Dracaena cambodiana high-efficiency tissue culture and rapid propagation method

Abstract

The application discloses a high-efficiency tissue culture and rapid propagation method of Dracaena cochinchinensis, and belongs to the field of plant biotechnology. The method comprises the steps of external explant disinfection, callus induction, cluster bud differentiation and proliferation, rooting culture, and seedling raising and transplanting. Through optimization of a culture medium formula and culture conditions, the callus induction rate is greater than or equal to 90%, the proliferation coefficient is greater than or equal to 8.0, the rooting rate is greater than or equal to 95%, and the transplanting survival rate is greater than or equal to 90%, and the method has the advantages of fast propagation speed, healthy and uniform seedlings, low contamination rate and suitability for factory production, and can be widely applied to rapid propagation of various Dracaena cochinchinensis, such as Hainan Dracaena cochinchinensis, sword-leaf Dracaena cochinchinensis and fragrant Dracaena cochinchinensis.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology breeding and rapid seedling propagation technology, specifically involving a tissue culture method for disinfecting explants of Dracaena cochinchinensis, inducing callus, differentiating and proliferating clustered buds, rooting culture, and hardening and transplanting seedlings. Background Technology

[0002] Dracaena spp. is a precious ornamental, medicinal, and ecologically protected tree species belonging to the genus Dracaena in the family Agavaceae. Important species include Dracaena hainanensis, Dracaena lanceolata, and Dracaena fragrans. Traditional propagation methods, primarily sowing and cutting, suffer from low propagation coefficients, long cycles, easy degradation of germplasm, and seasonal limitations, making it difficult to meet the demands of large-scale production.

[0003] Existing tissue culture techniques for Dracaena fragrans generally suffer from drawbacks such as high explant contamination rates, unstable callus induction, low proliferation rates of clustered shoots, difficulty in rooting, and low transplant survival rates, which limit the industrial-scale seedling production and promotion of Dracaena fragrans. Therefore, establishing an efficient, stable, and reproducible rapid propagation system for Dracaena fragrans tissue culture is of significant application value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for tissue culture of Dracaena fragrans with low pollution rate, high induction rate, high proliferation rate, robust rooting, and high transplant survival rate, which is suitable for the rapid industrial propagation of various types of Dracaena fragrans.

[0005] The technical solution of this invention is as follows: A method for efficient tissue culture and rapid propagation of Dracaena cochinchinensis includes the following steps: (1) Selection and disinfection of explants: Select healthy young stem segments or stem tips of Dracaena cochinchinensis, rinse with running water, disinfect with 75% ethanol, sterilize with 0.1% mercuric chloride solution, and rinse with sterile water to obtain sterile explants; (2) Callus induction: Sterile explants were inoculated into callus induction medium and cultured in the dark to obtain callus tissue; (3) Differentiation and proliferation of clustered shoots: The callus tissue was transferred into the differentiation and proliferation medium and cultured under light to obtain a large number of robust clustered shoots; (4) Rooting culture: Transfer healthy single buds into rooting culture medium and culture under light to form complete plants; (5) Hardening and transplanting: After hardening the tissue culture seedlings, transplant them into the substrate to obtain living Dracaena seedlings.

[0006] The culture medium formulations are as follows: Callus induction medium: MS + 6BA 2.0–3.0 mg / L + 2,4D 0.5–1.0 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8; Differentiation and proliferation medium: MS + 6BA 1.5–2.5 mg / L + NAA 0.1–0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8; Rooting medium: 1 / 2 MS + IBA 0.3–0.6 mg / L + NAA 0.2–0.4 mg / L + sucrose 25 g / L + agar 7.0 g / L, pH 5.8.

[0007] Culture conditions: temperature 25-28℃, light intensity 1500-2000 lx, light duration 12 h / d; callus induction was performed in dark culture.

[0008] The beneficial effects of this invention are: explant contamination rate is less than 5%, callus induction rate is ≥90%, proliferation coefficient is ≥8.0, rooting rate is ≥95%, transplant survival rate is ≥90%, propagation speed is fast, seedlings are uniform and strong, and it is suitable for factory seedling production. Detailed Implementation

[0009] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are used to understand the present invention and do not limit the scope of protection.

[0010] Example 1: Rapid propagation of Dracaena fragrans hainanensis through tissue culture 1. Explant selection and disinfection Select healthy, disease-free young stem segments of Hainan dragon blood tree, 1.5–2.0 cm in length. Rinse with running water for 2 hours → Immerse in 75% ethanol for 30 seconds in a clean bench → Sterilize with 0.1% mercuric chloride solution for 10 minutes → Rinse with sterile water 6 times, 3 minutes each time → Blot dry with sterile filter paper.

[0011] 2. Callus induction The sterilized stem segments were inoculated into MS medium containing 2.5 mg / L 6BA, 0.8 mg / L 2,4D, 30 g / L sucrose, and 7.0 g / L agar, at pH 5.8. Culture conditions: 25±1℃, dark culture for 18 days. Results: The cut site showed significant swelling, forming a dense, pale yellow callus with a callus induction rate of 93%.

[0012] 3. Differentiation and proliferation of clustered buds Callus tissue was transferred to MS medium containing 2.0 mg / L 6BA, 0.2 mg / L NAA, 30 g / L sucrose, and 7.0 g / L agar, at pH 5.8. Culture conditions: temperature 26±1℃, light intensity 2000 lx, light duration 12 h / d, cultured for 28 days. Results: The callus tissue differentiated into numerous clusters of shoots; the shoots were robust, with dark green leaves, and the proliferation coefficient was 9.2.

