Tissue culture rapid propagation method of olive and its application

Abstract

The present invention relates to the technical field of plant tissue culture, and is a method for rapid propagation of olive by tissue culture and its application; the steps include: taking mature, pest-free and undehiscent olive pods, cleaning and disinfecting them, then inoculating them into a primary culture medium and culturing them for 55 days, inoculating them into a proliferation culture and culturing them for 85 days, and finally inoculating them into a rooting culture medium and culturing them for 85 days. The present invention adds a certain concentration of GA3 during the primary culture to shorten the seed germination start-up time and proliferation time, and the germination rate reaches more than 99.5%; during the subculture proliferation culture, Huabao No. 2, peptone, potato and coconut milk are added based on CPPU, the subculture proliferation coefficient is as high as more than 10, and the vitrification is controlled within 1%; during the rooting culture, the concentrations of Huabao No. 2, peptone, potato and coconut milk are increased, and potassium dihydrogen phosphate is added at the same time, the rooting rate of the olive reaches 100%, the root system is thick, and the stem can also be promoted to swell.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of olive tissue culture and an application thereof. Background Art

[0002] Pholidota chinensis, also known as the Stone Fairy Peach, is a perennial herbaceous plant of the Orchidaceae family, growing year-round on rocky cliffs in the shade of deep mountain forests. Its rhizomes are creeping, with stems 3-8 mm or thicker. Its peduncle is 12-38 cm long, and its inflorescence often curves slightly outward. Its bracts persist when the flowers wither. Its flowers are white or light yellow, and its sepals are ovate-elliptical. Its capsule is obovate-oval. Pholidota chinensis has a sweet, slightly bitter flavor and a cooling nature. It enters the lung and kidney meridians. It nourishes yin, clears the lungs, promotes dampness, and eliminates blood stasis. It is used to treat dizziness, headaches, coughs, hematemesis, nocturnal emissions, dysentery, leucorrhea, and malnutrition. However, due to its continued reliance on wild resources, the availability of Pholidota chinensis is dwindling.

[0003] Artificial propagation has become a trend in the growth of olive trees. The main methods of propagation are rhizome branching and tissue culture. Branching involves severing the rhizome every two pseudobulbs. The leaves and old roots on the rhizome are then removed. Dormant lateral buds on the rhizome are then allowed to sprout, forming a complete plant. Rhizome branching is an economical and practical method of propagation; however, it requires a large amount of wild resources, has a low propagation coefficient, and cannot be readily adopted for large-scale cultivation.

