Blueberry radiation mutation breeding method

Abstract

The invention discloses a radiation mutation breeding method for blueberries. The method comprises the following steps: 1) performing radiation treatment on tissue culture seedlings growing in a culture medium A by using 60Co-gamma rays to obtain an explant material; wherein the intensity of the radiation treatment ranges from 0.5 Gy / min to 1.5 Gy / min, and the dosage of the radiation treatment ranges from 80 Gy to 120 Gy; (2) inoculating the explant material into a culture medium B, and carrying out primary illumination culture, so as to induce the generation of adventitious buds; (3) inoculating the culture obtained in the step (2) into a culture medium A, and carrying out secondary illumination culture to obtain regenerated seedlings; and 4) transplanting the regenerated seedlings into a culture medium C for third illumination culture to obtain blueberry plants. According to the method, radiation mutagenesis is applied to blueberries, a large number of radiated blueberry plants are obtained within 3-4 months, and technical support is provided for breeding and improvement of the blueberries.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to a blueberry radiation mutagenesis breeding method. Background Art

[0002] blueberry( Vaccinium spp.) is a woody berry plant of the genus Vaccinium in the family Ericaceae, with a relatively short domestication history of about 120 years. Blueberry fruits are soft blue-black berries when ripe, rich in a variety of bioactive ingredients that are beneficial to the human body, such as vitamins with antioxidant activity, polyphenols such as anthocyanins and flavanols. In recent years, rising consumer demand for healthier dietary options and increasing awareness of the nutritional benefits of blueberries have driven their popularity. This popularity, in turn, has stimulated extensive research on the breeding of new blueberry varieties. Hybridization remains the main breeding method for blueberry breeding. Hybrid breeding has made the most improvements to highbush blueberry varieties. Highbush blueberry ( Vaccinium corymbosum L.) originated from Canada and North America, and the highbush blueberry was selectively bred to produce the northern highbush blueberry variety. Vaccinium darrowii Camp) produced the southern highbush blueberry, expanding the growing range of northern highbush blueberries to subtropical and tropical regions. Subsequent hybridization of highbush blueberries with other varieties further improved the adaptability and berry quality of highbush blueberries, improving drought tolerance, high pH adaptability, berry firmness, size, taste and yield.

[0003] Blueberries have made significant progress in traditional hybrid breeding, but advanced breeding methods, such as mutagenesis, are rarely used in blueberries. Mutagenesis breeding is divided into physical mutagenesis and chemical mutagenesis. It uses physical and chemical mutagens to induce mutations in plants or plant seeds to induce random and multiple variations in the plant genome, including site mutations and large fragment changes. With the maturity of mutagenesis breeding technology, the use of mutagenesis breeding can effectively shorten the breeding time for new variety selection. At present, mutagenesis breeding has been widely used in a variety of fruit crops, such as pears, peaches, apples, bananas, grapes, plums and citrus species, to enhance the appearance, taste, aroma, tree shape and self-compatibility of fruits. Although mutagenesis breeding is very promising in cultivating excellent varieties required by the market, there have been no successful case reports on blueberries so far.

[0004] The growing market demand has led to an expansion of blueberry cultivation worldwide. In the absence of advanced breeding methods, hybridization between blueberry varieties has been widely used to screen seedlings adapted to local climates, including China, Chile, Japan, the United Kingdom, and New Zealand. China's blueberry planting area has increased more than 200 times in the past 20 years, but the soil in many areas of China is not suitable for growing blueberries from North America, and it is necessary to cultivate varieties that are better adapted to the local environment. The southern highbush blueberry 'Blue Beauty No. 1' is a commercially cultivated variety in my country with good soil adaptability and a high content of anthocyanins in its fruit. It is a recognized national excellent variety and is recommended for extensive cultivation in southern my country. At present, 'Blue Beauty No. 1' is the most widely cultivated local variety in my country and has high economic value in both the fresh berry market and industrial anthocyanin extraction. Compared with traditional hybrid breeding, the use of mutagenesis breeding to optimize the breeding of excellent varieties such as 'Blue Beauty No. 1' is more suitable. Hybrid breeding combines the genomes of two parents and may produce undesirable phenotypes in the F1 generation. In contrast, mutation breeding is more conducive to improving specific traits of superior varieties while maintaining other desirable traits. Therefore, establishing a mutation breeding system for blueberry breeding is crucial for cultivating superior varieties.

