A method for blueberry radiation-induced mutation breeding

Abstract

This invention discloses a method for blueberry radiation-induced mutation breeding. The method includes the following steps: 1) 60 Co-γ rays are used to irradiate tissue culture seedlings grown in culture medium A to obtain explant material; wherein the intensity of the irradiation treatment is 0.5-1.5 Gy / min, and the dose of the irradiation treatment is 80-120 Gy; 2) the explant material is inoculated into culture medium B for a first light culture to induce the generation of adventitious buds; 3) the culture from step 2) is inoculated into culture medium A for a second light culture to obtain regenerated seedlings; 4) the regenerated seedlings are transplanted into culture medium C for a third light culture to obtain blueberry plants. This invention utilizes radiation mutagenesis in blueberries to obtain a large number of irradiated blueberry plants within 3-4 months, providing technical support for blueberry breeding and improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to a blueberry radiation mutation breeding method. Background Technology

[0002] blueberry( Vaccinium Blueberries (Vaccinium spp.) are woody berry plants belonging to the genus Vaccinium in the family Ericaceae. They have a relatively short domestication history, approximately 120 years ago. When ripe, blueberries are soft, dark blue berries, rich in various bioactive components beneficial to human health, such as vitamins with antioxidant activity, anthocyanins, and polyphenols like flavanols. In recent years, rising consumer demand for healthier dietary choices and increasing awareness of the nutritional benefits of blueberries have fueled their popularity. This popularity, in turn, has stimulated extensive research into breeding new blueberry varieties. Hybridization remains the primary breeding method for blueberries. Hybridization has been most effective in improving highbush blueberry varieties. Highbush blueberries (Vaccinium spp.) Vaccinium corymbosum L.) originated in Canada and North America, and through selective breeding of highbush blueberries, the northern highbush blueberry variety was developed. (This is in contrast to evergreen blueberries.) Vaccinium darrowii The hybridization of North Highbush blueberries with Camp resulted in Southern Highbush blueberries, expanding their range to subtropical and tropical regions. Subsequent hybridization with other varieties further improved the adaptability and berry quality of Highbush blueberries, enhancing characteristics such as drought tolerance, high pH tolerance, berry firmness, size, flavor, and yield.

[0003] Significant progress has been made in traditional hybridization breeding of blueberries, but advanced breeding methods, such as mutation breeding, are rarely used. Mutation breeding, divided into physical and chemical mutagenesis, uses physical and chemical mutagens to induce random and diverse variations in the plant genome, including site mutations and large-segment changes. With the maturation of mutation breeding technology, it can effectively shorten the breeding time for new varieties. Currently, mutation breeding is widely used in various fruit crops, such as pears, peaches, apples, bananas, grapes, plums, and citrus fruits, to enhance characteristics such as appearance, taste, aroma, tree shape, and self-compatibility. Although mutation breeding shows great promise in developing superior varieties needed by the market, there are currently no reported successful cases for blueberries.

[0004] Growing market demand has led to an expansion of blueberry cultivation areas globally. In the absence of advanced breeding methods, crossbreeding between blueberry varieties has been widely used to screen for seedlings adapted to local climates, including in China, Chile, Japan, the UK, and New Zealand. China's blueberry cultivation area has increased more than 200-fold in the past 20 years, but the soil in many parts of China is unsuitable for growing North American blueberries, necessitating the development of varieties better adapted to local environments. The Southern Highbush blueberry 'Lanmei No. 1' is a commercially cultivated variety in my country with good soil adaptability and high anthocyanin content in its fruit, recognized as a superior national variety and recommended for widespread cultivation in southern my country. Currently, 'Lanmei No. 1' is the most widely cultivated native variety in my country, possessing high economic value in the fresh berry market and industrial anthocyanin extraction. Compared to traditional hybridization breeding, mutation breeding is more suitable for optimizing superior varieties like 'Lanmei No. 1'. Hybridization combines the genomes of two parents, potentially producing undesirable phenotypes in the F1 generation. In contrast, mutation breeding is more effective at improving specific traits of superior varieties while maintaining other desirable traits. Therefore, establishing a mutation breeding system for blueberries is crucial for cultivating superior varieties.