[0004] Tissue culture seedlings utilize seeds or buds from rhizomes of the plant as explants, using MS as the basic culture medium. This allows for the rapid production of large quantities of seedlings in a short period of time, meeting production needs and effectively addressing seedling shortages. Publication number CN104255497A, entitled "Method for Rapid Propagation of the Plant Using Rhizomes ... This method has a contamination rate of up to 30-50%, a sterilization survival rate of only 40-70%, a survival rate of 84% after primary induction, 6.2 buds at the primary induction rate, a survival rate of 85.82% after proliferation culture, 2.72 roots after rooting culture, and a seedling rate of 91.65%. The publication number is CN109673509A, and the name is: A method for rapid propagation of wild olives, steps: a. Harvesting wild olive seeds; b. Preparing 0.5-1.5g / L of Huabao No. 1, 0.1-1g / L of naphthaleneacetic acid, 3-5g / L of agar, and 12-18g / L of white sugar for sterile sowing; c. After 2 months, the seeds germinate to form a white protocorm, which further differentiates into a green rhizome. There are multiple growth points on the rhizome, and seedlings can germinate from the growth points of the rhizome. d. The proliferation culture medium can be transferred in large quantities on the basic culture medium. The culture medium formula is: 1 / 2M basic culture medium, 1.5-2.5g / L peptone, 25-35g / L banana, 8-12g / L sugar, and 0.1-1g / L activated carbon. e. After 5-6 months, when a large number of seedlings with leaves appear in the tissue culture bottles, move the seedlings from an environment with a constant temperature of 25-28°C and a constant humidity of 40-50% to a greenhouse with an environment of 5000lux-8000lux, plant them in humus soil, and apply exogenous nutrients to promote their rooting. Wu Yuhan et al. (Wu Yuhan, Yu Hanxi, Li Jing, et al. Effects of different culture media on germination of Schefflera seeds [J]. Agricultural Engineering, 2019(3):5. DOI:CNKI:SUN:NYGE.0.2019-03-025.) selected Schefflera seeds with a maturity of 80%, disinfected them with 75% alcohol for 30 seconds, and then soaked them in 0.1% mercuric chloride for 8 minutes. After seed germination culture, the seed protocorm was induced to proliferate and pseudobulbs were induced, and finally acclimated and transplanted.The seed germination medium combination of this method is 6-BA 1.0 mg / L + NAA 0.1 mg / L + KT 0.5 mg / L, the germination rate can reach 99.39%, and the germination period is 25 days. The optimal medium for inducing the proliferation of protocorms is MS + 6-BA 1.0 mg / L + KT 0.5 mg / L + NAA 0.5 mg / L + peptone 1 g / L, with a proliferation rate of 93.67%; the optimal medium for pseudobulb induction is 1 / 2MS + peptone 1.5 g / L + banana puree 40.0 g / L + potato puree 40.0 g / L + Huabao No. 1 1.0 g / L, with an induction rate of 656.70%. Wen Xiuping et al. (Wen Xiuping, Lin Qingqing, Yang Chengzi, et al. Preliminary report on the aseptic sowing and tissue culture rapid propagation technology of Shixiantao from Fujian [J]. Heilongjiang Agricultural Sciences, 2015(8):4. DOI:10.11942 / j.issn1002-2767.2015.08.0019.) used seeds as tissue culture materials and carried out subculture and rooting culture after seed germination. The optimal disinfection solution for this method was 0.1% mercuric chloride, and the treatment time was 8 minutes; the seed germination medium was N6 + 1.0 mg·L. -1 6-BA + 1.0 mg·L -1 NAA, seed germination rate was 100%, time was 25 days; proliferation medium was N6 + 1.5 mg·L -1 6-BA + 0.5 mg·L -1 NAA, the growth rate was 93%; the rooting medium was N6 + 0.5 mg·L -1 6-BA + 1.5 mg·L -1 NAA+75g·L -1 , cultured for about 42 days, the rooting rate reached 85%. Chen Yinling (Chen Yinling, Chen Dongyi, Jiang Xuejing, et al. Research on tissue culture and rapid propagation system of fine-leaved stone peach [J]. Acta Physiologica Sinica, 2025, 61(05): 613-618. DOI: 10.13592 / j.cnki.ppj.101233.) et al. used seeds as materials, and carried out subculture and rooting culture after seed germination. It was also disclosed that the germination culture medium was MS + 2.0 mg·L –1 6-BA+0.1mg·L –1 After 50 days of culture with NAA + 6% banana puree, the seed germination rate was 85.37%. The proliferation medium was MS + 4.0 mg·L –1 6-BA+0.5 mg·L –1 NAA + 3% banana puree, the proliferation coefficient reached 4.0; the rooting medium was 1 / 2MS + 1.0 mg·L –1 IBA + 0.6 g L –1Activated carbon, rooting rate is 90%; culture medium is sphagnum moss, survival rate is 98.4%. Liu Baocai (Liu Baocai, Huang Yingzhen, Zhao Yunqing, et al. Preliminary report on the sterile sowing and rapid propagation technology of fine-leaved philodendron [J]. Journal of Fujian Agricultural Sciences, 2014, 29(5):4. DOI:10.3969 / j.issn.1008-0384.2014.05.011.) et al. used seeds as culture materials and disclosed that the seed germination medium was MS + 20g·L -1 Sucrose + 8g·L -1 Agar, seed germination rate is more than 70%; MS + 50g·L -1 Potato + 20 g·L -1 Sucrose + 8 g·L -1 Agar medium is conducive to the differentiation of protocorms into seedlings. The combination of low concentrations of 6-BA and NAA is suitable for the proliferation of protocorms and the differentiation of buds. -1 NAA + 20 g·L -1 Sucrose + 8g·L -1 The rooting rate on agar medium was 100%.

