A method for improving transformation efficiency of grape embryo rescue breeding abnormal seedlings

Abstract

The application discloses a method for improving the transformation efficiency of grape embryo rescue breeding abnormal seedlings, and comprises the following steps: inoculating the embryo rescue abnormal seedlings in a transformation culture medium to culture, transferring the small seedlings with grown true leaves to a seedling culture medium to culture, and obtaining normal seedlings; for the abnormal seedlings which are not transformed into the normal seedlings, performing the rescue through an adventitious bud regeneration approach again; cutting the abnormal seedlings to obtain hypocotyls, inoculating the hypocotyls in an induction culture medium to perform the adventitious bud regeneration culture, and transferring the explants differentiated with the adventitious buds to the seedling culture medium to obtain the normal seedlings. The application provides a technical scheme for effectively transforming the abnormal seedlings generated in the process of the seed rescue breeding of the seedless grape into the normal seedlings, the abnormal seedlings generated in the process of the seed rescue of the seedless grape are transformed and reused by using two rescue methods, and then the seed rescue breeding efficiency of the seedless grape is improved, the seed rescue efficiency of the seedless grape can be improved from about 4-10% to about 25%.

Description

Technical Field

[0001] The present invention relates to a grape embryo rescue method, in particular to a method for improving the conversion efficiency of grape embryo rescue breeding deformed seedlings. Background Art

[0002] As a key component of the fresh and dried grape markets, seedless grapes play a crucial role in promoting the development of the grape industry. Seedless grapes can be divided into two categories based on their pollination and fruiting characteristics: parthenocarpic and pseudoparthenocarpic. Parthenocarpic grapes develop directly into fruit without pollination and fertilization during their growth and development. Pseudoparthenocarpic grapes undergo normal pollination and fertilization, but the fertilized embryo aborts midway, and the ovules fail to form normal seeds. Parthenocarpic grapes do not form zygotic embryos, and their genetic information cannot be passed on to offspring. Therefore, they are rarely used in seedless grape breeding. However, pseudoparthenocarpic grapes, whose seedless trait can be passed on to offspring, are the primary parent material for seedless grape breeding.

[0003] Because hybrid embryos using seedless grapes as the female parent often abort mid-development, the development of embryo rescue breeding technology has made it possible to use seedless grapes as the female parent. This has enriched hybrid combinations, increased the proportion of seedless offspring, shortened the breeding cycle, and is a primary method for creating new seedless grape germplasm. However, during embryo rescue breeding using seedless grapes as the female parent, approximately 40% of hybrids with malformed embryos germinate, severely impacting the effectiveness of embryo rescue in seedless grape breeding.

[0004] The occurrence of deformed seedlings is inevitable during seedless grape embryo rescue breeding, and the proportion of deformed seedlings is a major factor affecting seedless grape breeding efficiency. Transforming deformed seedlings from in vitro embryo germination into normal seedlings is a key approach to improving the efficiency of seedless grape embryo rescue breeding. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the conversion efficiency of grape embryo rescue breeding deformed seedlings, which can rescue grape deformed seedlings and improve the efficiency of seedless grape embryo rescue breeding.

[0006] To achieve the above object, the present invention provides a method for improving the conversion efficiency of grape embryo rescue breeding deformed seedlings, the method comprising: inoculating the embryo rescued deformed seedlings in a conversion medium for cultivation, transferring the seedlings that have grown true leaves to a seedling formation medium for cultivation to obtain normal seedlings; rescuing the deformed seedlings that have not been converted into normal seedlings by an adventitious bud regeneration pathway: cutting the deformed seedlings to obtain embryonic axes, inoculating them into an induction medium for adventitious bud regeneration culture, and transferring the explants that have differentiated adventitious buds to a seedling formation medium to obtain normal seedlings; wherein the conversion medium comprises the following components: WPM, 2 mg / L 6-BA, 2.0 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and 1.5 g / L activated carbon; the seedling formation medium comprises the following components: 1 / 2 MS, 0.3 mg / L IBA, 30 g / L sucrose, 3 g / L phytogel, and 1.5 g / L activated carbon; and the induction medium comprises the following components: MS, 0.2 mg / L 6-BA, 2.0 mg / L IBA, 20 g / L sucrose and 7 g / L agar.

[0007] Preferably, the seedlings that have grown true leaves are transferred to a seedling culture medium for cultivation, and normal seedlings are obtained after 30 days.

