Culture method for improving growth efficiency and medicinal value of astragalus membranaceus callus

Abstract

The invention provides a culture method for improving the growth efficiency and medicinal value of astragalus membranaceus calluses. The culture method comprises the following steps: S1, washing and cleaning astragalus membranaceus seeds, and soaking the astragalus membranaceus seeds in pure water; soaking the seeds with a 0.5% sodium hypochlorite solution, washing the seeds with sterile water, and inoculating the seeds to a culture medium with tweezers; s2, taking a sterile seedling which is cultured for one month and has a good growth state as a material, cutting an explant stem section, and carrying out subculture; s3, growth promoting agent treatment: respectively adding chitosan or brassinolide with different concentrations into the culture medium, and by limiting the ratio of the induction culture medium with different concentrations, the growth cycle of the callus and the dosage of the chitosan and the brassinolide, not only is the growth of the astragalus membranaceus callus greatly improved, but also the survival rate of the astragalus membranaceus callus is increased; and the medicinal value of the astragalus membranaceus callus is improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a culture method for improving the growth efficiency and medicinal value of astragalus callus. Background Art

[0002] Astragalus membranaceus ( Astragalus membranaceus Astragalus (Fisch.) Bunge. is a perennial herbaceous plant of the genus Astragalus in the Leguminosae family. It is primarily sourced from wild resources. Its roots are used as medicine and have high medicinal value. It is an important traditional Chinese medicine and widely used as a livestock feed additive. Its roots contain saponins, polysaccharides, flavonoids, various amino acids, vitamins, unsaturated fatty acids, and trace elements. Saponins, polysaccharides, flavonoids, and amino acids are the main active ingredients of Astragalus. Flavonoids include four major categories: flavonoids, isoflavones, isoflavans, and pteranodones; saponins include astragalosides, isoastragalosides, acetylastragalosides, and soybean saponins; and polysaccharides include glucans and heteropolysaccharides. In addition, Astragalus contains various amino acids and trace elements such as folic acid, vitamin D, linoleic acid, linolenic acid, vanillic acid, and ferulic acid.

[0003] Due to its immense medicinal value, wild Astragalus has long been overharvested, leading to the destruction of cultivated wild resources. Demand for Astragalus is increasing, but wild Astragalus resources are scarce and are a Class III protected plant in China. Artificial cultivation of Astragalus is now underway in many parts of the country, replacing wild Astragalus with cultivated varieties.

[0004] It is very difficult to find large-scale wild Astragalus. Nowadays, medicinal Astragalus is cultivated artificially, and there are some problems with artificial cultivation: due to global climate change, environmental pollution and other reasons, the yield of wild Astragalus has been significantly reduced; Astragalus membranaceus has a well-developed root system, a short main root and many lateral roots, and is called "chicken claw Astragalus". Astragalus has a long growth cycle, long and deep roots, and is difficult and costly to harvest; many planting areas do not have professional technical personnel to guide, pesticides are used continuously, and Astragalus is seriously diseased and pest-infected, resulting in reduced returns; the seeds in the planting areas are uneven, resulting in a serious decline in the quality of the medicinal materials, which cannot meet the medicinal quality grade standards. Summary of the Invention

[0005] The purpose of the present invention is to provide a culture method for improving the growth efficiency and medicinal value of astragalus callus, so as to solve the problems of slow growth and low content of medicinal ingredients in existing astragalus, and provide technical support for large-scale production and resource protection of astragalus.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a culture method for improving the growth efficiency and medicinal value of Astragalus callus, comprising the following steps: S1: Before sowing, wash the Astragalus seeds and soak them in pure water for 24 hours. Then, soak them in 0.5% sodium hypochlorite solution for 10-15 minutes, rinse with sterile water, and dry them. The seeds are then inoculated onto pre-sterilized MS medium (121°C, 20 minutes); the MS medium contains 30 g / L sucrose and 7.5 g / L agar, with a pH of 5.8. The seeds are then cultured in a tissue culture room. S2: Using sterile seedlings that have been cultured for one month and are in good growth condition, explant stem segments were cut and placed flat on MS medium containing 0.5-1.5 mg / L 6-BA, 0.5-1.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar; and cultured in a tissue culture room. S3: When the callus tissue grew to 35 days old, the callus tissue with good growth condition was inoculated on MS medium containing 0.5-1.5 mg / L 6-BA, 0.5-1.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for subculture. The subculture was carried out every 35 days for a total of three subcultures. S4: Select callus tissue with good growth condition, cut it, and inoculate it on MS medium containing 0.01-0.15 mg / L brassinolide, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for culture; alternatively, inoculate it on MS medium containing 50-200 mg / L chitosan, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for culture.

