Tissue culture and rapid propagation method for dragon flower

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for tissue culture and rapid propagation of dragon flower. The method comprises the steps of explant selection and disinfection, axillary bud growth induction, multiplication culture, rooting culture and acclimatization and transplantation. According to the method, by optimizing an explant disinfection process, an optimal culture medium formula of each culture stage is screened out, efficient, rapid and stable propagation of the dragon surface flowers is achieved, and the problems that progeny character separation and cutting propagation coefficients are low and are limited by seasons in traditional seed propagation are solved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for rapid propagation of *Lysimachia christinae* via tissue culture. Background Technology

[0002] Dragon Face Flower ( Nemesia strumosa Benth. is a biennial herbaceous plant belonging to the Scrophulariaceae family and the genus *Benth.*, growing to a height of 30-60 cm with numerous branches. Leaves are opposite; basal leaves are oblong-spatulate with entire margins, while stem leaves are lanceolate with 3-4 pairs of serrations. Racemes are borne at the tips of branches, forming corymbs with a pouch-like base. *Benth.* flowers come in a rich variety of colors, including white, yellow, and purple, often with yellow or dark spots or variegation in the throat. In landscaping, *Benth.* is renowned for its elegant flower shape and vibrant colors; when planted in large groups, it creates a visually striking effect, resembling a colorful cloud formation. It is an excellent material for flower beds, used alone or in combination with other plants, and is also suitable for potted plants or cut flowers, making it a favorite among gardening enthusiasts worldwide. Besides its ornamental value, some varieties of *Benth.* also possess a fragrant aroma, with potential applications in fragrance extraction and ecological horticulture.

[0003] However, the commercial production and promotion of *Lysimachia christinae* have long been severely constrained by its traditional propagation methods. *Lysimachia christinae* is conventionally propagated by seeds and cuttings. While seed propagation yields a large number of seedlings, it suffers from severe segregation of traits in offspring, failing to maintain the uniformity and stability of traits in superior varieties (especially F1 hybrids with unique colors or selected mutants), seriously affecting the uniformity of commercial flowers and their market competitiveness. While cutting propagation can preserve the superior traits of the parent plant, it is limited by seasonality, the availability of mother plant material, and a low propagation coefficient, making it unable to meet the market's demand for large-scale, year-round production of high-quality seedlings.

[0004] Plant tissue culture, as a key technology in modern agricultural production, boasts advantages such as rapid propagation, no seasonal limitations, and preservation of superior traits from the parent plant. It has become a core technology for the industrialized production of seedlings for major commercial flowers such as orchids, carnations, anthuriums, and gerberas. This technology can significantly enhance the value of *Scrophularia ningpoensis* and is an ideal way to overcome the bottlenecks in the industrialization of *Scrophularia ningpoensis* seedlings. Currently, although there are sporadic studies involving tissue culture of other plants in the Scrophulariaceae family, commercially available, efficient, and rapid tissue culture propagation techniques specifically for *Scrophularia ningpoensis* are still relatively scarce and immature both domestically and internationally.

[0005] Therefore, establishing a technical system that can efficiently, quickly, and stably propagate high-quality dragon face flower seedlings is of great significance for consolidating their superior traits, accelerating the promotion of new varieties, and realizing year-round standardized production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for rapid propagation of *Gynostemma pentaphyllum* via tissue culture.

[0007] To achieve the above-mentioned objectives of this invention, the specific technical solution adopted by this invention is as follows: A method for rapid propagation of *Gynostemma pentaphyllum* via tissue culture includes the following steps: (1) Explant disinfection: Take the stem segment with axillary buds of the dragon face flower, clean it, disinfect it in turn with ethanol solution and sodium hypochlorite solution, and then soak it in plant culture solution to obtain sterile explants; (2) Axillary bud growth induction: Sterile explants were inoculated into an induction medium to induce axillary bud growth; the induction medium was MS medium containing 1.0-1.5 mg / L 6-benzylaminopurine and 0.05-0.1 mg / L naphthaleneacetic acid; (3) Proliferation culture: The stem segments of the robust buds induced in step (2) were inoculated into the proliferation medium for the induction and proliferation of clustered buds. The proliferation medium was MS medium containing 0.05-0.1 mg / L thiamethoxam, 0.1-0.2 mg / L naphthaleneacetic acid and 0.5-1.0 mg / L gibberellin. (4) Rooting culture: The robust seedlings obtained in step (3) are inoculated into a rooting medium to induce rooting. The rooting medium is a 1 / 2 MS medium containing 0.8-1.2 g / L activated carbon, 1.0-1.5 mg / L indolebutyric acid and 0.1-0.2 mg / L naphthaleneacetic acid. (5) Hardening and transplanting: After hardening the complete tissue culture seedlings obtained in step (4), transplant them into the cultivation substrate.