[0005] Gamma rays have wide applications in plant mutation breeding. Currently, over 70% of new varieties produced using radiation-induced mutation breeding utilize gamma rays, including berry plants. CN108935088A discloses a method for improving the radiation-induced mutation rate of blueberry explants, comprising the following steps: placing blueberry embryogenic callus tissue in a cobalt... 60 Repeated irradiation at half the half-lethal dose yields embryogenic callus tissue; subculture and propagation generate M0 generation seedlings; these seedlings are cultivated until flowering and fruiting, then naturally hybridized with unirradiated target varieties to harvest hybrid fruits, obtaining M1 generation hybrid seeds with irradiated parents; these irradiated hybrid seeds are cultivated to obtain irradiated hybrid seedlings; after selecting the target traits, the target materials are used for rapid tissue culture propagation to obtain M2 generation blueberry irradiation lineages with different target traits. This method improves the radiation-induced mutagenesis rate, reduces the radiation lethality rate, increases the number of effective seedlings after irradiation, and improves the efficiency of radiation work. It solves problems encountered in blueberry radiation breeding, such as abundant original materials but few effective materials after irradiation, easy browning and death of irradiated materials, and low radiation-induced mutagenesis rate. The material used in this patent is blueberry embryogenic callus tissue, which is more difficult to obtain.

[0006] Therefore, this application uses different dosages. 60 The aim of this study was to establish a method for blueberry mutation breeding by using Co-γ rays as a radiation mutagenesis source to induce radiation mutagenesis in blueberry tissue culture seedlings. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for blueberry radiation-induced mutation breeding, comprising the following steps:

[0008] 1) 60 Co-γ rays were used to irradiate tissue culture seedlings grown in culture medium A to obtain explant material; wherein the intensity of the irradiation treatment was 0.5–1.5 Gy / min and the dose of the irradiation treatment was 80–120 Gy.

[0009] 2) The explant material was inoculated into culture medium B for the first light culture to induce the generation of adventitious shoots;

[0010] 3) The culture from step 2) is inoculated into culture medium A for a second light culture to obtain regenerated seedlings;

[0011] 4) The regenerated seedlings were transplanted into culture medium C for a third light culture to obtain blueberry plants.

[0012] This invention, in its initial experimental phase, collected published blueberry-related radiation-induced mutagenesis experiments and methods. It was found that existing blueberry radiation-induced mutagenesis experiments did not utilize tissue culture for propagation, and existing methods could not rapidly obtain a large number of blueberry plants. Therefore, this application uses radiation-induced mutagenesis in blueberries to identify a suitable median lethal dose of radiation, and combines this with tissue culture to obtain a large number of irradiated blueberry plants in a short time, providing technical support for the breeding of superior blueberry varieties. This patent utilizes tissue culture to obtain induced mutagenesis plants, using fewer raw materials and obtaining a large amount of effective post-irradiation material.

[0013] In some embodiments, in 1), the culture medium A comprises: WPM medium, 20–40 g / L sucrose, 7–10 g / L agar, 0.01–0.1 mg / L zeatin, and 0.01–0.1 mg / L indolebutyric acid. In a specific example, it is WPM medium, 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L zeatin, and 0.05 mg / L indolebutyric acid. This application also conducted research on the culture medium formulation for the regeneration of blueberry tissue culture seedlings, and established a culture medium formulation suitable for the growth of blueberry tissue culture seedlings, such as the study on zeatin in culture medium A. Zeatin (ZT), as a type of cytokinin, can promote the induction of adventitious buds, the proliferation and growth of lateral buds; indolebutyric acid (IBA) plays a role in rooting culture, and both have an important influence on the in vitro culture of blueberries. In the prior art, it is generally believed that the zeatin concentration of 0.5 to 1.0 mg / L has the best effect on promoting the proliferation of blueberry lateral buds. In this study, it was found that tissue culture seedlings could induce buds after being cultured in medium A with a zeatin concentration of 0.01 to 0.1 mg / L, but only callus tissue was produced and no buds were produced in medium A with a zeatin concentration of 0.5 to 2 mg / L.

[0014] In some embodiments, in 1), the growth conditions of the tissue culture seedlings are: 20–30°C under light for 12–16 h, with a light intensity of 50–150 μmol / m². -2 s -1 The humidity was 40–80%. In one specific example, the culture was carried out at 24°C under light for 16 h, with a light intensity of 100 μmol / m². -2 s -1 The humidity is 60%.

