Directional screening and cultivating method for high-polysaccharide dendrobium officinale variety in germination period

Abstract

The invention discloses a high-polysaccharide dendrobium officinale variety germination period directional screening cultivation method, and belongs to the technical field of tissue culture. The invention provides a high-polysaccharide dendrobium officinale variety germination period directional screening cultivation method aiming at the problems that existing dendrobium officinale germplasm is mixed and the polysaccharide content is unstable. According to the method, the concentration of a periodate solution adopted for acidolysis treatment is remarkably reduced, the acidolysis time is shortened, a chromogenic reaction is achieved outside the protocorm, the situation that epidermal cells of the protocorm are excessively damaged and a coloring agent enters the protocorm to cause tissue structure change is avoided, and it is guaranteed that the structure of dendrobium officinale is complete and the shape of dendrobium officinale is stable; meanwhile, the dyeing time is shortened, rapid and lossless identification of the dendrobium officinale high-polysaccharide germplasm in the protocorm stage is achieved, the breeding period is remarkably shortened, and the screening accuracy and efficiency are improved. The cultivated high-polysaccharide dendrobium officinale variety shows consistency and stability in the aspects of polysaccharide content and agronomic traits.

Description

Technical Field

[0001] This invention relates to a method for screening and directional cultivation of high-polysaccharide Dendrobium officinale, belonging to the field of tissue culture technology. Background Technology

[0002] Dendrobium is a perennial epiphytic herb belonging to the Orchidaceae family. In traditional Chinese medicine, Dendrobium officinale (iron-skin dendrobium) is used... Dendrobium officinale The *Dendrobium officinale* (Kimura et Migo) is widely used. As a precious resource used for both medicinal and edible purposes, the core active substance of *Dendrobium officinale* is its polysaccharide, which possesses traditional effects such as nourishing the stomach and promoting fluid production, nourishing yin and clearing heat, and soothing the liver and gallbladder. It also exhibits significant biological activity in immune regulation, anti-tumor activity, and antioxidant activity, making its medicinal and economic value outstanding. However, *Dendrobium officinale* has extremely strict requirements for its growth environment. Currently, although artificial cultivation has become the main method of market supply, it generally suffers from problems such as mixed germplasm, reduced genetic diversity, and degraded agronomic traits, leading to significant fluctuations in polysaccharide content (dry weight 12.3-38.7%) and unstable yield per acre (fresh weight 50-200 kg), among other quality defects. Therefore, breeding new *Dendrobium officinale* varieties with both high polysaccharide content and excellent stress resistance has become an urgent need for current germplasm innovation and industrial development.

[0003] Wild *Dendrobium officinale* from Danxia grows slowly, taking 4-5 years or longer from seed to first flowering, resulting in a low fruit set rate and sensitivity to environmental changes. While this slow growth rate ensures the accumulation of medicinal components to some extent, it also severely limits large-scale breeding and industrial development. Therefore, establishing a method for screening and targeted cultivation of a high-quality *Dendrobium officinale* variety with high polysaccharide content during its germination period has become a key scientific issue in overcoming industrial bottlenecks. Summary of the Invention

[0004] This invention addresses the problems of mixed germplasm and unstable polysaccharide content in existing Dendrobium officinale varieties by proposing a method for targeted screening and cultivation of high-polysaccharide Dendrobium officinale varieties during the germination period.