[0008] Preferably, the cleaning in step (1) is as follows: use dish soap to create foam and clean for 25-35 minutes, then seal with double-layer gauze and rinse with running water for 0.8-1.2 hours; the volume concentration of the ethanol solution is 70-80%, the ethanol solution disinfection time is 25-35 seconds, and after ethanol solution disinfection, rinse with sterile water for 2-4 minutes.

[0009] Preferably, the sodium hypochlorite solution has a concentration of 2 wt%, contains 0.1% Tween-80 by volume, and is disinfected 2-3 times with the sodium hypochlorite solution, each time for 3.5-4.5 minutes. After disinfection with the sodium hypochlorite solution, it is rinsed 2-3 times with sterile water, each time for 2-4 minutes.

[0010] Preferably, the volume concentration of the plant cultivation solution is 0.4-0.6%, the soaking time is 8-12 minutes, and after soaking, it is rinsed with sterile water 2-3 times, each time for 2-4 minutes.

[0011] Preferably, the induction medium in step (2) is MS medium containing 1.5 mg / L 6-benzylaminopurine and 0.05 mg / L naphthaleneacetic acid.

[0012] Preferably, the proliferation medium in step (3) is MS medium containing 0.1 mg / L thiamethoxam, 0.2 mg / L naphthaleneacetic acid and 1.0 mg / L gibberellin.

[0013] Preferably, in step (4), the rooting medium is a 1 / 2 MS medium containing 1 g / L activated carbon, 1.5 mg / L indolebutyric acid and 0.2 mg / L naphthaleneacetic acid.

[0014] Preferably, sucrose and agar are added to the culture medium in steps (2) to (4), wherein the sucrose concentration of the induction medium and the proliferation medium is 28-32 g / L, the sucrose concentration of the rooting medium is 18-22 g / L, the agar concentration is 6-8 g / L, and the pH value of the culture medium is 5.5-6.0.

[0015] Preferably, the seedling hardening in step (5) includes: first, closing the bottle and placing it under natural light and temperature conditions for 5-7 days, and then opening the bottle cap halfway for 2-3 days. The cultivation substrate is a mixture of humus, perlite and vermiculite in a volume ratio of 2:1:1, and has been sterilized.

[0016] Preferably, the cultivation conditions in steps (2) to (4) are: temperature 23-27℃, light intensity 1500-2500lx.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention optimizes the disinfection process based on the characteristics of the explant of *Lysimachia christinae*. It adopts a three-step disinfection method of "ethanol-sodium hypochlorite-plant disinfectant" to effectively control the browning rate while ensuring the disinfection effect, and significantly reduce the contamination rate of explants to below 15%, laying a good foundation for subsequent culture.

[0018] (2) Through systematic screening, this invention has determined the optimal ratio of plant growth regulators for each stage (initiation, proliferation, and rooting) of *Lysimachia christinae* tissue culture. In particular, the introduction of an appropriate concentration of GA3 during the proliferation stage effectively promotes the robust growth of clustered buds, avoids excessive vegetative growth, and significantly improves the proliferation coefficient (up to 5.0 or higher) and the quality of buds.

[0019] (3) The tissue culture rapid propagation system established by this invention has standardized operation, clear process and high propagation efficiency. It can realize the rapid propagation of the asexual line of the excellent varieties of Longmianhua, break through the limitations of traditional propagation methods, and provide a reliable technical solution for the large-scale, industrialized and year-round seedling production of Longmianhua. Attached Figure Description

[0020] Figure 1 This is a flowchart of the rapid propagation method of *Gynostemma pentaphyllum* tissue culture according to the present invention.

