Method for inducing somatic embryo regeneration system by hypocotyl of grape seedling

Abstract

The invention relates to the technical field of plant tissue culture, and particularly discloses an establishment method of a grape seedling hypocotyl induced somatic embryo regeneration system. Comprising the following steps: by taking a secondary embryo obtained from a hypocotyl of a seedless grape embryonic-handle malformed seedling as an explant, inducing in a KBN culture medium to obtain an embryonic callus, a non-embryonic callus and a secondary proembryo mass, inoculating the secondary proembryo mass into an X6 culture medium, and carrying out propagation culture to obtain the secondary proembryo mass with increased quality and a somatic embryo, performing secondary induction on the somatic embryos before the cotyledon stage to establish a somatic embryo maintenance and propagation circulating system, inoculating the somatic embryos into an X3 culture medium to differentiate hypocotyls and radicles, and transferring to a WPM culture medium to culture into seedlings. According to the method disclosed by the invention, the hypocotyl of the seedless grape embryonic malformation seedling is successfully induced to obtain a regeneration system, a secondary proembryonic mass maintenance and propagation circulation system is successfully established, and a new acceptor material can be provided for grape genetic transformation.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for inducing a somatic embryo regeneration system from hypocotyls of grape seedlings. Background Art

[0002] As one of the most important fruit trees in the world, grapes have significant benefits to the economy, society and ecology. Vitis genus plants are woody vines. Due to their long growth cycle and high heterozygosity of the genome, traditional breeding methods are time-consuming. The application of grape genetic transformation technology makes it possible to improve grape traits in a targeted manner, shorten the breeding cycle, and analyze and verify gene functions, while promoting the transfer of genes between different species. This technology has become a core area of ​​grape biotechnology research and application, and is also a key means of improving grape variety traits.

[0003] With the application of plant cell engineering technology in the field of grapes, researchers have established a regeneration system through somatic embryogenesis and organogenesis technology. However, there are relatively few successful cases of organogenesis, and the regeneration efficiency is generally not high. In contrast, the grape somatic embryo regeneration system has become a stable and efficient regeneration pathway due to its advantages such as the stability of genetic material, rapid and efficient reproduction ability, trait stability of single-cell regenerated plants, and fewer transgenic plant chimeras. It is considered to be the most suitable receptor in the genetic transformation system. However, the grape somatic embryo regeneration system is affected by multiple factors such as genotype, explant, and culture medium components, resulting in the pathway generally facing problems such as low induction efficiency, difficulty in somatic embryo germination and seedling formation, and high incidence of deformed embryos. Therefore, it is urgent to provide a method for establishing a stable grape somatic embryo regeneration system. Summary of the invention

[0004] In order to develop a stable and efficient method for establishing a grape somatic embryo regeneration system, the present invention provides a method for establishing a grape seedling hypocotyl-induced somatic embryo regeneration system. The method provided by the present invention utilizes seedless grape embryos to pull the hypocotyls of deformed seedlings to successfully obtain regenerated plants through the somatic embryo pathway, and establishes a circulation system for somatic embryo maintenance and propagation.

[0005] The present invention provides a method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings, comprising the following steps:

[0006] Secondary embryos obtained from the hypocotyls of seedless grape embryos were inoculated into KBN medium and cultured for 28-32 days to obtain embryonic callus, non-embryonic callus and secondary proembryonic masses.

[0007] The secondary proembryonic masses are then inoculated into X6 medium and cultured for 28 to 32 days to obtain secondary proembryonic masses and somatic embryos with increased mass. The secondary proembryonic masses with increased mass are used for subculture preservation and propagation of secondary proembryonic masses; somatic embryos before the cotyledon stage are selected for secondary induction to establish a circulatory system for somatic embryo maintenance and propagation.

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

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

[0010] The formula of X6 culture medium is: MS + 55g / L~65g / L sucrose + 1.0g / L~2.0g / L activated carbon;

[0011] The formula of X3 culture medium is: MS + 28g / L~32g / L sucrose + 1.0g / L~2.0g / L activated carbon;

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

[0013] The invention uses the hypocotyl of the seedless grape embryo-retained deformed seedling as an explant for induction culture, successfully obtains the regenerated plant, and establishes a stable circulation system for somatic embryo maintenance and propagation.