[0005] The present invention relates to a method for targeted screening and cultivation of high-polysaccharide Dendrobium officinale varieties during the germination period, which is carried out according to the following steps: I. Screening of high polysaccharide germplasm and preparation of materials: Dendrobium officinale plants of different groups were collected, and the total polysaccharide content was determined. Dendrobium officinale plants with a total polysaccharide content of more than 40% were screened. II. Obtaining self-pollinated capsules: The Dendrobium officinale plants selected in step one are artificially cultivated. During the artificial cultivation period, after reaching the flowering period, self-pollination is carried out to obtain mature capsules. III. Aseptic sowing and protocorm induction: Select plump and disease-free self-pollinating capsules obtained in step II, disinfect their surfaces, and sow them in plant tissue cell culture medium. Culture them under aseptic conditions until protocorms with a diameter of 0.2-0.3 cm are obtained. The plant tissue cell culture medium was 1 / 2 MS medium, with added sucrose: 20 g / L, potato starch: 30 g / L, agar: 5.2 g / L; pH value was 5.8; IV. Acid hydrolysis treatment of protocorms: Place the protocorms obtained in step 3 in periodic acid solution and soak them at room temperature for 1-2 minutes for acid hydrolysis; after acid hydrolysis, wash the protocorms. The periodic acid solution has a mass fraction of 0.1%. V. Protocorm staining: Take out the protocorms washed in step 4, place them in the staining solution, and stain in the dark for 3-5 minutes; after staining, wash the protocorms and screen out the stained protocorms; The preparation method of the dye is as follows: chitosan, sodium alginate, basic fuchsin, hydrochloric acid, sodium metabisulfite and activated carbon are mixed, calcium chloride crosslinking agent is added, and after stirring for 10-15 minutes, anhydrous ethanol is added to obtain the dye. The dye contains 0.5-1 wt.% chitosan, 1-2 wt.% sodium alginate, 4.5 wt.% basic fuchsin, 0.1 wt.% hydrochloric acid, 2.5 wt.% sodium metabisulfite, 0.1 wt.% anhydrous ethanol, and 20 wt.% activated carbon. VI. Induction of Differentiation and Cultivation of High-Polysaccharide Plants: The stained protocorms from step five were inoculated into the differentiation induction medium and cultured in the dark at 24-26℃ for 36-48 hours; finally, plant cultivation was carried out. The differentiation induction medium was 1 / 2 MS medium, with the following added ingredients: sucrose: 25 g / L, potato starch: 30 g / L, benzyladenine (6-BA): 1.0 mg / L, α-naphthaleneacetic acid (NAA): 0.1 mg / L, vitamin C (Vc): 100 mg / L, peptone: 1 g / L, agar: 5.2 g / L; pH value was 5.8.

[0006] The beneficial effects of this invention are as follows: 1. In this invention, after cultivating protocorms, acid hydrolysis is performed to release the polysaccharide components within the protocorms. The staining agent is a hydrogel system made of chitosan and sodium alginate. After the addition of anhydrous ethanol, the released polysaccharide components are adsorbed into the staining agent with the hydrogel system, achieving alcohol precipitation and enrichment. After staining reaction, color development is achieved, enabling rapid identification of high-polysaccharide Dendrobium officinale varieties. Compared with existing technologies, this invention not only significantly reduces the concentration of periodic acid solution used in acid hydrolysis and shortens the acid hydrolysis time, but also achieves the color development reaction on the outside of the protocorm, avoiding excessive damage to the epidermal cells of the protocorm and changes in tissue structure caused by the staining agent entering the protocorm. This ensures the structural integrity and shape stability of Dendrobium officinale, while shortening the staining time. This allows for rapid and non-destructive identification of high-polysaccharide Dendrobium officinale germplasm at the protocorm stage, significantly shortening the breeding cycle and improving screening accuracy and efficiency.

[0007] 2. The method of this invention optimizes the light, temperature and nutrient conditions during the critical period of polysaccharide synthesis through early screening combined with targeted environmental regulation, effectively promoting the biosynthesis and accumulation of Dendrobium polysaccharides, and enabling the cultivated high-polysaccharide Dendrobium officinale varieties to exhibit consistency and stability in terms of polysaccharide content and agronomic traits.

[0008] 3. The method of the present invention is simple to operate and has good repeatability, making it suitable for large-scale promotion and application. It can effectively reduce production costs, increase unit yield and quality, and provide a stable, high-quality and efficient seed source guarantee for the industrialization of Dendrobium officinale. Attached Figure Description

[0009] Figure 1 The following are examples of Dendrobium officinale protocorms aseptically cultured after staining: A is Dendrobium officinale protocorms stained in step five; B is Dendrobium officinale protocorms that were further cultured for 15 days after staining. Figure 2 This is a diagram showing the differentiation of Dendrobium officinale protobulbs after staining and cultivation for 40 days in the example. Figure 3 The image shows Dendrobium officinale plants that were stained and then cultured for 180 days in the example. A is the stained group, and B is the control group. Figure 4 The image shows the phenotypic pattern of Dendrobium officinale plants that were stained and then cultured for 9 months in the example. A represents the stained group; B represents the control group. Figure 5 The results of the determination of relevant indicators of Dendrobium officinale polysaccharide content in the stained group and the control group in the examples are shown below; Note: Indicates a significant difference ( P <0.05), This indicates that the difference is highly significant ( P <0.01). Detailed Implementation

[0010] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any reasonable combination of the specific embodiments.