[0007] Furthermore, in step S1, the sample is soaked in a 0.5% sodium hypochlorite solution for 10-15 minutes, and then rinsed with sterile water 7-10 times.

[0008] Preferably, the culture medium used in steps S2 and S3 is: MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar.

[0009] Preferably, the culture medium used in step S4 is: MS medium containing 0.01 mg / L brassinolide, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar.

[0010] Preferably, the culture medium used in step S4 is: MS medium containing 100 mg / L chitosan, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar.

[0011] Furthermore, the culture conditions of steps S1 to S4 are: temperature (25±1)°C, humidity 60%, illumination time 16h / d, and illumination intensity 3000lx.

[0012] Preferably, the astragalus is Astragalus membranaceus.

[0013] In a second aspect, the present invention provides the use of chitosan or brassinolide in Astragalus tissue culture technology.

[0014] Preferably, the astragalus is Astragalus membranaceus.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (1) Based on the characteristics of Astragalus membranaceus, given the near-exhaustion of wild resources and the numerous problems associated with artificial cultivation, this invention applies chitosan and brassinolide to Astragalus membranaceus cultivation. By adding varying concentrations of chitosan and brassinolide to the culture medium for inducing Astragalus membranaceus callus, the application period and dosage of different growth regulators can be precisely controlled, avoiding the adverse effects of blind application.

[0016] (2) The chitosan and brassinolide used in the present invention can significantly promote the growth of Astragalus callus and plants, significantly increasing their yield. They also significantly enhance the medicinal qualities of Astragalus, increasing its application value in medicine, health products, and other fields.

[0017] (3) Chitosan and brassinolide offer numerous advantages. Their widespread availability and ease of use improve the quality and yield of astragalus while also helping to reduce cultivation costs. Furthermore, they reduce reliance on chemical fertilizers, minimizing environmental pollution. By improving soil structure and increasing soil organic matter content, they significantly promote the sustainable development of astragalus cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the fresh weight and dry weight of the Astragalus membranaceus callus tissue growth of the present invention.

[0019] Figure 2 The figure is a graph showing the change in the content of total saponins during the growth of Astragalus membranaceus callus of the present invention.

[0020] Figure 3Figures show the effects of different chitosan and brassinolide concentrations on callus growth and antioxidant capacity of Astragalus membranaceus in a preferred embodiment of the present invention. (a)-(h) show the effects of different chitosan and brassinolide concentrations on callus fresh weight, dry weight, malondialdehyde, superoxide anion production rate, proline content, glutathione content, antioxidant enzyme activity, and superoxide dismutase activity. CK represents the control group; K1, K2, K3, and K4 represent samples treated with chitosan concentrations of 50, 100, 150, and 200 mg / L, respectively; and B1, B2, B3, and B4 represent samples treated with brassinolide concentrations of 0.01, 0.05, 0.10, and 0.15 mg / L, respectively. Values ​​are the mean ± standard error of three replicates. Different letters in the left and right panels indicate significant differences (P < 0.05) between treatments with the same plant growth regulator.

[0021] Figure 4 Figures show the effects of different chitosan and brassinolide concentrations on the medicinal value of Astragalus membranaceus callus tissue in a preferred embodiment of the present invention. (a)-(h) show the effects of different chitosan and brassinolide concentrations on soluble sugars, soluble protein, vitamin C, alkaloids, polysaccharides, total flavonoids, total saponins, and phenylalanine ammonia-lyase activity in Astragalus membranaceus callus tissue. CK represents the control group; K1, K2, K3, and K4 represent samples treated with chitosan concentrations of 50, 100, 150, and 200 mg / L, respectively; and B1, B2, B3, and B4 represent samples treated with brassinolide concentrations of 0.01, 0.05, 0.10, and 0.15 mg / L, respectively. Values ​​are the mean ± standard error of three replicates. Different letters in the left and right panels indicate significant differences (P < 0.05) between treatments with the same plant growth regulator. DETAILED DESCRIPTION

