Tissue culture and rapid propagation method for large cherries

Abstract

The invention provides a large cherry tissue culture and rapid propagation method which comprises the following steps: taking a large cherry rhizome as an explant, sterilizing, cutting into blocks, inoculating into an induction culture medium, and inducing to generate callus after 35-45 days; inoculating the callus onto a proliferation culture medium for proliferation culture; after the leaves grow to 2-3 cm, the leaves are inoculated into a rooting culture medium to be cultured into seedlings, and brown roots grow on the base parts of the seedlings after 25-35 days; transplanting when the seedlings have 3-5 roots and the height is more than 5cm, hardening the seedlings under sunlight for 6-8 days, taking out bottle seedlings, cleaning the rooting culture medium attached to the roots, airing and planting the seedlings in a sterilized culture medium to finish transplanting. By optimizing a culture medium formula and culture conditions, high-efficiency induction and rapid proliferation of the large cherries are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of plant propagation, and particularly relates to a tissue culture rapid propagation method for cherry. Background Art

[0002] Sweet cherries are popular among consumers for their bright colors, sweet taste and rich nutrition, and they occupy an important position in the fruit market. Traditional sweet cherry propagation methods have problems such as slow propagation speed and susceptibility to virus infection, which restricts the rapid development of the sweet cherry industry. Plant tissue culture technology provides a new way for sweet cherry propagation. Through tissue culture and rapid propagation, a large number of high-quality seedlings with consistent genetic traits and no viruses can be obtained in a short period of time, effectively solving the drawbacks of traditional propagation and promoting the large-scale and modern development of the sweet cherry industry. 1. Explant selection and processing

[0003] Explant selection: During the growing season of big cherries, select mother plants of excellent varieties that are strong and free of diseases and pests. Prioritize the collection of new shoots that are on the periphery of the crown, semi-lignified and with full buds. The length of the new shoots should be 5-10 cm. At this time, the new shoots have high physiological activity and strong cell division ability, which is conducive to the start of rapid propagation by tissue culture. Explant pretreatment: Bring the collected new shoots back to the laboratory, remove the excess leaves first, and only retain 2-3 young leaves on the top to reduce water evaporation and nutrient consumption. Then rinse the dust and impurities on the surface of the explant with running water for about 30 minutes. Then place the explant in a water solution with detergent, gently shake and wash for 10-15 minutes to remove surface oil and some microorganisms. Finally, rinse with running water to remove any detergent residue. Explant Disinfection: On a laminar flow hood, immerse the pretreated explants in a 70%-75% alcohol solution for 10-15 seconds. Remove immediately and then immerse in a saturated sodium hypochlorite solution for 8-10 minutes, gently shaking the container to ensure uniform disinfection. After disinfection, rinse the explants 5-6 times with sterile water to completely remove any residual disinfectant. Place the disinfected explants on sterile filter paper to blot dry before proceeding.

[0004] The existing tissue culture rapid propagation method of sweet cherry faces problems such as low proliferation rate and long culture cycle, which affects large-scale production and application. Summary of the Invention

[0005] The invention aims to provide a method for rapid propagation of sweet cherry by tissue culture, which uses sweet cherry rhizomes as explants and optimizes various culture medium formulas and culture condition parameters to significantly improve the rapid propagation of sweet cherry by tissue culture.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for rapid propagation of cherry by tissue culture, comprising the following steps:

[0008] S1: Explant preparation and sterilization: Rinse and place the cherry rhizomes as explants on a clean bench. Soak in sterile GA3 solution, sterilize with 75% alcohol and 0.1% mercuric chloride solution, and rinse several times with sterile water. Cut the sterilized explants into several pieces for later use.

[0009] S2: Induction culture: The explants cut in step S1 are inoculated into an induction medium and callus tissue is induced after 35-45 days;

[0010] S3: proliferation culture: inoculating the callus tissue in step S2 onto a proliferation culture medium for proliferation culture;

[0011] S4: Rooting culture: After the leaves in step S3 grow to 2-3 cm, they are inoculated into rooting medium and cultured into seedlings. After 25-35 days, brown roots grow at the base of the seedlings.