[0005] Gamma rays are widely used in plant mutagenesis breeding. Currently, more than 70% of new varieties produced by radiation mutagenesis breeding use gamma rays, including berry plants. CN108935088A discloses a method for increasing the radiation mutagenesis rate of blueberry explants, including the following steps: exposing blueberry embryonic callus to cobalt 60 Repeated radiation at half the half-lethal dose to obtain embryonic callus; subculture and multiply to regenerate M0 seedlings; cultivate until flowering and fruiting, naturally hybridize with target varieties that have not been irradiated, harvest hybrid fruits, and obtain M1 hybrid seeds with radiation as parents; cultivate radiation hybrid seeds to obtain radiation hybrid seedlings; select target traits after fruiting, take target materials for tissue culture and rapid propagation, and obtain M2 generation blueberry radiation pedigrees with different target traits, which increases the radiation mutagenesis rate, reduces the radiation lethality, increases the number of effective seedlings after radiation, improves the radiation work efficiency, and solves the problems encountered in the process of blueberry radiation breeding, such as more original materials and less effective materials after radiation, easy browning and death of radiation materials, and low radiation mutagenesis rate. The material used in this patent is blueberry embryonic callus, which is more difficult to obtain.

[0006] Therefore, this application uses different doses of 60 Co-γ rays were used as a radiation mutagenesis source to induce radiation mutagenesis in blueberry tissue culture seedlings in order to establish a method for blueberry mutation breeding. Summary of the invention

[0007] One of the purposes of the present invention is to provide a method for radiation-induced mutagenesis breeding of blueberries, comprising the following steps: 1) 60 Co-γ rays are used to irradiate the tissue culture seedlings grown in culture medium A to obtain explant materials; wherein the intensity of the irradiation is 0.5 to 1.5 Gy / min, and the dose of the irradiation is 80 to 120 Gy; 2) inoculating the explant material into culture medium B for the first light culture to induce the production of adventitious buds; 3) inoculating the culture of step 2) into medium A for a second light culture to obtain regenerated seedlings; 4) Transplanting the regenerated seedlings into culture medium C for a third light culture to obtain blueberry plants.

[0008] The present invention collected published radiation mutagenesis experiments and methods related to blueberries at the beginning of the experiment, and found that none of the existing blueberry radiation mutagenesis experiments used tissue culture methods for propagation, and the existing methods could not quickly obtain a large number of blueberry plants. Therefore, the present application performs radiation mutagenesis on blueberries, finds a semi-lethal dose of radiation suitable for blueberries, and combines it with tissue culture to obtain a large number of irradiated blueberry plants in a short period of time, providing technical support for the breeding of blueberry varieties. It is also applied to radiation mutagenesis, finds a semi-lethal dose of radiation suitable for blueberries, and obtains a large number of irradiated blueberry plants in a short period of time. This patent uses tissue culture to obtain mutated plants, uses less raw materials, and obtains more effective materials after radiation.

[0009] In certain embodiments, in 1), the culture medium A comprises: WPM culture 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 culture medium, 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L zeatin, and 0.05 mg / L indolebutyric acid. The present application also conducted a study on the culture medium formula for the regeneration of blueberry tissue culture seedlings, and established a culture medium formula suitable for the growth of blueberry tissue culture seedlings, such as the study of 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. It is generally believed in the prior art that the effect of promoting the proliferation of blueberry lateral buds is best when the concentration of zeatin is 0.5-1.0 mg / L. In the study of this application, it was found that tissue culture seedlings can be induced to sprout after being cultured in medium A containing zeatin concentration of 0.01-0.1 mg / L, but only callus tissue is produced without buds in medium A containing zeatin concentration of 0.5-2 mg / L.

