Method for rescuing Zijin early-growing grape embryos

Abstract

The invention relates to the field of grape cultivation, in particular to a Zijin early-growing grape embryo rescue method which comprises the following steps: 38-42 days after flowering, soaking clusters with brassinolide; after 48-52 days after flowering, young fruits are picked, seed embryos are taken and inoculated to an embryo development culture medium, after the embryos are cultured in the embryo development culture medium for 58-62 days, seed coats are cut open, the developed seed embryos are transferred to a germination culture medium, and after the seed embryos germinate into seedlings, the seedlings are transferred to a rooting culture medium. According to the method, the grape Zijin early embryo rescue technology is researched and innovated, the abortion of the Zijin early embryos is effectively reduced by using the post-flowering growth regulator for treatment, the development number of the embryos in fruits is increased, the grape embryo rescue efficiency is improved, an efficient breeding technology can be provided for seedless grapes, and the seedless grape breeding efficiency is improved.

Description

Technical Field

[0001] The invention relates to the field of grape breeding, and in particular to a method for rescuing Zijin early grape embryos. Background Art

[0002] The seedless characteristics of table grapes are increasingly favored by consumers. The planting area of ​​seedless grapes is increasing year by year, and seedless fruit has become an important direction and development trend for table grape breeding and consumption in the world. One of the breeding methods for seedless grapes is to use seeded grapes as the female parent and seedless grapes as the male parent for conventional hybridization. However, the probability of seedless offspring obtained by this method is relatively low, with an average of only 13.5%. Another method is embryo rescue technology, which uses seedless grapes as the female parent and obtains hybrid offspring through in vitro embryo culture, effectively increasing the seedless rate of hybrid offspring to more than 50%, and the seedless grape breeding cycle can be shortened by at least 5 years.

[0003] People have explored and optimized the embryo rescue system by studying the factors that affect embryo rescue, mainly including genotype, ovule inoculation period, integument treatment method, culture medium composition of young embryos after stripping, culture environment, and transplanting of tissue culture seedlings, so as to continuously enrich and improve the embryo rescue breeding technology system and improve the embryo development rate and seedling rate. In practical applications, the embryo development rate and seedling rate are affected by many factors. The optimal sampling period, culture medium type and added ingredients, and culture conditions of different parental genotypes are different. It is a necessary way to speed up the efficiency of seedless grape breeding by screening seedless grape parents with high seedling rate and studying and improving their embryo rescue technology system. However, the current grape embryo rescue method has the defects of complex operation and high cost. It is necessary to provide a scientific and reasonable grape embryo rescue method.

[0004] The 'Zi Jin Zao Sheng' grape variety has the advantages of being seedless, early maturing, high quality, and strong resistance. It is an excellent seedless grape breeding parent. By researching and innovating the embryo rescue technology of the 'Zi Jin Zao Sheng' grape, we can provide efficient breeding technology for seedless grapes and improve the breeding efficiency of seedless grapes. Summary of the invention

[0005] The invention discloses a technical method for rescuing offspring embryos of Zijin early grape breeding into seedlings.

[0006] The method disclosed by the invention comprises post-flowering treatment of plant growth regulator, embryo development medium formula and seed embryo tissue culture process.

[0007] The technical solution of the present invention is:

[0008] A method for rescuing embryos of Zijin Zaosheng grapes comprises the following steps: soaking fruit clusters with brassinolide 38 to 42 days after flowering; picking young fruits 48 to 52 days after flowering, taking embryos and inoculating them on an embryo development medium, culturing in the embryo development medium for 58 to 62 days, cutting the seed coat, transferring the developed embryos to a germination medium, and transferring the embryos to a rooting medium after the embryos germinate into seedlings.

[0009] Furthermore, the concentration of brassinolide is 5-7 mg / LBR.

[0010] Furthermore, the concentration of brassinolide is 6 mg / LBR.

[0011] Furthermore, the embryo development medium includes gibberellins, auxins, brassinolide, 6-benzylaminopurine, hydrolyzed casein, putrescine, spermidine and sugar.

[0012] Furthermore, the embryo development medium includes Nitsch medium, gibberellins, auxins, brassinolide, 6-benzylaminopurine, hydrolyzed casein, putrescine, spermidine, activated carbon and sugar.

[0013] Furthermore, the embryo development medium includes Nitsch medium, gibberellin GA0.4 mg / L, auxin IAA2.0 mg / L, brassinolide BR0.5 mg / L, 6-benzylaminopurine 6-BA0.5 mg / L, hydrolyzed casein 100 mg / L, putrescine 2 mmol / L, spermidine 0.5 mmol / L, activated carbon 1 g / L and sugar 20 g / L.