[0014] Furthermore, the KBN culture medium formula is: MS+1.1mg / L 6-BA+0.5mg / L2,4-D+0.55mg / LNOA+30g / L sucrose+3g / L phytogels+1g / L inositol+0.3g / L potassium nitrate;

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

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

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

[0018] Furthermore, the secondary embryos were inoculated into KBN medium for induction culture at 24°C to 25°C in the dark.

[0019] Furthermore, the secondary proembryo mass was inoculated into X6 medium and cultured in the dark at 24° C. to 25° C.

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

[0021] Furthermore, the somatic embryos before the cotyledon stage are selected for secondary induction process as follows: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, and then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for somatic embryo maintenance and propagation.

[0022] Furthermore, the induction culture condition is dark culture at 24°C to 25°C, with subculture once a month.

[0023] Furthermore, the culture conditions during propagation are dark culture at 24°C to 25°C, with subculturing once a month.

[0024] Furthermore, the culture temperature during the propagation was 25°C.

[0025] Furthermore, the seedless grape embryo deformity seedlings are obtained by hybridizing purple sweet seedless grapes and Molisa seedless grapes or by hybridizing Molisa seedless grapes and Green State Gem grapes.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a method for constructing a somatic cell regeneration induction system for the hypocotyls of seedless grape embryo-held deformed seedlings, including the induction, maintenance and propagation processes of proembryo masses, successfully inducing the hypocotyls of seedless grape embryo-held deformed seedlings to obtain a regeneration system, and successfully establishing a circulation system for maintaining and propagating secondary proembryo masses.

[0028] The method for establishing a somatic regeneration induction system for hypocotyls of seedless grape embryos with deformed seedlings provided by the present invention has high stability and efficiency. This provides high-quality receptor materials for the construction of grape genetic transformation systems, the verification of functional genes, and molecular breeding studies. In addition, the present invention successfully established a somatic embryo regeneration system through secondary embryo differentiation and secondary induction of hypocotyls, which not only adds a new way to induce somatic embryo regeneration of hypocotyls, but also enriches the types of explants available.

[0029] The invention realizes the establishment of a regeneration system for inducing somatic embryos from hypocotyls of seedless grape embryo-recovered deformed seedlings, and finally realizes a somatic embryo regeneration pathway in the hypocotyls of hybrid seedlings of Molisa×Green Oasis Gem and Purple Sweet Seedless×Molisa, which can shorten the time cost in the embryonic callus induction stage, provide new receptor materials for further carrying out subsequent grape genetic transformation, and simultaneously establish a circulation system for somatic embryo maintenance and propagation. The secondary proembryo masses directly produced by the induced embryos of somatic embryos before the cotyledon stage are the most, and the induction rate is about 85%-95%. Compared with the induced embryonic callus, the secondary embryo masses directly obtained can be more efficiently and stably preserved and propagated, and the somatic embryos germinated in the proembryo masses can continue to be induced in a cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is the phenomenon of secondary embryo differentiation from hypocotyl; in the figure, A is the secondary embryo differentiated at the junction of the hypocotyl and the radicle; B is the secondary embryo differentiated from the hypocotyl; C is the clustered secondary embryos with dense differentiation from the hypocotyl.

[0032] Figure 2 The process of establishing the somatic embryo regeneration system by inducing hypocotyl; in the figure, A: secondary embryo differentiated from the junction of hypocotyl and radicle; B: induced secondary proembryo mass; C: secondary proembryo mass; D: secondary proembryo mass develops into embryo; E: the mass of secondary proembryo mass increases; F: secondary proembryo mass is subcultured and preserved and propagated; G: embryos at different developmental stages (globby embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo); M: embryo further develops, hypocotyl and radicle elongate; N: seedling.

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

[0034] Figure 4 Embryogenic callus and secondary proembryonic masses grew in X6. In the figure, A: embryonic callus differentiated into embryos; B: some secondary proembryonic masses developed into embryos; C: ungerminated secondary embryonic masses continued to multiply.

