Strawberry tissue culture and rapid propagation method using flower stalk as explant

Abstract

The application discloses a strawberry tissue culture and rapid propagation method taking flower stalks as explants, and steps are as follows: 1) picking strawberry inflorescences, cutting young flower stalks, and placing the flower stalks in sterilized triangular bottles; 2) adding 2% sodium hypochlorite solution and dropwise adding Tween-80 in the triangular bottles on an ultra-clean workbench, and shaking to sterilize and rinsing with sterile water; 3) after sterilizing the flower stalks, cutting a small amount of bottom tissue, and inoculating the flower stalks on an induction medium, culturing for 6 weeks under the condition of 25 DEG C, 16h light / 8h darkness, and obtaining seedlings; and 4) transplanting the seedlings into a culture medium to proliferate, separating the seedlings into single plants after 3-4 weeks, and transplanting the single plants into a rooting culture medium, so that strawberry aseptic seedlings are obtained after 3-4 weeks. The application takes strawberry flower stalks as explants, makes up for the instability of creeping stem sprouting and the small number of stems in stem tip culture, has a simple whole tissue culture process, short culture time, high increment rate, and is easy to operate, and can be used for variety preservation, variety rejuvenation assistance and industrial production.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, specifically relating to a method for rapid propagation of strawberries using flower stalks as explants. Background Technology

[0002] Strawberries are perennial evergreen herbaceous plants belonging to the genus *Fragaria* in the family Rosaceae. Strawberry berries are fragrant, juicy, sweet and sour, and rich in nutrients, earning them the title of "Queen of Fruits." Cultivated strawberries are octoploid pineapple strawberries, which are susceptible to fungal and bacterial diseases. Strawberry production primarily relies on asexual reproduction via runners, making seedlings highly susceptible to carrying pathogens, severely impacting the strawberry industry's development. Healthy seedlings have become a crucial element of the entire strawberry industry. The promotion of aseptic tissue culture seedlings can effectively cut off the spread of pathogens, ensuring the production of healthy seedlings.

[0003] Currently, aseptic tissue culture seedlings are mainly obtained from strawberry shoot tips as explants, with a small amount produced from leaves, sepals, axillary buds, root tips, and runners. Strawberry runner emergence is concentrated in the warmer, longer-dayd spring and early summer, with fewer runners emerging during the flowering and fruiting period. The number of runners varies considerably from batch to batch, and the number of runners in perennial varieties is often lower than in uniseason varieties. Tissue culture using flower stalks as explants complements shoot tip culture using runners, overcoming the time limitations of material acquisition for tissue culture. To date, there are no mature literature reports on the technique of rapid propagation using strawberry flower stalks as explants. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a method for rapid propagation of strawberries using flower stalks as explants through tissue culture, specifically achieved through the following technical solution:

[0005] A method for rapid propagation of strawberries via tissue culture using flower stalks as explants includes the following steps:

[0006] 1) Selection of explants: Cut off healthy strawberry inflorescences from the field, cut off tender flower stalks, and place them in a sterilized 100mL Erlenmeyer flask;

[0007] 2) Sterilization treatment: Place the Erlenmeyer flask on the ultra-clean workbench, add an appropriate amount of freshly prepared 2% sodium hypochlorite solution to the Erlenmeyer flask, and add 1-2 drops of surfactant Tween-80 at the same time. After sterilization treatment by gently shaking, rinse 4-5 times with sterile water.

[0008] 3) Induction of seedlings: After removing a small amount of the bottom tissue of the flower stalk after sterilization in step 2), inoculate it onto the induction medium for culture. The culture conditions are: 25℃, 16h light / 8h dark for 6 weeks to obtain seedlings.

[0009] 4) Proliferation and rooting culture: The obtained seedlings are transferred to the same culture medium as in step 3) for propagation. After 3-4 weeks, the proliferated seedlings are separated into individual plants and transferred to the rooting culture medium for further cultivation. After 3-4 weeks, sterile strawberry seedlings are obtained.

[0010] Furthermore, in step 1), unopened buds and bracts are retained on the tender flower stalks.