[0008] Preferably, the deformed seedlings include: any one or more of the following: monocotyledon deformity, leafless and rootless deformity, twisted and wrinkled cotyledon deformity, fused cotyledon deformity, rootless cotyledon deformity, cotyledonless root deformity, developmental arrest deformity, multiple cotyledon deformity and tubular cotyledon deformity.

[0009] Preferably, the deformed seedlings are cut to obtain the embryonic axes, which are inoculated into an induction medium for culture. After 30 days, the explants that have differentiated into adventitious buds are transferred to a seedling culture medium, and normal seedlings are obtained after 30 days.

[0010] Preferably, the adventitious bud regeneration culture is cultured under light conditions of 16 h and dark conditions of 8 h.

[0011] The method of the present invention for improving the conversion efficiency of grape embryos for rescuing and breeding deformed seedlings has the following advantages:

[0012] The present invention proposes a technical solution for effectively converting deformed seedlings generated during seedless grape embryo rescue breeding into normal seedlings. Two rescue methods are used to convert and reuse the deformed seedlings generated by seedless grape embryo rescue, thereby improving the efficiency of seedless grape embryo rescue breeding. The embryo rescue efficiency can be increased from the original 4-10% to about 25%, thereby improving the seedless grape embryo rescue efficiency.

[0013] Compared with the existing single rescue method, the present invention combines two methods to transform and reuse seedless grape embryos to rescue deformed seedlings. The transformation culture medium reported in the existing technology can only rescue a few types of deformed seedlings. After the transformation culture medium of the rescue method one of the present invention is optimized, it can be applied to the rescue of most types of deformed seedlings with high rescue efficiency. The rescue method two of the present invention is to induce adventitious bud regeneration of deformed seedlings that are not directly transformed into normal seedlings in the method one to obtain normal plants, which adds a new way to rescue deformed seedlings and can further improve the utilization rate of deformed seedlings.

[0014] The rescue method 1 in the method of the present invention can be applied to 8 types of seedless grape embryos to rescue deformed seedlings (monocotyledon deformity, twisted and wrinkled cotyledon deformity, fused cotyledon deformity, root-without-cotyledon deformity, cotyledon-without-root deformity, developmental arrest deformity, multiple cotyledon deformity, and tubular cotyledon deformity) for direct transformation and reuse, and can rescue 35.69% of the deformed seedlings; the rescue method 2 is to obtain normal plants by inducing adventitious bud regeneration of deformed seedlings that are not directly transformed into normal seedlings in the method 1, and can rescue 33.33% of the deformed seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention shows the seedless grape embryo rescue breeding process and the process of transforming and reusing deformed seedlings; A to I: the seedless grape embryo rescue normal seedling breeding process; J: the seedless grape embryo rescue some deformed seedling types; ① and ② are two methods for transforming deformed seedlings rescued by embryos into normal seedlings.

[0016] Figure 2 This is the effect of different transformation culture media on the transformation of deformed seedlings into normal seedlings.

[0017] Figure 3 Thirteen seedling types emerged during the embryo rescue breeding process of the seedless grape of the present invention: a: normal seedling; b: monocotyledon deformity; c: wrinkled and twisted cotyledons; d: multicotyledon deformity; e: tubular cotyledons; f: leggy seedling; j: albino seedling; h: cotyledons but no roots; i: roots but no cotyledons; j: fused cotyledons; k: developmental arrest; l: no germination; m: no leaves and no roots.

[0018] Figure 4 To invent a rescue method ① The process of converting embryo-rescued deformed seedlings into normal seedlings; A: deformed seedlings are inoculated into transformation medium and cultured; a~d: deformed seedlings grow true leaves; B: deformed seedlings grow into normal seedlings.

[0019] Figure 5Figure 2 shows the adventitious bud induction effects of deformed seedlings in rescue method ② of the present invention in four culture media; A: callus tissue was differentiated in all cases in T1 culture medium; B: adventitious bud differentiation was accompanied by the occurrence of a few aerial roots in T2 culture medium; C: callus tissue was differentiated in all cases in T4 culture medium; D: adventitious bud differentiation was accompanied by the occurrence of a few aerial roots in T3 culture medium; a-d: adventitious bud differentiation was induced by hypocotyl. DETAILED DESCRIPTION

[0020] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0021] Experimental Example 1 Grape Embryo Rescue Breeding Deformity Rescue Method

[0022] Embryo rescue breeding was conducted by configuring 18 different hybrid combinations ( Figure 1 A~I), 3567 deformed seedlings ( Figure 1 J, Table 1) for rescue treatment.

