Grape somatic embryo regeneration method

Abstract

The invention provides a grape somatic embryo regeneration method, and belongs to the technical field of grape tissue culture. The grape somatic embryo regeneration method comprises the steps of seed germination, embryogenic callus induction, somatic embryo differentiation by the embryogenic callus, somatic embryo rooting induction and seedling hardening. According to the method, the hypocotyl of the grape plant is selected as the explant, and the specific plant growth regulator is added into the induction culture medium and the somatic embryo differentiation culture medium, so that the callus induction rate and the somatic embryo occurrence rate are increased. According to the method, a large number of grape somatic embryo regeneration plants with consistent hereditary basis can be obtained, rapid, stable and efficient seedling culture of grapes can be realized, the period of grape breeding is shortened, and a new method is provided for preparing the grape somatic embryo regeneration plants.

Description

Technical Field

[0001] The present invention relates to the technical field of grape tissue culture, in particular to a method for regenerating grape somatic embryos. Background Art

[0002] Grape (Vitis vinifera L.), a woody vine of the genus Vitis in the family Vitaceae, is one of the world's oldest fruit tree species. Native to western Asia, it is now widely cultivated worldwide. Vitis species are woody vines, and traditional breeding methods are time-consuming due to their long growth cycles and high genomic heterozygosity. Muscat, also known as muscat and purple rose, is a Eurasian species and one of the world's oldest grape varieties. Its oval, deep purple-red berries are densely packed, with a medium-thick skin and medium-textured flesh. It is sweet with a rich rose aroma and is of high quality. Native to the United Kingdom, it is renowned for its versatile uses, including fresh consumption, winemaking, and juice production. With the development of the winemaking industry, demand for Muscat is increasing.

[0003] Currently, the main pathways for grape regeneration include organogenesis and somatic embryogenesis. However, compared to organogenesis, somatic embryogenesis produces embryonic cells with better dispersion, vigorous growth, active physiological and biochemical metabolism, strong vitality, and easy differentiation. Using embryonic callus as a recipient for genetic transformation, the plant has high regeneration and better genetic manipulation results. Therefore, in the research of grape transgenic technology, the regeneration system mainly relies on somatic embryogenesis. Although there has been a large amount of research on grape somatic embryogenesis in the prior art, grape somatic embryo regeneration systems are affected by multiple factors such as genotype, explant, and culture medium composition. As a result, this pathway generally faces problems such as low induction efficiency, difficulty in somatic embryo germination and seedling formation, and a high incidence of malformed embryos. Therefore, there is an urgent need to provide a method for establishing a somatic embryo regeneration system for "Rose Fragrance" grapes. Summary of the Invention

[0004] In view of this, the present invention provides a method for regenerating grape somatic embryos to solve the problem of difficulty in germinating somatic embryos into seedlings.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for regenerating grape somatic embryos, comprising the following steps:

[0007] (1) Seed germination: Grape seeds were sown in a solid culture medium and cultured under light for 32 to 36 days to obtain grape plants that had not grown true leaves;

[0008] (2) Embryonic callus induction: Cut the embryonic axis of the plant, inoculate it into the induction medium, and culture it in the dark for 24 to 28 days to obtain callus;

[0009] (3) Differentiation of embryonic callus into somatic embryos: The callus is transferred to a somatic embryo differentiation medium and cultured in a dark environment; after the cotyledonary embryos grow out, the culture is switched to light culture, and somatic embryos are obtained after 30 to 40 days;

[0010] (4) Inducing rooting of somatic embryos: When the hypocotyl and radicle of the somatic embryos are differentiated, they are transferred to a rooting medium and cultured under light for 30 to 40 days to obtain rooted plants;

[0011] (5) Hardening of seedlings: Transfer the rooted plants to the cultivation medium and harden them for 20 to 30 days to obtain regenerated grape plants;

[0012] The somatic embryo differentiation medium comprises the following components: MS+2,4-D 0.5-1 mg / L+IBA 3-5 mg / L+sucrose 25-35 g / L+agar 6-8 g / L, pH=6.0±0.2.

[0013] Preferably, the solid culture medium comprises the following components: MS+sucrose 25-35 mg / L+agar 6-8 mg / L, pH=6.0±0.2.

[0014] Preferably, the induction medium comprises the following components: MS+6-BA 1-3 mg / L+sucrose 25-35 mg / L+agar 6-8 mg / L, pH=6.0±0.2.

