

[0056] Table 2. Effects of activated carbon on the germination rate of Bletilla striata seeds.

[0057]

[0058] Once the seedlings have developed a robust root system and sprouted bulbs, they can be transplanted into the field. Before transplanting, dig planting trenches 10cm deep, 30cm apart, and 25cm apart between plants, and manage them according to the standard management methods for Bletilla striata.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for breeding a white birch triploid, characterized by, It comprises the following steps: (1) screening Bletilla striata diploid; (2) Bletilla striata tetraploid artificial induction, identification and screening; (3) cultivating the identified tetraploid obtained in step (2) to flowering; (4) crossing the tetraploid obtained in step (3) with the diploid in step (1) to obtain Bletilla striata triploid; In step (2), the specific method of artificial induction of Bletilla striata tetraploid is: selecting white Bletilla striata as the mutagenic material of Bletilla striata tetraploid, sowing the seeds in the seedbed to obtain round corms, and before the green bud points of the protocorms emerge, artificial induction is carried out with mutagenic agents: 20-22℃, intermittent treatment for 3 times, 24-48 hours each time; the mutagenic agent is: a mixed aqueous solution of 10ppm p-dichlorobenzene and 5ppm α-bromonaphthalene.

2. The method of claim 1, wherein, The screening process is: taking Bletilla striata new roots, cutting the root part of the living tissue, treating with 0.002M 8-hydroxyquinoline at 20-22℃ for 3.5-4 hours, fixing with a fixing solution, preparing chromosome specimens by smear method, then staining with staining solution for 40 minutes, rinsing with tap water, and air drying.

3. The method of claim 2, wherein, The fixing solution is methanol: glacial acetic acid = 3:1; the staining solution is phosphate buffer solution: Giemsa mother liquor = 40:

1.

4. Use of the method according to any one of claims 1-3 in the preparation of Bletilla striata triploid.

5. A method for breeding seedlings of a white birch triploid, characterized by, First, the cultivation method of Bletilla striata triploid according to claim 1 is used to obtain triploid, then the seeds in the fruit pods of the triploid are uniformly mixed with activated carbon, the seeds are uniformly sown on the seedbed with a 20-mesh sieve, misty water is sprayed 2 times a day to keep the substrate of the seedbed moist, after the seeds germinate into seedlings, water is sprayed 1 time a day, water-soluble fertilizer is sprayed 1 time a week for 2 weeks, and ventilation is maintained to obtain triploid seedlings; In which, the ratio of seeds in the fruit pods of triploid to activated carbon is 1:20-30.

6. Use of the method according to claim 5 in the preparation of Bletilla striata triploid seedlings.

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