Breeding method and application of bletilla striata triploid plants

Abstract

The invention belongs to the technical field of cultivation of new varieties of plants, and particularly relates to a breeding method and application of a bletilla striata triploid plant. The cultivation method of the bletilla striata triploid comprises the following steps: screening a bletilla striata diploid; artificially inducing and identifying the rhizoma bletillae tetraploid; cultivating the identified tetraploid until the tetraploid blooms; hybridizing the tetraploid and the diploid to obtain a rhizoma bletillae triploid fruit; wherein a mutagenic agent used for induction is a mixed aqueous solution of 10 ppm of p-dichlorobenzene and 5 ppm of alpha-bromonaphthalene. According to the invention, a new bletilla striata triploid strain is created for the first time, and a set of complete bletilla striata triploid advantage utilization and breeding method is established. The new bletilla striata triploid germplasm is obtained for the first time, and a scientific foundation is laid for bletilla striata triploid breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant new variety breeding, and particularly relates to a breeding method of Bletilla striata triploid plants and application thereof. BACKGROUND

[0002] Bletilla striata (Thunb) Rchb.f. belongs to Orchidaceae and is a perennial herbaceous plant, mainly distributed in the Yangtze River Basin to the south and southwest, and widely distributed in Anhui, Jiangxi, Hubei, Sichuan, Guizhou, Yunnan, Shaanxi and Gansu in China, and cultivated in all places. There are about 6 species of Bletilla in the world, all of which are produced in East Asia. There are 4 species of Bletilla in China, including B. sinensis, B. formosana, B. striata and B. ochracea. Bletilla varieties can be roughly divided into two categories: one is purple flower Bletilla, with purple red flowers, including two small categories of large and small species; the other is yellow flower Bletilla, with yellow white flowers. There are also a small amount of Bletilla varieties with white, blue, yellow and pink flowers, mainly purple and red. Bletilla has high ornamental value due to its rich and beautiful flower color, elegant flower shape and beautiful leaf state.

[0003] Bletilla is one of the commonly used Chinese medicinal materials in China, and the dried tubers are used as traditional Chinese medicine, which is widely used in medicine. Bletilla tubers contain more than 100 active compounds such as steroids, terpenes, esters, ethers, biphenyls, dihydrophenyls and benzyls, as well as trace elements such as boron, molybdenum, manganese, iron, zinc and copper, and also contain rich starch, glucose, volatile oil and mucilage, which has the functions of stopping bleeding, promoting tissue regeneration, anti-inflammatory, protecting gastric mucosa, tonifying lung, improving skin nutrition and preventing wrinkle, and is widely used in medicine, food industry and daily chemical industry, and has high medicinal value and economic value.

[0004] Due to long-term human plundering and lack of protection, the resources of Bletilla are decreasing, and the medicinal raw materials are in serious shortage, and Bletilla has been listed as one of the 30 rare and endangered natural medicines in China. Hybridization and new variety breeding of Bletilla in China are still in the lagging stage, and long-term artificial cultivation is mainly used, which leads to degeneration of germplasm, reduction of effective component content and difficulty in increasing yield. It is urgent to protect Bletilla germplasm resources, genetic improvement and new variety breeding.

[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 placing protocorms in colchicine and culturing the 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 has not been reported. 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: white-flowered white-bark magnolia is selected as the mutagenic material for tetraploid, the seeds are sowed on a seedbed to obtain round corms, and before the green bud points of the corms emerge, artificial induction is carried out using the mutagen: 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: the new roots of white-bark magnolia are taken, the root part of the living tissue is cut, 0.002 M 8-hydroxyquinoline is used for treatment at 20-22 DEG C for 3.5-4 hours, a fixing solution is used for fixation, a smear method is used to prepare a chromosome specimen, then a staining solution is used for staining for 40 minutes, tap water is used for rinsing, and air drying is carried out.

[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 a white-bark magnolia triploid, and the method is characterized in that: seeds in a fruit pod of the triploid obtained in claim 1 are uniformly stirred with activated carbon, the seeds are uniformly sowed on a seedbed by using a 20-mesh sieve, misty water is sprayed 2 times a day to keep the seedbed substrate moist, after the seeds germinate into seedlings, water is sprayed 1 time a day, water-soluble fertilizer is sprayed 1 time a week, and ventilation is kept to obtain triploid seedlings.