[0015] In some embodiments, 1) further includes cutting leaves and / or stem segments of the tissue-cultured seedlings after radiation treatment to form partial tissues for use as explant material. In some specific embodiments, leaves are cut in half or stem segments are cut into 1-3 cm segments. In some specific embodiments, the cut leaves are laid flat on the surface of the subsequent culture medium B, with the upper surface of the leaves facing down. Cutting facilitates full contact with the subsequent culture medium B, improving the germination rate.

[0016] In some embodiments, in step 2), the explant is a leaf or stem segment of a tissue-cultured seedling. This involves cutting the irradiated tissue-cultured seedling into 1-3 cm stem segments and halving the leaves. The stem segments are preferably 2 cm long.

[0017] In some embodiments, in step 2), the culture medium B comprises: WPM medium, 5–10 g / L sucrose, 6–10 g / L agar, and 0.05–0.1 mg / L thidiazuron. In a specific example, it is WPM medium, 5 g / L sucrose, 8.5 g / L agar, and 0.05 mg / L thidiazuron. This application studies culture medium formulations suitable for the growth of blueberry tissue culture seedlings, such as the concentrations of thidiazuron and sucrose in culture medium B. Thidiazuron (TDZ) is a synthetic plant growth regulator, belonging to the phenylurea class of compounds, and has the effect of kinetin. It is mainly used to promote bud differentiation and proliferation in plants; sucrose is a commonly used carbon source, providing substrate and energy for cellular respiratory metabolism, thus enabling robust plant growth. This application investigated the concentration of thiamethoxam, finding that 0.05–0.1 mg / L thiamethoxam resulted in a blueberry budding rate exceeding 60%, while concentrations below 0.05 and above 0.1 mg / L resulted in budding rates consistently below 60%. The application also investigated the concentration of sucrose, finding that 10–20 g / L sucrose resulted in a blueberry budding rate exceeding 60%. Further research was conducted on the compatibility of different concentrations of sucrose and thiamethoxam, finding that a combination of 0.05–0.1 mg / L thiamethoxam and 10–20 g / L sucrose resulted in the best leaf budding rate of 62–95% after 60 days of radiation, with the highest budding rate (90–95%) achieved when 0.05 mg / L thiamethoxam was combined with 10 g / L sucrose. Furthermore, the application investigated culture media with identical concentrations of all other components, but with sucrose concentrations of 0.5 g / L and 10 g / L, finding that... When cultured in a medium containing g / L sucrose, more buds appear on individual leaves. The formulation of medium B in this application is beneficial for the formation of new adventitious buds and regeneration from callus tissues such as stem segments and leaves of tissue-cultured seedlings.

[0018] In some embodiments, in step 2), the conditions for the first photoculture are: a light intensity of 50–150 μmol / m². -2 s -1 The culture cycle consisted of alternating light and dark conditions, with the light culture time being 12–16 h and the dark culture time being 8–12 h. In a specific example, the light intensity was 100 μmol / m². -2 s -1 The light exposure time was 16 hours, and the dark incubation time was 8 hours.

[0019] In some embodiments, in step 2), the temperature for the first light incubation is 20–30°C, and the humidity is 40–80%. In a specific example, the temperature is 24°C, and the humidity is 60%.

[0020] In some embodiments, in 3), the culture is the adventitious bud induced in 2), and the height of the adventitious bud is 2-6 cm. In a specific example, it is 4 cm. An adventitious bud refers to a bud that differentiates directly from an abnormal location on a plant (such as a leaf, stem segment, root, callus, etc.) in tissue culture. It can be induced directly from an explant or through callus culture.

[0021] In some embodiments, in step 3), the conditions for the second light-induced culture are: a light intensity of 50–150 μmol / m². -2 s -1 The culture cycle consisted of alternating light and dark conditions, with the light culture time being 12–16 h and the dark culture time being 8–12 h. In a specific example, the light intensity was 100 μmol / m². -2 s -1 The light incubation time was 16 hours, and the dark incubation time was 8 hours.

[0022] In some embodiments, in step 3), the temperature for the second light incubation is 20–30°C, and the humidity is 40–80%. In a specific example, the temperature is 24°C, and the humidity is 60%.