[0010] In certain embodiments, in 1), the growth conditions of the tissue culture seedlings are: light culture at 20-30°C for 12-16 h, with a light intensity of 50-150 μmol m -2 s -1 In a specific example, the culture was carried out under light conditions at 24°C for 16 h with a light intensity of 100 μmol m -2 s -1 , humidity is 60%.

[0011] In some embodiments, in 1), after the radiation treatment, the method further comprises cutting the leaves and / or stem segments of the tissue culture seedlings to form partial tissues as explant materials. In some specific embodiments, the leaves are cut in half or the stem segments are cut into small segments of 1 to 3 cm. In some specific embodiments, the cut leaves are laid flat on the surface of the subsequent culture medium B, so that the front of the leaves face downward. After cutting, it is conducive to full contact with the subsequent culture medium B, thereby improving the germination rate.

[0012] In certain embodiments, in 2), the explant is a leaf or stem segment of a tissue culture seedling. That is, the tissue culture seedling after radiation treatment is cut into stem segments of 1 to 3 cm, and the leaf is cut in half. The stem segment is preferably 2 cm.

[0013] In certain embodiments, in 2), the culture medium B comprises: WPM culture 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 culture medium, 5 g / L sucrose, 8.5 g / L agar, and 0.05 mg / L thidiazuron. The present application studies the culture medium formula suitable for the growth of blueberry tissue culture seedlings, such as the concentration of thidiazuron and sucrose in culture medium B. Thidiazuron (TDZ) is a synthetic plant growth regulator belonging to the phenylurea class of compounds, which has the effect of kinetin and is mainly used to promote plant bud differentiation and proliferation; sucrose is a commonly used carbon source, which can provide substrates and energy for the respiratory metabolism of cells, and can make plants grow robustly. The present application has studied the concentration of thidiazuron and found that 0.05-0.1 mg / L thidiazuron makes the germination rate of blueberry higher than 60%, while the germination rates of thidiazuron below 0.05 and above 0.1 mg / L are uniformly lower than 60%; the present application has also studied the concentration of sucrose and found that 10-20 g / L sucrose makes the germination rate of blueberry higher than 60%; and further studied the compatibility of different concentrations of sucrose and different concentrations of thidiazuron, and found that the best germination rate of leaves after irradiation for 60 days is 62-95% when thidiazuron with a concentration of 0.05-0.1 mg / L and sucrose with a concentration of 10-20 g / L, among which the germination rate can reach 90-95% when thidiazuron with a concentration of 0.05 mg / L and sucrose with a concentration of 10 g / L; in addition, the culture medium with the same concentration of other components but sucrose concentration of 0.5 g / L and 10 g / L was further studied, and the results showed that 5 When cultured in a medium containing 100 g / L sucrose, more buds were found on a single leaf. The formula of the medium B in the present application is conducive to the formation of new adventitious buds in callus tissues such as stem segments and leaves of tissue culture seedlings for regeneration.

[0014] In certain embodiments, in 2), the conditions for the first light culturing are: the light intensity is 50 to 150 μmol m -2 s -1 The light cycle is alternating light and dark, the light culture time is 12 to 16 hours, and the dark culture time is 8 to 12 hours. In a specific example, the light intensity is 100 μmol m -2 s -1 The light duration was 16 h and the dark culture time was 8 h.

[0015] In certain embodiments, in 2), the temperature of the first light culturing is 20-30°C, and the humidity is 40-80%. In a specific example, the temperature is 24°C, and the humidity is 60%.

[0016] In certain embodiments, in 3), the culture is the adventitious bud induced in 2), and the height of the adventitious bud is 2 to 6 cm. In a specific example, it is 4 cm. Adventitious buds refer to buds directly differentiated from abnormal positions of plants (such as leaves, stem segments, roots, callus, etc.) in tissue culture, which can be directly induced from explants or induced by callus culture.

[0017] In certain embodiments, in 3), the conditions for the second illumination culture are: the illumination intensity is 50 to 150 μmol m -2 s -1 The light cycle is alternating light and dark, the light culture time is 12 to 16 hours, and the dark culture time is 8 to 12 hours. In a specific example, the light intensity is 100 μmol m -2 s -1 The light culture time is 16h, and the dark culture time is 8h.