[0012] S5: Transplanting tissue culture seedlings: When the seedlings in step S4 have 3-5 roots and are more than 5 cm in height, they are transplanted. After being hardened in sunlight for 6-8 days, the seedlings are taken out of the bottle, the rooting medium attached to the roots is washed off, dried and planted in a sterilized culture medium to complete the transplanting.

[0013] Preferably, step S1 specifically comprises: selecting a healthy, pest-free, and well-growing cherry rhizome as an explant, rinsing it with tap water, moving it into a clean bench, soaking it in a sterile GA3 solution for 4-6 minutes, then soaking it in 75% alcohol for 45-75 seconds, and sterilizing it with a 0.1% mercuric chloride solution for 3-6 minutes, rinsing it with sterile water 2-5 times, and cutting the sterilized explant into several pieces with a length of 0.3-0.5 cm for later use.

[0014] By using the rhizomes of sweet cherries as explants, adopting specific formulas of induction culture medium, proliferation culture medium and rooting culture medium, and optimizing parameters such as light intensity, time, temperature, and cycle, the reproduction efficiency and quality of sweet cherries have been significantly improved.

[0015] Preferably, the induction culture medium in step S2 is 1 / 10MS + TDZ 0.4-0.8 mg / L + NAA 0.4-0.6 mg / L + 6-BA 1-3 mg / L + hydrolyzed casein 200-400 mg / L + sucrose 35 g / L + agar 7.0 g / L, with a pH of 5.5-6.0.

[0016] Preferably, in step S2, the light intensity during the induction culture is 1500-2500 LX, the light duration is 10-14 h / d, and the temperature is 25-30°C.

[0017] Preferably, the proliferation culture medium in step S3 is: 1 / 3MS + 6-BA 0.5-1 mg / L + NAA 0.1-0.2 mg / L + IBA 1-2 mg / L + sucrose 30 g / L + agar 4.5 g / L, with a pH of 5.9-6.3.

[0018] Preferably, the proliferation culture in step S3 has a light intensity of 2600-3200 LX, a light duration of 14-18 h / d, and a temperature of 20-25°C.

[0019] Preferably, the rooting medium in step S4 is: 1 / 4MS + IBA 0.5-1 mg / L + NAA 0.1-0.2 mg / L + sucrose 10-20 g / L + activated carbon 0.8-1.1 g / L, with a pH of 5.7-6.1.

[0020] Preferably, the light intensity for rooting culture in step S4 is 2200-2500 LX, the light duration is 13-15 h / d, and the temperature is 22-26°C.

[0021] Preferably, the culture medium for transplanting tissue culture seedlings in step S5 is a mixture of coconut husk, peat soil and volcanic rock, with a mass ratio of the three being 1:1.3-1.5:1.1-1.3.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0023] (1) The present invention utilizes cherry rhizomes as explants, adopts specific induction culture medium, proliferation culture medium and rooting culture medium, and optimizes parameters such as light intensity, time, temperature and cycle, thereby significantly improving the reproduction efficiency and quality of cherry.

[0024] (2) The present invention reduces the browning degree of the explant material and improves the induction efficiency of adventitious buds by adopting an induction culture medium and an induction environment; effectively reduces the degree of vitrification and improves the induction efficiency through the proliferation culture medium and the induction environment, and the proliferation coefficient can reach 4-5; through the rooting culture medium and environment, the root induction rate reaches 98.5%; and by using a mixture of coconut husk, peat soil and volcanic rock as a planting matrix, the survival rate of seedlings after transplanting reaches more than 95.5%. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1

[0027] This embodiment provides a method for rapid propagation of sweet cherry through tissue culture, comprising the following steps:

[0028] S1: Explant preparation and sterilization: Select healthy, pest-free, and well-growing cherry rhizomes as explants. Rinse with tap water, transfer to a clean bench, and soak in sterile GA3 solution for 4 minutes. Then, soak in 75% alcohol for 75 seconds, sterilize with 0.1% mercuric chloride solution for 6 minutes, and rinse twice with sterile water. Cut the sterilized explants into pieces of 0.3 cm in length for later use.

[0029] S2: Induction culture: The explants cut in step S1 were inoculated into an induction medium containing 1 / 10MS, 0.4mg / L TDZ, 0.4mg / L NAA, 1mg / L 6-BA, 200mg / L hydrolyzed casein, 35g / L sucrose, and 7.0g / L agar, with a pH of 5.5. The culture medium was placed under a light intensity of 1500LX, a light duration of 10h / d, and a temperature of 25°C. Callus tissue was induced after 35 days.