[0023] In some embodiments, in step 4), the culture medium C comprises: WPM medium, 10–40 g / L sucrose, 6–10 g / L agar, 0.1–0.6 mg / L 3-indoleacetic acid, 0.5–1.5 mg / L kinetin, and 0.1–1 g / L charcoal powder. In a specific example, the culture medium C comprises: WPM medium, 30 g / L sucrose, 8.5 g / L agar, 0.3 mg / L 3-indoleacetic acid, 1 mg / L kinetin, and 0.5 g / L charcoal powder. 3-Indoleacetic acid (IAA), as an auxin, promotes blueberry rooting; kinetin (KT), also known as 6-furfurylaminopurine, is a non-natural cytokinin that promotes cell division and organ differentiation in blueberry culture; the charcoal powder is activated carbon powder, which creates a dark environment conducive to root induction and root growth. The culture medium C of this application is beneficial for blueberry rooting.

[0024] In some embodiments, in step 4), the conditions for the third photoculture are: a light intensity of 50–150 μmol / m². -2 s -1 The culture cycle consisted of alternating light and dark conditions, with the light exposure time being 12–16 h and the dark culture time being 8–12 h. In a specific example, the light intensity was 100 μmol / m². -2 s -1The light incubation time was 16 hours, and the dark incubation time was 8 hours.

[0025] In some embodiments, in step 4), the temperature for the third light incubation is 20–30°C, and the humidity is 40–80%. In a specific example, the temperature is 24°C, and the humidity is 60%.

[0026] In this application, the typical but not limited formulation of the WPM culture medium is, for example: 400 mg / L NH4NO3, 556 mg / L Ca(NO3)2·4H2O, 96 mg / L CaCl2·2H2O, 900 mg / L K2SO4, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 0.25 mg / L NaMoO4·2H2O, 22.4 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L CuSO4·5H2O, 27.8 mg / L FeSO4·5H2O, 37.3 mg / L Na2EDTA, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L pyridoxine phosphate (VB6), 0.5 mg / L thiamine phosphate (VB1), 2.0 mg / L glycine. Specifically, it is Lloyd & McCown Woody Plant Basal Medium with Vitamins (WPM) (Phyto Tech, L449).

[0027] In some implementations, in step 4), when the seedlings are grown under light until they reach 6-14 cm in height, they are transferred to seedling trays for further cultivation to obtain blueberry plants.

[0028] In some embodiments, the blueberry variety is selected from the superior southern highbush blueberry variety 'Lanmei No. 1' developed and bred by Zhejiang Lanmei Agriculture Co., Ltd.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) This invention establishes a blueberry regeneration system, from the irradiated tissue culture seedlings to the induction of buds by cutting leaves or stem segments, to rooting and planting in seedling trays, the cycle is 3 to 4 months.

[0031] 2) This invention uses different dosages 60 Radiation treatment of blueberry tissue culture seedlings with Co-γ rays was conducted to find the appropriate radiation median lethal dose for blueberries, laying the foundation for obtaining superior blueberry varieties.

[0032] 3) The method of the present invention can quickly obtain a large number of irradiated plants. Attached Figure Description

[0033] Figure 1 These are photographs showing the growth of stem segments induced by different radiation doses after 30 and 60 days, as described in Example 1. The scale bar is 1 cm.

[0034] Figure 2 The image shows leaves from culture medium B that induced adventitious buds 6 weeks after irradiation in Example 3. The scale bar is 1 mm.

[0035] Figure 3 The stem segments from Example 3 that induced numerous adventitious buds on culture medium B 4 weeks after irradiation. The scale bar is 1 cm.

[0036] Figure 4 Seedlings that were induced to root by culturing on culture medium C for one week 12 weeks after irradiation, as described in Example 3. The scale bar is 1 cm.

[0037] Figure 5 The radiated seedlings of 'Lanmei No. 1' were obtained after 15 weeks of hardening-off following radiation, as described in Example 3. The scale bar is 1 cm. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0041] In the following embodiments of this application, ZT represents zeatin, KT represents kinetin, IBA represents indolebutyric acid, TDZ represents thiabendazole, and IAA represents 3-indoleacetic acid.

[0042] The WPM medium used was Lloyd & McCown Woody Plant Basal Medium with Vitamins (WPM) (Phyto Tech, L449).

[0043] The leaf callus induction rate is calculated as follows: the number of leaves that produce callus after 60 days on culture medium B / the total number of leaves.

[0044] The leaf budding rate after 60 days is calculated as follows: the number of leaves that produce adventitious buds after 60 days on culture medium B / the total number of leaves. (A leaf with multiple buds is still counted as 1).

[0045] The budding rate of stem segments after 60 days is calculated as follows: the number of stem segments that produce adventitious buds after 60 days on culture medium B / the total number of stem segments. (A stem segment with multiple buds is still counted as 1).