[0035] Figure 5 This is the process of the proembryo mass developing into a seedling. In the figure, A is a spherical embryo; B is a heart-shaped embryo; C is a torpedo embryo; D is a cotyledon embryo; E is the elongation of the hypocotyl and radicle; FG is the seedling process. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0037] The present invention studies the somatic embryo regeneration system of grapes induced by hypocotyls of grape seedlings, and successfully obtains somatic embryos of two hybrid combinations, Molisa×Green Oasis Gem and Purple Sweet Seedless×Molisa, and uses the secondary embryo cycle regeneration system to induce, preserve and propagate the original embryo masses. It is found that the secondary original embryo masses directly produced by the somatic embryo induced embryos before the cotyledon stage are the most, with an induction rate of about 85-95%. Compared with the induced embryonic callus, directly obtaining the secondary embryo masses can be more efficiently and stably preserved and propagated, and the somatic embryos germinated in the original embryo masses can continue to be induced in a cycle.

[0038] The present invention rescues the 2023 seedless grape embryo deformed seedlings, inoculates the deformed seedlings in a transformation medium (WPM+2mg / L 6-BA+2.0mg / L IAA+30g / L sucrose+7g / L agar+1.5g / L activated carbon), and observes during the culture process that secondary embryos occur in the hypocotyls of the hybrid seedlings (e.g. Figure 1 The differentiated secondary embryos were used to construct a somatic embryo regeneration system through the following steps. Under sterile conditions, secondary embryos obtained from the hypocotyls of seedless grape embryo-induced deformed seedlings were induced for the second time. Figure 2 This is the process of establishing the hypocotyl-induced somatic embryo regeneration system of the present invention. The specific steps are:

[0039] The secondary embryo ( Figure 2 A) After being chopped and inoculated in KBN medium for one month, there will be embryonic callus, non-embryonic callus and secondary proembryonic masses (such as Figure 2The occurrence of B), embryonic callus and secondary proembryonic masses were inoculated into X6 medium for expansion and culture for 1 month. The mass of some secondary proembryonic masses increased by 3 times (i.e., Figure 2 The state of C changes to Figure 2 The mass of E) continues to grow. The ungerminated secondary proembryo masses can be inoculated into X6 medium for culture, and the secondary proembryo masses can be subcultured and propagated (such as Figure 2 F).

[0040] Another part of the secondary proembryo mass produces dense white somatic embryos (such as Figure 2 D), under the microscope, embryos at different developmental stages can be seen: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos ( Figure 2 G); somatic embryos before the cotyledon stage (globby embryos, heart-shaped embryos and torpedo embryos) can be selected for secondary induction: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, which are then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for somatic embryo maintenance and propagation.

[0041] Select any somatic embryos and inoculate them in X3 medium (MS + 30 g / L sucrose + 1.5 g / L activated carbon) for 1 month. When the cotyledon embryo, hypocotyl and radicle are differentiating, transfer them to WPM medium and culture them into seedlings ( Figure 2 M- Figure 2 of N).

[0042] The culture medium and its formula used in the present invention are as follows:

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

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

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

[0046] The formula of WPM culture medium is: WPM+0.2mg / L 6-BA+20g / L sucrose+7g / L agar+1.5g / 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 hypocotyls of grape seedlings.

[0049] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Molisa × Green State Gem as materials.

[0050] The specific steps are as follows: Under sterile conditions, the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Molisa × Green State Gem grape were induced for the second time, and the secondary embryos were cut into pieces and inoculated into KBN medium for induction culture for 1 month to produce embryonic callus ( Figure 3 B), non-embryonic callus ( Figure 3 A) and secondary proembryonic mass ( Figure 3 D), after the secondary proembryo masses were inoculated in X6 medium and cultured for 1 month, the mass of some secondary proembryo masses increased to 3 times of the original mass, and the mass continued to increase. The secondary proembryo masses that did not germinate could continue to be inoculated in X6 medium for culture, and the secondary proembryo masses could be subcultured and expanded ( Figure 4 C).

[0051] Embryogenic callus differentiates into somatic embryos ( Figure 4 A), another part of the secondary proembryo mass produces dense white somatic embryos ( Figure 4 B), somatic embryos can be observed under a microscope to see embryos at different developmental stages: spherical embryos ( Figure 5 A), heart-shaped embryo ( Figure 5 B), torpedo embryo ( Figure 5 C) and cotyledon embryo ( Figure 5 D); selecting somatic embryos before the cotyledon stage for secondary induction: inoculating the somatic embryos into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, then inoculating them into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, the secondary proembryonic masses with increased mass are used for subculture maintenance and expansion of the secondary proembryonic masses, selecting somatic embryos before the cotyledon stage from the somatic embryos for induction culture again, repeating the above steps, thereby establishing a cyclic system for somatic embryo maintenance and expansion.

