Culture method and application of strawberry leaf tissue rapid propagation system

Abstract

The invention discloses a culture method of a strawberry leaf tissue rapid propagation system, in the culture method, strawberry leaves are used for regeneration propagation, materials are convenient to obtain and are not limited by the number, and the number of strawberry seedlings is greatly increased; operation is simple, materials are saved, and the influence on plants and the environment is small; meanwhile, HgCl2 or NaClO can be used as a strawberry explant disinfection method, a very good disinfection effect can be achieved, and the disinfection time is short; according to the method, strawberry explant leaves are subjected to 14 days of callus induction, 14 days of clumpy bud induction and 21 days of rooting culture, breeding can be completed, the total growth cycle is short (49 days), the growth cycle of strawberry seedling culture is remarkably shortened, the breeding efficiency of virus-free seedlings is improved, and the method is suitable for factory breeding of strawberry seedlings.

Description

Technical Field

[0001] The invention relates to a plant tissue rapid propagation system culture technology, and in particular to a strawberry leaf tissue rapid propagation system culture method and application thereof. Background Art

[0002] Strawberry (Fragaria×ananassa Duch.) is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family. It has strong adaptability, juicy flesh, and high nutritional value. Strawberry berries also have high medicinal and medical value, and have good therapeutic effects on the prevention and treatment of atherosclerosis, coronary heart disease, and cerebral hemorrhage. Strawberry has a short growth cycle, quick results, and high economic benefits. It is becoming the fastest-growing emerging industry in my country's fruit industry and facility agriculture, and the planting area is increasing year by year. Therefore, the development of strawberry production not only provides people with rich nutrition and meets the needs of people's increasingly improved living standards, but also has important significance for increasing farmers' income, exporting and earning foreign exchange, and revitalizing the local economy.

[0003] Strawberries are susceptible to infection by viruses and pathogens. Strawberries are mainly propagated asexually by runners. Traditional self-propagation is still the main cultivation mode of strawberries in China. Many years of continuous planting without virus removal will lead to a decline in seedling quality, decreased disease resistance, decreased yield, fruit deformity, poor taste, etc., affecting the growth and quality of strawberries. The use of pesticides and post-management have increased production costs. At present, the main viruses that harm strawberries are strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV) and strawberry crinkle virus (SCrV).

[0004] In the factory breeding of virus-free strawberry seedlings, strawberry stem tips are often used for virus-free. The strawberry stem tip virus-free technology can not only ensure the uniformity of seedling genetic traits, but also improve the quality of strawberry seedlings. In 2020, the strawberry planting area in my country was 127,200 hm2. The market potential of virus-free tissue culture seedlings is great, but the number of strawberry stem tip tissues is limited, which is difficult to meet the growing market demand. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems existing in the prior art and provide a cultivation method and application of a strawberry leaf tissue rapid propagation system. The present invention uses leaves for regeneration and propagation, which is convenient to obtain and not limited in quantity, greatly increasing the number of strawberry seedlings; and the operation is simple, material-saving, and has little impact on plants and the environment, which is suitable for factory-based breeding of strawberry seedlings.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: a culture method of a strawberry leaf tissue rapid propagation system, comprising the following steps:

[0007] S1. Take strawberry leaves, wash them with soapy water, and then rinse them under running water for 1 hour. Use filter paper to absorb the surface moisture as explants.

[0008] S2, disinfect the explants;

[0009] S3, the explants after disinfection were cultured in callus induction medium under light for 14 days, the culture temperature was 25±2℃, the light intensity was 2000lx, and the light time was 12h / d;

[0010] S4, the callus cultured in S3 was transferred to the cluster bud induction medium for culture, with a light intensity of 2000lx and a light duration of 12h / d; after 14 days of culture, 4-5 cluster buds grew on each callus;

[0011] S5. The bud segments obtained in S4 were transferred to a rooting medium for cultivation at a temperature of 25±2°C, a light intensity of 2000 lx, and a light duration of 12 h / d. Complete plants were obtained after 21 days of cultivation.

[0012] Preferably, in S2, the disinfection method is: first disinfect with 75% ethanol for 2 minutes, shaking the bottle continuously during the disinfection, then disinfect with 0.1% HgCl2 for 5 minutes, shaking the bottle continuously during the disinfection, and finally rinse with sterile water 4 to 5 times.

[0013] Preferably, in S2, the disinfection treatment method is: first disinfect with 75% ethanol for 3 minutes, shaking the bottle continuously during the disinfection, then disinfect with 2% NaClO for 3 minutes, shaking the bottle continuously during the disinfection, and finally rinse with sterile water for 4 to 5 times.

[0014] Preferably, in S3, the callus induction medium is MS+TDZ0.5mg.L with a pH value of 5.8. －1 +NAA0.1mg.L －1 .

[0015] Preferably, in S4, the bud induction medium is MS+TDZ1.5mg.L －1 +NAA0.3mg.L －1 .

[0016] Preferably, in S5, the rooting medium is MS+IBA0.3mg.L －1 +NAA0.3mg.L －1 .