[0046] Example 1

[0047] In Example 1, radiation doses were screened to obtain suitable radiation doses for tissue culture seedlings. The details are as follows:

[0048] 1.1 The crop and nuclear technology provided by the Zhejiang Academy of Agricultural Sciences Research Institute 60 Co-γ rays were used to treat 'Lanmei No. 1' tissue culture seedlings with seven radiation doses of 10, 20, 40, 80, 120, and 150 Gy, with a radiation intensity of 1 Gy·min at each dose. -1 Four replicates were set up for each radiation dose, with 60 seedlings per replicate, as shown in Tables 1 and 2.

[0049] The tissue culture seedlings were 'Lanmei No. 1' seedlings that had been cultured in culture medium A for 8 weeks and exhibited good and uniform growth. The growth and culture conditions were as follows: 16 hours of light per day at 24℃ for 8 weeks, with a light intensity of 100 μmol / m². -2 s -1 The humidity was 60%; the composition of culture medium A was: WPM, 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L ZT and 0.05 mg / L IBA, pH=5.2±0.02.

[0050] 1.2 Using the tissue culture seedlings irradiated in step 1.1 as explant material, the leaves were cut in half and placed on culture medium B with the upper surface of the leaves facing down to induce the formation of adventitious buds. Simultaneously, stem segments were cut into 2 cm pieces and placed in culture medium B. The cut leaves and stem segments were then incubated at 24℃, 60% humidity, and under normal light (light intensity 100 μmol / m²). -2 s -1 Cultured in medium B for 16 h to ensure full contact, and subcultured every 14 days.

[0051] The components of culture medium B are: WPM, 5 g / L sucrose, 8.5 g / L agar and 0.05 mg / L TDZ, pH = 5.2 ± 0.02.

[0052] Observe the growth on the 30th and 60th days after radiation, see... Figure 1 The leaf lethality rate (%) was obtained by counting the number of surviving leaves and the total number of leaves in each replicate at each radiation dose on the 60th day after radiation, and the stem lethality rate (%) was obtained by counting the number of surviving axillary buds in each axillary bud in each replicate at each radiation dose on the 60th day after radiation, as shown in Tables 1 and 2.

[0053] Table 1 Leaf mortality rate (%)

[0054]

[0055] Table 2. Stem segment mortality rate (%)

[0056]

[0057] from Figure 1 As shown in Tables 1 and 2, the leaves treated with different radiation doses showed significant differences in leaf condition, callus formation, and the number of adventitious buds during cultivation; the growth rate and growth status of adventitious buds in stem segments treated with different radiation doses also varied.

[0058] As shown in Tables 1 and 2, the suitable radiation dose for tissue culture seedlings is 80–120 Gy. Further research will be conducted using a radiation dose of 80 Gy.

[0059] Example 2

[0060] In Example 2, the screening of culture medium A and culture medium B during the growth of tissue culture seedlings included the following:

[0061] 2.1 Screening of zeatin concentration in culture medium A

[0062] 1) To explore suitable culture media for blueberries, this example investigated a regeneration system represented by 'Lanmei No. 1'. The culture medium A used contained 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L indolebutyric acid, and different concentrations of zeatin in WPM medium. Based on the total volume of medium A, the zeatin concentrations were 0.01 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, 1.5 mg / L, and 2 mg / L, respectively. Radiation induction was applied to 'Lanmei No. 1' stem segments at a radiation intensity of 1 Gy·min⁻¹. -1 The radiation dose was 80 Gy, and all other aspects were the same as in Example 1.

[0063] Observe the growth of adventitious buds on stem segments over 60 days.

[0064] The results showed that in the culture media containing 0.5–2 mg / L zeatin, only callus tissue was produced, and no budding was induced; however, in the culture media containing 0.01–0.1 mg / L zeatin, both stem bud growth points and callus tissue produced budding. Therefore, subsequent studies were conducted using culture medium A with a zeatin concentration of 0.05 mg / L.

[0065] 2.2 Screening of different concentrations of thiamethoxam and different ratios of sucrose in culture medium B

[0066] 1) Using medium B, which is a WPM medium containing 0.05 mg / L thiamethoxam, 8.5 g / L agar, and different concentrations of sucrose, the sucrose concentrations were 10 g / L, 20 g / L, and 30 g / L based on the total volume of medium B. Radiation induction was performed on the leaves of 'Lanmei No. 1' at a radiation intensity of 1 Gy·min⁻¹. -1 The radiation dose was 80 Gy, and all other aspects were the same as in Example 1.