[0011] Specific Implementation Method 1: This implementation method for the directional screening and cultivation of high-polysaccharide Dendrobium officinale varieties during the germination period is carried out according to the following steps: I. Screening of high polysaccharide germplasm and preparation of materials: Dendrobium officinale plants of different groups were collected, and the total polysaccharide content was determined. Dendrobium officinale plants with a total polysaccharide content of more than 40% were screened. II. Obtaining self-pollinated capsules: The Dendrobium officinale plants selected in step one are artificially cultivated. During the artificial cultivation period, after reaching the flowering period, self-pollination is carried out to obtain mature capsules. III. Aseptic sowing and protocorm induction: Select plump and disease-free self-pollinating capsules obtained in step II, disinfect their surfaces, and sow them in plant tissue cell culture medium. Culture them under aseptic conditions until protocorms with a diameter of 0.2-0.3 cm are obtained. The plant tissue cell culture medium was 1 / 2 MS medium, with added sucrose: 20 g / L, potato starch: 30 g / L, agar: 5.2 g / L; pH value was 5.8; IV. Acid hydrolysis treatment of protocorms: Place the protocorms obtained in step 3 in periodic acid solution and soak them at room temperature for 1-2 minutes for acid hydrolysis; after acid hydrolysis, wash the protocorms. The periodic acid solution has a mass fraction of 0.1%. V. Protocorm staining: Take out the protocorms washed in step 4, place them in the staining solution, and stain in the dark for 3-5 minutes; after staining, wash the protocorms and screen out the stained protocorms; The preparation method of the dye is as follows: chitosan, sodium alginate, basic fuchsin, hydrochloric acid, sodium metabisulfite and activated carbon are mixed, calcium chloride crosslinking agent is added, and after stirring for 10-15 minutes, anhydrous ethanol is added to obtain the dye. The dye contains 0.5-1 wt.% chitosan, 1-2 wt.% sodium alginate, 4.5 wt.% basic fuchsin, 0.1 wt.% hydrochloric acid, 2.5 wt.% sodium metabisulfite, 0.1 wt.% anhydrous ethanol, and 20 wt.% activated carbon. VI. Induction of Differentiation and Cultivation of High-Polysaccharide Plants: The stained protocorms from step five were inoculated into the differentiation induction medium and cultured in the dark at 24-26℃ for 36-48 hours; finally, plant cultivation was carried out. The differentiation induction medium was 1 / 2 MS medium, with the following added ingredients: sucrose: 25 g / L, potato starch: 30 g / L, benzyladenine (6-BA): 1.0 mg / L, α-naphthaleneacetic acid (NAA): 0.1 mg / L, vitamin C (Vc): 100 mg / L, peptone: 1 g / L, agar: 5.2 g / L; pH value was 5.8.

[0012] This embodiment has the following beneficial effects: 1. In this embodiment, after obtaining the protocorms through cultivation, acid hydrolysis is performed to release the polysaccharide components within the protocorms. The staining agent is a hydrogel system made of chitosan and sodium alginate. After the addition of anhydrous ethanol, the released polysaccharide components are adsorbed into the staining agent with the hydrogel system, achieving alcohol precipitation and enrichment. After staining reaction, color development is achieved, enabling rapid identification of high-polysaccharide Dendrobium officinale varieties. Compared with existing technologies, this invention not only significantly reduces the concentration of periodic acid solution used in acid hydrolysis and shortens the acid hydrolysis time, but also achieves the color development reaction on the outside of the protocorms, avoiding excessive damage to the epidermal cells of the protocorms and changes in tissue structure caused by the staining agent entering the protocorms. This ensures the structural integrity and shape stability of Dendrobium officinale, while shortening the staining time. This allows for rapid and non-destructive identification of high-polysaccharide Dendrobium officinale germplasm at the protocorm stage, significantly shortening the breeding cycle and improving screening accuracy and efficiency.

[0013] 2. The method of this implementation optimizes the light, temperature and nutrient conditions during the critical period of polysaccharide synthesis through early screening combined with targeted environmental regulation, effectively promoting the biosynthesis and accumulation of Dendrobium officinale polysaccharides, and enabling the cultivated high polysaccharide Dendrobium officinale varieties to show consistency and stability in terms of polysaccharide content and agronomic traits.