[0021] In the figure: a. Explant of *Lysimachia christinae*; b. Initiation culture (axillary bud induction); cd. Stem segments and leaves inoculated on proliferation medium; ef. Callus tissue and shoot growth of stem segments and leaves; g. Rooting culture; h. Hardening and transplanting. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0023] Experimental materials: Healthy, disease-free, and flowering-normal single plants of *Lysimachia christinae* were selected from greenhouses. Young stem segments from the current year were collected as explants and immediately brought back to the laboratory for processing.

[0024] Experimental reagents: MS medium (Beijing Solarbio Science & Technology Co., Ltd., catalog number M8521), 1 / 2 MS medium (Beijing Solarbio Science & Technology Co., Ltd., catalog number M8526), ​​sucrose, agar (Beijing Solarbio Science & Technology Co., Ltd., catalog number A8190), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), indolebutyric acid (IBA), gibberellin (GA3), thidiazuron (TDZ), kinetin (KT), 2,4-dichlorophenoxyacetic acid (2,4-D), zeatin (ZT), sodium hypochlorite (NaClO), ethanol (75% v / v), plant growth regulator (Type II, purchased from Mumu Biotechnology Co., Ltd.), anhydrous ethanol, sterile water, Tween-80, and activated carbon. All reagents were of analytical grade.

[0025] Example 1 A method for rapid propagation of *Gynostemma pentaphyllum* via tissue culture is described below: (1) Explant disinfection: a. Pretreatment: Soak the stem segments in dish soap solution for 30 minutes and rinse with running water for 1 hour.

[0026] b. Aseptic disinfection: In a clean bench, immerse the stem segments in a 75% ethanol solution for 30 seconds, then rinse with sterile water for 3 minutes; next, immerse them in a 2wt% NaClO solution containing 0.1% (v / v) Tween-80 for 8 minutes (divided into two 4-minute intervals), then rinse with sterile water 3 times; finally, soak them in a 0.5% Plant Cultivation Cleaner Type II solution for 10 minutes, then rinse twice with sterile water. After blotting the surface moisture with sterile filter paper, cut them into 1-1.5 cm stem segments with axillary buds for later use.

[0027] (2) Axillary bud growth induction: Disinfected stem segments were inoculated into induction medium (MS + 1.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8). Culture conditions: temperature 25±2℃, light 2000 lx. Cultured for 20 days.

[0028] (3) Proliferation culture: Stem segments (1-1.5 cm) of sterile seedlings induced in step (2) were inoculated into proliferation medium (MS + 0.1 mg / L TDZ + 0.2 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar, pH 5.8). GA3 hormone is not resistant to high temperature and pressure; after autoclaving, the medium needs to be filtered and sterilized in a clean bench before adding the GA3 solution. After inoculation, the medium was first cultured in the dark for 5 days, then transferred to normal light culture at a temperature of 25℃±2℃ and a light intensity of 2000 lx for 40 days.

[0029] (4) Rooting culture: Healthy seedlings with a height of 2-3 cm and 3-4 true leaves were cut from the proliferation culture and inoculated into rooting medium (1 / 2MS + 1.5 mg / L IBA + 0.2 mg / L NAA + 1 g / L activated carbon + 20 g / L sucrose + 7 g / L agar, pH 5.8), cultured at 25℃±2℃, light intensity 2000 lx, for 20 days.

[0030] (5) Hardening off and transplanting: When the roots of the rooted seedlings reach 2-3 cm in length, move the culture bottles to a greenhouse and harden off for 7 days with the caps closed, then harden off for another 3 days with the caps partially open. Remove the seedlings, wash the culture medium from the roots with sterile water, soak them in a 0.1% carbendazim solution for 5 minutes, and transplant them into a sterilized substrate (humus: perlite: vermiculite = 2:1:1) after drying. Maintain humidity, provide 50% shade, keep the temperature at 25℃, and ventilate regularly. After 20 days, the survival rate of transplanted seedlings was 92%, and the plants were growing well.

[0031] Example 2 A method for rapid propagation of *Gynostemma pentaphyllum* via tissue culture is described below: (1) Explant disinfection: a. Pretreatment: Soak the stem segments in dish soap solution for 30 minutes and rinse with running water for 1 hour.