[0017] The invention also provides application of the culture method in strawberry breeding.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention uses strawberry leaves for regeneration and reproduction, which is convenient to obtain and is not limited in quantity, greatly increasing the number of strawberry seedlings; and the operation is simple, material-saving, and has little impact on plants and the environment, and is suitable for factory-based breeding of strawberry seedlings;

[0020] 2. The strawberry explant leaves of the present invention are subjected to callus induction for 14 days, cluster bud induction for 14 days, and rooting culture for 21 days to complete breeding, and the total growth cycle is short (49 days), which significantly shortens the growth cycle of strawberry seedling culture and improves the breeding efficiency of virus-free seedlings;

[0021] 3. The method for disinfecting strawberry explants in the present invention can use HgCl2 or NaClO, both of which can achieve good disinfection effects and short disinfection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the stem segment after callus induction culture in Example 1;

[0023] Figure 2 This is a picture after the bud induction culture in Example 1;

[0024] Figure 3 This is a picture after rooting and cultivation in Example 1;

[0025] Figure 4 This is a diagram of the stem segment after callus induction culture in Example 2;

[0026] Figure 5 This is a picture after the bud induction culture in Example 2;

[0027] Figure 6 This is a picture after rooting culture in Example 2. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0029] Example 1

[0030] Take strawberry leaves, wash them with soapy water, and then rinse them under running water for 1 hour (strawberry leaves have fluff and need to be rinsed for a long time). Use filter paper to absorb the surface moisture and place them as explants on a clean bench. First disinfect them with 75% ethanol for 2 minutes, shaking the bottle constantly during the process, then disinfect them with 0.1% HgCl2 for 5 minutes, shaking the bottle constantly during the disinfection process to ensure that the disinfectant is in full contact with the explants, and finally rinse them with sterile water for 4 to 5 times for later use.

[0031] MS+TDZ0.5mg.L －1 +NAA0.1 mg.L －1, pH value is 5.8, which is the callus induction medium. Culture under light at (25±2)℃, light intensity 2000lx, light time 12h / d. After 14 days of culture, observe the callus growth and transfer to bud differentiation medium.

[0032] MS+TDZ1.5mg.L －1 +NAA0.3mg.L －1 Bud induction medium, after the callus tissue is transferred to the cluster bud induction medium, the light intensity is 2000lx, the lighting time is 12h / d, after a period of time (14d), the callus tissue differentiates into buds, and a piece of leaf callus tissue has 4-5 buds.

[0033] MS+IBA0.3mg.L －1 +NAA0.3mg.L －1 The rooting medium was quickly prepared, and the bud segments were transferred to the rooting medium and cultured under light at (25±2)℃, with a light intensity of 2000lx and a light duration of 12h / d. After 21 days of culture, complete plants were obtained.

[0034] Example 2

[0035] Take strawberry leaves, wash them with soapy water, and then rinse them under running water for 1 hour (strawberry leaves have hair and need to be rinsed for a long time). Use filter paper to absorb the surface moisture and place them as explants on a clean bench. First disinfect them with 75% ethanol for 2 minutes, shaking the bottle constantly during the process, then disinfect them with 2% NaClO for 3 minutes, shaking the bottle constantly during the disinfection process to ensure that the disinfectant is in full contact with the explants, and finally rinse them with sterile water for 4 to 5 times for later use.

[0036] MS+TDZ0.5mg.L －1 +NAA0.1 mg.L －1 , pH value is 5.8, which is the callus induction medium. Culture under light at (25±2)℃, light intensity 2000lx, light time 12h / d. After 14 days of culture, observe the callus growth and transfer to bud differentiation medium.

[0037] MS+TDZ1.5mg.L －1 +NAA0.3mg.L －1 Bud induction medium, after the callus tissue is transferred to the cluster bud induction medium, the light intensity is 2000lx, the lighting time is 12h / d, after a period of time (14d), the callus tissue differentiates into buds, and a piece of leaf callus tissue has 4-5 buds.

[0038] MS+IBA0.3mg.L －1 +NAA0.3mg.L －1The rooting medium was quickly prepared, and the bud segments were transferred to the rooting medium and cultured under light at (25±2)℃, with a light intensity of 2000lx and a light duration of 12h / d. After 21 days of culture, complete plants were obtained.

[0039] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A method for cultivating a strawberry leaf tissue rapid propagation system, characterized in that: The following steps are involved: S1. Take strawberry leaves, wash them with soapy water, and then rinse them under running water for 1 hour. Use filter paper to absorb the surface moisture as explants. S2, disinfect the explants; S3, the explants after disinfection were cultured in callus induction medium under light for 14 days, the culture temperature was 25±2℃, the light intensity was 2000lx, and the light time was 12h / d; S4, the callus cultured in S3 was transferred to the cluster bud induction medium for culture, with a light intensity of 2000lx and a light duration of 12h / d; after 14 days of culture, 4-5 cluster buds grew on each callus; S5. The bud segments obtained in S4 were transferred to a rooting medium for cultivation at a temperature of 25±2°C, a light intensity of 2000 lx, and a light duration of 12 h / d. Complete plants were obtained after 21 days of cultivation.

2. The culture method according to claim 1, characterized in that In S2, the disinfection method is: first disinfect with 75% ethanol for 2 minutes, shaking the bottle continuously during the disinfection, then disinfect with 0.1% HgCl2 for 5 minutes, shaking the bottle continuously during the disinfection, and finally rinse with sterile water for 4 to 5 times.

3. The culture method according to claim 1, characterized in that In S2, the disinfection treatment method is: first disinfect with 75% ethanol for 3 minutes, shaking the bottle continuously during the disinfection, then disinfect with 2% NaClO for 3 minutes, shaking the bottle continuously during the disinfection, and finally rinse with sterile water for 4 to 5 times.

4. The culture method according to claim 1, characterized in that In S3, the callus induction medium is MS+TDZ0.5mg.L with a pH value of 5.

8. －1 +NAA0.1 mg.L －1 .

5. The culture method according to claim 1, characterized in that: In S4, the bud induction medium is MS+TDZ1.5mg.L －1 +NAA0.3mg.L －1 .

6. The culture method according to claim 1, characterized in that In S5, the rooting medium is MS+IBA0.3mg.L －1 +NAA0.3mg.L －1 .

7. Use of the culture method as claimed in claim 1 in strawberry breeding.

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