A method for inducing blueberry callus

Abstract

The application belongs to the technical field of blueberry cultivation, and discloses a blueberry callus induction method, which comprises the following steps: (1) selecting blueberry flower buds 1-3 days before field flowering, removing petals and stamens, and placing the ovary with the stigma and pedicel retained in a 75% ethanol solution for sterilization for 10-15 min; (2) under aseptic conditions, the ovary with the stigma and pedicel retained is cut to remove the stigma, 1 / 4 of the ovary and the receptacle, and the ovule surrounded on the placenta is carefully taken out with sterilized tweezers and inoculated into a callus induction medium; after 30 days of dark culture, callus is obtained; the application provides a blueberry callus induction method, and has the technical effect of improving the callus induction rate of blueberries.

Description

Technical Field

[0001] This invention relates to the field of blueberry cultivation technology, and in particular to a method for inducing blueberry callus. Background Technology

[0002] Blueberries have high nutritional and economic value. They are rich in vitamins, minerals, anthocyanins, and other antioxidants, and have the effects of improving human immunity, softening blood vessels, fighting cancer, strengthening the heart, and protecting the liver. They are listed by the Food and Agriculture Organization of the United Nations as one of the "five healthiest foods for mankind." Due to their high nutritional value, unique taste, and perfect balance of sweet and sour, blueberries are very popular among consumers, have a large market demand, and have good economic development prospects.

[0003] my country's blueberry industry is developing rapidly, but breeding efforts urgently need to be strengthened. In recent years, the domestic blueberry planting area has increased year by year; in 2020, my country's total blueberry planting area reached 66,400 hectares, with a total output of approximately 347,200 tons and a fresh fruit output of 234,700 tons, surpassing the United States to become the world's largest blueberry producer. Commercial blueberry cultivation in my country began in 2000, and existing blueberry cultivars are mainly from abroad, lacking independently developed varieties. Developing new, high-quality blueberry varieties with Chinese characteristics is an urgent need for developing the blueberry industry and participating in international market competition.

[0004] Haploid breeding is a shortcut to quickly obtain genetically pure lines, which can greatly shorten the breeding cycle and accelerate the breeding process. Blueberry callus induction is an important step in laying the material foundation for in vitro preservation, regeneration and related research of blueberry resources, so its research is very necessary. Summary of the Invention

[0005] The purpose of this invention is to provide a method for inducing blueberry callus, which has the technical effect of improving the blueberry callus induction rate.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0007] A method for inducing blueberry callus includes the following steps:

[0008] (1) Select blueberry buds 1-3 days before flowering in the field, remove the petals and stamens, and disinfect the ovary with the stigma and pedicel in 75% ethanol solution for 10-15 minutes.

[0009] (2) Under sterile conditions, remove the stigma, the upper 1 / 4 of the ovary and the receptacle from the ovary obtained in step (1) with the stigma and pedicel intact. Carefully remove the ovules surrounding the placenta with sterile forceps and inoculate them into callus induction medium. After culturing in the dark for 30 days, callus tissue is obtained.

[0010] As a further feature of the present invention, in step (1), the callus induction culture medium is WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, and the pH is 5.2-5.4.

[0011] As a further feature of the present invention, in step (1), the callus tissue is a light yellow clump.

[0012] As a further feature of the present invention, the dark culture conditions described in step (2) are 25±1℃.

[0013] The beneficial effects of this invention are:

[0014] 1. This invention uses blueberry ovules inoculated into callus induction medium and cultured in the dark for 30 days to obtain light yellow, lumpy callus tissue. Compared with the use of blueberry tissue culture seedling leaves (callus induction rate of 99.12%) and mature blueberry pulp to induce callus tissue (callus induction rate of 91.25%), this invention can significantly improve the callus induction rate, reaching 99.32%.

[0015] 2. This invention uses leaves from O'Neill tissue culture seedlings as material. The leaves are cut into two sections and inoculated into nine different culture media. After culturing in the dark for 30 days, the callus induction rate is calculated, and the optimal callus induction medium is selected as WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, with the pH adjusted to 5.2-5.4.

[0016] 3. This invention uses unfertilized blueberry ovules to culture to obtain haploid callus, which has the advantages of easy acquisition of sterile materials and high callus induction rate, providing a technical basis and experimental materials for further research on blueberry haploid breeding and related theories. Attached Figure Description

[0017] Figure 1 This is a photograph of a blueberry tissue culture seedling leaf that was used to induce callus tissue.