[0052] Partially developed somatic embryos were selected and inoculated into X3 medium for 1 month. When the cotyledon embryonic axis and radicle were differentiated ( Figure 5 E) were transferred to WPM medium and cultured into seedlings under light ( Figure 5 F- Figure 5 G).

[0053] Example 2: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0054] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa as materials.

[0055] The specific steps are as follows: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa grapes; the secondary embryos are chopped and inoculated in KBN medium for induction culture for 1 month to produce embryonic callus, non-embryonic callus and secondary proembryonic masses; the secondary proembryonic masses are inoculated in X6 medium and cultured for 1 month, and the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture for subculture preservation and propagation of the secondary proembryonic masses.

[0056] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, embryos of different developmental stages can be seen in the somatic embryos: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction, and the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, which are then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, and the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, and somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and propagation.

[0057] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0058] Example 3: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0059] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Molisa × Green State Gem as materials.

[0060] The specific steps are as follows: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Molisa × Oasis Gem Grape, the secondary embryos are chopped and inoculated in KBN medium for induction culture for 2 months, embryonic callus, non-embryonic callus and secondary proembryonic masses are produced, the secondary proembryonic masses are inoculated in X6 medium and cultured for 1 month, the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to increase in mass and have not germinated can continue to be inoculated in X6 medium for culture, and the secondary proembryonic masses can be subcultured, preserved and propagated.

[0061] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, somatic embryos can be seen in different developmental stages: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, and then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass. The secondary proembryonic masses with increased mass are used for subculture maintenance and expansion of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and expansion.

[0062] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0063] Example 4: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0064] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Molisa × Green State Gem as materials.

[0065] The specific steps are: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the deformed seedlings rescued from the embryos of Molisa × Oasis Gem Grape, the secondary embryos are chopped and inoculated in KBN medium for induction culture for 3 months, embryonic callus, non-embryonic callus and secondary proembryonic masses are produced, the secondary proembryonic masses are inoculated in X6 medium and cultured for 1 month, the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to increase in mass and have not germinated can continue to be inoculated in X6 medium for culture, and the secondary proembryonic masses can be subcultured, preserved and propagated.

[0066] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, somatic embryos can be seen in different developmental stages: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, and then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass. The secondary proembryonic masses with increased mass are used for subculture maintenance and expansion of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and expansion.

[0067] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0068] Example 5: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0069] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Molisa × Green State Gem as materials.

[0070] The specific steps are: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Molisa × Oasis Gem Grape, the secondary embryos are chopped and inoculated in KBN medium for induction culture for 1 month, embryonic callus, non-embryonic callus and secondary proembryonic masses are produced, the secondary proembryonic masses are inoculated in X6 medium and cultured for 2 months, the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture, and the secondary proembryonic masses can be subcultured, preserved and propagated.

[0071] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, somatic embryos can be seen in different developmental stages: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, and then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass. The secondary proembryonic masses with increased mass are used for subculture maintenance and expansion of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and expansion.

[0072] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0073] Example 6: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0074] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Molisa × Green State Gem as materials.

[0075] The specific steps are: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Molisa × Oasis Gem Grape, the secondary embryos are cut into pieces and inoculated in KBN medium for induction culture for 1 month to produce embryonic callus, non-embryonic callus and secondary proembryonic masses, the secondary proembryonic masses are inoculated in X6 medium and cultured for 3 months, the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture, and the secondary proembryonic masses can be subcultured, preserved and propagated.

[0076] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, somatic embryos can be seen in different developmental stages: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, and then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass. The secondary proembryonic masses with increased mass are used for subculture maintenance and expansion of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and expansion.

[0077] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0078] Example 7: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0079] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa as materials.

[0080] The specific steps are as follows: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa grapes, the secondary embryos are chopped and inoculated in KBN medium for induction culture for 2 months, embryonic callus, non-embryonic callus and secondary proembryonic masses are produced, the secondary proembryonic masses are inoculated in X6 medium and cultured for 1 month, the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture, and the secondary proembryonic masses can be subcultured, preserved and propagated.

[0081] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, embryos of different developmental stages can be seen in the somatic embryos: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction, and the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, which are then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, and the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, and somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and propagation.

[0082] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0083] Example 8: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0084] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa as materials.