[0005] The prior art discloses a large number of technical methods for propagation using seeds, but in actual operation, there are problems such as relatively high disinfection contamination rate and mortality rate, low proliferation coefficient and high vitrification. Summary of the Invention

[0006] The invention aims to provide a tissue culture rapid propagation method for olive and its application.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid propagation of olive by tissue culture, comprising the following steps:

[0008] (1) Material selection and cleaning: Take mature, pest-free and uncracked olive pods, dip a cotton ball in soapy water with alcohol, wipe the surface of the pods 4-5 times, then add 1-2 drops of Tween-80, add a small amount of water and mix well, then rinse with running water for 40-60 minutes;

[0009] (2) Disinfection: After removing the pods, absorb the surface moisture with sterile paper, then transfer them to a clean bench, wipe the surface of the pods 5 to 7 times with an alcohol cotton ball dipped in 75% alcohol, rinse with sterile water 3 to 4 times, then use a saturated bleach solution to disinfect for 60 to 80 minutes, rinse with sterile water 5 to 6 times, and finally shake with 0.2% mercuric chloride solution for 5 to 6 minutes, rinse with sterile water 7 to 8 times;

[0010] (3) Primary culture: Cut small holes in the sterilized pods and sow the seeds evenly in the primary culture medium. Culture them at 25±2℃ in complete darkness for 30-40 days, then transfer them to 25±2℃ with a light intensity of 2000-3000Lx and a single daylight of 12h for 50-60 days.

[0011] The formula of the primary culture medium is: MS + NAA 0.1-0.2 mg / L + KT 3.0-4.0 mg / L + GA30.05-0.08 mg / L + potato 30 g / L;

[0012] (4) Proliferation culture: After the seeds germinate and produce a small number of buds, they are inoculated into a proliferation medium in clusters of 3 to 5 plants. They are cultured for 80 to 90 days at a temperature of 25 ± 2 °C, a light intensity of 2000 to 3000 Lx, and a single daylight of 12 h.

[0013] The proliferation culture medium formula is: 1 / 2MS + Huabao No. 2 1.5-2.0g / L + CPPU 0.5-0.8mg / L + peptone 0.5-1.0g / L + potato 30.0-40.0g / L + coconut milk 30.0-40.0mL / L;

[0014] (5) Rooting culture: Cut the proliferated buds into single buds, inoculate them into rooting medium, and culture them for 80 to 90 days at a temperature of 25 ± 2 °C, a light intensity of 2000 to 3000 Lx, and a photoperiod of 12 h per day;

[0015] The formula of the rooting medium is: 1 / 2MS + Huabao No. 2 2.0-2.5g / L + NAA 0.3-0.4 mg / L + potassium dihydrogen phosphate 0.10-0.15g / L + peptone 3.0-4.0g / L + potato 60.0-80.0g / L + coconut water 60.0-80.0mL / L.

[0016] Furthermore, in step (3), the formula of the primary culture medium is: MS + NAA 0.15 mg / L + KT3.5 mg / L + GA3 0.07 mg / L + potato 30.0 g / L.

[0017] Furthermore, in step (4), the proliferation culture medium formula is: 1 / 2MS + Huabao No. 2 1.7g / L + CPPU 0.6mg / L + Peptone 0.75g / L + Potato 35.0g / L + Coconut milk 35.0mL / L.

[0018] Furthermore, in step (5), the formula of the rooting medium is: 1 / 2MS + Huabao No. 2 2.3g / L + NAA 0.3mg / L + potassium dihydrogen phosphate 0.13g / L + peptone 3.5g / L + potato 70.0g / L + coconut milk 70.0mL / L.