[0022] The present invention provides a culture method for improving the growth efficiency and medicinal value of astragalus callus, comprising the following steps: S1: Before sowing, wash the Astragalus seeds and soak them in pure water for 24 hours. Then soak them in 0.5% sodium hypochlorite solution for 10-15 minutes, rinse with sterile water and dry them. Inoculate the seeds onto pre-sterilized MS medium (121°C, 20 minutes). The MS medium contains 30g / L sucrose and 7.5g / L agar, with a pH of 5.8. Cultivate in a tissue culture room at a temperature of (25±1)°C, a humidity of 60%, a photoperiod of 16 hours per day, and a light intensity of 3000 lx. S2: Explant stem segments were cut from sterile, well-grown seedlings that had been cultured for one month and placed flat on MS medium containing 0.5-1.5 mg / L 6-BA, 0.5-1.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar. Cultured in a tissue culture room at (25 ± 1)°C, 60% humidity, 16 h / d of light, and 3000 lx. S3: When the callus tissue grew to 35 days old, the callus tissue with good growth condition was inoculated on MS medium containing 0.5-1.5 mg / L 6-BA, 0.5-1.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for subculture. The subculture was carried out every 35 days for a total of three subcultures. The culture temperature was (25±1)℃, the humidity was 60%, the light duration was 16 h / d, and the light intensity was 3000 lx. S4: Select callus tissue with good growth condition, cut it, and inoculate it on MS medium containing 0.01-0.15 mg / L brassinolide, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for culture; alternatively, inoculate it on MS medium containing 50-200 mg / L chitosan, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for culture; the culture temperature is (25±1)℃, the humidity is 60%, the light duration is 16 h / d, and the light intensity is 3000 lx.

[0023] Furthermore, in step S1, the sample is immersed in a 0.5% sodium hypochlorite solution for 10-15 minutes, and then rinsed with sterile water 7-10 times.

[0024] Furthermore, in step S2, callus tissue was induced using MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar.

[0025] Furthermore, in step S3, the obtained callus tissue was inoculated on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for subculture, and the subculture was performed every 35 days for a total of three times.

[0026] Further, in step S4, the Astragalus callus tissue is treated with an MS medium containing 0.01 mg / L brassinolide, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar; or, the Astragalus callus tissue is treated with an MS medium containing 100 mg / L chitosan, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar.

[0027] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0028] Brassinolide and chitosan used in the following examples were purchased from ANPEL Laboratory Technology.

[0029] Example 1 This embodiment provides a method for increasing the growth efficiency and medicinal value of Astragalus callus, comprising the following steps: S1. Inducing sterile seedlings: Rinse the Astragalus seeds and soak them in pure water. Then soak them in 0.5% sodium hypochlorite solution, rinse with sterile water, and inoculate the seeds with forceps onto pre-sterilized MS medium (121°C, 20 min) containing 30 g / L sucrose and 7.5 g / L agar, pH 5.8. S2. Callus Induction: Explant stem segments were cut from sterile, well-grown seedlings grown for one month and placed flat on Petri dishes containing callus induction medium containing 30 g / L sucrose and 7.5 g / L agar (pH 5.8) supplemented with various hormone concentrations (6-BA, NAA). Nine different hormone concentration combinations (6-BA, NAA) were used, as shown in Table 1. The Petri dishes were incubated in a tissue culture room at a temperature of (25 ± 1)°C, a humidity of 60%, and a photoperiod of 16 h / d at a light intensity of 3000 lx.

[0030] Table 1 Plant hormone ratio experimental design S3. Subculture: When the callus tissue grows to 35 days old, the callus tissue with good growth condition is inoculated into MS medium containing 30g / L sucrose and 7.5g / L agar, pH 5.8, and 9 different hormone concentration ratios for subculture. Subculture once every 35 days, for a total of three subcultures. The culture temperature is (25±1)℃, the humidity is 60%, the light duration is 16 h / d, and the light intensity is 3000lx. The experimental results of Example 1 are shown in Table 2.

[0031] Table 2 Effects of different plant hormone ratios on the callus growth of Astragalus membranaceus Table 1 shows that the medium formulation significantly influences callus induction, with 6-BA playing a crucial role in the induction of adventitious buds. The 6-BA to NAA ratio, within a certain concentration range, influences the callus induction rate. When the 6-BA concentration is below 1.0 mg / L, the induction rate is low. When the 6-BA concentration is above 1.0 mg / L and the NAA concentration is at a low level, a higher number of successful inductions are achieved. When the 6-BA concentration is above 1.0 mg / L or the NAA concentration is above 0.5 mg / L, the callus induction rate decreases. The browning rate of callus is also affected by hormones. Low 6-BA concentrations result in a lower browning rate. Using formulation N4, the induction rate reached 93.51%, with a lower browning rate than N5. The callus also showed good growth, and a comparison of fresh and dry weights revealed that N4 had the highest dry weight.