[0030] S3: Proliferation culture: The callus tissue in step S2 was inoculated onto a proliferation culture medium for proliferation culture; the proliferation culture medium was: 1 / 3MS + 0.5mg / L 6-BA + 0.1mg / L NAA + 1mg / L IBA + 30g / L sucrose + 4.5g / L agar, pH 5.9; the proliferation culture was controlled at a light intensity of 2600LX, a light duration of 14h / d, and a temperature of 20°C;

[0031] S4: Rooting culture: After the leaves in step S3 grow to 2-3 cm, they are inoculated into rooting medium and cultured into seedlings. The rooting medium consists of 1 / 4 MS + 0.5 mg / L IBA + 0.1 mg / L NAA + 10 g / L sucrose + 0.8 g / L activated carbon, with a pH of 5.7. The light intensity is controlled at 2200 LX, the photoperiod is 13 h / d, and the temperature is 22°C. After 25 days, brown roots grow at the base of the seedlings.

[0032] S5: Transplanting of tissue culture seedlings: When the seedlings in step S4 have 3-5 roots and are more than 5 cm in height, they are transplanted. After being placed in sunlight for 6 days, the seedlings are taken out of the bottle, the rooting medium attached to the roots is washed off, dried and planted in a sterilized culture medium (the culture medium is a mixture of coconut coir, peat soil and volcanic rock, with a mass ratio of 1:1.3:1.1) to complete the transplanting.

[0033] Example 2

[0034] This embodiment provides a method for rapid propagation of sweet cherry through tissue culture, comprising the following steps:

[0035] S1: Explant preparation and sterilization: Select healthy, pest-free, and well-growing cherry rhizomes as explants. Rinse with tap water, move to a clean bench, and soak in sterile GA3 solution for 6 minutes. Then, soak in 75% alcohol for 75 seconds, sterilize with 0.1% mercuric chloride solution for 6 minutes, and rinse five times with sterile water. Cut the sterilized explants into pieces of 0.5 cm in length for later use.

[0036] S2: Induction culture: The explants cut in step S1 were inoculated into an induction medium containing 1 / 10MS, 0.8mg / L TDZ, 0.6mg / L NAA, 3mg / L 6-BA, 400mg / L hydrolyzed casein, 35g / L sucrose, and 7.0g / L agar, with a pH of 6.0. The culture medium was placed under a light intensity of 2500LX, a light duration of 14h / d, and a temperature of 30°C. Callus tissue was induced after 45 days.

[0037] S3: Proliferation culture: The callus tissue in step S2 was inoculated onto a proliferation culture medium for proliferation culture; the proliferation culture medium was: 1 / 3MS + 6-BA 1mg / L + NAA 0.2mg / L + IBA 2mg / L + sucrose 30g / L + agar 4.5g / L, pH 6.3; the proliferation culture was controlled at a light intensity of 3200LX, a light duration of 18h / d, and a temperature of 25°C;

[0038] S4: Rooting culture: After the leaves in step S3 grow to 2-3 cm, they are inoculated into rooting medium and cultured into seedlings. The rooting medium consists of 1 / 4 MS + 1 mg / L IBA + 0.2 mg / L NAA + 20 g / L sucrose + 1.1 g / L activated carbon, with a pH of 6.1. The light intensity is controlled at 2500 LX, the photoperiod is 15 h / d, and the temperature is 26°C. After 35 days, brown roots grow at the base of the seedlings.

[0039] S5: Transplanting of tissue culture seedlings: When the seedlings in step S4 have 3-5 roots and are more than 5 cm in height, they are transplanted. After being placed in sunlight for 8 days, the seedlings are taken out of the bottle, the rooting medium attached to the roots is washed off, dried and planted in a sterilized culture medium (the culture medium is a mixture of coconut coir, peat soil and volcanic rock, with a mass ratio of 1:1.5:1.3) to complete the transplanting.

