A method for regeneration of grape somatic embryos

Abstract

The application 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, embryogenic callus differentiation somatic embryo, rooting of the induced somatic embryo and seedling hardening. The embryonic axis of grape plants is selected as the explant, and specific plant growth regulators are added in the induction medium and the somatic embryo differentiation medium, so that the callus induction rate and the somatic embryo generation rate are improved. A large number of grape somatic embryo regeneration plants with consistent genetic basis can be obtained, rapid, stable and efficient seedling raising of grapes can be realized, the cycle of grape breeding is shortened, and a new method for preparing grape somatic embryo regeneration plants is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of grape tissue culture, and particularly relates to a grape somatic embryo regeneration method. BACKGROUND

[0002] Grape (Vitis vinifera L.) is a woody vine plant of the genus Vitis in the family Vitaceae, is one of the oldest fruit tree species in the world, and is originally from the west of Asia and is now widely planted in various parts of the world. The Vitis plant belongs to a woody vine plant, and due to a long growth cycle and high heterozygosity of a genome, a traditional breeding method is time-consuming. Muscat, also known as musk grape and purple muscat, belongs to the European-Asian species, is one of the oldest grape varieties in the world, has medium tight fruiting, is oval, is deep purple red, has a medium thick fruit skin, has a medium flesh, tastes sweet, has a strong muscat flavor, has excellent quality, is originally from the United Kingdom, and is famous for the functions of fresh eating, wine making and juice making. With the development of the wine industry, the demand for muscat is increasing.

[0003] At present, the grape regeneration system is mainly generated by an organogenesis pathway and a somatic embryo generation pathway. However, compared with the organogenesis, the somatic embryo generation has good dispersibility of embryonic cells, vigorous growth, active physiological and biochemical metabolism, strong vitality and easy differentiation. The somatic embryo generation takes embryonic callus as a genetic transformation receptor, has high plant regeneration, and has good genetic operation effect. Therefore, in the process of grape transgenic technology research, the somatic embryo generation pathway is mainly used in the regeneration system. Although a large number of grape somatic embryo generation researches have been made in the prior art, the grape somatic embryo regeneration system is affected by various factors such as genotype, explant and medium composition, and thus the pathway generally faces problems such as low induction efficiency, difficulty in somatic embryo germination and seedling growth and high abnormal embryo generation rate. Therefore, it is urgent to provide a method for establishing a grape somatic embryo regeneration system for muscat. SUMMARY

[0004] Therefore, the present application provides a grape somatic embryo regeneration method, which solves the problem of difficulty in somatic embryo germination and seedling growth.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0006] The present application provides a grape somatic embryo regeneration method, which includes the following steps:

[0007] (1) Seed germination: grape seeds are sowed in a solid culture medium, and are cultured under light for 32-36 days to obtain grape plants without true leaves;

[0008] (2) Embryogenic callus induction: cutting the embryonic axis of the plant, inoculating into the induction medium, and culturing in the dark for 24-28 days to obtain callus;

[0009] (3) Embryogenic callus differentiation into somatic embryo: transferring the callus into somatic embryo differentiation medium and culturing in the dark; after the cotyledon embryo grows, transferring to light culture, and obtaining somatic embryo after 30-40 days;

[0010] (4) Inducing rooting of somatic embryo: when the embryonic axis and radicle of the somatic embryo differentiate, transferring to rooting medium and culturing under light for 30-40 days to obtain rooted plants;

[0011] (5) Seedling hardening: transferring the rooted plants to a cultivation substrate and hardening for 20-30 days to obtain grape regenerated 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 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 light illumination time of steps (3) and (4) is 14-18 h per day, and the light intensity is 1500-2000 Lx.

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

[0018] Preferably, the cultivation substrate comprises peat soil, perlite, and vermiculite, and the mass ratio of the peat soil, perlite, and vermiculite is 3-4:1:1.

[0019] Preferably, the seedling hardening is cultured and grown under natural light in a greenhouse.

[0020] By adopting the technical scheme, the grape somatic embryo regeneration method provided by the application comprises the steps of seed germination, embryogenic callus induction, embryogenic callus differentiation somatic embryo, rooting induction of the somatic embryo and seedling hardening. The embryonic axis of the grape plant is selected as the explant, and specific plant growth regulators are added in the induction medium and the somatic embryo differentiation medium, so that the callus induction rate and the somatic embryo generation rate are improved. A large number of grape somatic embryo regeneration plants with consistent genetic basis can be obtained, and rapid, stable and efficient seedling raising of the grape can be realized, the cycle of grape breeding is shortened, and a new method for preparing the grape somatic embryo regeneration plant is provided. DETAILED DESCRIPTION

[0021] The technical scheme provided by the application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the protection scope of the application.