[0018] In certain embodiments, in 3), the temperature of the second light culture is 20-30°C, and the humidity is 40-80%. In a specific example, the temperature is 24°C, and the humidity is 60%.

[0019] In certain embodiments, in 4), the culture medium C comprises: WPM culture 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 culture 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, can promote blueberry rooting; kinetin (KT), also known as 6-furfurylaminopurine, is a non-natural cytokinin that can promote cell division and organ differentiation in blueberry culture; charcoal powder is activated carbon powder, and the dark environment it creates is conducive to root induction and root growth. The culture medium C of the present application is conducive to blueberry rooting.

[0020] In certain embodiments, in 4), the conditions for the third light culturing are: the light intensity is 50 to 150 μmol m -2 s -1 The light cycle is alternating light and dark, the light duration is 12 to 16 hours, and the dark duration is 8 to 12 hours. In a specific example, the light intensity is 100 μmol m -2 s -1The light culture time is 16h, and the dark culture time is 8h.

[0021] In certain embodiments, in 4), the temperature of the third light culture is 20-30°C, and the humidity is 40-80%. In a specific example, the temperature is 24°C, and the humidity is 60%.

[0022] In the present application, the WPM medium formula is typically but not limited to: 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 NaMoO4·2 H2O, 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, Lloyd & McCown Woody Plant Basal Medium with Vitamins (WPM) (Phyto Tech, L449).

[0023] In certain embodiments, in 4), light culture is performed until the regenerated seedlings reach 6 to 14 cm, and then they are transferred and planted in plug trays to obtain blueberry plants.

[0024] In certain embodiments, the blueberry variety is selected from the southern highbush blueberry elite variety 'Lanmei No. 1' developed and bred by Zhejiang Lanmei Agriculture Co., Ltd.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1) The present invention establishes a regeneration system for blueberries, from inducing budding through cutting leaves or stem segments of tissue culture seedlings treated with radiation to rooting and planting in plug trays, with a cycle of 3 to 4 months.

[0026] 2) The present invention uses different doses 60 Blueberry tissue culture seedlings were irradiated with Co~γ rays, and the semi-lethal dose of radiation suitable for blueberries was found, providing a basis for obtaining excellent blueberry varieties.

[0027] 3) The method of the present invention can quickly obtain a large number of irradiated plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The following are actual photos of the growth status of stem segments induced by different radiation doses for 30 and 60 days in Example 1. The scale bar is 1 cm.

[0029] Figure 2 The leaves with adventitious buds induced on medium B 6 weeks after irradiation in Example 3. The scale bar is 1 mm.

[0030] Figure 3 This is the stem segment that induced a large number of adventitious buds on medium B 4 weeks after irradiation in Example 3. The scale bar is 1 cm.

[0031] Figure 4 The seedlings were cultured on medium C for one week to induce rooting 12 weeks after irradiation in Example 3. The scale bar is 1 cm.

[0032] Figure 5 This is the radiation-induced 'Lanmei No. 1' irradiated seedling obtained after hardening 15 weeks after irradiation in Example 3. The scale bar is 1 cm. DETAILED DESCRIPTION

[0033] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0034] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0036] In the following examples of the present application, ZT represents zeatin, KT represents kinetin, IBA represents indolebutyric acid, TDZ represents thidiazuron, and IAA represents 3-indoleacetic acid.

[0037] The WPM medium used was Lloyd & McCown Woody Plant Basal Medium with Vitamins (WPM) (Phyto Tech, L449).

[0038] The leaf callus induction rate was calculated as follows: the number of leaves producing callus after the leaves were placed on medium B for 60 days / the total number of leaves.

[0039] The calculation method of leaf budding rate after 60 days is: the number of leaves with adventitious buds after the leaves were placed on medium B for 60 days / the total number of leaves. (If there are multiple buds on one leaf, the number is still counted as 1).

