A method for inducing somatic embryogenesis in hypocotyls of grape seedlings

Abstract

The present application relates to the technical field of plant tissue culture, and particularly discloses a method for establishing a grape seedling hypocotyl induced somatic embryo regeneration system, which comprises the following steps: taking secondary embryos obtained by using seedless grape embryos to rescue deformed seedling hypocotyls as explants, inducing the explants in KBN medium to obtain embryogenic calli, non-embryogenic calli and secondary proembryo groups, inoculating the secondary proembryo groups in X6 medium to proliferate and culture the secondary proembryo groups and somatic embryos, performing secondary induction on the somatic embryos before cotyledon stage to establish a somatic embryo maintaining and proliferating circulation system, inoculating the somatic embryos into X3 medium to differentiate hypocotyls and radicles, and transferring the somatic embryos to WPM medium to grow into seedlings. The present application successfully induces the regeneration system of the secondary proembryo groups obtained by using seedless grape embryos to rescue deformed seedling hypocotyls, and successfully establishes a secondary proembryo group maintaining and proliferating circulation system, which can provide new recipient materials for grape genetic transformation.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for inducing somatic embryo regeneration in grape seedling hypocotyls. Background Technology

[0002] Grapes, as one of the world's most important fruit trees, offer significant economic, social, and ecological benefits. As woody vines, grapes have a long growth cycle and a highly heterozygous genome, making traditional breeding methods time-consuming. The application of grape genetic transformation technology has made it possible to target and improve grape traits, shorten breeding cycles, and analyze and verify gene function, while also promoting gene transfer between different species. This technology has become a core area of ​​grape biotechnology research and application, and a key means of improving grape variety traits.

[0003] With the application of plant cell engineering technology in the grape industry, researchers have established regeneration systems through somatic embryogenesis and organogenesis. However, successful cases of organogenesis are relatively few, and the regeneration efficiency is generally low. In contrast, the grape somatic embryo regeneration system, with its advantages of stable genetic material, rapid and efficient reproduction capacity, phenotypic stability of single-cell regenerated plants, and fewer chimeras in transgenic plants, has become a stable and efficient regeneration pathway, and is considered the most suitable recipient in genetic transformation systems. However, the grape somatic embryo regeneration system is affected by various factors such as genotype, explant, and culture medium composition, leading to problems such as low induction efficiency, difficulty in somatic embryo germination and seedling formation, and a high rate of deformed embryos. Therefore, there is an urgent need to provide a stable method for establishing a grape somatic embryo regeneration system. Summary of the Invention

[0004] To develop a stable and efficient method for establishing a grape somatic embryo regeneration system, this invention provides a method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system. The method provided by this invention utilizes the hypocotyl of seedless grape seedlings to successfully obtain regenerated plants via the somatic embryo pathway, and establishes a cyclical system for maintaining and propagating somatic embryos.

[0005] This invention provides a method for establishing a hypocotyl-induced somatic embryo regeneration system for grape seedlings, comprising the following steps:

[0006] Secondary embryos obtained from the hypocotyls of malformed seedlings of seedless grapes were inoculated into KBN medium and induced for 28-32 days to obtain embryogenic callus, non-embryonic callus, and secondary proto-embryo mass.

[0007] The secondary protoblastomas were then inoculated into X6 medium and cultured for 28–32 days to obtain secondary protoblastomas and somatic embryos with increased quality. The secondary protoblastomas with increased quality were used for subculture preservation and propagation. Somatic embryos before the cotyledon stage were selected for secondary induction to establish a cycle system for the preservation and propagation of somatic embryos.

[0008] Somatic embryos at any stage were inoculated into X3 medium and cultured in the dark for 28-32 days. When the hypocotyl and radicle of the somatic embryos differentiated, they were transferred to WPM medium and cultured at 24-25°C for 16 h of light and 8 h of darkness to produce seedlings.