[0014] 3. The method described in this implementation is simple to operate and has good repeatability, making it suitable for large-scale promotion and application. It can effectively reduce production costs, increase unit yield and quality, and provide a stable, high-quality and efficient seed source guarantee for the industrialization of Dendrobium officinale.

[0015] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the method for determining the total polysaccharide content in step one is as follows: Take stem segments of Dendrobium officinale plants, clean and grind them, then extract them with water using the heating reflux method, and filter to collect the extract; after cooling and adjusting the volume, filter the extract, add anhydrous ethanol to the filtrate for alcohol precipitation, refrigerate and let stand, then centrifuge, discard the supernatant, wash the precipitate and centrifuge again to obtain crude polysaccharide from Dendrobium officinale; dissolve the crude polysaccharide in hot water and adjust the volume to prepare the test solution, measure the absorbance at a wavelength of 490 nm, and calculate the total polysaccharide content using glucose as a standard.

[0016] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 1 or 2 in that the substrate used for artificial cultivation in step 2 is composed of wood fiber blocks, coconut coir, perlite, vermiculite, and charcoal wool mixed in a mass ratio of 4:2:2:1:1.

[0017] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the cultivation conditions for promoting flowering during the artificial cultivation period described in Step Two are: 16 hours of light per day, 8 hours of darkness per day; temperature of 24-26℃, light intensity of 20,000-25,000 lx, and continuous cultivation for 180-240 days.

[0018] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the cultivation conditions during the capsule development period in step two are as follows: 12 hours of light per day, 12 hours of darkness per day; temperature of 26-28℃, light intensity of 10000-15000 lx, and continuous cultivation for 150-180 days.

[0019] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that the surface disinfection method described in step three is as follows: First, wipe the epidermis with 75% alcohol, then soak it in a 30% sodium hypochlorite solution for 20-25 minutes for disinfection, then rinse it with sterile water 5-6 times, and finally absorb the moisture with sterile filter paper.

[0020] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that the cultivation conditions described in step three are: 12 hours of light per day, 12 hours of darkness per day; temperature of 24-26℃, light intensity of 1500-2000 lx, and continuous cultivation for 30-35 days.

[0021] Specific Implementation Method Eight: This implementation method differs from one of Specific Implementation Methods One to Seven in that the mass ratio of calcium chloride crosslinking agent to chitosan in step five is 0.1-0.15:1.

[0022] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that the cleaning process described in step four is as follows: wash with sterile water 2-3 times, 1-2 minutes each time, and then use sterile filter paper to absorb the surface moisture.

[0023] Specific Implementation Method 10: This implementation method differs from Specific Implementation Methods 1 to 9 in that the process of cleaning the original corm in step 5 is as follows: wash with sterile water 2-3 times, 1-2 minutes each time, and then use sterile filter paper to absorb the surface moisture.

[0024] Example 1: This embodiment describes a method for targeted screening and cultivation of high-polysaccharide Dendrobium officinale varieties during the germination period, which is carried out according to the following steps: I. Screening and Material Preparation of High Polysaccharide Germplasm: Twenty germplasm resources of Dendrobium officinale from Danxia, ​​Guangdong were collected, and their agronomic traits, including plant height, number of stem nodes, stem diameter, number of leaves, length and width of terminal leaf, and root length, were measured. The results are recorded in Table 1. Table 1

[0025] The total polysaccharide content was determined, and *Dendrobium officinale* plants with a total polysaccharide content exceeding 40% were screened. Accurately weigh glucose dried to constant weight at 105℃ and dissolved in water to prepare a 100 μg / mL glucose standard solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution into 10 mL stoppered test tubes, add water to each to bring the volume to 1.0 mL, accurately add 1.0 mL of 5% phenol solution, shake well, then accurately add 5 mL of concentrated sulfuric acid, shake well, and heat in a boiling water bath for 20 min. After heating, immediately cool in an ice-water bath for 5 min. Using the reagent as a blank, the absorbance value was measured at a wavelength of 490 nm. A glucose standard curve was plotted with glucose content (μg) on ​​the x-axis and absorbance value on the y-axis. The standard curve equation was obtained as: y = 0.4136X - 0.0084 (…). R 2 =0.9971).