[0040] The calculation method of stem segment budding rate after 60 days is: the number of stem segments that produce adventitious buds after the stem segments are placed on medium B for 60 days / the total number of stem segments. (If there are multiple buds on one stem segment, the number is still 1).

[0041] Example 1 In this Example 1, the radiation dose was screened to obtain a radiation dose suitable for tissue culture seedlings. The details are as follows: 1.1. Use the information provided by the Institute of Crop and Nuclear Technology, Zhejiang Academy of Agricultural Sciences 60 The tissue culture seedlings of 'Lanmei No. 1' were irradiated with Co-γ rays. The radiation doses were 10, 20, 40, 80, 120, and 150 Gy, with a total of 7 gradients. The radiation intensity at each radiation dose was 1 Gy·min -1 , 4 replicates were set for each radiation dose, with 60 seedlings in each replicate, see Tables 1 and 2.

[0042] The tissue culture seedlings were 'Lanmei No. 1' tissue culture seedlings that had been grown in medium A for 8 weeks and had good growth and consistent growth. The growth and culture conditions were: light culture for 16 h per day at 24°C for 8 weeks, with a light intensity of 100 μmol / m -2 s -1 , 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.

[0043] 1.2. Using the irradiated tissue culture seedlings in step 1.1 as explant materials, cut the leaves in half and place them on medium B with the front side of the leaves facing downward to induce the formation of adventitious buds. At the same time, cut the stem segments into 2 cm small segments and place them in medium B. Then, the cut leaves and stem segments were placed in medium B at 24°C, 60% humidity and normal light (light intensity of 100 μmol m -2 s -1 ) for 16 h to allow them to fully contact with medium B and subculture every 14 days.

[0044] The composition of medium B was WPM, 5 g / L sucrose, 8.5 g / L agar and 0.05 mg / L TDZ, pH = 5.2 ± 0.02.

[0045] Observe the growth on the 30th and 60th days after radiation. Figure 1 ; The number of surviving leaves and the total number in each replication at each radiation dose on the 60th day after radiation were counted to obtain the leaf mortality rate (%); the number of surviving axillary buds in each replication at each radiation dose on the 60th day after radiation were counted to obtain the stem mortality rate (%), see Tables 1 and 2.

[0046] Table 1 Leaf mortality rate (%)

[0047] Table 2 Stem segment mortality rate (%)

[0048] from Figure 1 As shown in Tables 1 and 2, the leaf states, callus tissues and adventitious bud numbers of leaves treated with different radiation doses were significantly different during culture; the growth rate and growth state of adventitious buds of stem segments treated with different radiation doses were also different.

[0049] From Tables 1 and 2, it can be seen that the appropriate radiation dose for tissue culture seedlings is 80-120 Gy. Further research will be conducted with a radiation dose of 80 Gy.

[0050] Example 2 In this embodiment 2, the medium A and the medium B during the growth of the tissue culture seedlings are screened, including the following: 2.1. Screening of Zeatin Concentration in Medium A 1) In order to explore a culture medium suitable for blueberry, a regeneration system represented by 'Lanmei No. 1' was studied in this example. The culture medium A used contained 30 g / L sucrose, 8.5 g / L agar, 0.05 mg / L indolebutyric acid, and WPM culture medium with different concentrations of zeatin, wherein the concentrations of zeatin 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, based on the total volume of culture medium A. The stem segments of 'Lanmei No. 1' were irradiated at an intensity of 1 Gy·min -1 , the radiation dose is 80 Gy, and the rest is the same as Example 1.

[0051] The growth of adventitious buds on the stem segments was observed for 60 days.

[0052] The results showed that in the medium with 0.5-2 mg / L zeatin, only callus tissue was produced, and no budding was induced; while in the medium with 0.01-0.1 mg / L zeatin, both the stem bud growth points and callus tissue sprouted. Therefore, the subsequent study was carried out with 0.05 mg / L zeatin in medium A.