[0085] The specific steps are as follows: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa grapes, the secondary embryos are chopped and inoculated in KBN medium for induction culture for 3 months, embryonic callus, non-embryonic callus and secondary proembryonic masses are produced, the secondary proembryonic masses are inoculated in X6 medium and cultured for 1 month, the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture, and the secondary proembryonic masses can be subcultured, preserved and propagated.

[0086] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, embryos of different developmental stages can be seen in the somatic embryos: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction, and the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, which are then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, and the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, and somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and propagation.

[0087] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0088] Example 9: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0089] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa as materials.

[0090] The specific steps are as follows: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa grapes; the secondary embryos are chopped and inoculated in KBN medium for induction culture for 1 month to produce embryonic callus, non-embryonic callus and secondary proembryonic masses; the secondary proembryonic masses are inoculated in X6 medium and cultured for 2 months, and the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture for subculture preservation and propagation of the secondary proembryonic masses.

[0091] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, embryos of different developmental stages can be seen in the somatic embryos: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction, and the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, which are then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, and the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, and somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and propagation.

[0092] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0093] Example 10: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0094] In this example, the somatic embryo regeneration system was established using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa as materials.

[0095] The specific steps are as follows: under sterile conditions, secondary induction is performed on the secondary embryos obtained from the hypocotyls of the embryo-rescued deformed seedlings of Purple Sweet Seedless × Molisa grapes; the secondary embryos are chopped and inoculated in KBN medium for induction culture for 1 month to produce embryonic callus, non-embryonic callus and secondary proembryonic masses; the secondary proembryonic masses are inoculated in X6 medium and cultured for 3 months, and the mass of a part of the secondary proembryonic masses increases to 3 times the original mass, and the secondary proembryonic masses that continue to grow in mass and have not germinated can continue to be inoculated in X6 medium for culture for subculture preservation and propagation of the secondary proembryonic masses.

[0096] Embryonic callus differentiates into somatic embryos, and dense white somatic embryos are produced around another part of the secondary proembryonic masses. Under a microscope, embryos of different developmental stages can be seen in the somatic embryos: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos; somatic embryos before the cotyledon stage are selected for secondary induction, and the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, which are then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, and the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, and somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a circulation system for somatic embryo maintenance and propagation.

[0097] Partially developed somatic embryos were selected and inoculated into X3 medium for culture for 1 month. When the cotyledon, embryo, hypocotyl and radicle were differentiating, they were transferred to WPM medium and cultured into seedlings under light.

[0098] The state presented in the subculture preservation and propagation of the secondary proembryo mass, the circulation system for somatic embryo maintenance and propagation, and the seedling formation process in the establishment of the induced grape somatic embryo regeneration system in Example 2-Example 10 is similar to that in Example 1, which can be referred to Figure 1-Figure 5 .

[0099] According to the above examples, the secondary embryos obtained from the hypocotyls of the seedless grape embryo-reinforced deformed seedlings of the present invention are chopped and inoculated in KBN medium to induce embryonic callus, and the following four states are observed: ① non-embryonic callus with a relatively soft texture and a water-soaked appearance; ② golden embryonic callus with a hard texture. After continuous subculture, the embryonic callus begins to swell and proliferate; ③ a chimera of embryonic callus and non-embryonic callus ( Figure 3C); ④ Secondary proembryo mass. After embryonic callus and secondary proembryo mass are selected and inoculated in X6 medium for culture, embryonic callus gradually differentiates into embryos from the periphery, while secondary proembryo mass has two states. Most of the secondary proembryo mass has not germinated, and the mass is 2-3 times that before inoculation. The mass of the proembryo mass will double with each subculture, and it can be preserved and expanded. Another small part of the proembryo mass will begin to germinate into embryos of different states as the subculture time increases, which can be used to repeat the steps of secondary induction of embryonic callus, establish a circulation system, and the somatic embryos before the cotyledon stage produce the most secondary proembryo mass, with an induction rate of 85%-95%. The present invention has maintained continuous induction of secondary proembryo mass for 6 months.

[0100] The developed somatic embryos are inoculated in X3 medium. One month later, embryos in different states can be clearly observed: spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos. With further development, when the hypocotyl and radicle of the cotyledon embryos differentiate, they can be transferred to WPM medium to develop into seedlings.