[0015] Preferably, the rooting medium comprises the following components: MS+NAA 1-2 mg / L+sucrose 25-35 g / L+agar 6-8 g / L, pH=6.0±0.2.

[0016] Preferably, the illumination time in step (3) and step (4) is 14 to 18 hours per day, and the illumination intensity is 1500 to 2000 Lx.

[0017] Preferably, the culture temperature of steps (1) to (5) is 22 to 26°C.

[0018] Preferably, the cultivation matrix includes peat soil, perlite and vermiculite, and the mass ratio of the peat soil, perlite and vermiculite is 3 to 4:1:1.

[0019] Preferably, the seedlings are hardened and grown in a greenhouse under natural light.

[0020] By employing the above-mentioned technical solution, the present invention has the following beneficial effects: The method for regenerating grape somatic embryos comprises the steps of seed germination, embryonic callus induction, differentiation of somatic embryos from embryonic callus, rooting of induced somatic embryos, and seedling hardening. The present invention selects the hypocotyl of a grape plant as an explant and adds specific plant growth regulators to the induction medium and somatic embryo differentiation medium, thereby increasing the callus induction rate and somatic embryogenesis rate. The present invention can produce a large number of grape somatic embryo-regenerated plants with a consistent genetic basis, enabling rapid, stable, and efficient grape seedling cultivation, shortening the grape breeding cycle, and providing a new method for producing grape somatic embryo-regenerated plants. DETAILED DESCRIPTION

[0021] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] The grape variety in the embodiment of the present invention is "Rose Fragrance".

[0023] Example 1

[0024] (1) Seed germination: After sterilizing, grape seeds were sown in a solid culture medium and cultured under light for 36 days to obtain grape plants without true leaves.

[0025] (2) Embryonic callus induction: The embryonic axis of the plant was cut and inoculated into the induction medium. The culture was carried out in the dark for 28 days to obtain callus tissue.

[0026] (3) Differentiation of embryonic callus into somatic embryos: The callus was transferred to a somatic embryo differentiation medium and cultured in a dark environment; after the cotyledonary embryos grew out, the culture was switched to light culture, and somatic embryos were obtained after 40 days;

[0027] (4) Inducing rooting of somatic embryos: When the hypocotyls and radicles of the somatic embryos are differentiated, they are transferred to a rooting medium and cultured under light for 40 days to obtain rooted plants;

[0028] (5) Seedling hardening: The rooted plants were transferred to the cultivation medium and hardened in a greenhouse under natural light for 30 days to obtain grape regenerated plants;

[0029] The culture temperature of steps (1) to (5) is 22 to 26° C., the illumination time of steps (3) and (4) is 14 hours per day, and the illumination intensity is 2000 Lx.

[0030] Solid culture medium includes the following components: MS + sucrose 25 mg / L + agar 6 mg / L, pH = 5.8;

[0031] The induction medium consisted of the following components: MS + 6-BA 1 mg / L + sucrose 25 mg / L + agar 6 mg / L, pH = 5.8;

[0032] Somatic embryo differentiation medium includes the following components: MS + 2,4-D 0.5 mg / L + IBA 3 mg / L + sucrose 25 g / L + agar 6 g / L, pH = 5.8;

[0033] The rooting medium includes the following components: MS+NAA 1mg / L+sucrose 25g / L+agar 6g / L, pH=5.8;

[0034] The cultivation matrix comprises peat soil, perlite and vermiculite, and the mass ratio of the peat soil, perlite and vermiculite is 3:1:1.

[0035] Example 2

[0036] (1) Seed germination: After sterilizing, grape seeds were sown in a solid culture medium and cultured under light for 36 days to obtain grape plants without true leaves.

[0037] (2) Embryonic callus induction: The embryonic axis of the plant was cut and inoculated into the induction medium. The culture was carried out in the dark for 28 days to obtain callus tissue.

[0038] (3) Differentiation of embryonic callus into somatic embryos: The callus was transferred to a somatic embryo differentiation medium and cultured in a dark environment; after the cotyledonary embryos grew out, the culture was switched to light culture, and somatic embryos were obtained after 40 days;

[0039] (4) Inducing rooting of somatic embryos: When the hypocotyls and radicles of the somatic embryos are differentiated, they are transferred to a rooting medium and cultured under light for 40 days to obtain rooted plants;

[0040] (5) Seedling hardening: The rooted plants were transferred to the cultivation medium and hardened in a greenhouse under natural light for 30 days to obtain grape regenerated plants;

[0041] The culture temperature of steps (1) to (5) is 22 to 26° C., the illumination time of steps (3) and (4) is 16 hours per day, and the illumination intensity is 1800 Lx.

