Strawberry seedling and rapid propagation method combining tissue culture and non-test-tube rooting

Abstract

The invention discloses a strawberry seedling and tissue culture and non-test tube rooting combined rapid propagation method, which comprises the following steps: selecting an explant, and disinfecting; inoculating and culturing; performing non-test-tube rooting culture; and hardening seedlings and transplanting. Through non-test-tube rooting culture, the process is simplified, the pollution risk is reduced, the transplanting difficulty of tissue culture seedlings is reduced, and the survival rate is increased; through the combination of tissue culture and a non-test-tube rooting technology, the survival rate of acclimatization and transplantation is as high as 91% or above; the method realizes efficient and low-cost propagation of strawberries, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to a strawberry seedling and a rapid propagation method combining tissue culture with non-test tube rooting. Background Art

[0002] Strawberry (Fragaria ananassa) is a fruit with high economic value and large market demand.

[0003] The traditional strawberry propagation method mainly relies on runner division, but this method has a low propagation coefficient, a long cycle, and is easily affected by diseases and pests.

[0004] Most existing technologies use tissue culture technology. Although it can achieve rapid reproduction of strawberry seedlings, it has problems such as high cost, complex operation, and low transplant survival rate. Summary of the invention

[0005] The present invention can solve the above-mentioned problems existing in the existing strawberry propagation technology by providing a strawberry seedling and a rapid propagation method combining tissue culture with non-test tube rooting, and realizes efficient and low-cost propagation of strawberries.

[0006] In order to solve the above technical problems, the present invention provides a rapid propagation method combining strawberry seedling tissue culture with non-test tube rooting, comprising the following steps:

[0007] (1) Select explants and sterilize them;

[0008] (2) Inoculation and tissue culture: The explants after the above disinfection treatment are cut in a sterile environment and inoculated into callus tissue culture medium for primary culture. After the primary culture, they are inoculated into subculture culture medium for subculture;

[0009] (3) Non-test tube rooting culture: The strawberry seedlings obtained by subculture are taken out and inserted into a matrix containing a rooting agent for non-test tube rooting culture;

[0010] (4) Hardening and transplanting: After the rooting, the strawberry seedlings are moved to the greenhouse for hardening. After the hardening is completed, they are transplanted to the field or greenhouse.

[0011] In a preferred embodiment of the present invention, in step (2), the callus tissue culture medium is MS basal medium + 0.5-1.0 mg / L 6-BA + 0.1-0.2 mg / L NAA.

[0012] In a preferred embodiment of the present invention, in step (2), the subculture culture medium is MS basal culture medium + 0.2-0.5 mg / L 6-BA + 0.05-0.1 mg / L NAA.

[0013] In a preferred embodiment of the present invention, in step (2), the conditions of the primary culture and the subculture are: temperature 25±2°C, light intensity 2000-3000 lux, and illumination time 12 hours / day;

[0014] The period of the primary culture is 4-6 weeks; the period of the secondary culture is 3-4 weeks.

[0015] In a preferred embodiment of the present invention, in step (3), the rooting agent is 100-200 mg / L IBA solution.

[0016] In a preferred embodiment of the present invention, the amount of the rooting agent is 100-200 mg per liter of substrate.

[0017] In a preferred embodiment of the present invention, the matrix includes perlite and vermiculite, and the mixing mass ratio of the perlite and vermiculite is 1:1-3.

[0018] In a preferred embodiment of the present invention, the non-test tube rooting culture conditions are 80%-90% humidity, 25±2°C temperature, 3000-4000 lux light intensity, 12 hours / day light time, and 2-3 weeks of culture.

[0019] In a preferred embodiment of the present invention, in step (4), the conditions for hardening the seedlings are: humidity 60-70%, light 5000-6000 lux, and time 1-2 weeks.

[0020] In order to solve the above technical problems, the present invention also provides a strawberry seedling cultivated by adopting the above rapid propagation method.

[0021] The beneficial effects of the present invention are as follows: the present invention provides a rapid propagation method combining strawberry seedling tissue culture with non-test tube rooting, which simplifies the process and reduces the risk of pollution through non-test tube rooting culture, reduces the difficulty of transplanting tissue culture seedlings, and improves the survival rate; through the combination of tissue culture and non-test tube rooting technology, the survival rate of seedling hardening and transplanting is as high as more than 91%; the present invention realizes efficient and low-cost reproduction of strawberries, and is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The invention is a schematic diagram of a rapid propagation method combining strawberry seedling tissue culture with non-test tube rooting. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0024] Example 1

[0025] The invention discloses a rapid propagation method combining strawberry seedling tissue culture with non-test tube rooting, and the specific steps are as follows:

[0026] (1) Explant selection and disinfection

[0027] Select healthy and disease-free strawberry plants, take their stem tips as explants, rinse the explants with tap water to remove surface dirt.