[0022] The grape variety in the embodiment of the application is “Rose”.

[0023] Embodiment 1

[0024] (1) Seed germination: after the grape seeds are sterilized, the seeds are sowed in a solid culture medium, and the grape plants without true leaves are obtained after 36 days of illumination culture;

[0025] (2) Embryogenic callus induction: the embryonic axis of the plant is cut and inoculated into an induction medium, and the callus is obtained after 28 days of culture in the dark;

[0026] (3) Embryogenic callus differentiation somatic embryo: the callus is transferred into a somatic embryo differentiation medium and cultured in the dark; after the cotyledon embryo grows out, the culture is changed to illumination, and the somatic embryo is obtained after 40 days;

[0027] (4) Rooting induction of the somatic embryo: when the embryonic axis and the radicle of the somatic embryo differentiate, the somatic embryo is transferred into a rooting medium and cultured under illumination for 40 days, and the plant with roots is obtained;

[0028] (5) Seedling hardening: the plant with roots is transferred into a cultivation substrate and hardened in the greenhouse under natural illumination for 30 days, and the grape regeneration plant is obtained;

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

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

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

[0032] The somatic embryo differentiation medium comprises 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 comprises the following components: MS+NAA 1 mg / L+sucrose 25 g / L+agar 6 g / L, pH=5.8;

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

[0035] Example 2

[0036] (1) Seed germination: after disinfection, the grape seeds are sowed in the solid medium, and are cultured under light for 36 days to obtain grape plants without true leaves;

[0037] (2) Embryogenic callus induction: the hypocotyls of the plants are cut and inoculated into the induction medium, and are cultured in the dark for 28 days to obtain callus;

[0038] (3) Embryogenic callus differentiation into somatic embryo: the callus is transferred into the somatic embryo differentiation medium and is cultured in the dark; after the cotyledon embryo grows out, the culture is switched to light, and after 40 days, somatic embryos are obtained;

[0039] (4) Inducing the somatic embryo to root: when the hypocotyl and the radicle of the somatic embryo differentiate, the somatic embryo is transferred into the rooting medium and is cultured under light for 40 days to obtain the plant after rooting;

[0040] (5) Seedling hardening: the plant after rooting is transferred into the cultivation substrate and is hardened in the greenhouse under natural light for 30 days to obtain the regenerated grape plant;

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

[0042] The solid medium comprises the following components: MS+sucrose 30 mg / L+agar 7 mg / L, pH=6.0;

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

[0044] The somatic embryo differentiation medium comprises 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 substrate comprises peat soil, perlite and vermiculite, and the mass ratio of the peat soil, the perlite and the vermiculite is 3.5:1:1.

[0047] Example 3

[0048] (1) Seed germination: after disinfection, grape seeds are sowed in a solid medium, and are cultured under light for 36 days to obtain grape plants without true leaves;

[0049] (2) Embryogenic callus induction: the hypocotyls of the plants are cut and inoculated into an induction medium, and are cultured in the dark for 28 days to obtain callus;

[0050] (3) Embryogenic callus differentiation somatic embryo: the callus is transferred into a somatic embryo differentiation medium and is cultured in the dark; after cotyledon embryos grow out, the culture is switched to light, and 40 days later, somatic embryos are obtained;

[0051] (4) Inducing rooting of somatic embryos: when the hypocotyls and radicles of the somatic embryos differentiate, the somatic embryos are transferred into a rooting medium and are cultured under light for 40 days to obtain plants with roots;

[0052] (5) Seedling hardening: the plants with roots are transferred into a cultivation substrate and are hardened in a greenhouse under natural light for 30 days to obtain regenerated grape plants;

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

[0054] The solid medium comprises the following components: MS+sucrose 35 mg / L+agar 8 mg / L, pH=6.2;

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

[0056] The somatic embryo differentiation medium comprises 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 comprises the following components: MS + NAA 2 mg / L + sucrose 35 g / L + agar 8 g / L, pH = 6.2.

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

[0059] Comparative Example 1

[0060] Different from Example 2, cotyledons of the plant were selected as the explants.