[0053] 2.2. Screening of different concentrations of thidiazuron and sucrose ratios in medium B 1) Using medium B, which is WPM medium containing 0.05 mg / L thidiazuron, 8.5 g / L agar, and different concentrations of sucrose, the sucrose concentrations are 10 g / L, 20 g / L, and 30 g / L based on the total volume of medium B, the leaves of 'Lanmei No. 1' were irradiated at an intensity of 1 Gy·min -1 , the radiation dose is 80 Gy, and the rest is the same as Example 1.

[0054] The leaf germination rate was counted for 60 days.

[0055] The results showed that the leaf germination rate of medium B with 10 g / L, 20 g / L, and 30 g / L sucrose added was higher than 60%.

[0056] 2) Using medium B, which is WPM medium containing 10 g / L sucrose and 8.5 g / L agar and different concentrations of thidiazuron, where the concentrations of thidiazuron are 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, the leaves of 'Lanmei No. 1' were irradiated at an intensity of 1 Gy min -1 , the radiation dose is 80 Gy, and the rest is the same as Example 1.

[0057] The leaf germination rate was counted 60 days after radiation.

[0058] The results showed that the leaf germination rate of medium B supplemented with 0.05 mg / L and 0.1 mg / L thidiazuron was higher than 60%, while other concentrations of thidiazuron resulted in leaf germination rates lower than 60%, indicating that the appropriate concentration of thidiazuron added is 0.05-0.1 mg / L.

[0059] 3) The concentrations of thidiazuron selected in 2) were 0.05 mg / L and 0.1 mg / L, and the concentrations of sucrose selected in 1) were 10 g / L, 20 g / L, and 30 g / L, respectively. The specific formula is shown in Table 3. The leaves of Lanmei No. 1 were irradiated at an intensity of 1 Gy·min -1 The radiation dose is 80 Gy, and the remaining steps are the same as in Example 1.

[0060] The leaf germination rate was statistically analyzed 60 days after radiation. The results are shown in Table 3.

[0061] Table 3

[0062] As shown in Table 3, the following three formulas all have more adventitious buds: the germination rate of formula 1 containing 0.05 mg / L thidiazuron and 10 g / L sucrose is 90% to 95%; the germination rate of formula 5 containing 0.1 mg / L thidiazuron and 20 g / L sucrose is 62% to 85%; the germination rate of formula 3 containing 0.05 mg / L thidiazuron and 30 g / L sucrose is 85% to 95%. Among them, the leaves of formula 1 containing 0.05 mg / L thidiazuron and 10 g / L sucrose have the best growth and the highest germination rate.

[0063] 2.3. Further optimization of the formula of medium B Subsequently, we further optimized the regeneration system of 'Lanmei No. 1'. The formulas of medium B were WPM medium containing 0.05 mg / L thidiazuron, 5 g / L sucrose and 8.5 g / L agar and WPM medium containing 0.05 mg / L thidiazuron, 10 g / L sucrose and 8.5 g / L agar. The leaves of 'Lanmei No. 1' were irradiated at an intensity of 1 Gy·min -1 , the radiation dose is 80 Gy, and the rest is the same as Example 1.

[0064] The leaf germination rate was statistically analyzed 60 days after radiation. The results are shown in Table 3.

[0065] In comparison, the germination rates of the above two mediums were similar, but the WPM medium containing 0.05 mg / L thidiazuron, 5 g / L sucrose and 8.5 g / L agar had more buds on a single leaf. Therefore, the WPM medium containing 0.05 mg / L thidiazuron, 5 g / L sucrose and 8.5 g / L agar, pH=5.2±0.02 was selected as the medium B for the optimal induction of adventitious buds in the regeneration system of 'Lanmei No. 1'.

[0066] Example 3 Based on the radiation dose determined in Example 1 and the zeatin concentration in medium A and the sucrose and thidiazuron concentrations in medium B determined in Example 2, this Example 3 further investigated the feasibility of the method for radiation-induced mutagenesis breeding of blueberries. The specific steps are as follows: 3.1 Adoption 60 The tissue culture seedlings of 'Lanmei No. 1' were irradiated with Co-γ rays at a dose of 80 Gy and an intensity of 1 Gy / min.