[0023] Table 1 Statistics of deformed seedlings produced by 18 embryo rescue hybrid combinations

[0024]

[0025] Rescue method ① is as follows:

[0026] (1) Three transformation media for deformed seedlings were set up (Medium A: MS + 2 mg / L 6-BA + 0.5 mg / L IBA + 30 g / L sucrose + 7 g / L agar + 1.5 g / L activated carbon, Medium B: 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, Medium C: WPM + 2 mg / L 6-BA + 2.0 mg / L IBA + 30 g / L sucrose + 7 g / L agar + 1.5 g / L activated carbon), and the medium with the highest transformation rate was screened out;

[0027] (2) The deformed seedlings of 18 embryo rescue hybrid combinations were classified, and the 9 types of deformed seedlings with the largest number (monocotyledon deformity, leafless and rootless deformity, twisted and wrinkled cotyledon deformity, fused cotyledon deformity, rootless cotyledon deformity, cotyledonless root deformity, developmental arrest deformity, multiple cotyledon deformity, and tubular cotyledon deformity) were selected. A total of 3365 plants were inoculated into the optimal culture medium selected in step (1) for culture. After 30 days, the number of normal seedlings transformed from the 9 types of deformed seedlings of different hybrid combinations was counted.

[0028] Rescue method ② is as follows:

[0029] For the deformed seedlings that were not directly transformed into normal seedlings, the deformed seedlings were cut into cotyledons, hypocotyls, and radicles, and inoculated into four induction media (T1: MS + 2 mg / L 6-BA + 1.0 mg / L 2,4-D + 60 g / L sucrose + 3 g / L phytagel; T2: MS + 2 mg / L 6-BA + 1.0 mg / L IAA + 60 g / L sucrose + 3 g / L phytagel; T3: MS + 0.2 mg / L 6-BA + 2.0 mg / L IBA + 20 g / L sucrose + 7 g / L agar; T4: MS + 2 mg / L ZT + 0.2 mg / L 2,4-D+20g / L sucrose+7g / L agar) for adventitious bud regeneration culture, T1 and T2 culture media were cultured in the dark, and T3 and T4 culture media were cultured in 16h light / 8h dark (if dark culture was carried out first, the cycle of adventitious bud regeneration would be longer, and fewer aerial roots would occur around the adventitious buds induced after dark treatment). 60 deformed seedlings were inoculated in each treatment, and after 30 days of adventitious bud differentiation, they were transferred to the seedling culture medium 1 / 2MS+0.3mg / L IBA+30g / L sucrose+3g / L plant gel+1.5g / L activated carbon for culture, and the number of seedlings was counted after 30 days.

[0030] The results are as follows Figure 2 As shown, the transformation rate of Medium C was significantly higher than that of Medium A and Medium B. The deformed seedlings of 18 embryo rescue hybrid combinations were classified into 12 types of deformed seedlings ( Figure 3 ), the 9 types of deformed seedlings with the largest number (monocotyledon deformity, leafless and rootless deformity, twisted and wrinkled cotyledon deformity, fused cotyledon deformity, rootless cotyledon deformity, cotyledonless root deformity, developmental arrest deformity, multiple cotyledon deformity, and tubular cotyledon deformity) were selected, and a total of 3365 plants were inoculated into Medium C for culture. After 30 days, the number of normal seedlings transformed from the 9 types of deformed seedlings in different hybrid combinations was counted ( Figure 4 ). As shown in Table 2 and Table 3, a total of 3365 deformed seedlings were obtained from the 18 embryo rescue hybrid combinations, and 1201 normal seedlings were obtained, with a conversion rate of 35.69%. Analysis of the conversion efficiency of the 9 types of deformed seedlings in the 18 embryo rescue hybrid combinations showed that no normal seedlings were obtained from the leafless and rootless deformed seedlings. Figure 3 It can be found that the young embryos turned brown and died after germinating for a period of time. The average conversion rates of the other eight types of deformed seedlings (monocotyledon deformity, twisted and wrinkled cotyledon deformity, fused cotyledon deformity, root-without-cotyledon deformity, cotyledon-without-root deformity, developmental arrest deformity, multiple cotyledon deformity, and tubular cotyledon deformity) were 38.74%, 41.67%, 32.47%, 14.29%, 9.18%, 51.28%, 59.13%, and 23.07%, respectively.