[0040] Example 3

[0041] This embodiment provides a method for rapid propagation of sweet cherry through tissue culture, comprising the following steps:

[0042] S1: Explant preparation and sterilization: Select healthy, pest-free, and well-growing cherry rhizomes as explants. Rinse with tap water, transfer to a clean bench, and soak in sterile GA3 solution for 5 minutes. Then, soak in 75% alcohol for 65 seconds, sterilize with 0.1% mercuric chloride solution for 4 minutes, and rinse three times with sterile water. Cut the sterilized explants into pieces of 0.4 cm in length for later use.

[0043] S2: Induction culture: The explants cut in step S1 were inoculated into an induction medium containing 1 / 10 MS, 0.6 mg / L TDZ, 0.5 mg / L NAA, 2 mg / L 6-BA, 350 mg / L hydrolyzed casein, 35 g / L sucrose, and 7.0 g / L agar, with a pH of 5.7. The culture medium was placed under a light intensity of 2000 LX, a light duration of 12 h / d, and a temperature of 27°C. Callus tissue was induced after 42 days.

[0044] S3: Proliferation culture: The callus tissue from step S2 was inoculated onto a proliferation culture medium for proliferation culture; the proliferation culture medium consisted of 1 / 3MS + 0.7 mg / L 6-BA + 0.15 mg / L NAA + 1.5 mg / L IBA + 30 g / L sucrose + 4.5 g / L agar, with a pH of 6.1; the proliferation culture was controlled at a light intensity of 3000 LX, a light duration of 16 h / d, and a temperature of 23°C;

[0045] S4: Rooting culture: After the leaves in step S3 grow to 2-3 cm, they are inoculated into rooting medium and cultured into seedlings. The rooting medium consists of 1 / 4 MS + 0.8 mg / L IBA + 0.15 mg / L NAA + 15 g / L sucrose + 1 g / L activated carbon, with a pH of 5.8. The light intensity is controlled at 2400 LX, the photoperiod is 14 h / d, and the temperature is 24°C. After 32 days, brown roots grow at the base of the seedlings.

[0046] S5: Transplanting of tissue culture seedlings: When the seedlings in step S4 have 3-5 roots and are more than 5 cm in height, they are transplanted. After being placed in sunlight for 7 days, the seedlings are taken out of the bottle, the rooting medium attached to the roots is washed off, dried and planted in a sterilized culture medium (the culture medium is a mixture of coconut coir, peat soil and volcanic rock, with a mass ratio of 1:1.4:1.2) to complete the transplanting.

[0047] The comprehensive performance of Example 3 is the most outstanding. However, because Comparative Examples 1-6 do not adopt the necessary technical solutions, their corresponding performance tests are significantly worse than those of the Examples. The above experimental results further demonstrate the importance of the technical solutions defined in the present invention for its technical effects.

[0048] Comparative Example 1

[0049] The difference between this comparative example and Example 1 is that in step S2, the induction medium contains MS+TDZ0.4 mg / L+NAA0.4 mg / L+6-BA1 mg / L, and the other steps are the same.

[0050] Comparative Example 2

[0051] The difference between this comparative example and Example 1 is that in step S2, the induction environment temperature is 35° C., the illumination duration is 2 h / d, the illumination intensity is 500 Lx, and the induction is 10 days. The other steps are the same.

[0052] Comparative Example 3

[0053] The difference between this comparative example and Example 1 is that in step S3, 1 / 3MS+6-BA0.5mg / L+NAA0.1mg / L+IBA1mg / L are added to the proliferation culture medium, and the other steps are the same.

[0054] Comparative Example 4

[0055] The difference between this comparative example and Example 1 is that in step S3, the proliferation environment temperature is 35° C., the illumination duration is 20 h / d, the illumination intensity is 2000 lux, and the culture is carried out for 50 days. The other steps are the same.

[0056] Comparative Example 5

[0057] The difference between this comparative example and Example 1 is that in step S4, the rooting medium is MS+IBA0.5 mg / L+NAA0.1 mg / L, and the other steps are the same.

[0058] Comparative Example 6

[0059] The difference between this comparative example and Example 1 is that the ambient temperature of the rooting culture in step S4 is 10° C., the illumination duration is 5 h / d, the illumination intensity is 15,000 lux, and the culture is carried out for 20 days. The other steps are the same.

[0060] The root induction rate and seedling survival rate were tested using the preparation methods of the various embodiments and comparative examples. The results are shown in Table 1 below.