[0067] The tissue culture seedlings were 'Lanmei No. 1' tissue culture seedlings that had been grown in medium A for 8 weeks and had good growth and consistent growth. The growth and culture conditions were: light culture for 16 h per day at 24°C for 8 weeks, with a light intensity of 100 μmol / m -2 s -1 , 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.

[0068] 3.2. Use the irradiated tissue culture seedlings from step 3.1 as explant material, cut the leaves in half and place them on medium B with the front side of the leaves facing downward to induce the formation of adventitious buds (such as Figure 2 ), which is a real picture taken on medium B 6 weeks after irradiation. At the same time, the stem segments were cut into 2 cm small segments and placed in medium B. The leaves and stem segments were kept at 24 °C, 60% humidity and normal light (light intensity was 100 μmol m -2 s -1 ) for 16 h to allow them to fully contact with medium B and subculture every 14 days.

[0069] The composition of medium B was WPM, 5 g / L sucrose, 8.5 g / L agar and 0.05 mg / L TDZ, pH = 5.2 ± 0.02.

[0070] After cultivation, callus tissue grows from leaves, and bud-like growth points appear on callus tissue after cultivation. After 4 to 5 weeks, obvious small buds appear. Among them, the budding rate of leaves that successfully survive and grow small buds is about 15%; after 3 to 4 weeks, new buds sprout from stem segments or grow from callus tissue (such as Figure 3 ), which is a real picture taken on culture medium B 4 weeks after radiation, with a germination rate of about 50%.

[0071] 3.3. After the new shoots in step 3.2 grow to 4 cm, cut them from the base and transfer them to a culture bottle containing culture medium A to obtain regenerated seedlings.

[0072] The culture conditions were as follows: 24°C for 16 h per day, 60% humidity, and 100 μmol m -2 s -1 ; Subculture every 30 days.

[0073] 3.4. Transfer the regenerated seedlings obtained in step 3.3 to medium C for rooting culture (such as Figure 4), which is a photo of seedlings cultured on rooting medium C for one week 12 weeks after irradiation; subcultured every 30 days until the seedlings grew to 10 cm, then transferred to plug trays for culture (as shown in Figure 5 As shown in the figure, that is, a real shot of blueberry plants obtained by transferring tissue culture seedlings from tissue culture bottles to plug trays (hardening of seedlings 15 weeks after radiation).

[0074] The composition of medium C was 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°C, 16 h of light, 60% humidity, and 100 μmol m -2 s -1 .

[0075] Experimental results: The growth cycle from inducing buds from cutting leaves or stem segments to rooting and planting in plug trays is 3 to 4 months. The irradiated tissue culture seedlings were cultivated using regeneration culture, and a large number of seedlings were obtained in a short period of time.

[0076] Comparative Example 1 The difference between this comparative example 1 and example 3 is that: 60 The tissue culture seedlings were not irradiated with Co-γ rays, but with ordinary light. The germination rate of leaves reached 90%, and the germination rate of stem segments reached 95%.

[0077] Ordinary light is used to replace radiation mutagenesis for blueberry breeding. The specific steps are as follows: 1) Ordinary light was used to irradiate the tissue culture seedlings at an intensity of 100 μmol m -2 s -1 ; The rest is the same as step 3.1 in Example 3.

[0078] 2) Same as step 3.2 in Example 3.

[0079] After cultivation, callus tissue grows out of the leaves. After cultivation, bud-like growth points appear on the callus tissue, and obvious small buds appear after 3 weeks. The budding rate of the leaves reaches 90%. After 2 to 3 weeks, new buds sprout from the stem segments or grow new buds from the callus tissue, and the budding rate is about 95%.

[0080] 3) After the new shoots grow to 4 cm, they are cut from the base and transferred to a culture bottle for culture; the rest is the same as step 1.3 in Example 3.

[0081] 4) Same as step 3.4 in Example 3.

[0082] Results: When the non-irradiated tissue culture seedlings were used for regeneration, the time for adventitious buds on leaves and stem segments was 1 to 2 weeks shorter than that on irradiated tissue culture seedlings. Compared with the irradiated regenerated seedlings, the non-irradiated regenerated seedlings took 1 to 2 weeks less time to grow to the same height, and the regenerated seedlings had thicker stems and larger leaves.