[0067] The leaf budding rate was statistically analyzed over 60 days.

[0068] The results showed that the leaf budding rate was higher than 60% when medium B was supplemented with 10 g / L, 20 g / L, and 30 g / L sucrose.

[0069] 2) Using medium B, which is a WPM medium containing 10 g / L sucrose, 8.5 g / L agar, and different concentrations of thiamethoxam, the thiamethoxam concentrations were 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.15 mg / L, and 0.2 mg / L (based on the total volume of medium B), radiation induction was performed on the leaves of 'Lanmei No. 1' at a radiation intensity of 1 Gy·min. -1 The radiation dose was 80 Gy, and all other aspects were the same as in Example 1.

[0070] The budding rate of leaves was statistically analyzed 60 days after radiation.

[0071] The results showed that the leaf germination rate was higher than 60% in culture medium B with 0.05 mg / L and 0.1 mg / L thiamethoxam, while other concentrations of thiamethoxam resulted in a leaf germination rate of less than 60%; indicating that the appropriate concentration of thiamethoxam is 0.05–0.1 mg / L.

[0072] 3) Using the thiamethoxam concentrations of 0.05 mg / L and 0.1 mg / L selected in 2) above, and the sucrose concentrations of 10 g / L, 20 g / L, and 30 g / L selected in 1), a mixture was prepared, as shown in Table 3. Radiation induction was then applied to the leaves of 'Lanmei No. 1' at a radiation intensity of 1 Gy·min⁻¹. -1 The radiation dose was 80 Gy, and the remaining steps were the same as in Example 1.

[0073] The budding rate of leaves after 60 days of radiation was statistically analyzed, and the results are shown in Table 3.

[0074] Table 3

[0075]

[0076] Table 3 shows that the following three formulations all produced a significant number of adventitious buds: Formulation 1, containing 0.05 mg / L thiamethoxam and 10 g / L sucrose, had a germination rate of 90%–95%; Formulation 5, containing 0.1 mg / L thiamethoxam and 20 g / L sucrose, had a germination rate of 62%–85%; and Formulation 3, containing 0.05 mg / L thiamethoxam and 30 g / L sucrose, had a germination rate of 85%–95%. Among them, Formulation 1, containing 0.05 mg / L thiamethoxam and 10 g / L sucrose, exhibited the best leaf growth and the highest germination rate.

[0077] 2.3 Further optimization of the culture medium B formulation.

[0078] Subsequently, we further optimized the regeneration system of 'Lanmei No. 1'. Culture medium B consisted of WPM medium containing 0.05 mg / L thiazuron, 5 g / L sucrose, and 8.5 g / L agar, and WPM medium containing 0.05 mg / L thiazuron, 10 g / L sucrose, and 8.5 g / L agar. Radiation induction was then performed on 'Lanmei No. 1' leaves at a radiation intensity of 1 Gy·min⁻¹. -1 The radiation dose was 80 Gy, and all other aspects were the same as in Example 1.

[0079] The budding rate of leaves after 60 days of radiation was statistically analyzed, and the results are shown in Table 3.

[0080] In comparison, the germination rates of the two culture medium formulations were similar, but the WPM medium containing 0.05 mg / L thiamethoxam, 5 g / L sucrose, and 8.5 g / L agar produced more buds per leaf. Therefore, the WPM medium containing 0.05 mg / L thiamethoxam, 5 g / L sucrose, and 8.5 g / L agar, with a pH of 5.2 ± 0.02, was selected as the optimal medium B for inducing adventitious buds in the 'Lanmei No. 1' regeneration system.

[0081] Example 3

[0082] Based on the radiation dose determined in Example 1 and the zeatin concentration in culture medium A and the sucrose and thidiazuron concentrations in culture medium B determined in Example 2, Example 3 further investigates the feasibility of the blueberry radiation-induced mutation breeding method. The specific steps are as follows:

[0083] 3.1, Adopt 60 Co-γ rays were used to treat 'Lanmei No. 1' tissue culture seedlings with a radiation dose of 80 Gy and a radiation intensity of 1 Gy / min.

[0084] The tissue culture seedlings were 'Lanmei No. 1' seedlings that had been cultured in culture medium A for 8 weeks and exhibited good and uniform growth. The growth and culture conditions were as follows: 16 hours of light per day at 24℃ for 8 weeks, with a light intensity of 100 μmol / m². -2 s -1 The humidity was 60%; the composition of culture medium A was: WPM, 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L ZT and 0.05 mg / L IBA, pH=5.2±0.02.