[0061] Table 1 Performance test results

[0062]

[0063]

[0064] The performance test results above demonstrate that the cherry tissue culture and rapid propagation methods of Examples 1-3 exhibit high root induction rates and seedling survival rates; in particular, Example 3 exhibits the most outstanding overall performance. Comparative Examples 1-6, however, lack the necessary technical solutions, resulting in significantly inferior performance to the Examples in the corresponding performance tests. These experimental results further demonstrate the importance of the technical solutions defined in the present invention for its technical effectiveness.

[0065] By adopting induction culture medium and induction environment, the browning degree of explant material is reduced and the induction efficiency of adventitious buds is improved; by using value-added culture medium and induction environment, the degree of vitrification is effectively reduced and the value-added efficiency is improved, and the proliferation coefficient can reach 4-5; by using rooting culture medium and environment, the root induction rate reaches 98.5%; by using a mixture of coconut coir, peat soil and volcanic rock as the planting matrix, the survival rate of seedlings after transplanting reaches more than 95.5%.

[0066] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of cherry by tissue culture, characterized in that: The steps include: S1: Explant preparation and sterilization: Rinse and place the cherry rhizomes as explants on a clean bench. Soak in sterile GA3 solution, sterilize with 75% alcohol and 0.1% mercuric chloride solution, and rinse several times with sterile water. Cut the sterilized explants into several pieces for later use. S2: Induction culture: The explants cut in step S1 are inoculated into an induction medium and callus tissue is induced after 35-45 days; S3: proliferation culture: inoculating the callus tissue in step S2 onto a proliferation culture medium for proliferation culture; S4: Rooting culture: After the leaves in step S3 grow to 2-3 cm, they are inoculated into rooting medium and cultured into seedlings. After 25-35 days, brown roots grow at the base of the seedlings. S5: Transplanting tissue culture seedlings: When the seedlings in step S4 have 3-5 roots and are more than 5 cm in height, they are transplanted. After being hardened in sunlight for 6-8 days, the seedlings are taken out of the bottle, the rooting medium attached to the roots is washed off, dried and planted in a sterilized culture medium to complete the transplanting.

2. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, Step S1 specifically comprises: selecting a healthy, pest-free, and well-growing cherry rhizome as an explant, rinsing it with tap water, moving it into a clean bench, soaking it in a sterile GA3 solution for 4-6 minutes, then soaking it in 75% alcohol for 45-75 seconds, sterilizing it with a 0.1% mercuric chloride solution for 3-6 minutes, rinsing it with sterile water 2-5 times, and cutting the sterilized explant into several pieces with a length of 0.3-0.5 cm for later use.

3. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, The induction medium in step S2 is 1 / 10MS + TDZ 0.4-0.8 mg / L + NAA 0.4-0.6 mg / L + 6-BA 1-3 mg / L + hydrolyzed casein 200-400 mg / L + sucrose 35 g / L + agar 7.0 g / L, with a pH of 5.5-6.

0.

4. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, In step S2, the light intensity during the induction culture is 1500-2500 LX, the light exposure time is 10-14 h / d, and the temperature is 25-30°C.

5. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, The proliferation culture medium in step S3 is: 1 / 3MS + 6-BA 0.5-1 mg / L + NAA 0.1-0.2 mg / L + IBA 1-2 mg / L + sucrose 30 g / L + agar 4.5 g / L, with a pH of 5.9-6.

3.

6. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, Step S3 proliferation culture light intensity 2600-3200LX, illumination time 14-18h / d, temperature 20-25°C.

7. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, The rooting medium in step S4 is: 1 / 4MS+IBA0.5-1mg / L+NAA0.1-0.2mg / L+sucrose 10-20g / L+0.8-1.1g / L activated carbon, with a pH of 5.7-6.

1.

8. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, The light intensity for rooting culture in step S4 is 2200-2500 LX, the light duration is 13-15 h / d, and the temperature is 22-26°C.

9. A method for rapid propagation of cherry tissue culture according to claim 1, characterized in that, In step S5, the culture medium for transplanting tissue culture seedlings is a mixture of coconut husk, peat soil and volcanic rock, with the mass ratio of the three being 1:1.3-1.5:1.1-1.3.

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