[0009] The KBN medium formulation is as follows: MS + 1.0 mg / L ~ 1.2 mg / L 6-BA + 0.4 mg / L ~ 0.6 mg / L 2,4-D + 0.53 mg / L ~ 0.57 mg / L NOA + 28 g / L ~ 32 g / L sucrose + 3 g / L plant gel + 0.9 g / L ~ 1.1 g / L inositol + 0.2 g / L ~ 0.4 g / L potassium nitrate;

[0010] The X6 medium formulation is: MS medium + 55 g / L to 65 g / L sucrose + 1.0 g / L to 2.0 g / L activated carbon;

[0011] The X3 medium formulation is: MS + 28 g / L to 32 g / L sucrose + 1.0 g / L to 2.0 g / L activated carbon;

[0012] The WPM medium formula is: WPM + 0.1 mg / L to 0.3 mg / L 6-BA + 18 g / L to 22 g / L sucrose + 6 g / L to 8 g / L agar + 1.0 g / L to 2.0 g / L activated carbon.

[0013] This invention uses the hypocotyl of seedless grape embryo-derived malformed seedlings as explants for induction culture, successfully obtaining regenerated plants, and establishing a stable cycle system for maintaining and proliferating somatic embryos.

[0014] Furthermore, the KBN culture medium formula is: MS + 1.1 mg / L 6-BA + 0.5 mg / L 2,4-D + 0.55 mg / L NOA + 30 g / L sucrose + 3 g / L plant gel + 1 g / L inositol + 0.3 g / L potassium nitrate;

[0015] The X6 medium formulation is: MS + 60 g / L sucrose + 1.5 g / L activated carbon;

[0016] The X3 medium formulation is: MS + 30 g / L sucrose + 1.5 g / L activated carbon;

[0017] The WPM medium formula is: WPM + 0.2 mg / L 6-BA + 20 g / L sucrose + 7 g / L agar + 1.5 g / L activated carbon.

[0018] Furthermore, the secondary embryos were inoculated in KBN medium and induced under dark culture conditions of 24℃~25℃.

[0019] Furthermore, the secondary protozoa were inoculated into X6 medium and cultured in the dark at 24℃~25℃.

[0020] Furthermore, somatic embryos include globular embryos, heart-shaped embryos, torpedo embryos, and cotyledonary embryos.

[0021] Furthermore, the secondary induction process for somatic embryos before the cotyledon stage is as follows: somatic embryos are inoculated into KBN embryogenic callus induction medium and induced to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium and cultured to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation and propagation of secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from somatic embryos and induced to culture again. The above steps are repeated to establish a cycle system for the preservation and propagation of somatic embryos.

[0022] Furthermore, the induction culture conditions are dark culture at 24℃~25℃, with subculture once a month.

[0023] Furthermore, the propagation process involves dark culture at 24℃~25℃, with subculture once a month.

[0024] Furthermore, the culture temperature during propagation is 25℃.

[0025] Furthermore, the seedless grape embryo deformed seedlings are obtained by hybridization of Purple Sweet Seedless Grape and Morissa Seedless Grape or by hybridization of Morissa Seedless Grape and Oasis Gem Grape.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention provides a method for constructing a hypocotyl somatic cell regeneration induction system for seedless grape embryoid malformed seedlings, including the induction, maintenance and propagation of protocotyls, successfully inducing the hypocotyl of seedless grape embryoid malformed seedlings to obtain a regeneration system, and successfully establishing a cyclic system for the maintenance and propagation of secondary protocotyls.

[0028] This invention provides a method for establishing a somatic embryo regeneration induction system for hypocotyls in seedless grape seedlings with embryogenic deformities, exhibiting high stability and efficiency. This provides high-quality recipient materials for research such as the construction of grape genetic transformation systems, verification of functional genes, and molecular breeding. Furthermore, this invention successfully established a somatic embryo regeneration system through secondary embryo differentiation and induction of the hypocotyl, not only adding a new pathway for hypocotyl-induced somatic embryo regeneration but also enriching the types of explants available.