[0022] Further, the ratio of the seeds in the fruit pod 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 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 Figure a is a diploid Baihe white flower, figure b is a genome chromosome of the diploid Baihe white flower, 2n=2x=36; figure c is a diploid Baihe purple flower, and figure d is a genome chromosome of the diploid Baihe purple flower, 2n=2x=36.

[0028] Figure 2 Figure a is a diploid Baihe white flower, figure b is a genome chromosome of the diploid Baihe white flower, 2n=2x=36; figure c is a diploid Baihe purple flower, and figure d is a genome chromosome of the diploid Baihe purple flower, 2n=2x=36.

[0029] Figure 3 Figure a is a diploid Baihe white flower, figure b is a genome chromosome of the diploid Baihe white flower, 2n=2x=36; figure c is a diploid Baihe purple flower, and figure d is a genome chromosome of the diploid Baihe purple flower, 2n=2x=36.

[0030] Figure 4 Figure a is a diploid Baihe white flower, figure b is a genome chromosome of the diploid Baihe white flower, 2n=2x=36; figure c is a diploid Baihe purple flower, and figure d is a genome chromosome of the diploid Baihe purple flower, 2n=2x=36.

[0031] Figure 5For Example 2, the roots and corms of diploid, triploid and tetraploid Bletilla hyacinthina, and the comparison of leaves (1 year old) are shown in Figures a-c, wherein Figures a and c are the roots and corms of diploid Bletilla hyacinthina seedlings; Figures b and d are the roots and corms of triploid Bletilla hyacinthina seedlings; and Figure e is a comparison of the leaves of diploid, triploid and tetraploid Bletilla hyacinthina seedlings. DETAILED DESCRIPTION

[0032] Example 1

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

[0034] Three main cultivars in Yunnan were collected, ① Bletilla ochracea Schltr., ② Bletilla striata war. alba, and ③ 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, Bletilla striata war. alba was selected as the tetraploid mutation material. First, the seeds of Bletilla striata war. alba were directly sown in the seedbed. When the seeds turned green and became green small nodules, i.e. protocorms, before the green bud points of the protocorms emerged, a mixed chemical mutagen was used to induce tetraploidy of the protocorms. 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 Bletilla hyacinthina: When 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 Bletilla hyacinthina seedling treated by the mutagen was treated with 0.002 M 8-hydroxyquinoline for 3.5-4 hours, and then fixed with a 3:1 (methanol: glacial acetic acid) fixing solution. 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 homozygous tetraploid Bletilla hyacinthina 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. Hybridization and breeding of triploid Bletilla striata

[0041] With the tetraploid white-flowered Bletilla striata as the female parent (4x♀) and the diploid purple-flowered Bletilla striata as the male parent (2x♂), artificial hybridization was performed 3-5 days after the tetraploid and diploid Bletilla striata bloomed, i.e., 4x♀×2x♂, to obtain the triploid Bletilla striata hybrid ( Figure 3 cd).

[0042] 2. Seedling propagation of triploid Bletilla

[0043] Take the triploid Bletilla hybrid and first mix it evenly with activated carbon. The ratio of seeds to activated carbon is 1:20. Use a 20-mesh sieve to evenly sow the seeds on the seedbed. The seedbed matrix is: forest humus: vermiculite: nutrient soil (1:1:1). The temperature is maintained at 20-25 ℃, the humidity is 60%-80%, and the light transmittance is about 25%. Spray water twice a day. After the seeds germinate into seedlings, the humidity is maintained at 50%-60%, and spray water once a day. The results are as follows Figure 4 As shown, the germination rate of seeds reached more than 80%. From the comparison of the roots, bulbs and leaves of one-year-old two, three and four ploid Bletilla striata, it can be seen that the triploid Bletilla striata showed obvious polyploidy and hybrid vigor ( Figure 3 af, Figure 5 ae).

[0044] Planting and management of triploid Bletilla striata: When the root system of triploid Bletilla striata is lush and bulbs appear on the roots, it can be transplanted into the field. Planting and field management should be carried out according to the conventional methods for Bletilla striata.