[0011] Furthermore, in step 2), an appropriate amount of sodium hypochlorite solution should be used to submerge the flower stalks, and the sterilization time is 15 minutes.

[0012] Further, in step 3), the induction medium is: MS basal medium, 0.3 mg / L 6-BA + 0.025 mg / L NAA, pH 5.8.

[0013] Furthermore, the rooting medium in step 4) is: 1 / 2 MS basal medium, 0.02 mg / L NAA, pH 5.8.

[0014] In conventional tissue culture, different culture media are often used for the induction, differentiation, and propagation stages. However, using flower stalks as explants results in less callus formation and a shorter seedling emergence time, allowing for a one-step seedling emergence method where all three stages use the same culture medium. This simplifies the entire tissue culture process, makes it easier to operate, shortens the seedling emergence time, and facilitates industrial production.

[0015] This invention uses strawberry flower stalks as explants, which can overcome the shortcomings of unstable and low-quantity stolon emergence in shoot tip culture. Simultaneously, callus formation is minimal, and the induction differentiation process is short, allowing for one-step seedling production using the same culture medium. The entire tissue culture process is simple, with short culture time, high multiplication rate, and easy operation, making it suitable for variety preservation, variety rejuvenation assistance, and industrial production. Attached Figure Description

[0016] Figure 1 This describes the tissue culture seedling formation process using the flower stalks of *Streptomyces rubra* as explants.

[0017] Figure 2 Different phenotypes were produced by tissue culture using different organs of Strawberry as explants;

[0018] Figure 3 Phenotypic results of tissue culture using flower stalks as explants for different strawberry varieties. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments and accompanying drawings in order to better understand the technical solution.

[0020] Example 1: Taking the "Red Cheek" strawberry as an example, an observation experiment was conducted on the tissue culture seedlings at different stages:

[0021] 1) Preparation of explants: Cut the "Red Cheek" strawberry inflorescences from the field, put them in a plastic bag and bring them back to the laboratory. Cut off the tender flower stalks of the strawberry, keeping the unopened flower buds and bracts on the flower stalks, and place them in a sterilized 100ml Erlenmeyer flask;

[0022] 2) Sterilization: Add an appropriate amount of freshly prepared 2% sodium hypochlorite solution to the laminar flow hood, ensuring the flower stalks are submerged; simultaneously add 1-2 drops of surfactant Tween-80, and gently shake to sterilize for 15 minutes. Rinse 4-5 times with sterile water;

[0023] 3) Induction of seedlings: After removing a small amount of basal tissue from the sterilized flower stalks, inoculate them onto culture medium (MS basal medium, 0.3 mg / L 6-BA + 0.025 mg / L NAA, pH 5.8), two flower stalks per bottle (250 ml glass bottle). Culture at 25℃ under 16h light / 8h dark conditions. After 1, 3, 4, and 6 weeks of culture, similar inflorescences are harvested and photographed under a dissecting microscope to compare the flower stalks used in the explant tissue culture seedling process. Figure 1 As shown. The experiment was repeated 3 times. From Figure 1 It can be seen that the rapid propagation of strawberries using the method of the present invention greatly shortens the tissue culture time because there is almost no callus formation.

[0024] Example 2: Comparison of tissue culture using different flower stalk parts as explants, taking "Pink Jade" strawberry as an example.

[0025] Experimental procedure:

[0026] 1) Preparation of explants: Cut the 'Pink Jade' strawberry inflorescences from the field, put them in a plastic bag and bring them back to the laboratory. Cut off the tender flower stalks of the strawberry, retaining the unopened buds and bracts on the flower stalks, and place them in a sterilized 100ml Erlenmeyer flask;

[0027] 2) Sterilization: Add an appropriate amount of freshly prepared 2% sodium hypochlorite solution to the laminar flow hood, ensuring the flower stalks are submerged; simultaneously add 1-2 drops of surfactant Tween-80, and gently shake to sterilize for 15 minutes. Rinse 4-5 times with sterile water;