[0029] This invention establishes a regeneration system for somatic embryos induced from hypocotyls in seedless grape seedlings with embryogenic deformities. The somatic embryo regeneration pathway was ultimately achieved in hypocotyls of hybrid seedlings of Morissa × Oasis Gem and Purple Sweet Seedless × Morissa. This reduces time and costs during the induction of embryogenic callus and provides new recipient materials for further grape genetic transformation. It also establishes a cyclical system for the preservation and propagation of somatic embryos. The secondary protoblastomere produced directly from somatic embryos before the cotyledon stage yields the highest induction rate (approximately 85%-95%). Compared to induced embryogenic callus, directly obtaining the secondary protoblastomere allows for more efficient and stable preservation and propagation. Somatic embryos germinating from the protoblastomere can continue to be cyclically induced. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This illustrates the differentiation of secondary embryos from the hypocotyl. In the figure, A represents the differentiation of secondary embryos at the junction of the hypocotyl and radicle; B represents the differentiation of secondary embryos from the hypocotyl; and C represents the differentiation of densely clustered secondary embryos from the hypocotyl.

[0032] Figure 2 This diagram illustrates the process of establishing a hypocotyl-induced somatic embryo regeneration system. In the diagram, A: secondary embryo differentiates at the junction of the hypocotyl and radicle; B: induced secondary protoblastomere; C: secondary protoblastomere; D: secondary protoblastomere develops into an embryo; E: increase in the mass of the secondary protoblastomere; F: subculture and propagation of the secondary protoblastomere; G: embryos at different developmental stages (globose, heart-shaped, torpedo-shaped, and cotyledonous embryos); M: further embryo development, with elongation of the hypocotyl and radicle; N: seedling formation.

[0033] Figure 3 This is a secondary induction process; in the figure, A: non-embryonic callus; B: hard, golden-yellow embryonic callus tissue; C: a chimera of embryonic and non-embryonic callus tissue; D: secondary protozoan.

[0034] Figure 4 The growth of embryogenic callus and secondary protoblastomere in X6; In the figure, A: embryogenic callus differentiates into embryo; B: some secondary protoblastomere develops into embryo; C: ungerminated secondary protoblastomere continues to proliferate.

[0035] Figure 5 The diagram shows the process of a proto-embryo mass developing into a seedling. In the diagram, A is a spherical embryo; B is a heart-shaped embryo; C is a torpedo-shaped embryo; D is a cotyledonous embryo; E is the elongation of the hypocotyl and radicle; and FG represents the seedling process. Detailed Implementation

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0037] This invention studies a grape seedling hypocotyl-induced somatic embryo regeneration system and successfully obtained somatic embryos from two hybrid combinations: Morissa × Oasis Gem and Purple Sweet Seedless × Morissa. A secondary embryo recycling system was used to induce, preserve, and propagate their protoblastomas. It was found that the secondary protoblastomas directly generated from somatic embryos before the cotyledon stage were the most abundant, with an induction rate of approximately 85-95%. Compared to induced embryogenic callus, directly obtaining the secondary protoblastomas allows for more efficient and stable preservation and propagation. Furthermore, somatic embryos germinating from the protoblastomas can be continuously induced.

[0038] This invention rescues seedlings with deformed seedless grape embryos from 2023. The deformed seedlings were inoculated into transformation medium (WPM + 2 mg / L 6-BA + 2.0 mg / L IAA + 30 g / L sucrose + 7 g / L agar + 1.5 g / L activated carbon). During the culture process, secondary embryos were observed on the hypocotyl of the hybrid seedlings (e.g., ...). Figure 1 (As shown). A somatic embryo regeneration system was constructed using differentiated secondary embryos through the following steps: Under aseptic conditions, secondary embryos obtained from the hypocotyls of malformed seedlings of seedless grapes were subjected to secondary induction. Figure 2 This invention outlines the process for establishing a hypocotyl-induced somatic embryo regeneration system. The specific steps are as follows:

[0039] Secondary embryo ( Figure 2 A) After being chopped and inoculated into KBN medium for induction culture for 1 month, embryogenic callus, non-embryonic callus, and secondary protozoa (such as...) will appear. Figure 2The occurrence of B) involves the propagation of embryogenic callus and secondary protozoa in X6 medium for one month. A portion of the secondary protozoa increased in mass to three times their original size (i.e., from B to C). Figure 2 The state changes in C are Figure 2 Secondary protozoa that have not yet germinated and whose quality continues to improve can be further cultured in X6 medium for subculturing and propagation (e.g., ...). Figure 2 (F).