[0045] Test Example 1

[0046] 1. From May to June 2020, a survey of Bletilla striata germplasm resources was conducted. The main cultivated varieties distributed in Yunnan were collected, morphologically analyzed, and chromosome number identification was performed. New roots of Bletilla striata were taken, and the root meristem was cut. The root tissue was treated with 0.002 M 8-hydroxyquinoline at 20-22 °C for 3.5-4 hours, fixed with a 3:1 (methanol: glacial acetic acid) fixative, and chromosome specimens were prepared by smear method. Then, the specimens were stained with a 40:1 volume ratio (phosphate buffer solution: Giemsa mother solution) staining solution for 40 minutes, rinsed with tap water, air-dried, and observed and analyzed under a microscope. The results are as follows: Figure 1 As shown, Bletilla striata war.alba and Bletilla striata (Thunb.) Reichb.f are both diploid, 2n=2x=36( Figure 1 ), and the homozygous (non-hybrid) diploid Bletilla striata was selected as the tetraploid mutagenesis material.

[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 Baiji with 2n=4x=72 was screened. 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. 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] 6. From March to April 2024, triploid hybridization was carried out. The tetraploid white flower Bletilla striata was used as the female parent (4x♀) and the diploid purple flower three-forked Bletilla striata was used as the male parent (2x♂), i.e. 4x♀×2x♂, and triploid hybrid Bletilla striata was obtained. The results are as follows: Figure 3 cd, Figure 4 shown.

[0054] 7. From July to August 2024, harvest the triploid Bletilla striata fruits, and start sowing in September. Use direct seeding to raise seedlings. Take the seeds from the fruit pods of the triploid Bletilla striata, first mix them evenly with activated carbon, with the ratio of seeds to activated carbon being 1:20, and then use a 20-mesh sieve to evenly sow the seeds on the seedbed. The seedbed matrix is: forest humus: vermiculite: nutrient soil (1:1:1). Keep the temperature at 20-25 ℃, humidity at 60%-80%, and light transmittance at about 25%. Mist water twice a day to keep the seedbed matrix moist. After the seeds germinate into seedlings, spray water once a day, spray water-soluble fertilizer once every two weeks, and maintain ventilation. The results showed that the germination rate of triploid Bletilla striata seeds was above 80% ( Figure 4 ).

[0055] Table 2 Effects of activated carbon on the germination rate of Bletilla striata seeds

[0056] When the seedlings have lush roots and sprouted bulbs, they can be transplanted into the field. Before transplanting the seedlings, dig planting trenches with a depth of 10 cm, a distance of 30 cm between trenches, and a spacing of 25 cm between plants. Manage them according to the conventional management methods for Bletilla striata.

[0057] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for cultivating triploid Bletilla striata, characterized in that: The steps include: (1) Screening of diploid Bletilla striata; (2) Artificial induction, identification and screening of tetraploid Bletilla striata; (3) cultivating the identified tetraploid obtained in step (2) until flowering; (4) hybridizing the tetraploid obtained in step (3) with the diploid obtained in step (1) to obtain a triploid Bletilla striata; The mutagen used for induction in step (2) is a mixed aqueous solution of 10 ppm p-dichlorobenzene and 5 ppm α-bromonaphthalene.

2. The method according to claim 1, characterized in that The specific method for artificially inducing tetraploid Bletilla striata in step (2) is as follows: selecting white flower Bletilla striata as the mutagenic material for tetraploid Bletilla striata, sowing the seeds in a nursery bed to obtain round corms, and artificially inducing the original corms before green buds emerge: 20-22°C, intermittent treatment 2-3 times, each time for 24-48 hours.

3. The method according to claim 1, characterized in that The screening process is as follows: taking new roots of Bletilla striata, cutting the root mesenchyme, treating with 0.002 M 8-hydroxyquinoline at 20-22° C. for 3.5-4 hours, fixing with a fixative, preparing chromosome specimens by a smear method, then staining with a staining solution for 40 minutes, rinsing with tap water, and air-drying.

4. The method according to claim 3, characterized in that The fixing solution is methanol: glacial acetic acid = 3:1; the staining solution is phosphate buffer solution: Giemsa mother solution = 40:

1.

5. Use of the method according to any one of claims 1 to 4 in preparing triploid Bletilla striata.

6. A method for breeding triploid seedlings of Bletilla striata, characterized in that: The seeds in the triploid fruit pods obtained in claim 1 are mixed evenly with activated carbon, the seeds are evenly sown on the seedbed using a 20-mesh sieve, and mist sprayed with water 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 every two weeks, and ventilation is maintained to obtain triploid seedlings. Among them, the ratio of seeds to activated carbon in the triploid pods is 1:20-30.

7. Use of the method according to claim 6 in preparing triploid seedlings of Bletilla striata.

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