Tissue culture method of hemerocallis middendorfii'white swan '

Abstract

The invention discloses a tissue culture method of hemerocallis middendorfii'white swan '. The method comprises the following steps: (1) obtaining a sterile material; (2) callus induction; (3) bud differentiation; (4) rooting culture; and (5) seedling hardening and transplanting. According to the method, underground buds serve as explants, complete plants are cultured through a series of culture medium formulas including explant disinfection, callus induction, bud differentiation and rooting culture, seedlings can be formed after 1-2 months of seedling hardening and transplanting, and the survival rate reaches 98%. The tissue culture method of the hemerocallis middendorfii'white swan 'is efficient and rapid, and lays a foundation for rapid propagation, popularization and application of the hemerocallis middendorfii.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a tissue culture method for the daylily 'White Swan'. Background Technology

[0002] Daylilies (Hemerocallis hybridus), a perennial herbaceous plant belonging to the genus Hemerocallis in the family Asphodelaceae, are abundant in varieties, with large, brightly colored flowers and a long flowering period. They are highly adaptable and have high ornamental value, demonstrating great practicality in landscaping and ecological conservation. In recent years, new varieties of daylilies have been introduced to my country, and researching simple and rapid propagation methods will help further promote and apply them in the country. Tissue culture, with its advantages of rapid propagation and stable plant traits, is the best way to mass-produce daylilies and meet market demand. Summary of the Invention

[0003] The purpose of this invention is to provide a tissue culture method for the large-flowered daylily 'White Swan', which has the advantages of good sterilization effect, low mortality rate, short cultivation cycle and good quality, and plays an important role in the propagation and promotion of superior varieties of large-flowered daylilies.

[0004] This invention is achieved through the following technical solution:

[0005] The tissue culture method for the daylily 'White Swan' includes the following steps:

[0006] (1) Obtaining sterile materials: In spring, underground shoots of daylilies were collected as explants for tissue culture. The collected explants were aseptically treated and cut into 1 cm pieces. 2 (1) Place the tissues in the callus induction medium. (2) Induction of callus: In the callus induction medium, under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, callus began to appear after 15 days. After subculture for 35 days, 1 cm of callus was obtained. 2 (3) Bud differentiation: The callus tissue obtained above is placed in the bud differentiation induction culture. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, the callus tissue begins to differentiate after 10 days and grows clustered buds. (4) Rooting culture: The clustered buds obtained above are cut into individual plants and placed in the rooting culture medium. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, the seedlings begin to grow roots at the base after 10 days of culture. (5) Hardening and transplanting: The tissue culture seedlings that have grown to 10 cm in height and have ≥5 roots are hardened in the bottle with the cap on for 2-3 days in a greenhouse with temperature 25-28 ℃, humidity 70-80%, and light intensity 3000 Lx. They are then transplanted into the substrate and cultured for 1-2 months to become seedlings.

[0007] Preferably, the callus induction medium is MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 7.8 g / L agar.

[0008] Preferably, the subculture medium is MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 7.8 g / L agar.

[0009] Preferably, the bud differentiation medium is MS + 0.6 mg / L 6-BA + 7.8 g / L agar.

[0010] Preferably, the rooting medium is MS + 0.5 mg / L NAA + 7.8 g / L agar.

[0011] Preferably, the volume ratio of the substrate is nutrient soil: vermiculite: perlite = 1:1:1.

[0012] This invention provides a tissue culture method for the daylily 'White Swan'.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention successfully obtained a culture medium for the induction of callus and rooting of Hemerocallis 'White Swan', which is faster than other Hemerocallis propagation methods.

[0015] This invention is of certain significance for the propagation and promotion of superior varieties of daylily. Attached Figure Description

[0016] In Figure 1, A, B, C, and D are explants taken from underground shoots of daylilies as tissue culture materials. After aseptic treatment, the collected explants were cut into 1 cm pieces. 2 A photo of the left and right sides, placed in the primary culture medium.

[0017] Appendix Figure 2 Photographs A, B, C, and D show plant tissues that have induced callus formation in callus induction medium after callus induction.

[0018] Appendix Figure 3 Photos A, B, C, and D show the buds induced from the callus tissue after it was placed in a bud differentiation induction culture.

[0019] Appendix Figure 4 Photos A and B show the seedlings placed in rooting culture medium, where the plant tissues begin to grow roots.

[0020] Appendix Figure 5Photos A, B, C, D, and E show plants that have already developed roots being transplanted into the substrate after hardening off. Photo E is a detail shot.