[0040] Another part of the secondary protozoan produces dense white somatic embryos (such as...) Figure 2 Under a microscope, embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonary embryos. Figure 2 (G); Selecting pre-cotyledonary somatic embryos (globose, heart-shaped, and torpedo-shaped embryos) allows for secondary induction: Somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomas. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomas and somatic embryos with increased quality. The secondary protoblastomas with increased quality are used for subculture, preservation, and propagation of secondary protoblastomas. Pre-cotyledonary somatic embryos are selected from the somatic embryos for induction culture again. The above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0041] Random somatic embryos were inoculated into X3 medium (MS + 30 g / L sucrose + 1.5 g / L activated carbon) and cultured for 1 month. When the cotyledonary hypocotyl and radicle differentiated, they were transferred to WPM medium and cultured into seedlings. Figure 2 M- Figure 2 (N).

[0042] The culture medium and its formulation used in this invention are as follows:

[0043] The KBN medium formula is: MS + 1.1 mg / L 6-BA + 0.5 mg / L 2,4-D + 0.55 mg / L NOA + 30 g / L sucrose + 3 g / L plant gel + 1 g / L inositol + 0.3 g / L potassium nitrate.

[0044] The X6 medium formula is: MS + 60 g / L sucrose + 1.5 g / L activated carbon.

[0045] The X3 medium formula is: MS + 30 g / L sucrose + 1.5 g / L activated carbon.

[0046] The WPM medium formula is: WPM + 0.2 mg / L 6-BA + 20 g / L sucrose + 7 g / L agar + 1.5 g / L activated carbon.

[0047] The following are specific embodiments of the present invention:

[0048] Example 1: A method for inducing somatic embryo regeneration system from hypocotyl in grape seedlings.

[0049] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyl of malformed seedlings salvaged by Morissa × Oasis Gem as the material.

[0050] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of Morissa × Oasis Gem grape seedlings rescued from malformation are subjected to secondary induction. The secondary embryos are then chopped and inoculated into KBN medium for induction culture for one month to produce embryogenic callus. Figure 3 B), non-embryonic callus ( Figure 3 A) and secondary protozoan ( Figure 3 (D) After inoculating the secondary protozoa into X6 medium and culturing for 1 month, some of the secondary protozoa increased in weight to 3 times their original size. Secondary protozoa that continued to increase in weight but did not germinate can be further inoculated into X6 medium for subculture preservation and propagation. Figure 4 (C).

[0051] Embryogenic callus differentiates into somatic embryos ( Figure 4 A), and another part of the secondary protozoan produces dense white somatic embryos ( Figure 4 (B) Somatic embryos, when observed under a microscope, can be seen to be embryos at different developmental stages: globular embryos ( Figure 5 A) Heart-shaped embryo ( Figure 5 B), torpedo embryo ( Figure 5 C) and cotyledon embryo ( Figure 5 (D) Select somatic embryos before the cotyledon stage for secondary induction: Somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomas. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomas and somatic embryos with increased quality. The secondary protoblastomas with increased quality are used for subculture, preservation and propagation of secondary protoblastomas. Somatic embryos before the cotyledon stage are selected from somatic embryos for induction culture again. The above steps are repeated to establish a cycle system for the preservation and propagation of somatic embryos.

[0052] Select partially developed somatic embryos and inoculate them into X3 medium and culture for 1 month. At the time of differentiation of the cotyledonary hypocotyl and radicle (… Figure 5 E) was transferred to WPM medium and cultured under light to grow seedlings. Figure 5 F- Figure 5 (G).

[0053] Example 2: A method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system.

[0054] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained by rescuing hypocotyls of malformed seedlings from the embryo of Zitian Seedless × Morissa as material.

[0055] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of seedless seedlings of Purple Sweet × Morissa grapes are induced a second time. The secondary embryos are chopped and inoculated into KBN medium for induction culture for 1 month, resulting in embryogenic callus, non-embryonic callus and secondary protoblasts. The secondary protoblasts are inoculated into X6 medium and cultured for 1 month. The mass of some secondary protoblasts increases to 3 times the original mass. Secondary protoblasts that continue to increase in mass but have not germinated can be inoculated into X6 medium for subculture preservation and propagation.

[0056] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction. The somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of the secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again. The above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0057] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0058] Example 3: A method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system.