[0028] 3) Tissue culture induction of different parts: After removing a small amount of basal tissue from the sterilized flower stalks, they were divided into 4 groups for further processing. Group A consisted of flowers + flower stalks with bracts; Group B consisted of flowers + flower stalks without bracts; Group C consisted of flower stalks with bracts; and Group D consisted of lateral flowers + flower stalks with bracts. The stalks were inoculated onto medium (MS basal medium, 0.3 mg / L 6-BA + 0.025 mg / L NAA, pH 5.8), with 2 flower stalks per bottle (250 ml glass bottle). The culture was carried out at 25℃ under 16h light / 8h darkness conditions. Similar inflorescences were harvested at 1, 3, 4, and 6 weeks of culture and photographed under a dissecting microscope. Figure 2As shown. The experiment was repeated 3 times. From Figure 2 It can be seen that, within the same time frame, the seedlings produced using the flower stalks with buds and bracts of this invention have a better seedling growth rate.

[0029] Example 3: Phenotypic results of different strawberry varieties in tissue culture using flower stalks as explants

[0030] Experimental procedure:

[0031] 1) Preparation of explants: Five varieties of strawberry inflorescences, namely “Fenyu”, “Yuexiu”, “Yuexin”, “Zhangji” and “Hongjia”, were cut and placed in plastic bags and brought back to the laboratory. The tender flower stalks of the strawberries were cut off, and the unopened flower buds and bracts on the flower stalks were retained and placed in sterilized 100ml Erlenmeyer flasks.

[0032] 2) Sterilization: Add an appropriate amount of freshly prepared 2% sodium hypochlorite solution to the laminar flow hood, ensuring the flower stalks are submerged; simultaneously add 1-2 drops of surfactant Tween-80, and gently shake to sterilize for 15 minutes. Rinse 4-5 times with sterile water;

[0033] 3) Tissue Culture Induction: After removing a small amount of basal tissue from the sterilized strawberry flower stalks, the tissue was divided into 5 groups and inoculated onto culture medium (MS basal medium, 0.3 mg / L 6-BA + 0.025 mg / L NAA, pH 5.8), with 2 flower stalks per bottle (250 ml glass bottle). Cultured at 25℃ under 16h light / 8h dark conditions. Similar inflorescences were harvested after 3 and 6 weeks of culture and photographed under a dissecting microscope, as shown... Figure 3 As shown. The experiment was repeated 3 times. From Figure 3 It can be seen that the method of the present invention is applicable to different varieties of strawberries and has strong applicability.

Claims

1. A method for rapid propagation of strawberries via tissue culture using flower stalks as explants, characterized in that... Includes the following steps: 1) Selection of explants for flower stalks: Cut off healthy strawberry inflorescences from the field, cut off tender flower stalks, and place them in a sterilized 100mL Erlenmeyer flask; the tender flower stalks should retain unopened buds and bracts; 2) Sterilization treatment: Place the Erlenmeyer flask on the ultra-clean workbench, add freshly prepared 2% sodium hypochlorite solution to the Erlenmeyer flask, and add 1-2 drops of surfactant Tween-80 at the same time. After sterilization treatment, gently shake and rinse with sterile water 4-5 times. 3) Seedling induction: After removing a small amount of the base tissue from the flower stalks that have undergone sterilization in step 2), inoculate them onto the induction medium for culture. The culture conditions are: 25℃, 16h light / 8h dark for 6 weeks to obtain seedlings. The induction medium is: MS basal medium, 0.3mg / L 6-BA + 0.025mg / L NAA, pH 5.

8. 4) Proliferation and rooting culture: The obtained seedlings are transferred to the same culture medium as in step 3) for propagation. After 3-4 weeks, the proliferated seedlings are separated into individual plants and transferred to the rooting culture medium for further cultivation. After 3-4 weeks, sterile strawberry seedlings are obtained.

2. The method for rapid propagation of strawberry tissue culture using flower stalks as explants as described in claim 1, characterized in that... In step 2), the sodium hypochlorite solution covers the flower stalks, and the sterilization time is 15 minutes.

3. The method for rapid propagation of strawberry tissue culture using flower stalks as explants as described in claim 1, characterized in that... In step 4), the rooting medium is: 1 / 2 MS basal medium, 0.02 mg / L NAA, pH 5.8.

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