[0032] b. Aseptic disinfection: In a clean bench, immerse the stem segments in a 75% ethanol solution for 30 seconds, then rinse with sterile water for 3 minutes; next, immerse them in a 2wt% NaClO solution containing 0.1% (v / v) Tween-80 for 8 minutes (divided into two 4-minute intervals), then rinse with sterile water 3 times; finally, soak them in a 0.5% Plant Cultivation Cleaner Type II solution for 10 minutes, then rinse twice with sterile water. After blotting the surface moisture with sterile filter paper, cut them into 1-1.5 cm stem segments with axillary buds for later use.

[0033] (2) Axillary bud growth induction: Disinfected stem segments were inoculated into induction medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8). Culture conditions: temperature 25±2℃, light 2000 lx, culture for 20 days.

[0034] (3) Proliferation culture: Stem segments (1-1.5 cm) of sterile seedlings induced in step (2) were inoculated into proliferation medium (MS + 0.1 mg / L TDZ + 0.2 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar, pH 5.8). GA3 hormone is not resistant to high temperature and pressure; after autoclaving, the medium needs to be filtered and sterilized in a clean bench before adding the GA3 solution. After inoculation, the medium was first cultured in the dark for 5 days, then transferred to normal light culture at a temperature of 25℃±2℃ and a light intensity of 2000 lx for 40 days.

[0035] (4) Rooting culture: Cut healthy seedlings with a height of 2-3 cm and 3-4 true leaves from the proliferation culture, and inoculate them into rooting medium (1 / 2MS + 1.5 mg / L IBA + 0.1 mg / L NAA + 1 g / L activated carbon + 20 g / L sucrose + 7 g / L agar, pH 5.8), culture temperature 25℃±2℃, light intensity 2000lx, culture for 20 days.

[0036] (5) Hardening off and transplanting: When the roots of the rooted seedlings reach 2-3 cm in length, move the culture bottles to a greenhouse and harden off for 7 days with the caps closed, then harden off for another 3 days with the caps partially open. Remove the seedlings, wash the culture medium from the roots with sterile water, soak them in a 0.1% carbendazim solution for 5 minutes, and transplant them into a sterilized substrate (humus: perlite: vermiculite = 2:1:1) after drying. Maintain humidity, provide 50% shade, keep the temperature at 25℃, and ventilate regularly. After 20 days, the survival rate of transplanted seedlings was 90%, and the plants were growing well.

[0037] Example 3 A method for rapid propagation of *Gynostemma pentaphyllum* via tissue culture is described below: (1) Explant disinfection: a. Pretreatment: Soak the stem segments in dish soap solution for 30 minutes and rinse with running water for 1 hour.

[0038] b. Aseptic disinfection: In a clean bench, immerse the stem segments in a 75% ethanol solution for 30 seconds, then rinse with sterile water for 3 minutes; next, immerse them in a 2wt% NaClO solution containing 0.1% (v / v) Tween-80 for 10 minutes (divided into two 5-minute intervals), rinsing with sterile water three times; finally, soak them in a 0.5% Plant Cultivation Cleaner Type II solution for 10 minutes, rinsing twice with sterile water. After blotting the surface moisture with sterile filter paper, cut them into 1-1.5 cm stem segments with axillary buds for later use.

[0039] (2) Axillary bud growth induction: Disinfected stem segments were inoculated into induction medium (MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8). Culture conditions: temperature 25±2℃, light 2000 lx, culture for 20 days.

[0040] (3) Proliferation culture: Stem segments (1-1.5 cm) of sterile seedlings induced in step (2) were inoculated into proliferation medium (MS + 0.05 mg / L TDZ + 0.2 mg / L NAA + 0.5 mg / L GA3 + 30 g / L sucrose + 7 g / L agar, pH 5.8). GA3 hormone is not resistant to high temperature and pressure; after autoclaving, the medium needs to be filtered and sterilized in a clean bench before adding the GA3 solution. After inoculation, the medium was first cultured in the dark for 5 days, then transferred to normal light culture at a temperature of 25℃±2℃ and a light intensity of 2000 lx for 40 days.

[0041] (4) Rooting culture: Healthy seedlings with a height of 2-3 cm and 3-4 true leaves were cut from the proliferation culture and inoculated into rooting medium (1 / 2MS + 1.0 mg / L IBA + 0.2 mg / L NAA + 1 g / L activated carbon + 20 g / L sucrose + 7 g / L agar, pH 5.8), cultured at 25℃±2℃, light intensity 2000lx, for 20 days.