[0059] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyl of malformed seedlings salvaged by Morissa × Oasis Gem as the material.

[0060] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of Morissa × Oasis Gem grape seedlings were induced a second time. The secondary embryos were chopped and inoculated into KBN medium for induction culture for 2 months, resulting in embryogenic callus, non-embryonic callus, and secondary protoblasts. The secondary protoblasts were inoculated into X6 medium and cultured for 1 month. The mass of some secondary protoblasts increased to 3 times the original mass. Secondary protoblasts that continued to increase in mass but did not germinate could be inoculated into X6 medium for subculture preservation and propagation.

[0061] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction: somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere, and then inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0062] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0063] Example 4: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0064] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyl of malformed seedlings salvaged by Morissa × Oasis Gem as the material.

[0065] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of Morissa × Oasis Gem grape seedlings were induced a second time. The secondary embryos were chopped and inoculated into KBN medium for 3 months of induction culture to produce embryogenic callus, non-embryonic callus and secondary protoblasts. The secondary protoblasts were inoculated into X6 medium and cultured for 1 month. The mass of some secondary protoblasts increased to 3 times the original mass. Secondary protoblasts that continued to increase in mass but did not germinate could be inoculated into X6 medium for subculture preservation and propagation.

[0066] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction: somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere, and then inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0067] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0068] Example 5: A method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system.

[0069] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyl of malformed seedlings salvaged by Morissa × Oasis Gem as the material.

[0070] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of Morissa × Oasis Gem grape seedlings were induced a second time. The secondary embryos were chopped and inoculated into KBN medium for induction culture for 1 month, resulting in embryogenic callus, non-embryonic callus, and secondary protoblasts. The secondary protoblasts were inoculated into X6 medium and cultured for 2 months. The mass of some secondary protoblasts increased to 3 times the original mass. Secondary protoblasts that continued to increase in mass but did not germinate could be inoculated into X6 medium for subculture preservation and propagation.

[0071] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction: somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere, and then inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0072] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0073] Example 6: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0074] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyl of malformed seedlings salvaged by Morissa × Oasis Gem as the material.

[0075] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of Morissa × Oasis Gem grape seedlings were induced a second time. The secondary embryos were chopped and inoculated into KBN medium for induction culture for 1 month, resulting in embryogenic callus, non-embryonic callus and secondary protoblasts. The secondary protoblasts were inoculated into X6 medium and cultured for 3 months. The mass of some secondary protoblasts increased to 3 times the original mass. Secondary protoblasts that continued to increase in mass but did not germinate could be inoculated into X6 medium for subculture preservation and propagation.

[0076] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction: somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere, and then inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0077] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0078] Example 7: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0079] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained by rescuing hypocotyls of malformed seedlings from the embryo of Zitian Seedless × Morissa as material.

[0080] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of seedless seedlings of Purple Sweet × Morissa grapes are induced a second time. The secondary embryos are chopped and inoculated into KBN medium for induction culture for 2 months, resulting in embryogenic callus, non-embryonic callus, and secondary protoblasts. The secondary protoblasts are inoculated into X6 medium and cultured for 1 month. The mass of some secondary protoblasts increases to 3 times the original mass. Secondary protoblasts that continue to increase in mass but have not germinated can be inoculated into X6 medium for subculture preservation and propagation.

[0081] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction. The somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of the secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again. The above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0082] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0083] Example 8: A method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system.

[0084] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained by rescuing hypocotyls of malformed seedlings from the embryo of Zitian Seedless × Morissa as material.

[0085] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of seedless seedlings of Purple Sweet × Morissa grapes are induced a second time. The secondary embryos are chopped and inoculated into KBN medium for induction culture for 3 months to produce embryogenic callus, non-embryonic callus and secondary protoblasts. The secondary protoblasts are inoculated into X6 medium and cultured for 1 month. The mass of some secondary protoblasts increases to 3 times the original mass. Secondary protoblasts that continue to increase in mass but have not germinated can be inoculated into X6 medium for subculture preservation and propagation.

[0086] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction. The somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of the secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again. The above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0087] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0088] Example 9: A method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system.