[0083] Comparative Example 2 The difference between this comparative example 2 and example 3 is that a radiation dose of 120 Gy is used. 60 When tissue culture seedlings were irradiated with Co-γ rays, the leaf survival rate was less than 20%, the leaf budding rate was about 5%, and the stem segment budding rate was less than 30%.

[0084] The specific steps are as follows: 1) Adoption 60 The tissue culture seedlings were irradiated with Co-γ rays, the radiation dose was 120 Gy, and the radiation intensity was 1 Gy / min; the rest was the same as step 3.1 in Example 3.

[0085] 2) Same as step 3.2 in Example 3.

[0086] After cultivation, callus tissue grew out of leaves, bud-like growth points appeared on the callus tissue, and obvious small buds appeared after 5 weeks. 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, new buds sprouted from the stem segments or grew new buds from the callus tissue, and the budding rate was about 30%.

[0087] 3) After the new shoots grow to 4 cm, they are cut from the base and transferred to a culture bottle for culture; the rest is the same as step 1.3 in Example 3.

[0088] (4) Same as step 3.4 in Example 1.

[0089] From Table 4, we can see that the use of 120 Gy dose 60 After Co-γ-ray treatment of 'Lanmei No. 1' tissue culture seedlings, the mortality rate of leaf materials was high, the callus induction rate was low, the leaf budding rate was low; the survival rate and budding rate of stem segments were low. Therefore, using a dose of 120Gy to treat 'Lanmei No. 1' tissue culture seedlings will reduce the regeneration efficiency, and the number of regenerated seedlings obtained is too small, so it is not suitable to use too high a dose for radiation mutagenesis.

[0090] Table 4

Claims

1. A method for blueberry radiation-induced mutagenesis breeding, characterized in that: The steps include: 1) 60 The tissue culture seedlings grown in the culture medium A are irradiated with Co-γ rays to obtain explant materials; wherein the intensity of the radiation is 0.5 to 1.5 Gy / min, and the radiation dose is 80 to 120 Gy; 2) inoculating the explant material into culture medium B for the first light culture to induce the production of adventitious buds; 3) inoculating the culture of step 2) into medium A for a second light culture to obtain regenerated seedlings; 4) Transplanting the regenerated seedlings into culture medium C for a third light culture to obtain blueberry plants.

2. The breeding method according to claim 1, characterized in that The culture medium A comprises: WPM culture 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.

3. The breeding method according to claim 1, characterized in that The growth conditions of the tissue culture seedlings are: light culture for 12 to 16 hours at 20 to 30°C, with a light intensity of 50 to 150 μmol m -2 s -1 , humidity is 40-80%.

4. The breeding method according to claim 1, characterized in that The culture medium B comprises: WPM culture medium, 5-10 g / L sucrose, 6-10 g / L agar and 0.01-0.1 mg / L thidiazuron; And / or, the conditions of the first illumination culture are: the illumination intensity is 50-150 μmol m -2 s -1 The light cycle is alternating light and dark, the light culture time is 12 to 16 hours; the dark culture time is 8 to 12 hours; And / or, the temperature of the first light culture 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 second light culture are: the light intensity is 50-150 μmol m -2 s -1 The light cycle is alternating light and dark, the light culture time is 12 to 16 hours; the dark culture time is 8 to 12 hours; And / or, the temperature of the second light culture is 20-30° C., and the humidity is 40-80%.

6. The breeding method according to claim 1, characterized in that The culture medium C comprises: WPM culture 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; And / or, the conditions for the third light culture are: the light intensity is 50-150 μmol m -2 s -1 The light cycle is alternating light and dark, the light culture time is 12 to 16 hours; the dark culture time is 8 to 12 hours; and / or, the temperature of the third light culture is 20 to 30°C and the humidity is 40 to 80%.

7. The breeding method according to claim 1, characterized in that: 2), the explant material is selected from leaves or stem segments.

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