[0061] Comparative Example 2

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

[0063] Comparative Example 3

[0064] Different from Example 2, the concentration of 6-BA in the induction medium was 0.5 mg / L.

[0065] Comparative Example 4

[0066] Different from Example 2, the concentration of 6-BA in the induction medium was 4 mg / L.

[0067] Comparative Example 5

[0068] Different from Example 2, IBA in the somatic embryo differentiation medium was replaced by NAA.

[0069] Comparative Example 6

[0070] Different from Example 2, 2,4-D in the somatic embryo differentiation medium was replaced by 6-BA.

[0071] Experimental Example 1

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

[0073] Callus induction rate = number of callus plants formed / total number of inoculated explants x 100%

[0074] Table 1 Callus induction rate of each group

[0075] Group Rate of callus induction (%) 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] From Examples 1-3, it can be seen that, in the embryonic callus induction stage, the radicle is used as the explant, and the induction and differentiation are carried out by using the induction medium of the present application, and the induction rate is above 96%; from Example 2 and Comparative Example 1, it can be seen that the cotyledon is selected as the explant, and the callus induction rate is reduced, and is only 65%; from Comparative Examples 2-4, it can be seen that the type or concentration of the plant growth regulator in the induction medium is changed, and the callus induction rate is reduced.

[0077] Experimental Example 2

[0078] The experiment is divided into 5 groups, and the grape somatic embryo regeneration culture is carried out according to the methods in Examples 1-3 and Comparative Examples 5 and 6, the explants are inoculated into the induction medium, and are cultured in the dark condition, the callus is obtained, 100 calluses in each group are transferred into the somatic embryo differentiation medium, and are germinated in the dark environment; after the cotyledon embryo grows out, it is transferred to the light culture, and after 40 days, the somatic embryo generation rate is counted, and the results are shown in Table 2.

[0079] Somatic embryo generation rate = number of somatic embryo plants formed / total number of calluses inoculated × 100%

[0080] Table 2: Somatic embryo generation rate of each group

[0081]

[0082]

[0083] According to the results in Table 2, the somatic embryo generation rate of Examples 1-3 of the present application is higher than that of Comparative Examples 5 and 6, which shows that the somatic embryo differentiation medium of the present application is a better medium formula for callus induction, and is beneficial to the formation of grape somatic embryos.

[0084] From the above examples, the present application provides a grape somatic embryo regeneration method, a large number of grape somatic embryo regeneration plants with consistent genetic basis can be obtained, and rapid, stable and efficient grape seedling raising can be realized.

[0085] The above only describes the preferred embodiments of the present application, and it should be pointed out that, for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for regenerating grape somatic embryos, characterized in that, Includes the following steps: (1) Seed germination: Grape seeds are sown in a solid culture medium and cultured under light for 32-36 days to obtain grape plants that have not yet grown true leaves; (2) Induction of embryogenic callus: The hypocotyl of the plant was cut and inoculated into the induction medium. It was cultured in the dark for 24-28 days to obtain callus. (3) Differentiation of embryogenic callus into somatic embryos: The callus was transferred into somatic embryo differentiation medium and cultured in the dark; after the cotyledon embryos grew, the culture was switched to light, and somatic embryos were obtained after 30-40 days. (4) Inducing rooting of somatic embryos: When the hypocotyl and radicle of the somatic embryo differentiate, transfer them to a rooting medium and culture them under light for 30 to 40 days to obtain rooted plants. (5) Seedling hardening: Transfer the rooted plants to the cultivation substrate and harden them for 20-30 days to obtain regenerated grape plants. The somatic embryo differentiation medium was: 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; The induction medium was: MS + 6-BA 1~3 mg / L + sucrose 25~35 mg / L + agar 6~8 mg / L, pH=6.0±0.

2.

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

2.

3. The method according to claim 1, characterized in that, The rooting medium was: MS + NAA 1~2 mg / L + sucrose 25~35 g / L + agar 6~8 g / L, pH=6.0±0.

2.

4. The method according to claim 1, characterized in that, In steps (3) and (4), the daily light exposure time is 14-18 hours and the light intensity is 1500-2000 Lx.

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

6. The method according to claim 1, characterized in that, The cultivation substrate includes peat moss, perlite, and vermiculite, with a mass ratio of 3-4:1:

1.

7. The method according to claim 1, characterized in that, The seedlings were cultivated and grown in a greenhouse under natural light.

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