[0089] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained by rescuing hypocotyls of malformed seedlings from the embryo of Zitian Seedless × Morissa as material.

[0090] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of seedless seedlings of Purple Sweet × Morissa grapes are induced a second time. The secondary embryos are chopped and inoculated into KBN medium for induction culture for 1 month, resulting in embryogenic callus, non-embryonic callus, and secondary protoblasts. The secondary protoblasts are inoculated into X6 medium and cultured for 2 months. The mass of some secondary protoblasts increases to 3 times the original mass. Secondary protoblasts that continue to increase in mass but have not germinated can be inoculated into X6 medium for subculture preservation and propagation.

[0091] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction. The somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of the secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again. The above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0092] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0093] Example 10: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0094] In this embodiment, a somatic embryo regeneration system was established using secondary embryos obtained by rescuing hypocotyls of malformed seedlings from the embryo of Zitian Seedless × Morissa as material.

[0095] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from hypocotyls of seedless seedless × Morissa grape seedlings are induced a second time. The secondary embryos are chopped and inoculated into KBN medium for induction culture for 1 month, resulting in embryogenic callus, non-embryonic callus and secondary protoblasts. The secondary protoblasts are inoculated into X6 medium and cultured for 3 months. The mass of some secondary protoblasts increases to 3 times the original mass. Secondary protoblasts that continue to increase in mass but have not germinated can be inoculated into X6 medium for subculture preservation and propagation.

[0096] Embryogenic callus differentiates into somatic embryos, while dense white somatic embryos are generated around a portion of the secondary protoblastomere. Under a microscope, somatic embryos at different developmental stages can be observed: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonous embryos. Somatic embryos before the cotyledon stage are selected for secondary induction. The somatic embryos are inoculated into KBN embryogenic callus induction medium for induction culture to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium for culture to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, preservation, and propagation of the secondary protoblastomere. Somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again. The above steps are repeated to establish a cyclic system for the preservation and propagation of somatic embryos.

[0097] Select partially developed somatic embryos and inoculate them in X3 medium for 1 month. When the cotyledonary embryo, hypocotyl, and radicle differentiate, transfer them to WPM medium and culture them under light to grow seedlings.

[0098] Examples 2-10 show a similar cycle system for the preservation and propagation of secondary protoblastomas, the maintenance and propagation of somatic embryos, and the seedling formation process in the grape somatic embryo regeneration system. These systems are similar to those in Example 1 and can be referenced. Figures 1-5 .

[0099] As can be seen from the above embodiments, when the secondary embryos obtained from the hypocotyls of seedless grape seedlings with malformed embryogenesis are chopped and inoculated onto KBN medium to induce embryogenic callus, the following four states can be observed: ① soft, water-soaked non-embryonic callus; ② hard, golden-yellow embryogenic callus, which begins to swell and proliferate after continuous subculturing; ③ a chimera of embryogenic and non-embryonic callus (… Figure 3(C); ④ Secondary protoplasts. After selecting embryogenic callus and secondary protoplasts and inoculating them in X6 medium, the embryogenic callus differentiates into embryos from the periphery, while the secondary protoplasts have two states. Most of the secondary protoplasts do not germinate and their mass is 2-3 times that before inoculation. With each subculture, the mass of the protoplasts doubles, and they can be preserved and propagated. Another small portion of the protoplasts will begin to germinate into embryos of different states as the subculture time increases. This can be used to repeat the secondary induction of embryogenic callus and establish a cycle system. The secondary protoplasts produced by somatic embryos before the cotyledon stage are the most abundant, with an induction rate of 85%-95%. This invention has been able to continuously induce the production of secondary protoplasts for 6 months.

[0100] Developing somatic embryos are inoculated in X3 medium, and after one month, different embryos in different states can be clearly observed: globular embryos, heart-shaped embryos, torpedo embryos, and cotyledon embryos. As they develop further, when the hypocotyl and radicle of the cotyledon embryo differentiate, they can be transferred to WPM medium to develop into seedlings.

[0101] Comparative Example 1: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0102] This comparative study used secondary embryos obtained from the hypocotyl rescue of malformed seedlings by Zheng Yan's nucleusless × Italian embryos as material to investigate the somatic embryo regeneration system.