[0028] First, soak the explants in 70% ethanol by volume for 30 seconds; then soak them in a sodium hypochlorite solution (i.e., 10% bleach by volume) for 10 minutes, shaking them gently; finally, rinse them with sterile water for 3-5 times to remove residual disinfectant.

[0029] (2) Inoculation and tissue culture

[0030] In an ultra-clean workbench or sterile operating box, use sterile tools to cut the disinfected stem tip into 1 cm size.

[0031] Primary culture: The above-mentioned cut stem tips were inoculated into the prepared callus tissue culture medium, cultured for 4 weeks at a temperature of 25±2°C, a light intensity of 2000 lux, and a light duration of 12 hours / day, and clustered buds grew from the stem tips.

[0032] The components of the callus tissue culture medium are MS basic culture medium+1.0 mg / L 6-BA+0.2 mg / L NAA.

[0033] Subculture: Cut the clustered buds obtained from the primary culture into single buds, inoculate them into the subculture medium, and culture them for 3 weeks at a temperature of 25±2℃, a light intensity of 2000lux, and a lighting time of 12 hours / day. The single buds will grow into strawberry seedlings.

[0034] The ingredients of the grade treatment are: MS basal medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA.

[0035] (3) Non-test tube rooting culture

[0036] The strawberry seedlings obtained by subculture were taken out from the culture medium and the culture medium remaining on the roots was washed off with clean water.

[0037] Insert the strawberry seedlings into a substrate containing a rooting agent, and then place them in a greenhouse environment, maintain a humidity of 80%-90%, a temperature of 25±2°C, a light intensity of 3000lux, a light time of 12 hours / day, and cultivate for 2 weeks, the rooting rate reaches more than 95%.

[0038] Specifically, the matrix is ​​a mixture of perlite and vermiculite in a mass ratio of 1:1;

[0039] The rooting agent is a 150 mg / L IBA solution, and its dosage is 200 mg per liter of substrate. Specifically, the substrate used for each strawberry seedling is 50 ml.

[0040] The use of non-test tube rooting culture instead of test tube rooting simplifies the process, reduces the risk of contamination, and is conducive to improving the survival rate.

[0041] (4) Hardening and transplanting

[0042] Move the rooted strawberry seedlings to the greenhouse for hardening, gradually reduce the humidity to 60%-70%, increase the light to 5000 lux, and the hardening cycle is 1 week.

[0043] After hardening is completed, transplant the strawberry seedlings to the field or greenhouse, ensuring sufficient light for 8 hours a day, a temperature of around 20°C, and a soil moisture of 70%. At the same time, apply fertilizers reasonably and prevent and control pests and diseases in a timely manner.

[0044] The survival rate of strawberry seedlings after transplanting reached over 91%.

[0045] Example 2

[0046] The invention discloses a rapid propagation method combining strawberry seedling tissue culture with non-test tube rooting, and the specific steps are as follows:

[0047] (1) Explant selection and disinfection

[0048] Select healthy and disease-free strawberry plants, take their axillary buds as explants, rinse the explants with tap water to remove surface dirt.

[0049] First, soak the explants in 70% ethanol by volume for 30 seconds; then soak them in a sodium hypochlorite solution (i.e., 10% bleach by volume) for 15 minutes, shaking them gently; finally, rinse them with sterile water for 3-5 times to remove residual disinfectant.

[0050] (2) Inoculation and tissue culture

[0051] In an ultra-clean workbench or sterile operating box, use sterile tools to cut the disinfected stem tip into 1 cm size.

[0052] Primary culture: The above-mentioned cut stem tips were inoculated into the prepared callus tissue culture medium, cultured for 6 weeks at a temperature of 25±2°C, a light intensity of 2000 lux, and a light duration of 12 hours / day, and clustered buds grew from the stem tips.

[0053] The components of the callus tissue culture medium are MS basic culture medium+1.0 mg / L 6-BA+0.2 mg / L NAA.

[0054] Subculture: Cut the clustered buds obtained from the primary culture into single buds, inoculate them into the subculture medium, and culture them for 3 weeks at a temperature of 25±2℃, a light intensity of 2000lux, and a lighting time of 12 hours / day. The single buds will grow into strawberry seedlings.

[0055] The components of the subculture culture medium are: MS basal culture medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA.

[0056] (3) Non-test tube rooting culture

[0057] The strawberry seedlings obtained by subculture were taken out from the culture medium and the culture medium remaining on the roots was washed off with clean water.

[0058] Insert the strawberry seedlings into a substrate containing a rooting agent, and then place them in a greenhouse environment, maintain a humidity of 80%-90%, a temperature of 25±2°C, a light intensity of 3000lux, a light time of 12 hours / day, and cultivate for 2 weeks, the rooting rate reaches more than 98%.

[0059] Specifically, the matrix is ​​a mixture of perlite and vermiculite in a mass ratio of 1:1.