[0014] Furthermore, the germination medium includes WPM medium, 6-benzylaminopurine, hydrolyzed casein, activated carbon and sugar.

[0015] Furthermore, the germination medium includes WPM medium, 6-benzylaminopurine 6-BA 0.3 mg / L, hydrolyzed casein 100 mg / L, activated carbon 1 g / L and sugar 30 g / L.

[0016] Furthermore, the rooting medium includes 1 / 2MS medium, activated carbon and sugar.

[0017] Furthermore, the rooting medium includes 1 / 2MS medium, 1 g / L activated carbon and 30 g / L sugar.

[0018] A 'Zijin Zaosheng' grape embryo rescue technology method comprises the following steps:

[0019] (1) 40 days after flowering, soak the fruit clusters with 6 mg / LBR.

[0020] (2) 50 days after flowering, young fruits were collected and embryos were inoculated on embryo development medium. The embryo development medium formula was: Nitsch medium + gibberellin GA 0.4 mg / L + auxin IAA 2.0 mg / L + brassinolide BR 0.5 mg / L + 6-benzylaminopurine 6-BA 0.5 mg / L + hydrolyzed casein 100 mg / L + putrescine 2 mmol / L + spermidine 0.5 mmol / L + activated carbon 1 g / L + sugar 20 g / L.

[0021] (3) After 60 days of culture in the embryo development medium, the seed coat was cut and the developed embryos were transferred to the germination medium. The germination medium formula was: WPM medium + 6-benzylaminopurine 6-BA 0.3 mg / L + hydrolyzed casein 100 mg / L + activated carbon 1 g / L + sugar 30 g / L.

[0022] (4) After the embryos germinate and grow into seedlings, transfer them to rooting medium: 1 / 2MS medium + 1g / L activated carbon + 30g / L sugar.

[0023] Beneficial Effects

[0024] The Zijin Early Grape variety has the advantages of being seedless, early maturing, high quality, and strong in resistance, and is an excellent seedless grape breeding parent. The present invention, through research and innovation of grape Zijin Early Embryo Rescue Technology, effectively reduces the abortion of Zijin Early Embryos by using post-flowering growth regulators, increases the number of embryos developed in the fruit, improves the grape embryo rescue efficiency, and increases the seedling rate to 3 to 4 times the original. This invention can provide efficient breeding technology for seedless grapes and improve the breeding efficiency of seedless grapes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of the embryo rescue technology of the present invention;

[0026] Figure 2 This is a comparison of the embryos in Example 2, where the left picture shows the browned embryos in the Eth treatment 20 days after flowering, and the right picture shows the normal embryos;

[0027] Figure 3 This is a comparison chart between BR treatment and control group. DETAILED DESCRIPTION

[0028] To make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the embodiments of the present invention will be described below.

[0029] Example 1

[0030] Effects of post-flowering treatment with different growth regulators on early embryo rescue in Zijin

[0031] Zijin Zaosheng was used as the test material, the experimental time: 2022, the experimental location: the orchard of Jiangsu Academy of Agricultural Sciences; 25 days after flowering and 40 days after flowering, the fruit clusters were soaked with 6-BA, CCC, ABA, BR, and Eth respectively. The specific treatments are shown in Table 1. 50 days after flowering, young fruits were picked, and embryos were taken and inoculated into embryo development medium (Nitsch medium + IAA 2.0 mg / L + GA 0.4 mg / L + BR 0.5 mg / L + 6-BA 0.5 mg / L + hydrolyzed casein 100 mg / L + putrescine 2 mmol / L + spermidine 0.5 mmol / L + activated carbon 1 g / L + sugar 20 g / L). The number of fruits and embryos was counted during inoculation, and the average number of fruit embryos was calculated as the number of embryos / the number of fruits. The fruits were cultured in the embryo development medium for 60 days, the seed coat was cut, the number of developed embryos was counted, and the embryo development rate was calculated as the number of developed embryos / the number of inoculated embryos × 100%. The developed embryos were transferred to germination medium WPM + 6-BA 0.3 mg / L + hydrolyzed casein 100 mg / L + activated carbon 1 g / L + sugar 30 g / L, the number of embryo seedlings was counted, and the embryo seedling rate was calculated as the number of seedling embryos / the number of inoculated embryos × 100%. Transfer to rooting medium: 1 / 2MS medium + activated carbon 1g / L + sugar 30g / L.

