Induction and culture method of adventitious roots of dalbergia odorifera
By germinating seeds with cotyledons of the fragrant-reducing seeds and germinated, the problem of unbalanced supply and demand of raw materials for fragrance reduction is solved, and the efficient production of active ingredients for fragrance reduction is achieved.
Patent Information
- Application Number
- CN202510289423.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-06-03
AI Technical Summary
The natural ingredient cycle of reducing fragrance has long lead to imbalance in supply and demand for medicinal raw materials, and it is difficult for the existing technology to produce its effective ingredients on a rapid and large scale through biotechnology.
By germinating seed cotyledons as explants, intractable roots are induced and subcultured. The induction rate and stability of intractable roots are improved by using specific culture media and conditions.
It has achieved efficient induction and stable succession of incense-reducing incense roots, improved the induction rate of incense roots, solved the contradiction between supply and demand of raw materials for incense-reducing incense materials, and laid the foundation for large-scale production of effective ingredients for incense-reducing.
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Figure CN120077951A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant biotechnology, and particularly relates to an induction and cultivation method for adventitious roots of Dalbergia odorifera. Background Art
[0002] Adventitious roots are roots that occur on the stems or leaves of plants. In most cases, adventitious roots occur due to injury to plant organs or stimulation by external factors such as hormones and pathogenic microorganisms, which manifests as a regeneration response of the plant. The occurrence and formation of adventitious roots is an important way of asexual reproduction of plants. Adventitious root culture has the advantages of short growth cycle, small error in controllable conditions, single source of materials, consistent genetic background, economic convenience, strong repeatability, high efficiency, and year-round testing or growth. Applying plant adventitious root culture to medicinal plants can quickly and massively obtain adventitious roots and secondary metabolites of medicinal plants, thereby effectively solving problems such as insufficient wild resources. In addition, adventitious roots are not genetically modified materials, which have certain advantages for their later development and application. Moreover, the adventitious root culture of medicinal plants can overcome the shortcomings of low content and instability of effective ingredients in cell suspension culture. Therefore, the adventitious root culture of medicinal plants is an effective way to develop Chinese medicine resources.
[0003] Dalbergia odorifera T.Chen, also known as Hainan Huanghuali, fragrant wood, and Dalbergia odorifera, is a plant of the leguminous family. The dried heartwood of its trunk and roots can be used as medicine, which has the effects of promoting blood circulation and dispersing blood stasis, stopping bleeding and relieving pain. It is mainly used to treat traumatic bleeding, liver depression and flank pain. In addition, the growth period of Dalbergia odorifera is relatively long, and the natural formation of heartwood is very slow. It takes more than 30 years to form heartwood for medicinal use. How to alleviate the contradiction between the supply and demand market of Dalbergia odorifera has become a key issue that needs to be solved in current production. The use of biotechnology to propagate Chinese medicine resources and mass-produce its effective ingredients has become a new development path, and there is no report on the use of biotechnology to produce the effective ingredients of Dalbergia odorifera. Summary of the invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for inducing and culturing adventitious roots of Dalbergia odorifera.
[0005] The technical solution of the present invention is summarized as follows:
[0006] The induction and cultivation method of the adventitious roots of Dalbergia odorifera comprises the following steps:
[0007] 1) Scrub the Dalbergia odorifera seeds clean, soak them in distilled water for 2 - 4 h, soak and disinfect them in an ethanol aqueous solution with a volume fraction of 75% in a laminar flow hood, rinse them 2 - 3 times with sterilized distilled water, then soak and disinfect them in a sodium hypochlorite aqueous solution with a mass concentration of 20% for 5 - 15 min, rinse them 2 - 3 times with sterilized distilled water, remove the outer seed coat and then soak and disinfect them in an ethanol aqueous solution with a volume fraction of 75%, rinse them 2 - 3 times with sterilized distilled water, blot the moisture with filter paper, inoculate them into the initiation medium, and culture them for germination at 25 ± 2 °C with a light intensity of 1000 - 2000 lux for 2 - 5 weeks to obtain germinated seeds;
[0008] 2) Inoculate the cotyledons of the germinated seeds obtained in step 1) into the adventitious root induction medium, and induce culture them at 25 ± 2 °C with a light intensity of 1000 - 2000 lux for 3 - 6 weeks to obtain Dalbergia odorifera adventitious roots;
[0009] 3) Inoculate the Dalbergia odorifera adventitious roots obtained in step 2) into the liquid medium, and culture them on a shaker at a rotational speed of 1000 - 1200 rpm for 30 d to obtain Dalbergia odorifera adventitious roots that can be stably subcultured.
[0010] Preferably, the initiation medium is: 1 / 2MS + agar 7.0 - 8.0 g / L + sucrose 15 - 30 g / L + GA 3 0.5 - 1.5 mg / L + Vc 50 - 100 mg / L, with a pH of 5.0 - 5.5.