[0019] The beneficial technical effects of the present invention are:

[0020] 1. In the process of olive tissue culture, it generally takes 20 to 30 days for seed germination to start, and when it reaches the time for subculture and proliferation, it is about 80 to 90 days. The time period is long, and the seed germination is extremely uneven, resulting in the culture time being too short during the later transfer, a large loss rate, and a long culture time, and some seedlings aging and possibly forming dead seedlings. The present invention adds a certain concentration of GA3 in the primary culture, and at the same time cooperates with NAA, KT and potatoes, which can shorten the seed germination start time to 15 to 20 days, shorten the subculture and proliferation time to 60 to 70 days, and the seed germination rate reaches more than 99.5%, and the seed germination is more uniform.

[0021] 2. After germination of olive seeds, the seedlings are relatively thin and weak. When high concentrations of cytokinins are used, the seedlings are very likely to undergo vitrification, resulting in weak or even dead seedlings. When low concentrations of cytokinins are purchased, the seedlings are relatively strong, but the proliferation coefficient is greatly reduced. During subculture proliferation culture, the present invention uses a conventional MS basic culture medium with an appropriate concentration of CPPU as the basis, and adds a certain concentration of Huabao No. 2, peptone and potatoes. At the same time, an innovative addition of coconut milk at an appropriate concentration can make the subculture proliferation coefficient as high as 10 or more, and the seedlings are strong with almost no vitrification, and the vitrification phenomenon is controlled within 1%.

[0022] 3. During rooting culture, the present invention increases the concentrations of Huabao No. 2, peptone, potatoes and coconut milk on the basis of the subculture proliferation culture medium, and creatively adds potassium dihydrogen phosphate at an appropriate concentration, which can make the rooting rate of the stone olive reach 100%, the root system is strong, and the stem can also be promoted to swell. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0024] Figure 1 This is Example 1 of the present invention. 15 days after seed sowing, it can be seen that 70% of the seeds have turned green and started to germinate.

[0025] Figure 2 This is a view of the propagated seedlings of Example 1 of the present invention, in which no vitrification occurs and the seedlings grow robustly.

[0026] Figure 3 This is a view of the rooted seedling of Example 1 of the present invention, which has thick leaves, a thick stem, a well-developed root system, and a stem that is visibly enlarged. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0028] Example 1

[0029] A method for rapid propagation of olives by tissue culture, comprising the following steps:

[0030] (1) Material selection and cleaning: Take mature, pest-free and uncracked olive pods, dip a cotton ball in a soapy water solution with alcohol, wipe the surface of the pods 4 times, then add 1 drop of Tween-80, add a small amount of water to mix, and rinse with tap water for 50 minutes.

[0031] (2) Disinfection: After removing the pods, absorb the surface moisture with sterile paper, then transfer them to a clean bench, wipe the surface of the pods 6 times with an alcohol cotton ball dipped in 75% alcohol, rinse with sterile water 3 times, then use a saturated bleach solution to disinfect for 70 minutes, rinse with sterile water 6 times, and finally shake with 0.2% mercuric chloride solution for 6 minutes and rinse with sterile water 8 times.

[0032] (3) Primary culture: Cut small holes in the sterilized pods and sow the seeds evenly in the primary culture medium (formula: MS + NAA 0.15 mg / L + KT 3.5 mg / L + GA3 0.07 mg / L + potato 30.0 g / L), and culture at a temperature of 25 ± 2 ° C in complete darkness for 35 days (e.g. Figure 1 70% of the seeds turned green and started to germinate 15 days after sowing. The seeds were then transferred to a temperature of 25±2°C, a light intensity of 2500 Lx, and a single day of 12 hours for incubation for 55 days.