[0103] The specific steps are as follows: Under aseptic conditions, secondary embryos obtained from the hypocotyls of Zheng Yan seedless × Italian grape hybrid seedlings (embryo rescued malformed seedlings) were subjected to secondary induction. The secondary embryos were then chopped and inoculated onto KBN medium (MS + 1.1 mg / L 6-BA + 0.5 mg / L 2,4-D + 0.55 mg / L NOA + 30 g / L sucrose + 3 g / L plant gel + 1 g / L inositol + 0.3 mg ...3 After one month of induction culture with potassium nitrate (g / L), embryogenic and non-embryonic callus tissues will appear. During the three-month culture period, a small number of secondary protoblastomas will appear. The small number of secondary protoblastomas obtained will be inoculated into X6 medium for propagation. It was observed that the secondary protoblastomas could not be stably preserved and began to develop after one month of culture. When the somatic embryos developing in the pre-cotyledon stage were induced a second time, a small number of secondary protoblastomas were obtained after two months of culture. Similarly, they continued to develop for two months after being inoculated into X6 medium and could not maintain the state of secondary protoblastomas. Finally, they were inoculated into X3 medium and cultured, and subsequently developed into seedlings.

[0104] Comparative Example 2: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0105] The steps are basically the same as those in Comparative Example 1, except that the induction culture lasts for 2 months.

[0106] Comparative Example 3: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0107] The steps are basically the same as those in Comparative Example 1, except that the induction culture lasts for 3 months.

[0108] Comparative Example 4: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0109] The steps were basically the same as those in Comparative Example 1, except that the small number of secondary protozoa obtained were inoculated into X6 medium for propagation culture for 2 months.

[0110] Comparative Example 5: A method for establishing a grape seedling hypocotyl-induced grape somatic embryo regeneration system.

[0111] The steps were basically the same as those in Comparative Example 1, except that the small number of secondary protozoa obtained were inoculated into X6 medium for propagation culture for 3 months.

[0112] Using Examples 1-10 and Comparative Examples 1-5 as subjects, the effects of induction time on the induction rate of secondary protozoa were studied, as well as the effects of the culture time of secondary protozoa inoculated in X6 medium on the protozoa state. The experimental results are recorded in Tables 1 and 2.

[0113] 1. Effect of induction culture time on primary induction efficiency: Secondary embryos derived from hypocotyl differentiation of malformed seedlings using Morissa × Oasis Gem, Zitian seedless × Morissa, and Zhengyan seedless × Italian seedless embryos were chopped and inoculated into KBN medium. The induction rates of embryogenic callus, non-embryonic callus, and secondary protoblasts were counted for three consecutive months.

[0114] Embryogenic callus induction rate = number of embryogenic callus / number of inoculated tissues; Non-embryogenic callus induction rate = number of non-embryogenic callus / number of inoculated tissues; Secondary protozoan induction rate = number of secondary protozoan tissues / number of inoculated tissues.

[0115] 2. Description of the effect of culture time on the culture effect of secondary protozoa in X6 medium: The culture status of secondary protozoa of three materials in X6 medium was recorded for three consecutive months.

[0116] Table 1. Effect of induction culture time on initial induction efficiency

[0117]

[0118] As shown in Table 1, somatic embryos obtained from hypocotyl differentiation by salvage of Morissa × Oasis Gem, Zitian Nucleus-free × Morissa, and Zhengyan Nucleus-free × Italian embryos were minced and inoculated into KBN medium for initial induction. After three months of continuous culture and observation, it can be seen that the minced somatic embryos of Morissa × Oasis Gem and Zitian Nucleus-free × Morissa can obtain a small amount of secondary protoblastomere in the first month, with an induction rate of 30%. By the third month of culture, the secondary protoblastomere induction rate can reach 60%-70%. However, the secondary protoblastomere induction rate of Zhengyan Nucleus-free × Italian embryos is 0% in the first two months of culture, and only 10% secondary protoblastomere induction rate is obtained by the third month of culture. Moreover, the induction rate of non-embryonic callus tissue is very high throughout the entire culture period.

[0119] Table 2. Description of the effect of culture time on the culture of secondary proto-macromolecules in X6 medium.