[0032] Table 1 Growth regulator post-flowering treatment plan

[0033]

[0034] The effects of post-flowering treatments of different growth regulators on the embryo rescue efficiency of Zijin Zaosheng are shown in Table 2. The results show that the average number of embryos in fruits treated with all growth regulators is higher than that of the control, indicating that the post-flowering growth regulator treatment effectively reduces the abortion of Zijin Zaosheng embryos and increases the number of embryos developed in fruits, among which treatments 6, 3, and 9 have better effects. From the perspective of embryo development rate, treatments 9, 7, and 4 have better effects, which are significantly higher than the control, and the highest treatment 9 (40d BR 6mg / L) is 18.13% higher than the control. From the perspective of seedling rate, Zijin Zaosheng treatment 5 (25dEth 100mg / L) and treatment 9 (40d BR 6mg / L) have good effects, and the seedling rates are 4.8 times and 3.9 times that of the control, respectively.

[0035] Table 2 Effects of post-flowering treatment with different growth regulators on the efficiency of embryo rescue in Zijin

[0036]

[0037] Example 2

[0038] Effect of post-flowering treatment time of growth regulators on embryo rescue

[0039] Zijin Zaosheng was used as the experimental object, the experimental time was 2023, and the experimental location was the orchard of Jiangsu Academy of Agricultural Sciences; the ears were treated with growth regulators Eth and BR at different times after flowering, and the specific treatment scheme is shown in Table 3. Embryo rescue culture was carried out 50 days after flowering, and the medium formula and culture process were the same as in Example 1. The experimental treatments are shown in Table 3.

[0040] Table 3 Growth regulator post-flowering treatment plan

[0041]

[0042] Table 4 Effect of post-flowering treatment time of growth regulators on embryo rescue

[0043]

[0044] As can be seen from Table 4, Eth treatment 25 days after flowering increased the embryo development rate and seedling rate during embryo rescue. However, the embryo rescue efficiency of the three treatment times was quite different. Too early or too late treatment would reduce the efficiency of grape embryo rescue, especially the fruit embryos treated with Eth 20 days after flowering had browning and death, which led to a decrease in the number of embryos, development rate and seedling rate in the fruit, which was much greater than the control. This shows that the Eth treatment time is relatively strict and difficult to control in actual operation, so it is not suitable as a treatment agent to improve the efficiency of embryo rescue.

[0045] BR treatment 30 days after flowering can significantly increase the number of embryos in the fruit, which is 1.48 times that of the control and 1.29 times that of treatment 5, but the embryo development rate and embryo seedling rate are much lower than those of treatment 5. The seedling rate of treatment 5 is 3.87 times that of the control and 2.31 times that of treatment 4. Comprehensive comparison shows that BR treatment 40 days after flowering of Zijin Zaosheng has the best effect.

[0046] Example 3

[0047] Effect of sugar content in embryo development medium on embryo development rate

[0048] Zijin Zaosheng was used as the test material. The experimental time was 2024 and the experimental location was the orchard of Jiangsu Academy of Agricultural Sciences. 40 days after flowering, the fruit clusters were soaked with 6mg / L BR. 50 days after flowering, the young fruits were picked, disinfected with 75% alcohol for 1 minute, 15% sodium hypochlorite for 20 minutes, rinsed with sterile water twice, and the ovules were taken out and inoculated into the embryo development medium Nitsch medium + IAA 2.0mg / L + GA 0.4 mg / L + BR 0.5mg / L + 6-BA 0.5mg / L + hydrolyzed casein 100mg / L + putrescine 2mmol / L + spermidine 0.5mmol / L + activated carbon 1g / L, medium 1 was added with sugar 20g / L, medium 2 was added with sugar 60g / L, after 60 days of culture, the seed coat was cut, the embryo development rate was counted, and it was transferred to the germination medium. When the embryos germinated into seedlings, they were transferred to the rooting medium: 1 / 2MS medium + activated carbon 1g / L + sugar 30g / L.

[0049] Table 5 Effect of sugar content in embryo development medium on embryo development rate.

[0050]

[0051] It can be seen from Table 5 that Zijin Early Growth Medium 1 has a good effect on embryo development rate.

[0052] Example 4.