[0011] Preferably, the adventitious root induction medium is: 1 / 2MS + agar 7.0 - 8.0 g / L + sucrose 15 - 30 g / L + NAA 1.5 - 3 mg / L + Vc 50 - 100 mg / L, with a pH of 5.0 - 5.5.
[0012] Preferably, the liquid medium is: B5 + IBA 0.5 - 3 mg / L + sucrose 10 - 30 g / L, with a pH = 4.5 - 5.5.
[0013] In the present invention, by using the cotyledons of Dalbergia odorifera germinated seeds as explants to induce adventitious roots and conduct subculture, the induction rate of adventitious roots reaches 72.3% - 86.7%, laying a foundation for the future use of Dalbergia odorifera adventitious roots for important active ingredients in industrial production, etc., and at the same time solving the contradiction between supply and demand of Dalbergia odorifera raw materials. Description of the Drawings
[0014] Figure 1 Germinated seeds of Dalbergia odorifera cultured for 2 weeks.
[0015] Figure 2 Dalbergia odorifera adventitious roots obtained by culturing with the cotyledons of Dalbergia odorifera germinated seeds as explants.
[0016] Figure 3The obtained final adventitious roots of Dalbergia odorifera T. Chen Specific Embodiments
[0017] The present invention will be further described below in conjunction with embodiments. However, they should not be construed as limiting the protection scope of the present invention.
[0018] Example 1
[0019] The starting medium is: 1 / 2MS + 7.5 g / L agar + 20 g / L sucrose + GA 3 1 mg / L + 75 mg / L Vc, with a pH of 5.3.
[0020] Example 2
[0021] The starting medium is: 1 / 2MS + 7.0 g / L agar + 30 g / L sucrose + GA 3 0.5 mg / L + 50 mg / L Vc, with a pH of 5.0.
[0022] Example 3
[0023] The starting medium is: 1 / 2MS + 8.0 g / L agar + 15 g / L sucrose + GA 3 1.5 mg / L + 100 mg / L Vc, with a pH of 5.5.
[0024] Example 4
[0025] The adventitious root induction medium is: 1 / 2MS + 7.5 g / L agar + 20 g / L sucrose + 2 mg / L NAA + 75 mg / L Vc, with a pH of 5.3.
[0026] Example 5
[0027] The adventitious root induction medium is: 1 / 2MS + 7.0 g / L agar + 30 g / L sucrose + 1.5 mg / L NAA + 50 mg / L Vc, with a pH of 5.0.
[0028] Example 6
[0029] The adventitious root induction medium is: 1 / 2MS + 8.0 g / L agar + 15 g / L sucrose + 3 mg / L NAA + 100 mg / L Vc, with a pH of 5.5.
[0030] Example 7
[0031] The liquid medium is: B5 + 2 mg / L IBA + 20 g / L sucrose, pH = 5.0.
[0032] Example 8
[0033] The liquid medium is: B5 + IBA 0.5 mg / L + sucrose 30 g / L, pH = 4.5.
[0034] Example 9
[0035] The liquid medium is: B5 + IBA 3 mg / L + sucrose 10 g / L, pH = 5.5.
[0036] Example 10
[0037] An induction and culture method for adventitious roots of Dalbergia odorifera T.Chen, comprising the following steps:
[0038] 1) Scrub the Dalbergia odorifera seeds clean, soak them in distilled water for 3 h, soak and disinfect them in a 75% (v / v) ethanol aqueous solution for 30 s in a laminar flow hood, rinse them twice with sterilized distilled water, then soak and disinfect them in a 20% (w / v) sodium hypochlorite aqueous solution for 10 min, rinse them twice with sterilized distilled water, remove the outer seed coat, soak and disinfect them in a 75% (v / v) ethanol aqueous solution for 30 s, rinse them twice with sterilized distilled water, blot dry with filter paper, and inoculate them into the initiation medium (prepared in Example 1). Incubate them at 25 °C with a light intensity of 1500 lux for 2 weeks of germination culture to obtain germinated seeds, see Figure 1 ;
[0039] 2) Inoculate the cotyledons of the germinated seeds obtained in step 1) into the adventitious root induction medium (prepared in Example 4). Incubate them at 25 °C with a light intensity of 1500 lux for 4 weeks of induction culture to obtain adventitious roots of Dalbergia odorifera, see Figure 2 ; The induction rate is 86.7%;
[0040] 3) Inoculate the adventitious roots of Dalbergia odorifera obtained in step 2) into the liquid medium (prepared in Example 7), and culture them on a shaker at a rotational speed of 1100 rpm for 30 d to obtain adventitious roots of Dalbergia odorifera that can be stably subcultured, see Figure 3 .