[0042] (5) Hardening off and transplanting: When the roots of the rooted seedlings reach 2-3 cm in length, move the culture bottles to a greenhouse and harden off for 7 days with the caps closed, then harden off for another 3 days with the caps partially open. Remove the seedlings, wash the culture medium from the roots with sterile water, soak them in a 0.1% carbendazim solution for 5 minutes, and transplant them into a sterilized substrate (humus: perlite: vermiculite = 2:1:1) after drying. Maintain humidity, provide 50% shade, keep the temperature at 25℃, and ventilate regularly. After 20 days, the survival rate of transplanted seedlings was 85%, and the plants were growing well.

[0043] Comparative Example 1 The only difference between this comparative example and Example 1 is the duration of disinfection with NaClO solution (containing 0.1% Tween-80) in step (1), specifically: Group A1: Disinfect with 2% NaClO solution for 5 min, rinse with sterile water 3 times, 3 min each time.

[0044] Group A2: Disinfect with 2% NaClO solution for 12 min (disinfect 3 times, 4 min each time), then rinse 3 times with sterile water, then soak in 0.5% Plant Cultivation Clean Type II solution for 10 min, and rinse twice with sterile water.

[0045] Comparative Example 2 The only difference between this comparative example and Example 1 is that the hormone ratio in the induction culture medium in step (2) is different, specifically: Group B1: MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar; Group B2: MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar; Group B3: MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar; Group B4: MS + 1.0 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar; Group B5: MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar; Group B6: MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

[0046] Comparative Example 3 The only difference between this comparative example and Example 1 is that the hormone in the induction culture medium in step (2) is different, specifically: MS + 1.0 mg / L 6-BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar (pH 5.8).

[0047] Comparative Example 4 The only difference between this comparative example and Example 1 is that the hormone ratio in the proliferation medium in step (3) is different, specifically: Group C1: MS + 0 mg / L TDZ + 0 mg / L NAA + 0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar; Group C2: MS + 0.05 mg / L TDZ + 0.1 mg / L NAA + 0.5 mg / L GA3 + 30 g / L sucrose + 7 g / L agar; Group C3: MS + 0.05 mg / L TDZ + 0.2 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar; Group C4: MS + 0.05 mg / L TDZ + 0.1 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar; Group C5: MS + 0.1 mg / L TDZ + 0.1 mg / L NAA + 0.5 mg / L GA3 + 30 g / L sucrose + 7 g / L agar; Group C6: MS + 0.1 mg / L TDZ + 0.1 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar; Group C7: MS + 0.1 mg / L TDZ + 0.2 mg / L NAA + 0.5 mg / L GA3 + 30 g / L sucrose + 7 g / L agar.

[0048] Comparative Example 5 The only difference between this comparative example and Example 1 is that the hormones in the proliferation medium in step (3) are different, specifically: MS + 1.5 mg / L KT + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

[0049] Comparative Example 6 The only difference between this comparative example and Example 1 is that the hormones in the proliferation medium in step (3) are different, specifically: MS + 0.5 mg / L ZT + 0.1 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar.

[0050] Comparative Example 7 The only difference between this comparative example and Example 1 is that the hormones in the proliferation medium in step (3) are different, specifically: MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 1.0 mg / L GA3 + 30 g / L sucrose + 7 g / L agar.

[0051] Comparative Example 8 The only difference between this comparative example and Example 1 is the hormone ratio in the rooting medium in step (4), which is different: Group D1: 1 / 2 MS + 0 mg / L IBA + 0 mg / L NAA + 1 g / L activated charcoal + 20 g / L sucrose + 7 g / L agar; Group D2: 1 / 2 MS + 0.5 mg / L IBA + 0.1 mg / L NAA + 1 g / L activated charcoal + 20 g / L sucrose + 7 g / L agar; Group D3: 1 / 2 MS + 0.5 mg / L IBA + 0.2 mg / L NAA + 1 g / L activated charcoal + 20 g / L sucrose + 7 g / L agar; Group D4: 1 / 2MS + 1.0 mg / L IBA + 0.1 mg / L NAA + 1 g / L activated charcoal + 20 g / L sucrose + 7 g / L agar.