[0053] Effects of hormone formula of embryo development medium on embryo rescue efficiency

[0054] Zijin Zaosheng was used as the test material, the experimental time: 2024, the experimental location: Jiangsu Academy of Agricultural Sciences orchard; the embryo development medium was Nitsch medium + hydrolyzed casein 100mg / L + putrescine 2mmol / L + spermidine 0.5mmol / L + activated carbon 1g / L + sugar 20g as the basic medium, and two different hormone formulas were added:

[0055] Medium 1: GA 0.4 mg / L + IAA 1.8 mg / L + 6-BA 0.5 mg / L;

[0056] Culture medium 2: GA0.4mg / L+IAA2.0mg / L+BR0.5mg / L+6-BA0.5mg / L.

[0057] The remaining conditions are the same as those in Example 1.

[0058] Table 6 Effect of hormone content in embryo development medium on embryo development rate.

[0059]

[0060] The results of embryo rescue are shown in Table 6. The embryo development rate and seedling rate of medium 2 were high.

[0061] Example 5

[0062] Effects of Polyamines in Embryo Development Medium on Embryo Development Rate

[0063] Zijin Zaosheng was used as the test material, the experimental time was 2024, and the experimental location was the orchard of Jiangsu Academy of Agricultural Sciences; the embryo development medium was Nitsch medium + hydrolyzed casein 100 mg / L + activated carbon 1 g / L + sugar 20 g + GA 0.4 mg / L + IAA 2.0 mg / L + BR 0.5 mg / L + 6-BA 0.5 mg / L, and different polyamine formulas were added, medium 1: putrescine 3 mmol / L, medium 2: spermidine 1 mmol / L, medium 3: putrescine 2 mmol / L + spermidine 0.5 mmol / L, and the control did not add polyamines. The other conditions were the same as in Example 1.

[0064] Table 7 Effect of sugar content in embryo development medium on embryo development rate

[0065]

[0066] The experimental results are shown in Table 7. The embryo development rate of the embryo development medium supplemented with putrescine 2mmol / L + spermidine 0.5mmol / L was significantly higher than that of the control, and the effect was the best.

Claims

1. A method for rescuing embryos of Zijin early grape, characterized in that: The following steps are involved: 38 to 42 days after flowering, soak the fruit clusters with brassinolide; 48 to 52 days after flowering, pick the young fruits, take the embryos and inoculate them on the embryo development medium. After 58 to 62 days of culture in the embryo development medium, cut the seed coat and transfer the developed embryos to the germination medium. After the embryos germinate into seedlings, transfer them to the rooting medium.

2. The method for rescuing Zijin Zaosheng grape embryos according to claim 1, characterized in that: The concentration of brassinolide is 5-7 mg / LBR.

3. The method for rescuing embryos of Zijin Zaosheng grape according to claim 1, characterized in that: The concentration of brassinolide is 6 mg / LBR.

4. The method for rescuing embryos of Zijin Zaosheng grape according to claim 1, characterized in that: The embryo development medium comprises gibberellins, auxins, brassinolide, 6-benzylaminopurine, hydrolyzed casein, putrescine, spermidine and sugar.

5. The method for rescuing embryos of Zijin Zaosheng grape according to claim 1, characterized in that: The embryo development medium comprises Nitsch medium, gibberellins, auxins, brassinolide, 6-benzylaminopurine, hydrolyzed casein, putrescine, spermidine, activated carbon and sugar.

6. The method for rescuing embryos of Zijin Zaosheng grape according to claim 1, characterized in that: The embryo development medium includes Nitsch medium, gibberellin GA0.4 mg / L, auxin IAA2.0 mg / L, brassinolide BR0.5 mg / L, 6-benzylaminopurine 6-BA0.5 mg / L, hydrolyzed casein 100 mg / L, putrescine 2 mmol / L, spermidine 0.5 mmol / L, activated carbon 1 g / L and sugar 20 g / L.

7. The method for rescuing embryos of Zijin Zaosheng grape according to claim 1, characterized in that: The germination medium comprises WPM medium, 6-benzylaminopurine, hydrolyzed casein, activated carbon and sugar.

8. The method for rescuing Zijin Zaosheng grape embryos according to claim 1, characterized in that: The germination medium comprises WPM medium, 0.3 mg / L of 6-benzylaminopurine 6-BA, 100 mg / L of hydrolyzed casein, 1 g / L of activated carbon and 30 g / L of sugar.

9. The method for rescuing Zijin Zaosheng grape embryos according to claim 1, characterized in that: The rooting medium comprises 1 / 2MS medium, activated carbon and sugar.

10. The method for rescuing embryos of Zijin Zaosheng grape according to claim 1, characterized in that: The rooting medium comprises 1 / 2MS medium, 1 g / L activated carbon and 30 g / L sugar.

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