[0041] Example 11
[0042] An induction and culture method for adventitious roots of Dalbergia odorifera T.Chen, comprising the following steps:
[0043] 1) Scrub the Dalbergia odorifera seeds clean, soak them in distilled water for 2 h, soak and disinfect them in a 75% (v / v) ethanol aqueous solution for 30 s in a laminar flow hood, rinse them twice with sterilized distilled water, then soak and disinfect them in a 20% (w / v) sodium hypochlorite aqueous solution for 5 min, rinse them three times with sterilized distilled water, remove the outer seed coat, soak and disinfect them in a 75% (v / v) ethanol aqueous solution for 30 s, rinse them twice with sterilized distilled water, blot dry with filter paper, and inoculate them into the initiation medium (prepared in Example 2). Incubate them at 27 °C with a light intensity of 1000 lux for 2 weeks of germination culture to obtain germinated seeds;
[0044] 2) Inoculate the cotyledons of the germinated seeds obtained in step 1) into the adventitious root induction medium (prepared in Example 5), and culture them at 27 °C with a light intensity of 1000 lux for 3 weeks to obtain Dalbergia odorifera adventitious roots; the induction rate is 80.3%.
[0045] 3) Inoculate the Dalbergia odorifera adventitious roots obtained in step 2) into the liquid medium (prepared in Example 8), and culture them on a shaker at a rotation speed of 1000 rpm for 30 d to obtain Dalbergia odorifera adventitious roots that can be stably subcultured.
[0046] Example 12
[0047] A method for inducing and culturing Dalbergia odorifera adventitious roots, comprising the following steps:
[0048] 1) Scrub the Dalbergia odorifera seeds clean, soak them in distilled water for 4 h, soak and disinfect them in an aqueous ethanol solution with a volume fraction of 75% for 30 s in a laminar flow cabinet, rinse them 3 times with sterilized distilled water, then soak and disinfect them in an aqueous sodium hypochlorite solution with a mass concentration of 20% for 15 min, rinse them 2 times with sterilized distilled water, remove the outer seed coat, soak and disinfect them in an aqueous ethanol solution with a volume fraction of 75% for 30 s, rinse them 3 times with sterilized distilled water, blot the water with filter paper, and inoculate them into the initiation medium (prepared in Example 3), and culture them at 23 °C with a light intensity of 2000 lux for 5 weeks to obtain germinated seeds;
[0049] 2) Inoculate the cotyledons of the germinated seeds obtained in step 1) into the adventitious root induction medium (prepared in Example 6), and culture them at 23 °C with a light intensity of 2000 lux for 6 weeks to obtain Dalbergia odorifera adventitious roots; the induction rate is 72.3%.
[0050] 3) Inoculate the Dalbergia odorifera adventitious roots obtained in step 2) into the liquid medium (prepared in Example 9), and culture them on a shaker at a rotation speed of 1200 rpm for 30 d to obtain Dalbergia odorifera adventitious roots that can be stably subcultured.
[0051] Comparative Experiment 1
[0052] Replace the adventitious root induction medium in step 2) of Example 10 with each medium in Table 1, and keep the others the same as in Example 10 to determine the effect of different adventitious root induction media on the adventitious root induction rate. The results are shown in Table 1.
[0053] Table 1 Effect of different adventitious root induction media on the adventitious root induction rate
[0054]
Claims
1. A method for inducing and cultivating adventitious roots of Dalbergia odorifera, characterized in that The steps include: 1) Clean the Dalbergia odorifera seeds, soak them in distilled water for 2-4 hours, soak them in a 75% ethanol aqueous solution in a clean bench for disinfection, rinse them with sterile distilled water for 2-3 times, soak them in a 20% sodium hypochlorite aqueous solution for disinfection for 5-15 minutes, rinse them with sterile distilled water for 2-3 times, peel off the outer seed coat, soak them in a 75% ethanol aqueous solution for disinfection, rinse them with sterile distilled water for 2-3 times, dry them with filter paper, inoculate them into a start-up culture medium, and culture them for germination for 2-5 weeks at 25±2° C. and a light intensity of 1000-2000 lux to obtain germinated seeds; 2) inoculating the cotyledons of the germinated seeds obtained in step 1) into an adventitious root induction medium, inducing and culturing at 25±2° C. and a light intensity of 1000-2000 lux for 3-6 weeks to obtain adventitious roots of Dalbergia odorifera; 3) The Dalbergia odorifera adventitious roots obtained in step 2) are inoculated into a liquid culture medium, and cultured in a shaking table at a rotation speed of 1000-1200 rpm to obtain Dalbergia odorifera adventitious roots that can be stably subcultured.
2. The method according to claim 1, characterized in that The starting culture medium is: 1 / 2MS+agar 7.0-8.0 g / L+sucrose 15-30 g / L+GA3 0.5-1.5 mg / L+Vc 50-100 mg / L, and the pH value is 5.0-5.
5.
3. The method according to claim 1, characterized in that The adventitious root induction medium is: 1 / 2MS+agar 7.0-8.0 g / L+sucrose 15-30 g / L+NAA 1.5-3 mg / L+Vc 50-100 mg / L, and the pH is 5.0-5.
5.
4. The method according to claim 1, characterized in that The liquid culture medium is: B5+IBA 0.5-3 mg / L+sucrose 10-30 g / L, pH=4.5-5.5.