[0101] Comparative Example 1: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0102] In this comparative example, the somatic embryo regeneration system was studied using secondary embryos obtained from the hypocotyls of embryo-rescued deformed seedlings of Zheng Yan Seedless × Italy as materials.

[0103] The specific steps are as follows: Under sterile conditions, secondary embryos obtained from the hypocotyls of Zhengyan seedless × Italian stone grape hybrid seedlings (embryo rescue deformed seedlings) were induced for the second time, and the secondary embryos were cut into pieces and inoculated in KBN medium (MS+1.1mg / L 6-BA+0.5mg / L 2,4-D+0.55mg / L After one month of induction culture with NOA+30g / L sucrose+3g / L phytogel+1g / L inositol+0.3g / L potassium nitrate), embryonic callus and non-embryonic callus will occur. During the three-month culture period, a small amount of secondary proembryonic masses will occur. The obtained small amount of secondary proembryonic masses will be inoculated in X6 medium for propagation. It was found that the secondary proembryonic masses could not be stably preserved and began to develop after one month of culture. When the somatic embryos developing in the early stage of cotyledonary embryo were induced for the second time, a small amount of secondary proembryonic masses were obtained after 2 months of culture. Similarly, they would continue to develop during 2 months of inoculation in X6 medium and could not maintain the state of secondary proembryonic masses. Finally, they were inoculated in X3 medium for culture and could develop into seedlings later.

[0104] Comparative Example 2: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

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

[0106] Comparative Example 3: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

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

[0108] Comparative Example 4: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0109] The steps are basically the same as those in Comparative Example 1, except that a small amount of secondary proembryo masses obtained are inoculated into X6 medium for expansion and culture for 2 months.

[0110] Comparative Example 5: A method for establishing a grape somatic embryo regeneration system by inducing grape seedling hypocotyls.

[0111] The steps are basically the same as those in Comparative Example 1, except that a small amount of secondary proembryo masses obtained are inoculated into X6 medium for expansion and culture for 3 months.

[0112] The effects of induction time on the induction rate of secondary proembryo masses and the effects of the culture time of secondary proembryo masses inoculated in X6 medium on the state of proembryo masses were studied using Examples 1 to 10 and Comparative Examples 1 to 5. The experimental results are recorded in Tables 1 and 2.

[0113] 1. Effect of induction culture time on the efficiency of initial induction: The secondary embryos differentiated from the hypocotyls of deformed seedlings rescued by Molisa × Green Oasis Gem, Purple Sweet Seedless × Molisa and Zheng Yan Seedless × Italy Seedless embryos were used as materials. They were chopped and inoculated into KBN medium, and the induction rates of embryonic callus, non-embryonic callus and secondary proembryonic masses were counted for three consecutive months.

[0114] Embryogenic callus induction rate = embryonic callus number / inoculation number; non-embryonic callus induction rate = non-embryonic callus number / inoculation number; secondary proembryonic mass induction rate = secondary proembryonic mass number / inoculation number.

[0115] 2. Description of the effect of culture time on the culture of secondary proembryo masses in X6 medium: The culture conditions of secondary proembryo masses of three materials inoculated in X6 medium were recorded for three consecutive months.

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

[0117]

[0118] As shown in Table 1, the somatic embryos obtained by embryo-rescued hypocotyl differentiation of Molisa × Green Oasis Gem, Purple Sweet Seedless × Molisa and Zheng Yan Seedless × Italy were chopped and inoculated into KBN medium for initial induction. After three consecutive months of cultivation and observation, it was concluded that a small amount of secondary proembryonic masses could be obtained from the chopped somatic embryos of Molisa × Green Oasis Gem and Purple Sweet Seedless × Molisa in the first month, with an induction rate of 30%. By the third month, the secondary proembryonic masses could reach 60%-70%. In the first two months of cultivation, the induction rate of secondary proembryonic masses of Zheng Yan Seedless × Italy was 0, and by the third month, only 10% of the secondary proembryonic masses were obtained. In addition, the induction rate of non-embryonic callus accounted for a high proportion during the entire cultivation period.

[0119] Table 2 Description of the effect of culture time on the culture of secondary primary groups in X6 medium

[0120]

[0121] As shown in Table 2, the present invention has established a somatic embryo regeneration system for the hypocotyls of Molisa × Green State Gem and Purple Sweet Seedless × Molisa seedlings, including a cycle of secondary induction, maintenance and propagation of secondary proembryos. It can provide receptor materials for grape genetic transformation on a sustainable basis. However, the hypocotyls of Zhengyan Seedless × Italy seedlings can induce somatic embryos to regenerate seedlings, but their secondary proembryos cannot be stably maintained and propagated.