[0042] Solid culture medium includes the following components: MS + sucrose 30 mg / L + agar 7 mg / L, pH = 6.0;

[0043] The induction medium included the following components: MS + 6-BA 2 mg / L + sucrose 30 mg / L + agar 7 mg / L, pH = 6.0;

[0044] Somatic embryo differentiation medium includes the following components: MS + 2,4-D 0.8 mg / L + IBA 4 mg / L + sucrose 30 g / L + agar 7 g / L, pH = 6.0;

[0045] The rooting medium comprises the following components: MS+NAA 1.5 mg / L+sucrose 30 g / L+agar 7 g / L, pH=6.0;

[0046] The cultivation matrix comprises peat soil, perlite and vermiculite, and the mass ratio of the peat soil, perlite and vermiculite is 3.5:1:1.

[0047] Example 3

[0048] (1) Seed germination: After sterilizing, grape seeds were sown in a solid culture medium and cultured under light for 36 days to obtain grape plants without true leaves.

[0049] (2) Embryonic callus induction: The embryonic axis of the plant was cut and inoculated into the induction medium. The culture was carried out in the dark for 28 days to obtain callus tissue.

[0050] (3) Differentiation of embryonic callus into somatic embryos: The callus was transferred to a somatic embryo differentiation medium and cultured in a dark environment; after the cotyledonary embryos grew out, the culture was switched to light culture, and somatic embryos were obtained after 40 days;

[0051] (4) Inducing rooting of somatic embryos: When the hypocotyls and radicles of the somatic embryos are differentiated, they are transferred to a rooting medium and cultured under light for 40 days to obtain rooted plants;

[0052] (5) Seedling hardening: The rooted plants were transferred to the cultivation medium and hardened in a greenhouse under natural light for 30 days to obtain grape regenerated plants;

[0053] The culture temperature of steps (1) to (5) is 26° C., the illumination time of steps (3) and (4) is 18 hours per day, and the illumination intensity is 1500 Lx.

[0054] Solid culture medium includes the following components: MS + sucrose 35 mg / L + agar 8 mg / L, pH = 6.2;

[0055] The induction medium consisted of the following components: MS + 6-BA 3 mg / L + sucrose 35 mg / L + agar 8 mg / L, pH = 6.2;

[0056] Somatic embryo differentiation medium includes the following components: MS + 2,4-D 1 mg / L + IBA 5 mg / L + sucrose 35 g / L + agar 8 g / L, pH = 6.2;

[0057] The rooting medium includes the following components: MS+NAA 2 mg / L+sucrose 35 g / L+agar 8 g / L, pH=6.2;

[0058] The cultivation matrix comprises peat soil, perlite and vermiculite, and the mass ratio of the peat soil, perlite and vermiculite is 4:1:1.

[0059] Comparative Example 1

[0060] The difference from Example 2 is that the cotyledons of the plants were selected as explants.

[0061] Comparative Example 2

[0062] The difference from Example 2 is that 6-BA in the induction medium was replaced by 2,4-D.

[0063] Comparative Example 3

[0064] The difference from Example 2 is that the 6-BA concentration in the induction medium is 0.5 mg / L.

[0065] Comparative Example 4

[0066] The difference from Example 2 is that the 6-BA concentration in the induction medium is 4 mg / L.

[0067] Comparative Example 5

[0068] The difference from Example 2 is that the IBA in the somatic embryo differentiation medium is replaced by NAA.

[0069] Comparative Example 6

[0070] The difference from Example 2 is that 2,4-D in the somatic embryo differentiation medium is replaced by 6-BA.

[0071] Experimental Example 1

[0072] The experiment was divided into 7 groups, and grape somatic embryo regeneration culture was carried out according to the methods of Examples 1 to 3 and Comparative Examples 1 to 4, respectively. 100 explants were inoculated into the induction medium in each group. After culturing in the dark for 28 days, the callus induction rate was calculated. The results are shown in Table 1.