[0052] Effect test Test Example 1: Contamination and Browning Rate Tests of Explants After Disinfection Select healthy, disease-free stem segments with axillary buds from the current year's growth of *Gynostemma pentaphyllum* in a greenhouse. Remove leaves and flower buds, place the segments in a beaker, and wash with detergent to create foam for 30 minutes. Then seal the beaker with double-layered gauze and rinse with running water for 1 hour to prevent explant leakage. Place the washed explants in a laminar flow hood, which has been pre-wiped with alcohol swabs and sterilized with UV light for 30 minutes. Place the *Gynostemma pentaphyllum* stem segments in a sterile tissue culture bottle, disinfect with 75% alcohol for 30 seconds, shake rapidly, and rinse with sterile water for 3 minutes. Then place the *Gynostemma pentaphyllum* stem segments in another sterile tissue culture bottle and disinfect using the methods described in Examples 1, 3, and Comparative Examples 1 (groups A1 and A2). After disinfection, blot dry the surface moisture of the stem segments with sterile filter paper and set aside. The stem segments of *Lysimachia christinae* were cut into 1-1.5 cm segments with axillary buds and inoculated onto MS basal medium. Forty explants were inoculated for each treatment. After 10 days of culture, the contamination rate and browning rate were counted.

[0053] Contamination rate = (Number of contaminated bottles / Number of inoculated bottles) × 100%; Browning rate = (brown explants / total number of explants inoculated) × 100%.

[0054] The test results are shown in Table 1.

[0055] Table 1. Results of contamination and browning rates of explants after disinfection.

[0056] Test Example 2: Effects of different hormone ratios on the induction of axillary buds on stem segments of *Lysimachia christinae* The effects of hormone treatments on axillary bud growth induction in Examples 1-3 and Comparative Examples 2-3 were tested. Sterilized stem segments were inoculated into the induction medium of each group in Examples 1-3 and Comparative Examples 2-3 at a depth of 0.5 cm. Twenty explants were inoculated per treatment, with three replicates. The plants were placed in a culture room at 25℃±2℃ and 2000 lx light intensity for 20 days. The axillary bud induction rate and average number of axillary buds were calculated for different hormone treatment combinations.

[0057] Axillary bud induction rate = (Number of explants that induced axillary buds / Total number of explants inoculated) × 100%; Average number of axillary buds = total number of axillary buds / total number of explants inoculated.

[0058] The test results are shown in Table 2.

[0059] Table 2. Results of tests on the effects of different hormone ratios on axillary bud induction in *Lysimachia christinae* stem segments.

[0060] Test Example 3: Effects of different hormone ratios on the induction and proliferation of clustered shoots The effects of proliferation culture in Examples 1, 3, and Comparative Examples 4-5 were tested. Healthy, uncontaminated seedlings from the induction culture were selected. Leaves and stem segments were separated; leaves were cut into small pieces, and stem segments into 1-1.5 cm segments. These were inoculated into the proliferation culture media of Examples 1, 3, and Comparative Examples 4-7, respectively. Four leaves were inoculated per bottle, for a total of 40 leaves; two stem segments were inoculated per bottle, for a total of 20 stem segments. After inoculation, the plants were first treated in darkness for 5 days, then placed in a light-controlled culture chamber for normal culture at a temperature of 25℃±2℃ and a light intensity of 2000 lx for 40 days. The induction of callus tissue and shoot clusters was observed.

[0061] Callus induction rate (%) = (Number of explants that induced callus / Number of inoculated explants) × 100%; Proliferation coefficient = (Total number of new shoots / Total number of explants) × 100%.

[0062] The test results are shown in Table 3.

[0063] Table 3. Results of tests on the effects of different hormone ratios on the induction and proliferation of shoot clusters.

[0064] Test Example 4: Effects of Different Hormone Ratios on Adventitious Bud Rooting The effects of axillary bud growth induction in Examples 1-3 and Comparative Example 8 were tested. Healthy seedlings with a height of 2-3 cm and 3-4 true leaves, obtained from propagation culture, were selected. Excess callus tissue at the base was removed, and the seedlings were inoculated into the rooting medium of each group in Examples 1-3 and Comparative Example 8, with 2 seedlings inoculated per bottle, for a total of 20 seedlings. The seedlings were placed in a culture room at a temperature of 25℃±2℃ and a light intensity of 2000 lx for 20 days. Rooting rate, average number of roots, and root growth status were observed.