[0122] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0123] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings, characterized in that: The steps include: Secondary embryos obtained from the hypocotyls of seedless grape embryos were inoculated into KBN medium and cultured for 28-32 days to obtain embryonic callus, non-embryonic callus and secondary proembryonic masses. Then the secondary proembryo masses are inoculated in X6 medium and cultured for 28-32 days to obtain secondary proembryo masses and somatic embryos with increased mass; the secondary proembryo masses with increased mass are used for subculture maintenance and propagation of secondary proembryo masses; somatic embryos before the cotyledon stage are selected for secondary induction to establish a circulation system for somatic embryo maintenance and propagation; Somatic embryos at any stage were inoculated into X3 medium and cultured in the dark for 28-32 days. When the hypocotyls and radicles of the somatic embryos were differentiated, they were transferred to WPM medium and cultured into seedlings at 24-25°C, 16 h light / 8 h dark. The KBN culture medium formula is: MS+1.0mg / L-1.2mg / L 6-BA+0.4mg / L-0.6mg / L 2,4-D+0.53mg / L-0.57mg / L NOA+28g / L-32g / L sucrose+3g / L phytogels+0.9g / L-1.1g / L inositol+0.2g / L-0.4g / L potassium nitrate; The formula of X6 culture medium is: MS + 55g / L~65g / L sucrose + 1.0g / L~2.0g / L activated carbon; The formula of X3 culture medium is: MS + 28g / L~32g / L sucrose + 1.0g / L~2.0g / L activated carbon; The formula of WPM culture medium is: WPM + 0.1mg / L~0.3mg / L 6-BA + 18g / L~22g / L sucrose + 6g / L~8g / L agar + 1.0g / L~2.0g / L activated carbon.

2. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 1, characterized in that: The KBN culture medium formula is: MS+1.1mg / L 6-BA+0.5mg / L2,4-D+0.55mg / L NOA+30g / L sucrose+3g / L phytogels+1g / L inositol+0.3g / L potassium nitrate; The formula of X6 culture medium is: MS+60g / L sucrose+1.5g / L activated carbon; The formula of X3 culture medium is: MS+30g / L sucrose+1.5g / L activated carbon; The formula of WPM culture medium is: WPM+0.2mg / L 6-BA+20g / L sucrose+7g / L agar+1.5g / L activated carbon.

3. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 1, characterized in that: The secondary embryos were inoculated into KBN medium and cultured in the dark at 24℃~25℃.

4. The method for inducing somatic embryo regeneration system from hypocotyls of grape seedlings according to claim 1, characterized in that: The secondary proembryo mass was inoculated into X6 medium and cultured at 24°C to 25°C in the dark.

5. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 1, characterized in that: Somatic embryos include globular embryos, heart-shaped embryos, torpedo embryos and cotyledonary embryos.

6. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 5, characterized in that: The process of selecting somatic embryos before the cotyledon stage for secondary induction is as follows: the somatic embryos are inoculated into KBN embryonic callus induction medium for induction culture to obtain embryonic callus and secondary proembryonic masses, and then inoculated into X6 medium for culture to obtain secondary proembryonic masses and somatic embryos with increased mass, the secondary proembryonic masses with increased mass are used for subculture maintenance and propagation of the secondary proembryonic masses, somatic embryos before the cotyledon stage are selected from the somatic embryos for induction culture again, and the above steps are repeated to establish a cyclic system for somatic embryo maintenance and propagation.

7. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 6, characterized in that: The induction culture condition is dark culture at 24° C. to 25° C., and subculture is performed once a month.

8. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 6, characterized in that: The culture conditions during propagation are dark culture at 24℃~25℃, with subculture once a month.

9. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 8, characterized in that: The culture temperature during propagation was 25°C.

10. The method for establishing a somatic embryo regeneration system by inducing somatic embryos from hypocotyls of grape seedlings according to claim 1, characterized in that: The seedless grape embryo deformity seedlings are obtained by hybridizing purple sweet seedless grapes and Molisa seedless grapes or by hybridizing Molisa seedless grapes and Green State Gem grapes.

Images