[0026] Stem segments of Dendrobium officinale were collected, washed, ground, and then extracted with water using the reflux method. The extract was collected by filtration. After cooling and dilution, the extract was filtered again. Anhydrous ethanol was added to the filtrate for alcohol precipitation. After refrigeration and standing, the precipitate was centrifuged, and the supernatant was discarded. The precipitate was washed and centrifuged again to obtain crude polysaccharide from Dendrobium officinale. The crude polysaccharide was dissolved in hot water and diluted to a final volume to prepare a test solution. The absorbance was measured at a wavelength of 490 nm. The total polysaccharide content was calculated using glucose as a standard. The results are recorded in Table 2.

[0027] Table 2

[0028] II. Obtaining self-pollinated capsules: The Dendrobium officinale plants selected in step one are artificially cultivated. During the artificial cultivation period, after reaching the flowering period, self-pollination is carried out to obtain mature capsules. The substrate used for artificial cultivation is composed of wood fiber blocks, coconut coir, perlite, vermiculite, and charcoal wool mixed in a mass ratio of 4:2:2:1:1. The artificial cultivation conditions for promoting flowering were as follows: 16 hours of light per day, 8 hours of darkness per day; temperature of 25℃, light intensity of 20,000 lx, and continuous cultivation for 200 days. The cultivation conditions for the capsule development period during artificial cultivation were as follows: 12 hours of light per day, 12 hours of darkness per day; temperature of 27℃; light intensity of 10000 lx; ​​and continuous cultivation for 180 days. III. Aseptic sowing and protocorm induction: Select plump and disease-free self-pollinating capsules obtained in step II, disinfect their surfaces, and sow them in plant tissue cell culture medium. Culture them under aseptic conditions until protocorms with a diameter of 0.2-0.3 cm are obtained. The surface disinfection method is as follows: first wipe the surface with 75% alcohol, then soak it in 30% sodium hypochlorite solution for 23 minutes, then rinse it with sterile water 6 times, and finally dry it with sterile filter paper. The plant tissue cell culture medium was 1 / 2 MS medium, with added sucrose: 20 g / L, potato starch: 30 g / L, agar: 5.2 g / L; pH value was 5.8; The cultivation conditions were: 12 hours of light per day, 12 hours of darkness per day; temperature of 25°C; light intensity of 2000 lx; ​​and continuous cultivation for 35 days. IV. Acid hydrolysis treatment of protocorms: Place the protocorms obtained in step 3 in periodic acid solution and soak them at room temperature for 1 minute for acid hydrolysis; after acid hydrolysis, wash the protocorms. The cleaning process is as follows: wash with sterile water 3 times, 2 minutes each time, and then use sterile filter paper to absorb the surface moisture. The periodic acid solution has a mass fraction of 0.1%. V. Protocorm Staining: Take out the protocorms washed in step four, place them in the staining solution, and stain for 3 minutes in the dark; after staining, wash the protocorms and screen out the stained protocorms, see... Figure 1 ; The dyeing agent is prepared by mixing chitosan, sodium alginate, basic fuchsin, hydrochloric acid, sodium metabisulfite and activated carbon, adding calcium chloride crosslinking agent, stirring for 10 minutes and then adding anhydrous ethanol to obtain the dyeing agent. The dye contains 0.5 wt.% chitosan, 1 wt.% sodium alginate, 4.5 wt.% basic fuchsin, 0.1 wt.% hydrochloric acid, 2.5 wt.% sodium metabisulfite, and 0.1 wt.% anhydrous ethanol. The mass ratio of calcium chloride crosslinking agent to chitosan is 0.1:1, and the activated carbon content is 20 wt.%. The process for cleaning the protocorms is as follows: wash twice with sterile water for 1 minute each time, and then use sterile filter paper to absorb the surface moisture. VI. Induction of Differentiation and Cultivation of High-Polysaccharide Plants: The stained protocorms from step five were inoculated into the differentiation induction medium and cultured in the dark at 25°C for 48 hours; finally, plant cultivation was carried out. The differentiation induction medium was 1 / 2 MS medium, supplemented with sucrose: 25 g / L, potato starch: 30 g / L, benzyladenine (6-BA): 1.0 mg / L, α-naphthaleneacetic acid (NAA): 0.1 mg / L, vitamin C (Vc): 100 mg / L, peptone: 1 g / L, agar: 5.2 g / L; pH value was 5.8. The conditions for plant cultivation were: 12 hours of light per day, 12 hours of darkness per day; temperature 25℃; light intensity 2500 lx. The stained protocorms from step five were designated as the staining group, while the unstained protocorms from step three were designated as the control group. The staining group and the control group were cultured under the same conditions and placed in different culture bottles.