[0120]

[0121] As shown in Table 2, this invention establishes a hypocotyl-induced somatic embryo regeneration system for Morissa × Oasis Gem and Purple Sweet Seedless × Morissa seedlings, including a cyclical process of secondary induction, maintenance, and propagation of the secondary protoplast. This system can sustainably provide recipient materials for grape genetic transformation. While the hypocotyl of Zheng Yan Seedless × Italian seedlings can induce somatic embryo regeneration into seedlings, its secondary protoplast cannot be stably maintained or propagated.

[0122] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0123] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for establishing a somatic embryogenesis system of hypocotyls of grape seedlings, characterized by, Includes the following steps: Secondary embryos obtained from hypocotyls of seedless grape embryo-truncation malformed seedlings were inoculated into KBN medium and induced for 28-32 days to obtain embryogenic callus, non-embryonic callus, and secondary proto-embryonic masses; the seedless grape embryo-truncation malformed seedlings were obtained by hybridization of Purple Sweet Seedless Grape and Morissa Seedless Grape or by hybridization of Morissa Seedless Grape and Oasis Gem Grape. The secondary protoblastomas were then inoculated into X6 medium and cultured for 28-32 days to obtain secondary protoblastomas and somatic embryos with increased quality. The secondary protoblastomas with increased quality were used for subculture, maintenance and propagation. Somatic embryos before the cotyledon stage were selected for secondary induction to establish a cycle system for maintaining and propagating somatic embryos. The secondary induction process for somatic embryos before the cotyledon stage is as follows: Somatic embryos are inoculated into KBN embryogenic callus induction medium and induced to obtain embryogenic callus and secondary protoblastomere. Then, they are inoculated into X6 medium and cultured to obtain secondary protoblastomere and somatic embryos with increased quality. The secondary protoblastomere with increased quality is used for subculture, maintenance and propagation. Somatic embryos before the cotyledon stage are selected from somatic embryos and induced to undergo induction culture again. The above steps are repeated to establish a cycle system for maintaining and propagating somatic embryos. Somatic embryos at any stage were inoculated into X3 medium and cultured in the dark for 28-32 days. When the hypocotyl and radicle of the somatic embryos differentiated, they were transferred to WPM medium and cultured at 24-25°C for 16 h of light and 8 h of darkness to produce seedlings. The KBN medium formulation is as follows: MS + 1.1 mg / L 6-BA + 0.5 mg / L 2,4-D + 0.55 mg / L NOA + 30 g / L sucrose + 3 g / L plant gel + 1 g / L inositol + 0.3 g / L potassium nitrate; The X6 medium formulation is: MS + 60 g / L sucrose + 1.5 g / L activated carbon; The X3 medium formulation is: MS + 30 g / L sucrose + 1.5 g / L activated carbon; The WPM medium formula is: WPM + 0.2 mg / L 6-BA + 20 g / L sucrose + 7 g / L agar + 1.5 g / L activated carbon.

2. The method for establishing a somatic embryo regeneration system induced by hypocotyl in grape seedlings according to claim 1, characterized in that, Secondary embryos were inoculated into KBN medium and induced under dark culture conditions of 24℃~25℃.

3. The method for inducing somatic embryo regeneration in grape seedling hypocotyls according to claim 1, characterized in that, The secondary protozoa were inoculated into X6 medium and cultured in the dark at 24℃~25℃.

4. The method for establishing a somatic embryo regeneration system induced by hypocotyl in grape seedlings according to claim 1, characterized in that, Somatic embryos include globular embryos, heart-shaped embryos, torpedo embryos, and cotyledonary embryos.

5. The method for establishing a somatic embryo regeneration system induced by hypocotyl in grape seedlings according to claim 1, characterized in that, The induction culture conditions are dark culture at 24℃~25℃, with subculture once a month.

6. The method for establishing a somatic embryo regeneration system induced by hypocotyl in grape seedlings according to claim 1, characterized in that, The culture conditions for propagation are dark culture at 24℃~25℃, with subculture once a month.

7. The method for establishing a somatic embryo regeneration system induced by hypocotyl in grape seedlings according to claim 1, characterized in that, The culture temperature during propagation is 25℃.

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