[0018] Figure 2 These are real photos of blueberry buds at different stages;

[0019] Figure 3 It is an actual picture of the ovary with the stigma and pedicel preserved;

[0020] Figure 4 This is a photograph of a callus tissue induced using unfertilized blueberry ovules;

[0021] Figure 5 This is a photograph of a specimen showing how callus tissue is induced using ripe blueberry pulp. Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] (1) Screening of culture medium for inducing callus tissue using blueberry tissue culture seedling leaves

[0024] Young leaves from healthy and uniformly growing aseptic tissue culture seedlings of blueberry O'Neill (originating from the Fruit and Tea Research Institute of Hubei Academy of Agricultural Sciences) were cut into two sections and inoculated into nine different culture media at pH 5.2-5.4. Each culture medium was replicated in three groups, with three dishes per group. The culture media were placed in a tissue culture incubator at (25±1)℃ and cultured in the dark for 30 days. The callus induction rate and growth status of the leaves were statistically analyzed and observed.

[0025] Wherein: Leaf callus induction rate = Number of surviving leaves producing callus / Total number of inoculated leaves × 100%

[0026] Table 1 Composition of WPM culture medium

[0027]

[0028]

[0029] Table 2 Results of leaf callus experiments in different culture media

[0030]

[0031]

[0032] As shown in Table 2, the optimal callus induction medium is WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, with the pH adjusted to 5.2-5.4. The callus induction rate is 99.12%, and the callus is mostly pale yellow and granular.

[0033] (2) Induction of callus tissue using unfertilized blueberry ovules

[0034] Select blueberry buds from the field 1-3 days before flowering (e.g. Figure 2 As shown in the image, select blueberry buds at stages 7-9 (when the buds are 1.08–1.42 cm tall), remove the petals and stamens, and retain the ovary with the stigma and pedicel (as shown in the image). Figure 3(As shown) Disinfect in 75% ethanol solution for 10-15 min; under aseptic conditions, remove the stigma, the upper 1 / 4 of the ovary, and the receptacle; carefully remove the ovules surrounding the placenta with sterile forceps; inoculate into callus induction medium WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, and adjust the pH to 5.2-5.4; place in a tissue culture incubator and culture at (25±1)℃. After 30 days of dark culture, statistical analysis and observation of the callus induction rate and growth status of the leaves were performed.

[0035] Wherein: Callus induction rate = Number of surviving ovules producing callus / Total number of inoculated ovules × 100%

[0036] Experimental results: The callus induction rate using unfertilized blueberry ovules was 99.32%, and the callus was a light yellow clump.

[0037] (3) Inducing callus tissue using ripe blueberry pulp

[0038] Ripe blueberries were picked and sterilized in 75% ethanol solution for 5-8 minutes. Under aseptic conditions, the peel was removed with a sterilized blade, and the pulp was cut into small pieces and inoculated into callus induction medium WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, with the pH adjusted to 5.2-5.4. The medium was then placed in a tissue culture incubator at (25±1)℃ and cultured in the dark for 30 days. The callus induction rate and growth status of the leaves were statistically analyzed and observed.

[0039] Wherein: Callus induction rate = Number of surviving pulp pieces that produced callus / Total number of inoculated pulp pieces × 100%

[0040] Experimental results: Callus tissue was induced using mature blueberry pulp, with a callus induction rate of 91.25%, and the callus was a pale yellow clump.

[0041] In summary: 1. The optimal callus induction medium is WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, with the pH adjusted to 5.2-5.4;

[0042] 2. The highest induction rate of callus tissue was achieved using unfertilized blueberry ovules, with a callus tissue induction rate of 99.32%. The callus tissue was a light yellow clump.

[0043] 3. Unfertilized blueberry ovules can be cultured to obtain haploid callus, which has the advantages of easy acquisition of sterile materials and high callus induction rate, providing a technical basis and experimental materials for further research on blueberry haploid breeding and related theories.

[0044] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0045] The above description is merely a description of preferred embodiments of the present invention and is not intended to limit the scope of the present invention in any way. Any changes or modifications made by those skilled in the art based on the above disclosure shall fall within the protection scope of the claims.

Claims

1. A method for inducing blueberry callus, characterized in that, Includes the following steps: (1) Select blueberry buds 1-3 days before flowering in the field, remove the petals and stamens, and disinfect the ovary with the stigma and pedicel in 75% ethanol solution for 10-15 min. (2) Under aseptic conditions, the stigma and pedicel of the ovary obtained in step (1) are removed, along with the upper 1 / 4 of the ovary and the receptacle. The ovules surrounding the placenta are carefully removed with sterile forceps and inoculated into callus induction medium. The callus induction medium is WPM + 1.0 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar powder, with a pH of 5.2-5.

4. After culturing in the dark for 30 days, callus tissue is obtained.

2. The method for inducing blueberry callus according to claim 1, characterized in that: The dark culture conditions described in step (2) are 25±1℃.

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