[0029] After 60 days of proliferation culture, the protocorms of the stained group sprouted and differentiated into leaves, growing to about 2-3 cm with 2-4 leaves, thus obtaining differentiated tissue culture seedlings. Figure 2 The differentiated tissue culture seedlings were then transferred to rooting medium (1 / 2 MS medium + 25 g / L sucrose + 30 g / L potato starch + 0.5 mg / L NAA + 1 g / L peptone + 5.2 g / L agar, pH 5.8) for rooting culture. The seedlings were placed on culture racks in the tissue culture laboratory and cultured for 60 days under the same photoperiod, temperature of 25℃, and light intensity of 5000 lx to obtain rooted tissue culture seedlings. The rooted tissue culture seedlings were then placed in a constant temperature seedling room for further culture, with the photoperiod controlled at 12 h / d, temperature at 28℃, and light intensity at 6000 lx. After approximately 30 days of hardening off, the seedlings reached a height of about 5-6 cm, making them ready for transplanting. Remove the tissue culture seedlings suitable for transplanting, wash the culture medium from the roots with clean water, and transplant the stained group (52 seedlings) and the control group (48 seedlings) into 11cm diameter pots with 12-13 individual seedlings per clump. The stained group had 4 pots and the control group had 3 pots each. The cultivation substrate was sphagnum moss. Figure 3 The planted Dendrobium officinale seedlings were then nurtured in a constant-temperature nursery under the same light and temperature conditions.

[0030] Identification of high-polysaccharide plants: Nine months after cultivation, the growth and agronomic traits of *Dendrobium officinale* seedlings in both the stained group and the control group were measured, including stem length, number of stem nodes, stem diameter, number of leaves, length of the terminal leaf, leaf width, root length, and weight per plant. The results are shown in Table 3. Four plants from each of the stained group and the control group were randomly selected. Figure 4 Take 0.1g of fresh leaves and use a kit to determine the soluble sugar content, glucose content, fructose content, sucrose content, as well as the activities of sucrose synthase (decomposition direction) and soluble acid invertase. The average value of the results for each group is taken. Results are shown in […]. Figure 5The soluble sugar content assay kit (catalog number G0501F), glucose content assay kit (catalog number G0535F), fructose content assay kit (catalog number G0505F), sucrose content assay kit (catalog number G0506F), sucrose synthase (decomposition direction) activity assay kit (colorimetric method, G0513F), and soluble acid invertase activity assay kit (colorimetric method, G0517F) were all purchased from Greens Biotechnology (Suzhou) Co., Ltd.

[0031] Table 3

[0032] Continued from Table 3

[0033] Continued from Table 3

[0034] Results analysis: From Table 3 and Figure 4 It can be seen that the stained group was significantly better than the control group in agronomic traits such as plant height, stem diameter, and single plant weight. Furthermore, from Figure 5 The results showed that the soluble sugar content, fructose content, glucose content, and soluble acid invertase activity in the stained group were 34.64%, 35.96%, 43.86%, and 22.55% higher than those in the control group, respectively, reaching a highly significant level. The sucrose content and sucrose synthase activity were 20.15% and 17.83% higher than those in the control group, respectively, reaching a significant level. These results indicate that this invention, through germination-stage staining combined with directional cultivation, can effectively screen for Dendrobium officinale germplasm with high polysaccharide content and excellent agronomic traits. This is an effective method for breeding superior high-polysaccharide Dendrobium officinale germplasm, providing reliable technical support for industrial promotion.