[0085] 3.2 Using the tissue culture seedlings irradiated in step 3.1 as explant material, cut the leaves in half and place them on culture medium B with the upper surface of the leaves facing down to induce the generation of adventitious buds (e.g. Figure 2 As shown in the image, this is a photograph taken on culture medium B 6 weeks after irradiation; simultaneously, stem segments were cut into 2 cm pieces and placed in culture medium B. Both leaves and stem segments were kept at 24°C, 60% humidity, and under normal light (light intensity of 100 μmol / m²). -2 s -1 Cultured in medium B for 16 h to ensure full contact, and subcultured every 14 days.

[0086] The components of culture medium B are: WPM, 5 g / L sucrose, 8.5 g / L agar and 0.05 mg / L TDZ, pH = 5.2 ± 0.02.

[0087] After being cultured, leaves develop callus tissue, which then develops bud-like growth points and produces distinct small buds after 4-5 weeks. The budding rate of successfully surviving leaves and producing small buds is approximately 15%. Stem segments sprout new buds or grow from the callus tissue after 3-4 weeks (e.g., ...). Figure 3 As shown in the image, this is a photograph taken on culture medium B 4 weeks after irradiation, with a germination rate of about 50%.

[0088] 3.3 After the new shoots from step 3.2 have grown to 4 cm, cut them off from the base and transfer them to a culture bottle containing culture medium A to obtain regenerated seedlings.

[0089] The cultivation conditions were: 16 h of light per day at 24°C, 60% humidity, and 100 μmol / m² light intensity. -2 s -1 Subspecies are replaced every 30 days.

[0090] 3.4. Transfer the regenerated seedlings obtained in step 3.3 to culture medium C for rooting culture (e.g., ...). Figure 4 As shown in the image, this is a photograph of seedlings cultured on rooting medium C for one week, 12 weeks after irradiation; subcultured every 30 days until the seedlings reach 10 cm in height, at which point they are transferred to plug trays for further cultivation (e.g., ...). Figure 5 As shown in the image, this is a real photo of blueberry plants obtained after tissue culture seedlings have been transferred from tissue culture bottles to seedling trays (hardening off 15 weeks after irradiation).

[0091] The components of culture medium C were: WPM, 30 g / L sucrose, 8.5 g / L agar, 0.3 mg / L IAA, 1 mg / L KT, and 0.5 g / L charcoal powder, with a pH of 5.2 ± 0.02. The rooting culture conditions were: 24℃ per day, 16 h of light per day, 60% humidity, and a light intensity of 100 μmol / m². -2 s -1 .

[0092] Experimental results: The growth cycle from inducing buds by cutting leaves or stem segments to rooting and planting in seed trays is 3 to 4 months. The tissue culture seedlings after radiation were cultivated by regeneration culture, and a large number of seedlings were obtained in a short period of time.

[0093] Comparative Example 1

[0094] The difference between Comparative Example 1 and Example 3 is that: no method was used. 60 Instead of using Co-γ rays to treat the tissue culture seedlings, ordinary light irradiation was used. The germination rate of leaves reached 90%, and the germination rate of stem segments reached 95%.

[0095] The specific steps for using ordinary light instead of radiation-induced mutagenesis in blueberry breeding are as follows:

[0096] 1) Tissue culture seedlings were irradiated with ordinary light at an intensity of 100 μmol / m². -2 s -1 The rest are the same as step 3.1 in Example 3.

[0097] 2) Same as step 3.2 in Example 3.

[0098] After being cultured, the leaves grow callus tissue, and the callus tissue develops bud-like growth points. After 3 weeks, obvious small buds appear, and the budding rate of the leaves reaches 90%. The stem segments sprout new buds or grow new buds from the callus tissue after 2 to 3 weeks, with a budding rate of about 95%.

[0099] 3) After the new shoots grow to 4 cm, cut them off from the base and transfer them to a culture bottle for cultivation; the rest are the same as steps 1.3 in Example 3.

[0100] 4) Same as step 3.4 in Example 3.

[0101] Results: Regeneration using unirradiated tissue culture seedlings resulted in a 1-2 week shorter time for the formation of adventitious buds in leaves and stem segments compared to irradiated tissue culture seedlings. Compared to irradiated seedlings, unirradiated seedlings took 1-2 weeks less to grow to the same height, and the regenerated seedlings had thicker stem segments and larger leaves.