[0032] These results indicate that N4 is the optimal culture medium formulation for Astragalus membranaceus, which is an MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar.

[0033] Example 2 This embodiment provides a culture method for improving the growth efficiency and medicinal value of Astragalus callus, comprising the following steps: S1. Inducing sterile seedlings: Rinse the Astragalus seeds and soak them in pure water. Then soak them in 0.5% sodium hypochlorite solution, rinse with sterile water, and inoculate the seeds with forceps onto pre-sterilized MS medium (121°C, 20 min) containing 30 g / L sucrose and 7.5 g / L agar, pH 5.8. S2. Callus Induction: Using sterile, well-grown seedlings from one month of culture, explant stem segments were cut and placed flat on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar. Culture plates were placed in a tissue culture room at (25 ± 1)°C, 60% humidity, and a photoperiod of 16 h / d at 3000 lx. S3. Subculture: Callus tissue with good growth condition was inoculated on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for subculture. Nine subculture times were set (0d, 5d, 10d, 15d, 20d, 25d, 30d, 35d, 40d), and subculture was carried out three times in total. The culture temperature was (25±1)℃, the humidity was 60%, the light duration was 16 h / d, and the light intensity was 3000lx.

[0034] The experimental results of Example 2 are as follows Figure 1-Figure 2 shown.

[0035] Depend on Figure 1 As can be seen, during callus culture, callus growth slowed from 1 to 5 days, likely due to the callus needing to adapt to a new growth environment, resulting in slightly slower growth. This period is the lag phase of callus growth. From 20 to 30 days, the callus rapidly absorbed nutrients from the culture medium and grew rapidly, with fresh weight increasing rapidly, representing the logarithmic growth phase. From 30 to 35 days, callus growth slowed. From 35 to 40 days, callus fresh weight growth plateaued and began to show signs of senescence and browning, likely due to nutrient depletion in the culture medium. With extended culture time, callus dry weight accumulation generally showed an increasing trend, similar to fresh weight. Growth was relatively slow from 1 to 5 days, followed by a significant increase from 5 to 25 days. From 25 to 30 days, dry weight slowly increased, reaching its maximum on 35 days. This suggests that with extended culture time, nutrients in the culture medium gradually depleted, hindering callus growth.

[0036] Depend on Figure 2 Saponin content increases slowly from 1 to 5 days, then accelerates from the 5th day and enters a logarithmic growth phase. The highest growth rate occurs from 15 to 25 days, during which saponin synthesis is rapid. By 30 days, the growth rate gradually slows, stagnating or even decreasing after 35 days, and the callus tissue also exhibits browning.

[0037] It can be seen that the changes in fresh weight and dry weight content are roughly the same as the changing trends of total saponins. It was found that when the callus tissue was cultured for 35 days, both the fresh and dry weight and the saponin content reached the maximum value, and it was determined that the best effect was achieved when the callus tissue was grown for 35 days.

[0038] Example 3 This embodiment provides a culture method for improving the growth efficiency and medicinal value of Astragalus callus, comprising the following steps: S1. Inducing sterile seedlings: Rinse the Astragalus seeds and soak them in pure water. Then soak them in 0.5% sodium hypochlorite solution, rinse with sterile water, and inoculate the seeds with forceps onto pre-sterilized MS medium (121°C, 20 min) containing 30 g / L sucrose and 7.5 g / L agar, pH 5.8. S2. Callus Induction: Using sterile, well-grown seedlings from one month of culture, explant stem segments were cut and placed flat on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar. Culture plates were placed in a tissue culture room at (25 ± 1)°C, 60% humidity, and a photoperiod of 16 h / d at 3000 lx. S3. Subculture: Callus tissue with good growth was inoculated onto MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar for subculture. Subculture was performed every 35 days for a total of three times. The culture temperature was (25 ± 1) °C, the humidity was 60%, the photoperiod was 16 h / d, and the light intensity was 3000 lx. S4. Add brassinolide: Select loose, light yellow, non-browning callus for cutting, and inoculate it on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar + 4 different concentrations of brassinolide culture medium for culture. There are 4 concentrations of brassinolide (0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.15 mg / L), 20 bottles for each treatment, 3 replicates, culture temperature (25±1)℃, humidity 60%, light duration 16 h / d, light intensity 3000 lx, and harvest after 35 days.