Claims

1. A method for directional screening and cultivation of high-polysaccharide Dendrobium officinale varieties during the germination period, characterized in that: The application relates to a high-polymer iron-bark dendrobium variety germination period directional screening and cultivation method, which is carried out according to the following steps. First, high-polymer variety screening and material preparation: different groups of iron-bark dendrobium plants are collected, and the total polymer content is determined, and iron-bark dendrobium plants with a total polymer content exceeding 40% are screened out; Second, self-crossing of the obtained capsules: the iron-bark dendrobium plants screened out in step one are artificially cultivated, and self-crossing pollination is carried out after the plants reach the flowering stage, so that mature capsules are obtained; Third, aseptic sowing and prothallial induction: the full and disease-free self-crossing capsules obtained in step two are surface-disinfected and sowed in a plant tissue cell culture medium, and are cultured under aseptic conditions until prothallia with a diameter of 0.2-0.3 cm are obtained; The plant tissue cell culture medium is 1 / 2MS culture medium, and sucrose, potato powder and agar are added in an amount of 20 g / L, 30 g / L and 5.2 g / L respectively; and the pH value is 5.8; Fourth, acidolysis treatment of the prothallia: the prothallia obtained in step three are soaked in a high-iodic acid solution at room temperature for 1-2 min for acidolysis, and the acidolysed prothallia are cleaned; The mass fraction of the high-iodic acid solution is 0.1%; Fifth, prothallium staining: the cleaned prothallia in step four are taken out and placed in a staining agent for 3-5 min of dark staining; the stained prothallia are cleaned, and the stained prothallia are screened out; The preparation method of the staining agent is as follows: chitosan, sodium alginate, basic fuchsin, hydrochloric acid, sodium metabisulfite and activated carbon are mixed, calcium chloride crosslinking agent is added, stirring is carried out for 10-15 min, and then anhydrous ethanol is added, so that the staining agent is obtained; The content of chitosan in the staining agent is 0.5-1 wt.%, the content of sodium alginate is 1-2 wt.%, the content of basic fuchsin is 4.5 wt.%, the content of hydrochloric acid is 0.1 wt.%, the content of sodium metabisulfite is 2.5 wt.%, the content of anhydrous ethanol is 0.1 wt.%, and the content of activated carbon is 20 wt.%; Sixth, induction differentiation and high-polymer plant cultivation: The stained prothallia in step five are inoculated in an induction differentiation culture medium, are dark-cultured at a temperature of 24-26 DEG C for 36-48 h, and finally are subjected to plant cultivation. The induction differentiation culture medium is 1 / 2MS culture medium, and sucrose, potato powder, benzyladenine, alpha-naphthaleneacetic acid, vitamin C, protein peptone and agar are added in an amount of 25 g / L, 30 g / L, 1.0 mg / L, 0.1 mg / L, 100 mg / L, 1 g / L and 5.2 g / L respectively; and the pH value is 5.

8.

2. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The determination method of the total polymer content in step one is as follows: the stem tissue of the iron-bark dendrobium plant is cleaned and ground, and then is subjected to water extraction by a heating reflux method; the extracted solution is cooled and fixed in volume, filtered, and then is subjected to alcohol precipitation by adding anhydrous ethanol; after cold storage and standing, centrifugation is carried out, the supernatant is discarded, and the obtained precipitate is washed and centrifuged again, so that the crude polymer of the iron-bark dendrobium is obtained; the crude polymer is dissolved in hot water and fixed in volume, so that a to-be-tested solution is prepared; the absorbance is determined at a wavelength of 490 nm, and the total polymer content is calculated by taking glucose as a standard.

3. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The substrate for the artificial cultivation in step two is composed of wood fiber block, coconut bran, perlite, vermiculite and carbon cotton in a mass ratio of 4:2:2:1:

1.

4. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The culture conditions for promoting flowering during the artificial cultivation in step two are: light for 16 h / d, darkness for 8 h / d, temperature of 24-26℃, light intensity of 20000-25000 lx, and continuous culture for 180-240 d.

5. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The culture conditions for the capsule development period during the artificial cultivation in step two are: light for 12 h / d, darkness for 12 h / d, temperature of 26-28℃, light intensity of 10000-15000 lx, and continuous culture for 150-180 d.

6. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The surface disinfection method in step three is: first wiping the epidermis with 75% alcohol, then soaking in a 30% sodium hypochlorite solution for 20-25 min, then washing with sterile water for 5-6 times, and finally drying the surface with sterile filter paper.

7. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The culture conditions in step three are: light for 12 h / d, darkness for 12 h / d, temperature of 24-26℃, light intensity of 1500-2000 lx, and continuous culture for 30-35 d.

8. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The mass ratio of calcium chloride crosslinking agent to chitosan in step five is 0.1-0.15:

1.

9. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The washing process in step four is: washing with sterile water for 2-3 times, each time for 1-2 min, and then drying the surface with sterile filter paper.

10. The high polysaccharide Dendrobium candidum variety germination stage directional screening cultivation method according to claim 1, characterized in that: The washing process of the original protocorm in step five is: washing with sterile water for 2-3 times, each time for 1-2 min, and then drying the surface with sterile filter paper.

Images