[0033] (4) Proliferation culture: After the seeds germinate and produce a small number of buds, they are inoculated into a proliferation medium (formula: 1 / 2MS + Huabao No. 2 1.7g / L + CPPU 0.6mg / L + Peptone 0.75g / L + Potato 35.0g / L + Coconut juice 35.0mL / L) in a cluster of 4 plants. They are cultured at a temperature of 25±2℃, a light intensity of 2500Lx, and a single day of light for 12h for 85 days. During the culture process, the seedlings do not undergo vitrification and grow robustly. Figure 2 shown.

[0034] (5) Rooting culture: Cut the proliferated buds into single buds and inoculate them into rooting medium (formula: 1 / 2MS + Huabao No. 2 2.3g / L + NAA 0.3mg / L + potassium dihydrogen phosphate 0.13g / L + peptone 3.5g / L + potato 70.0g / L + coconut milk 70.0mL / L). Culture them at a temperature of 25±2℃, a light intensity of 2000-3000Lx, and a single day light of 12h for 85 days. The culture results are as follows: Figure 3 As shown, its leaves are relatively thick, the stems are sturdy, the root system is well developed, and the stems are visibly enlarged.

[0035] Example 2

[0036] A method for rapid propagation of olives by tissue culture, comprising the following steps:

[0037] (1) Material selection and cleaning: Take mature, pest-free and uncracked olive pods, dip a cotton ball in a soapy water solution with alcohol, wipe the surface of the pods 4 times, then add 1 drop of Tween-80, add a small amount of water to mix, and rinse with tap water for 60 minutes.

[0038] (2) Disinfection: After removing the pods, absorb the surface moisture with sterile paper, and then transfer them to a clean bench. Use an alcohol cotton ball dipped in 75% alcohol to wipe the surface of the pods 5 times, rinse with sterile water 3 times, then use a saturated bleach solution to disinfect for 60 minutes, rinse with sterile water 5 times, and finally shake with 0.2% mercuric chloride solution for 5 minutes and rinse with sterile water 7 times.

[0039] (3) Primary culture: Cut small holes in the sterilized pods and sow the seeds evenly in the primary culture medium (formula: MS + NAA 0.1mg / L + KT 3.0mg / L + GA3 0.05mg / L + potato 30g / L). Culture at 25±2℃ in complete darkness for 30 days, then transfer to 25±2℃, light intensity 2000Lx, and single-day light conditions for 12h for 50 days.

[0040] (4) Proliferation culture: After the seeds germinate and produce a small number of buds, they are inoculated into a proliferation medium (formula: 1 / 2MS + Huabao No. 2 1.5g / L + CPPU 0.5mg / L + peptone 0.5g / L + potato 30.0g / L + coconut milk 30.0mL / L) in a cluster of 3 plants. Culture for 80 days at a temperature of 25±2℃, a light intensity of 2000Lx, and a single-day light condition of 12h.

[0041] (5) Rooting culture: Cut the proliferated buds into single buds and inoculate them into rooting medium (formula: 1 / 2MS + Huabao No. 2 2.0g / L + NAA 0.3mg / L + potassium dihydrogen phosphate 0.10g / L + peptone 3.0g / L + potato 60.0g / L + coconut milk 60.0mL / L), and culture them at a temperature of 25±2℃, a light intensity of 2000Lx, and a single day light of 12h for 80d.

[0042] Example 3

[0043] A method for rapid propagation of olives by tissue culture, comprising the following steps:

[0044] (1) Material selection and cleaning: Take mature, pest-free and uncracked olive pods, dip a cotton ball in a soapy water solution with alcohol, wipe the surface of the pods 5 times, then add 2 drops of Tween-80, add a small amount of water to mix, and rinse with tap water for 60 minutes.

[0045] (2) Disinfection: After removing the pods, absorb the surface moisture with sterile paper, and then transfer them to a clean bench. Use an alcohol cotton ball dipped in 75% alcohol to wipe the surface of the pods 7 times, rinse with sterile water 4 times, then use a saturated bleach solution to disinfect for 80 minutes, rinse with sterile water 6 times, and finally shake with 0.2% mercuric chloride solution for 6 minutes and rinse with sterile water 8 times.