[0031] For the deformed seedlings that were not directly transformed into normal seedlings, the deformed seedlings were cut into cotyledons, hypocotyls, and radicles, and inoculated into four induction media for adventitious bud regeneration. It was found that only the hypocotyls could induce adventitious bud regeneration, while the cotyledons and radicles were basically callus or brown. After 30 days of differentiation of adventitious buds, they were transferred to seedling culture medium, as shown in Table 4 and Figure 4 As shown, among the four induction media, callus tissue differentiated in T1 and T4 media, and adventitious buds occurred in T2 and T3 media, accompanied by aerial roots. Among them, the adventitious bud regeneration efficiency in T3 medium was the highest, with a conversion rate of 33.33%, and 20 normal seedlings were obtained.

[0032] Table 2 shows the statistical results of the transformation of 9 kinds of deformed seedlings into normal seedlings.

[0033]

[0034] Table 3 shows the statistical results of the transformation of 9 kinds of deformed seedlings into normal seedlings.

[0035]

[0036] Table 4 shows the induction effect of deformed seedlings in four culture media

[0037]

[0038] Therefore, improving the transformation efficiency of grape embryo rescue breeding deformed seedlings can be achieved by the following method: embryo rescued deformed seedlings are directly inoculated in transformation medium WPM+2mg / L 6-BA+2.0mg / L IBA+30g / L sucrose+7g / L agar+1.5g / L activated carbon for culture, and the seedlings that have grown true leaves are successively transferred to seedling medium 1 / 2MS+0.3mg / L IBA+30g / L sucrose+3g / L phytogel+1.5g / L activated carbon for culture, and normal seedlings are obtained after 30 days; for deformed seedlings that are not directly transformed into normal seedlings, the deformed seedlings are cut into cotyledons, hypocotyls, and radicles, and inoculated into induction medium MS+0.2mg / L 6-BA+2.0mg / L IBA+20g / L sucrose+7g / L agar for culture, and after 30 days, the explants that have differentiated adventitious buds are transferred to seedling medium (same as above), and normal seedlings are obtained after 30 days.

[0039] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for improving the efficiency of grape embryo rescue and breeding of deformed seedlings, characterized in that: The method includes: The embryo-rescued deformed seedlings were inoculated into a transformation medium for cultivation, and the seedlings that grew true leaves were transferred to a seedling culture medium for cultivation to obtain normal seedlings; For the deformed seedlings that have not been transformed into normal seedlings, adventitious bud regeneration is used to rescue them: the embryonic axis of the deformed seedlings is cut to obtain the embryonic axis, which is inoculated into an induction medium for adventitious bud regeneration culture. The explants that have differentiated adventitious buds are transferred to a seedling culture medium to obtain normal seedlings. The transformation medium consists of the following components: WPM, 2 mg / L 6-BA, 2.0 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and 1.5 g / L activated carbon; The seedling culture medium is composed of the following components: 1 / 2 MS, 0.3 mg / L IBA, 30 g / L sucrose, 3 g / L plant gel and 1.5 g / L activated carbon; The induction medium consisted of the following components: MS, 0.2 mg / L 6-BA, 2.0 mg / L IBA, 20 g / L sucrose and 7 g / L agar.

2. The method according to claim 1, characterized in that The seedlings that have grown true leaves are transferred to seedling culture medium for cultivation, and normal seedlings are obtained after 30 days.

3. The method according to claim 1, characterized in that The deformed seedlings include: any one or more of monocotyledon deformity, leafless and rootless deformity, twisted and wrinkled cotyledon deformity, fused cotyledon deformity, rootless cotyledon deformity, cotyledonless root deformity, developmental arrest deformity, multiple cotyledon deformity and tubular cotyledon deformity.

4. The method according to claim 1, wherein The deformed seedlings were cut to obtain the embryonic axes, which were inoculated into the induction medium for culture. After 30 days, the explants that had differentiated into adventitious buds were transferred to the seedling medium, and normal seedlings were obtained after 30 days.

5. The method according to claim 1, wherein The adventitious bud regeneration culture is cultured under light conditions of 16 hours and darkness of 8 hours.

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