[0021] Appendix Figure 6 This is a flowchart of the tissue culture process for the daylily 'White Swan'. Detailed Implementation

[0022] The following examples are provided to further describe the technical solution of the present invention, but are not intended to limit the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and researchers in the art can achieve the results by following conventional tissue culture techniques. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0024] A tissue culture method for Daylily 'White Swan', comprising the following steps: (1) Obtaining sterile materials: In spring, underground shoots of daylilies were collected as explants for tissue culture. The collected explants were washed with detergent water and rinsed under running tap water for 20 min to ensure that the detergent was completely removed. They were then disinfected on a clean bench with 75% alcohol for 30 s, rinsed 5 times with sterile water, disinfected with 10% sodium hypochlorite solution for 15 min, rinsed 5 times with sterile water, and cut into 1 cm pieces. 2 Left and right size. (2) Induction of callus: The cut material was placed in callus induction medium MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 7.8 g / L agar. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, callus grew after 15 days. It was then placed in subculture medium MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 7.8 g / L agar. After 35 days, it grew to 1 cm. 2(3) Bud differentiation: The callus obtained above was placed in differentiation medium MS + 0.6 mg / L 6-BA + 7.8 g / L agar. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, the callus began to differentiate after 10 days and grew clustered buds. (4) Rooting culture: The clustered buds obtained above were cut into single plants and placed in rooting medium MS + 0.5 mg / L NAA + 7.8 g / L agar. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, roots began to grow after 10 days in the culture medium. (5) Hardening and transplanting: Tissue culture seedlings that have grown to 10 cm in height and have ≥5 roots are moved to a regular greenhouse with their bottles. After 3-5 days, the bottle caps are opened and the seedlings are hardened for 3-5 days at a temperature of 25-28 ℃, humidity of 70-80%, and light intensity of 3000 Lx. After hardening, the culture medium is washed off the roots of the tissue culture seedlings. The roots are soaked in a 1:1000 carbendazim solution for 30-60 seconds and then transplanted into a mixed substrate of peat and perlite. The seedlings are covered with plastic film and shade netting. They are cultivated for 1 week at a temperature of 25-30 ℃, humidity of 80-95%, and light intensity that is initially weak and then gradually increased. The film is then gradually opened. After 2 weeks, the seedlings are put into routine management and cultivated for 1-2 months to become seedlings.

[0025] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. The present invention extends to any new feature or combination disclosed in this specification, as well as any new method or process step or combination disclosed herein.

Claims

1. A tissue culture method of Hemerocallis 'White Swan', characterized by, The method includes the following steps: (1) Obtaining of sterile materials: The underground buds of Hemerocallis citrina Baroni were collected in spring as explants. The explants were cut into 1 cm2 pieces after sterile treatment and then put into callus induction medium. 2 left and right size, into callus induction medium; (2) Inducing callus: under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light time 12 h / d, callus appeared after 15 days in the callus induction medium, and 1 cm callus was obtained after 35 days 2 left and right. (3) Bud differentiation: The callus tissue obtained above was placed in the bud differentiation induction culture. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx and light time 12 h / d, the callus tissue began to differentiate after 10 days and grew into clustered buds. (4) Rooting culture: After cutting the above-obtained clustered buds into individual plants, they were placed in a rooting culture medium. Under the conditions of temperature 25±2 ℃, light intensity 2000 Lx, and light duration 12 h / d, roots began to grow from the base of the seedlings after 10 days of culture. (5) Hardening and transplanting: Tissue culture seedlings that have grown to 10 cm in height and have ≥5 roots are hardened in a greenhouse with a temperature of 25-28 ℃, humidity of 70-80% and light intensity of 3000 Lx for 2-3 days. They are then transplanted into the substrate and cultivated for 1-2 months to become seedlings.

2. The tissue culture method of Hemerocallis 'White Swan' according to claim 1, characterized by, The callus induction medium was MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 7.8 g / L agar.

3. The tissue culture method of Hemerocallis 'White Swan' according to claim 1, wherein The subculture medium was MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 7.8 g / L agar.

4. The tissue culture method of Hemerocallis 'White Swan' according to claim 1, wherein The bud differentiation medium was MS + 0.6 mg / L 6-BA + 7.8 g / L agar.

5. The tissue culture method of Hemerocallis 'White Swan' according to claim 1, wherein The rooting medium consisted of MS medium containing 0.5 mg / L NAA and 7.8 g / L agar.

6. The tissue culture method of Hemerocallis 'White Swan' according to claim 1, wherein The volume ratio of the substrate is nutrient soil: vermiculite: perlite = 1:1:1.

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