[0046] (3) Primary culture: Cut small holes in the sterilized pods and sow the seeds evenly in the primary culture medium (formula: MS + NAA 0.2 mg / L + KT 4.0 mg / L + GA3 0.08 mg / L + potato 30 g / L). Culture at 25 ± 2 °C in complete darkness for 40 days, then transfer to 25 ± 2 °C, light intensity 3000 Lx, and 12 h per day for 60 days.

[0047] (4) Proliferation culture: After the seeds germinate and produce a small number of buds, they are inoculated into a proliferation medium (formula: 1 / 2MS + Huabao No. 2 2.0g / L + CPPU 0.8mg / L + peptone 1.0g / L + potato 40.0g / L + coconut milk 40.0mL / L) in a cluster of 5 plants. Culture for 90 days at a temperature of 25±2℃, a light intensity of 3000Lx, and a single-day light condition of 12h.

[0048] (5) Rooting culture: Cut the proliferated buds into single buds and inoculate them into rooting medium (formula: 1 / 2MS + Huabao No. 2 2.5g / L + NAA 0.4 mg / L + potassium dihydrogen phosphate 0.15g / L + peptone 4.0g / L + potato 80.0g / L + coconut milk 80.0mL / L), and culture them at a temperature of 25±2℃, a light intensity of 3000Lx, and a single-day light condition of 12h for 90d.

[0049] Comparative Example 1

[0050] Take the seeds treated in steps 1-2 of Example 1 of the present invention, and perform the primary generation, subgeneration and rooting steps according to the publication number CN104255497A and the title: Method for rapid propagation of Echeveria using Echeveria rhizomes as explants.

[0051] Comparative Example 2

[0052] The operation is carried out according to the publication number: CN109673509A, and the name: A rapid propagation method for wild olive.

[0053] Comparative Example 3

[0054] The operation was performed according to the method published by Wu Yuhan et al. (Wu Yuhan, Yu Hanxi, Li Jing, et al. Effects of different culture media on germination of Schefflera seeds [J]. Agricultural Engineering, 2019(3):5. DOI:CNKI:SUN:NYGE.0.2019-03-025.).

[0055] Comparative Example 4

[0056] The operation was performed according to the method published by Wen Xiuping et al. (Wen Xiuping, Lin Qingqing, Yang Chengzi, et al. Preliminary report on the aseptic sowing and tissue culture rapid propagation technology of Prunus mume in Fujian [J]. Heilongjiang Agricultural Science, 2015(8):4. DOI:10.11942 / j.issn1002-2767.2015.08.0019.).

[0057] Comparative Example 5

[0058] The operation was performed according to the method published by Chen Yinling et al. (Chen Yinling, Chen Dongyi, Jiang Xuejing, et al. Research on tissue culture and rapid propagation system of fine-leaved philodendron [J]. Journal of Plant Physiology, 2025, 61(05): 613-618. DOI: 10.13592 / j.cnki.ppj.101233.).

[0059] Comparative Example 6

[0060] The operation was performed according to the method published by Liu Baocai et al. (Liu Baocai, Huang Yingzhen, Zhao Yunqing, et al. Preliminary report on the aseptic sowing and rapid propagation technology of Lithospermum tenuifolium [J]. Fujian Journal of Agricultural Sciences, 2014, 29(5):4. DOI:10.3969 / j.issn.1008-0384.2014.05.011.).

[0061] The disinfection contamination rate, disinfection mortality rate, seed germination rate, proliferation coefficient, vitrification rate and rooting rate of Examples 1-3 and Comparative Examples 1-6 were statistically compared, and the results are shown in the following table.