[0013] 4. Rooting culture Healthy single buds, 2–3 cm in height, were selected and transferred to: 1 / 2 MS medium + IBA 0.5 mg / L + NAA 0.3 mg / L + sucrose 25 g / L + agar 7.0 g / L, pH 5.8. Culture conditions: temperature 25±1℃, light intensity 1800 lx, light duration 12 h / d, culture for 22 days. Results: Each plantlet produced 4–6 roots, which were white, robust, and 2–4 cm long, with a rooting rate of 96%.

[0014] 5. Hardening off seedlings and transplanting Rooted seedlings were hardened off for 4 days under natural diffused light indoors. The tissue culture seedlings were carefully removed, and the agar on the roots was washed away. They were then transplanted into a substrate mixture of peat moss, perlite, and vermiculite in a ratio of 3:1:1. After transplanting, the air humidity was maintained at 85%–90%, and the seedlings were shaded for 7 days, after which normal management was implemented. Results: The transplant survival rate was 92%, and the seedlings grew uniformly with no variation.

[0015] Example 2: Rapid propagation of Dracaena fragrans by tissue culture 1. Explant selection and disinfection Select stem tip meristems of *Dracaena fragrans*, approximately 0.5 cm in length. Rinse with running water for 2 h → sterilize with 75% ethanol for 30 s → sterilize with 0.1% mercuric chloride for 9 min → rinse 5 times with sterile water → blot dry with sterile filter paper.

[0016] 2. Callus induction Culture medium: MS medium + 6BA 2.0 mg / L + 2,4D 0.6 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8. After 16 days of dark culture, callus induction rate was 91%.

[0017] 3. Differentiation and proliferation of clustered buds Culture medium: MS medium + 6BA 1.8 mg / L + NAA 0.15 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8. Light intensity: 2000 lx, 12 h / d, temperature: 26℃, cultured for 26 days. Results: The clustered shoots differentiated uniformly, with a proliferation coefficient of 8.5.

[0018] 4. Rooting culture Culture medium: 1 / 2 MS medium + IBA 0.4 mg / L + NAA 0.25 mg / L + sucrose 25 g / L + agar 7.0 g / L, pH 5.8. After 20 days of culture, each plantlet produced 3-5 roots, with a rooting rate of 95%.

[0019] 5. Hardening off seedlings and transplanting After 3 days of hardening off, the transplanting substrate was the same as above, and the survival rate of transplanted seedlings was 90%.

[0020] Example 3: Rapid propagation of Dracaena fragrans (Dracaena sanderiana) through tissue culture 1. Explant selection and disinfection Select healthy stem segments with axillary buds, 1.5 cm in length. Rinse with running water for 2 h → sterilize with 75% ethanol for 30 s → sterilize with 0.1% mercuric chloride for 10 min → rinse 6 times with sterile water → blot dry with sterile filter paper.

[0021] 2. Callus induction Culture medium: MS medium + 6BA 3.0 mg / L + 2,4D 1.0 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8. After 19 days of dark culture, callus induction rate was 92%.

[0022] 3. Differentiation and proliferation of clustered buds Culture medium: MS medium + 6BA 2.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8. Light intensity 2000 lx, 12 h / d, temperature 26℃, cultured for 28 days. Results: Proliferation coefficient 9.6, robust buds.

[0023] 4. Rooting culture Culture medium: 1 / 2 MS medium + IBA 0.6 mg / L + sucrose 25 g / L + agar 7.0 g / L, pH 5.8. After 21 days of culture, the rooting rate was 97%, and the root system was well-developed.

[0024] 5. Hardening off seedlings and transplanting After 5 days of hardening off, the transplant survival rate was 93%.

[0025] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for high efficient tissue culture and rapid propagation of Dracaena cochinchinensis, characterized in that, Includes the following steps: (1) Selection and disinfection of explants: Select tender stem segments or shoot tips of Dracaena cochinchinensis, rinse with running water, disinfect with 75% ethanol, sterilize with 0.1% mercuric chloride, and rinse with sterile water to obtain sterile explants; (2) Callus induction: Inoculate the sterile explants into the callus induction medium and culture in the dark to obtain callus tissue; (3) Differentiation and proliferation of clustered shoots: Transfer the callus tissue into the differentiation and proliferation medium and culture under light to obtain clustered shoots; (4) Rooting culture: Transfer the healthy single shoots into the rooting medium and culture under light to form complete plants; (5) Hardening and transplanting: After hardening the tissue culture seedlings, transplant them into the substrate to obtain surviving seedlings; Among them: the callus induction medium is MS + 6BA 2.0~3.0 mg / L + 2,4D 0.5~1.0 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8; the differentiation and proliferation medium is MS + 6BA 1.5–2.5 mg / L + NAA 0.1–0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8; Rooting medium was 1 / 2 MS + IBA 0.3–0.6 mg / L + NAA 0.2–0.4 mg / L + sucrose 25 g / L + agar 7.0 g / L, pH 5.8; Culture temperature was 25–28℃, light intensity was 1500–2000 lx, light duration was 12 h / d, and callus induction was performed in the dark.

2. The method of claim 1, wherein, Step (1) Disinfection is as follows: soak in 75% ethanol for 30 seconds, sterilize with 0.1% mercuric chloride for 8 to 12 minutes, and rinse with sterile water 5 to 6 times.

3. The method of claim 1, wherein, Step (5) The transplanting substrate is peat moss: perlite: vermiculite = 3:1:

1.

4. The method of claim 1, wherein, The dragon blood trees mentioned include Hainan dragon blood tree, sword-leaf dragon blood tree, and fragrant dragon blood tree.