[0073] Callus induction rate = number of callus-forming plants / total number of inoculated explants × 100%

[0074] Table 1 Callus induction rate of each group

[0075] Group Callus induction rate (%) Example 1 96 Example 2 98 Example 3 99 Comparative Example 1 65 Comparative Example 2 76 Comparative Example 3 80 Comparative Example 4 88

[0076] As shown in Examples 1 to 3, in the embryonic callus induction stage, when the radicle is used as the explant and the induction medium of the present invention is used for induction differentiation, the induction rate is above 96%; as shown in Example 2 and Comparative Example 1, when the cotyledon is selected as the explant, the callus induction rate is reduced to only 65%; as shown in Comparative Examples 2 to 4, when the type or concentration of the plant growth regulator in the induction medium is changed, the callus induction rate is reduced.

[0077] Experimental Example 2

[0078] The experiment was divided into five groups, and grape somatic embryo regeneration culture was performed according to the methods of Examples 1 to 3 and Comparative Examples 5 and 6. Explants were inoculated into an induction medium and cultured in the dark to obtain callus tissue. 100 calli in each group were transferred to a somatic embryo differentiation medium and germinated in the dark. After the cotyledonary embryos grew, the culture was switched to light. After 40 days, the somatic embryogenesis rate was counted. The results are shown in Table 2.

[0079] Somatic embryogenesis rate = number of plants forming somatic embryos / total number of inoculated callus tissues × 100%

[0080] Table 2 Somatic embryogenesis rate in each group

[0081]

[0082]

[0083] According to the results in Table 2, the somatic embryogenesis rates of Examples 1 to 3 of the present invention were higher than those of Comparative Examples 5 and 6, indicating that the somatic embryo differentiation medium of the present invention is an optimal medium formula for callus induction and is conducive to the formation of grape somatic embryos.

[0084] As can be seen from the above examples, the present invention provides a method for regenerating grape somatic embryos, which can obtain a large number of grape somatic embryo regenerated plants with a consistent genetic basis, and can achieve rapid, stable and efficient grape seedling cultivation.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for regenerating grape somatic embryos, characterized in that: The following steps are involved: (1) Seed germination: Grape seeds were sown in a solid culture medium and cultured under light for 32 to 36 days to obtain grape plants that had not grown true leaves; (2) Embryonic callus induction: Cut the embryonic axis of the plant, inoculate it into the induction medium, and culture it in the dark for 24 to 28 days to obtain callus; (3) Differentiation of embryonic callus into somatic embryos: The callus is transferred to a somatic embryo differentiation medium and cultured in a dark environment; after the cotyledonary embryos grow out, the culture is switched to light culture, and somatic embryos are obtained after 30 to 40 days; (4) Inducing rooting of somatic embryos: When the hypocotyl and radicle of the somatic embryos are differentiated, they are transferred to a rooting medium and cultured under light for 30 to 40 days to obtain rooted plants; (5) Hardening of seedlings: Transfer the rooted plants to the cultivation medium and harden them for 20 to 30 days to obtain regenerated grape plants; The somatic embryo differentiation medium comprises the following components: MS+2,4-D 0.5-1 mg / L+IBA 3-5 mg / L+sucrose 25-35 g / L+agar 6-8 g / L, pH=6.0±0.

2.

2. The method according to claim 1, characterized in that The solid culture medium comprises the following components: MS+sucrose 25-35 mg / L+agar 6-8 mg / L, pH=6.0±0.

2.

3. The method according to claim 1, characterized in that The induction culture medium includes the following components: MS+6-BA 1-3 mg / L+sucrose 25-35 mg / L+agar 6-8 mg / L, pH=6.0±0.

2.

4. The method according to claim 1, wherein The rooting medium comprises the following components: MS+NAA 1-2 mg / L+sucrose 25-35 g / L+agar 6-8 g / L, pH=6.0±0.

2.

5. The method according to claim 1, wherein The illumination time of step (3) and step (4) is 14 to 18 hours per day, and the illumination intensity is 1500 to 2000 Lx.

6. The method according to claim 1, characterized in that The culture temperature of steps (1) to (5) is 22 to 26°C.

7. The method according to claim 1, characterized in that The cultivation matrix comprises peat soil, perlite and vermiculite, and the mass ratio of the peat soil, perlite and vermiculite is 3-4:1:

1.

8. The method according to claim 1, characterized in that The hardened seedlings are cultured and grown in a greenhouse under natural light.

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