[0062] Table 1 Statistical results

[0063] Disinfection contamination rate Disinfection mortality rate Seed germination rate proliferation coefficient Glass transition rate Rooting rate Example 1 7.4% 0.5% 100.0% 10.3 0.5% 100.0% Example 2 8.2% 0.8% 99.8% 10.1 0.8% 100.0% Example 3 8.6% 0.7% 99.5% 10.0 0.7% 100.0% Comparative Example 1 7.8% 0.5% - 5.6 5.9% 90.3% Comparative Example 2 - - 68.2% 4.9 4.7% 86.3% Comparative Example 3 6.8% 3.2% 99.4% 5.8 8.1% 100.0% Comparative Example 4 9.3% 8.9% 100.0% 6.9 6.8% 85.0% Comparative Example 5 10.8% 5.3% 85.4% 4.0 8.9% 90.0% Comparative Example 6 5.0% 2.4% 70.1% 5.9 7.6% 100.0%

[0064] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of olives by tissue culture, characterized in that the steps include: (1) Material selection and cleaning: Take mature, pest-free and uncracked olive pods, dip a cotton ball in soapy water with alcohol, wipe the surface of the pods 4-5 times, then add 1-2 drops of Tween-80, add a small amount of water and mix well, then rinse with running water for 40-60 minutes; (2) Disinfection: After removing the pods, absorb the surface moisture with sterile paper, then transfer them to a clean bench, wipe the surface of the pods 5 to 7 times with an alcohol cotton ball dipped in 75% alcohol, rinse with sterile water 3 to 4 times, then use a saturated bleach solution to disinfect for 60 to 80 minutes, rinse with sterile water 5 to 6 times, and finally shake with 0.2% mercuric chloride solution for 5 to 6 minutes, rinse with sterile water 7 to 8 times; (3) Primary culture: Cut small holes in the sterilized pods and sow the seeds evenly in the primary culture medium. Culture them at 25±2℃ in complete darkness for 30-40 days, then transfer them to 25±2℃ with a light intensity of 2000-3000Lx and a single daylight of 12h for 50-60 days. The formula of the primary culture medium is: MS + NAA 0.1-0.2 mg / L + KT 3.0-4.0 mg / L + GA3 0.05-0.08 mg / L + potato 30 g / L; (4) Proliferation culture: After the seeds germinate and produce a small number of buds, they are inoculated into a proliferation medium in clusters of 3 to 5 plants. They are cultured for 80 to 90 days at a temperature of 25 ± 2 °C, a light intensity of 2000 to 3000 Lx, and a single daylight of 12 h. The proliferation culture medium formula is: 1 / 2MS + Huabao No. 2 1.5-2.0g / L + CPPU 0.5-0.8mg / L + Peptone 0.5-1.0g / L + Potato 30.0-40.0g / L + Coconut milk 30.0-40.0mL / L; (5) Rooting culture: Cut the proliferated buds into single buds, inoculate them into rooting medium, and culture them for 80 to 90 days at a temperature of 25 ± 2 °C, a light intensity of 2000 to 3000 Lx, and a photoperiod of 12 h per day; The formula of the rooting medium is: 1 / 2MS + Huabao No. 2 2.0-2.5g / L + NAA 0.3-0.4 mg / L + potassium dihydrogen phosphate 0.10-0.15g / L + peptone 3.0-4.0g / L + potato 60.0-80.0g / L + coconut water 60.0-80.0mL / L.

2. The method according to claim 1, characterized in that In step (3), the formula of the primary culture medium is: MS + NAA 0.15 mg / L + KT 3.5 mg / L + GA3 0.07 mg / L + potato 30.0 g / L.

3. The method according to claim 1, characterized in that In step (4), the proliferation medium formula is: 1 / 2MS + Huabao No. 2 1.7g / L + CPPU 0.6mg / L + peptone 0.75g / L + potato 35.0g / L + coconut milk 35.0mL / L.

4. The method according to claim 1, characterized in that In step (5), the formula of the rooting medium is: 1 / 2MS + Huabao No. 2 2.3g / L + NAA 0.3mg / L + potassium dihydrogen phosphate 0.13g / L + peptone 3.5g / L + potato 70.0g / L + coconut milk 70.0mL / L.

5. The method according to claim 1 is used in the rapid propagation of olive tissue culture.

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