[0102] Comparative Example 2

[0103] The difference between Comparative Example 2 and Example 3 is that 120 Gy of radiation was used. 60 When tissue culture seedlings were irradiated with Co-γ rays, the leaf survival rate was found to be less than 20%, the leaf budding rate was about 5%, and the stem budding rate was less than 30%.

[0104] The specific steps are as follows:

[0105] 1) Adopt 60 The tissue culture seedlings were irradiated with Co-γ rays at a dose of 120 Gy and an intensity of 1 Gy / min; all other steps were the same as in step 3.1 of Example 3.

[0106] 2) Same as step 3.2 in Example 3.

[0107] After being cultured, the leaves grew callus tissue, and the callus tissue developed bud-like growth points. After 5 weeks, obvious small buds appeared. The leaf mortality rate was high, the callus induction rate was less than 20%, and the budding rate was close to 5%. After 5 weeks, the stem segments sprouted new buds or grew new buds from the callus tissue, with a budding rate of about 30%.

[0108] 3) After the new shoots grow to 4 cm, cut them off from the base and transfer them to a culture bottle for cultivation; the rest are the same as steps 1.3 in Example 3.

[0109] (4) Same as step 3.4 in Example 1.

[0110] Table 4 shows that using a dose of 120 Gy... 60Treatment of 'Lanmei No. 1' tissue culture seedlings with Co-γ rays resulted in high mortality rates in leaf material, low callus induction rates, and low leaf budding rates; stem segment survival and budding rates were also low. Therefore, treating 'Lanmei No. 1' tissue culture seedlings with a dose of 120 Gy would reduce regeneration efficiency, yielding too few regenerated seedlings, making it unsuitable to use excessively high doses for radiation-induced mutagenesis.

[0111] Table 4

[0112]

Claims

1. A method for blueberry radiation-induced mutation breeding, characterized in that, Includes the following steps: 1) 60 Co-γ rays were used to irradiate tissue culture seedlings grown in culture medium A to obtain explant material; wherein the intensity of the radiation was 0.5–1.5 Gy / min and the dose of the radiation was 80–120 Gy. 2) The explant material was inoculated into culture medium B for the first light culture to induce the generation of adventitious shoots; 3) The culture from step 2) is inoculated into culture medium A for a second light culture to obtain regenerated seedlings; 4) The regenerated seedlings were transplanted into culture medium C for a third light culture to obtain blueberry plants; The blueberry variety mentioned is 'Lanmei No. 1'; The explant material is selected from leaves or stem segments; The composition of culture medium A is: WPM medium, 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L zeatin and 0.05 mg / L indolebutyric acid; The composition of culture medium B is: WPM medium, 5 g / L sucrose, 8.5 g / L agar and 0.05 mg / L thiamethoxam; The composition of culture medium C is as follows: WPM medium, 30 g / L sucrose, 8.5 g / L agar, 0.3 mg / L 3-indoleacetic acid, 1 mg / L kinetin and 0.5 g / L charcoal powder.

2. The breeding method according to claim 1, characterized in that, The growth conditions for the tissue culture seedlings were: 20–30℃ under light for 12–16 h, with a light intensity of 50–150 μmol / m². -2 s - 1. Humidity is 40-80%.

3. The breeding method according to claim 1, characterized in that, The conditions for the first photoculture were: light intensity of 50–150 μmol / m³. -2 s -1 The light cycle consists of alternating light and dark culture, with the light culture time being 12–16 h and the dark culture time being 8–12 h. And / or, the temperature of the first light culture is 20-30°C and the humidity is 40-80%.

4. The breeding method according to claim 1, characterized in that, The conditions for the second photoculture were: light intensity of 50–150 μmol / m³. -2 s -1 The light cycle consists of alternating light and dark culture, with the light culture time being 12–16 h and the dark culture time being 8–12 h. And / or, the temperature of the second light incubation is 20-30°C and the humidity is 40-80%.

5. The breeding method according to claim 1, characterized in that, The conditions for the third light-induced culture were as follows: light intensity of 50–150 μmol m⁻² s⁻¹, light-dark alternation culture, light-induced culture time of 12–16 h, and dark-induced culture time of 8–12 h. And / or, the temperature for the third light incubation is 20–30°C and the humidity is 40–80%.

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