[0065] Rooting rate = (Number of rooted plants / Total number of inoculated plants) × 100%; Average number of roots = total number of roots / total number of rooted plants × 100%.

[0066] The test results are shown in Table 4.

[0067] Table 4. Results of tests on the effects of different hormone ratios on adventitious shoot rooting.

[0068] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for tissue culture rapid propagation of Dracaena angulosa, characterized in that, The method comprises the following steps: (1) disinfecting the explant: taking the stem section with axillary buds of the Longmian flower, washing it, and then disinfecting it with ethanol solution, sodium hypochlorite solution, and then soaking it in the plant culture solution, to obtain the sterile explant; (2) inducing the growth of the axillary buds: inoculating the sterile explant into the induction medium to induce the growth of the axillary buds; the induction medium is MS medium containing 1.0-1.5 mg / L 6-benzylaminopurine and 0.05-0.1 mg / L naphthalene acetic acid; (3) proliferating culture: inoculating the stem section of the healthy seedling induced in step (2) into the proliferation medium to induce and proliferate the multiple shoots; the proliferation medium is MS medium containing 0.05-0.1 mg / L thidiazuron, 0.1-0.2 mg / L naphthalene acetic acid and 0.5-1.0 mg / L gibberellin; (4) rooting culture: inoculating the healthy seedling obtained in step (3) into the rooting medium to induce rooting; the rooting medium is 1 / 2MS medium containing 0.8-1.2 g / L activated carbon, 1.0-1.5 mg / L indole-3-butyric acid and 0.1-0.2 mg / L naphthalene acetic acid; (5) hardening and transplanting: hardening the complete tissue culture seedling obtained in step (4), and then transplanting it into the culture medium.

2. The method of tissue culture rapid propagation of Dracaena sanderiana according to claim 1, characterized in that, In step (1), the washing is as follows: washing for 25-35 min with the washing-up liquid to generate foam, then sealing with double-layer gauze, and then washing for 0.8-1.2 h with running water; the volume concentration of the ethanol solution is 70-80%, the ethanol solution disinfection time is 25-35 s, and the ethanol solution is washed with sterile water for 2-4 min after disinfection.

3. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in claim 1, characterized in that, The concentration of the sodium hypochlorite solution is 2 wt%, the sodium hypochlorite solution contains 0.1% (by volume) Tween-80, the sodium hypochlorite solution is disinfected for 2-3 times, each time for 3.5-4.5 min, and the sodium hypochlorite solution is washed with sterile water for 2-3 times, each time for 2-4 min after disinfection.

4. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in claim 1, characterized in that, The volume concentration of the plant culture solution is 0.4-0.6%, the soaking time is 8-12 min, and the plant culture solution is washed with sterile water for 2-3 times, each time for 2-4 min after soaking.

5. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in any one of claims 1-4, characterized in that, In step (2), the induction medium is MS medium containing 1.5 mg / L 6-benzylaminopurine and 0.05 mg / L naphthalene acetic acid.

6. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in any one of claims 1-4, characterized in that, In step (3), the proliferation medium is MS medium containing 0.1 mg / L thidiazuron, 0.2 mg / L naphthalene acetic acid and 1.0 mg / L gibberellin.

7. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in any one of claims 1-4, characterized in that, In step (4), the rooting medium is 1 / 2MS medium containing 1 g / L activated carbon, 1.5 mg / L indole-3-butyric acid and 0.2 mg / L naphthalene acetic acid.

8. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in any one of claims 1-4, characterized in that, In steps (2)-(4), the medium is added with sucrose and agar, wherein the sucrose concentration of the induction medium and the proliferation medium is 28-32 g / L, the sucrose concentration of the rooting medium is 18-22 g / L, the agar concentration is 6-8 g / L, and the pH value of the medium is 5.5-6.

0.

9. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in any one of claims 1-4, characterized in that, The seedling raising in step (5) comprises: first culturing for 5-7 days under natural light and temperature conditions with the bottle closed, and then culturing for 2-3 days with the bottle half opened.

10. The method of tissue culture rapid propagation of Dracaena sanderiana as claimed in any one of claims 1-4, characterized in that, The culture conditions in steps (2)-(4) are as follows: temperature 23-27℃, and light intensity 1500-2500 lx.

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