[0060] The rooting agent is a 200 mg / L IBA solution, and the dosage is 100 mg per liter of substrate. The substrate used for each strawberry seedling is 100 ml.

[0061] (4) Hardening and transplanting

[0062] Move the rooted strawberry seedlings to the greenhouse for hardening, gradually reduce the humidity to 60%-70%, increase the light to 5000 lux, and the hardening cycle is 1 week.

[0063] After hardening is completed, transplant the strawberry seedlings to the field or greenhouse, ensuring sufficient light for 8 hours a day, a temperature of around 20°C, and a soil moisture of 70%. At the same time, apply fertilizers reasonably and prevent and control pests and diseases in a timely manner.

[0064] By hardening the seedlings and gradually adjusting the environmental conditions, the strawberry seedlings can smoothly transition from the artificial cultivation environment to the natural environment, enhancing their environmental adaptability, stress resistance and physiological functions, and ultimately improving the survival rate of transplanting.

[0065] The survival rate of strawberry seedlings after transplanting reached over 94%.

[0066] Comparative Example 1

[0067] Compared with Example 1, the difference lies in that the strawberry seedlings obtained by subculture are inoculated into a rooting medium for rooting culture.

[0068] The rooting medium is: 1 / 2MS basic medium + 0.2mg / L IBA

[0069] The conditions for rooting culture are: maintain humidity at 80%-90%, temperature at 25±2℃, light intensity at 3000lux, light time at 12 hours / day, culture for 3 weeks, rooting rate at about 87%

[0070] For the strawberry seedlings after rooting, the culture conditions for hardening and transplanting to the field or greenhouse are the same as those in Example 1, and the survival rate of the seedlings is between 70-80%.

[0071] Comparative Example 2

[0072] Compared with Example 1, the difference is that after the selected strawberry seedlings are disinfected, inoculated and tissue cultured, and non-test tube rooting cultured, they are directly transplanted to the field or greenhouse for growth management without seedling hardening, and the seedling survival rate is 86%.

[0073] The invention has the following advantages: by combining tissue culture with non-test tube rooting technology, the process is simplified, the strawberry reproduction cycle is shortened to about 70 days; the pollution risk is reduced, the difficulty of transplanting tissue culture seedlings is reduced, and the survival rate is increased to more than 91%; the efficient and low-cost reproduction of strawberries is achieved, and it is suitable for large-scale promotion and application. In addition, after the seedling hardening treatment, the adaptability of the strawberry seedlings to the growth environment is improved, and the survival rate is further improved.

[0074] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A rapid propagation method combining strawberry seedling tissue culture with non-test tube rooting, characterized in that: The steps include: (1) Select explants and sterilize them; (2) Inoculation and tissue culture: The explants after the above disinfection treatment are cut in a sterile environment and inoculated into callus tissue culture medium for primary culture. After the primary culture, they are inoculated into subculture culture medium for subculture; (3) Non-test tube rooting culture: The strawberry seedlings obtained by subculture are taken out and inserted into a matrix containing a rooting agent for non-test tube rooting culture; (4) Hardening and transplanting: After the rooting, the strawberry seedlings are moved to the greenhouse for hardening. After the hardening is completed, they are transplanted to the field or greenhouse.

2. The rapid propagation method according to claim 1, characterized in that In step (2), the callus tissue culture medium is MS basal culture medium + 0.5-1.0 mg / L 6-BA + 0.1-0.2 mg / L NAA.

3. The rapid propagation method according to claim 1, characterized in that In step (2), the secondary culture medium is MS basal medium + 0.2-0.5 mg / L 6-BA + 0.05-0.1 mg / L NAA.

4. The rapid propagation method according to claim 1, characterized in that In step (2), the conditions for the primary culture and the secondary culture are: temperature 25±2°C, light intensity 2000-3000 lux, and illumination time 12 hours / day; The period of the primary culture is 4-6 weeks; the period of the secondary culture is 3-4 weeks.

5. The rapid propagation method according to claim 1, characterized in that In step (3), the rooting agent is 100-200 mg / LIBA solution.

6. The rapid propagation method according to claim 5, characterized in that The rooting agent is used in an amount of 100-200 mg per liter of substrate.

7. The rapid propagation method according to claim 6, characterized in that The matrix includes perlite and vermiculite, and the mixing mass ratio of the perlite to the vermiculite is 1:1-3.

8. The rapid propagation method according to claim 1, characterized in that The non-test tube rooting culture conditions are as follows: humidity 80%-90%, temperature 25±2° C., light intensity 3000-4000 lux, light duration 12 hours / day, and culture for 2-3 weeks.

9. The rapid propagation method according to claim 1, characterized in that In step (4), the conditions for hardening the seedlings are: humidity 60-70%, light 5000-6000 lux, and time 1-2 weeks.

10. A strawberry seedling, characterized in that: The rapid propagation method according to any one of claims 1 to 9 is used for cultivation.

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