[0039] Example 4 This example provides a culture method for improving the growth efficiency and medicinal value of Astragalus callus. The method is basically the same as Example 1, except that: callus with good growth condition is selected for excision, and the excision is inoculated into MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar, and cultured in medium containing different concentrations of chitosan. The chitosan concentrations are set at four levels (50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L), with 20 bottles per treatment and three replicates.

[0040] The details are as follows: The following steps are involved: S1. Inducing sterile seedlings: Rinse the Astragalus seeds and soak them in pure water. Then soak them in 0.5% sodium hypochlorite solution, rinse with sterile water, and inoculate the seeds with forceps onto pre-sterilized MS medium (121°C, 20 min) containing 30 g / L sucrose and 7.5 g / L agar, pH 5.8. S2. Callus Induction: Using sterile, well-grown seedlings from one month of culture, explant stem segments were cut and placed flat on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar. Culture plates were placed in a tissue culture room at (25 ± 1)°C, 60% humidity, and a photoperiod of 16 h / d at 3000 lx. S3. Subculture: Callus tissue with good growth was inoculated onto MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar for subculture. Subculture was performed every 35 days for a total of three times. The culture temperature was (25 ± 1) °C, the humidity was 60%, the photoperiod was 16 h / d, and the light intensity was 3000 lx. S4. Add chitosan: Select loose, light yellow, non-browning callus tissue for cutting, and inoculate it on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar + 4 concentrations of chitosan medium for culture. Chitosan is set at 4 concentrations (50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L), 20 bottles for each treatment, 3 replicates are set, culture temperature is (25±1)℃, humidity is 60%, light time is 16 h / d, light intensity is 3000 lx, and harvest after 35 days.

[0041] Example 5 This example provides a culture method for improving the growth efficiency and medicinal value of Astragalus callus. The method is substantially the same as that of Example 1, except that callus tissue with good growth conditions is selected for excision and inoculated into MS medium (without chitosan and brassinolide) containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar for culture. Each treatment involves 20 bottles with three replicates.

[0042] The details are as follows: The following steps are involved: S1. Inducing sterile seedlings: Rinse the Astragalus seeds and soak them in pure water. Then soak them in 0.5% sodium hypochlorite solution, rinse with sterile water, and inoculate the seeds with forceps onto pre-sterilized MS medium (121°C, 20 min) containing 30 g / L sucrose and 7.5 g / L agar, pH 5.8. S2. Callus Induction: Using sterile, well-grown seedlings from one month of culture, explant stem segments were cut and placed flat on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar. Culture plates were placed in a tissue culture room at (25 ± 1)°C, 60% humidity, and a photoperiod of 16 h / d at 3000 lx. S3. Subculture: Callus tissue with good growth was inoculated onto MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar for subculture. Subculture was performed every 35 days for a total of three times. The culture temperature was (25 ± 1) °C, the humidity was 60%, the photoperiod was 16 h / d, and the light intensity was 3000 lx. S4: Select loose, light yellow, non-browning callus tissue for cutting, inoculate it on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for culture, and harvest after 35 days.

[0043] Example 5 was used as a control (CK) and compared with Examples 3-4. The comparison results are as follows: Figure 3-Figure 4 shown.

[0044] Depend on Figure 3 As shown in (a), chitosan and brassinolide treatments had significant effects on the fresh weight of callus (P<0.05). Under different concentrations of chitosan treatment, the fresh weight increased with the increase of concentration, and compared with the control group, the fresh weight increased by 20.10%, 28.45%, 49.44%, and 49.52%, respectively, and reached the maximum at K4 ( Figure 3 (a)). Under different concentrations of brassinolide treatment, the fresh weight showed a trend of increasing first and then decreasing with the increase of concentration. Compared with the control group, the fresh weight increased by 15.18%, 28.71%, 65.25%, and 12.38%, respectively, and reached the maximum at B3 ( Figure 3 (a)).

[0045] Depend on Figure 3As shown in (b), chitosan and brassinolide treatments had significant effects on callus dry weight (P<0.05). Under different concentrations of chitosan treatment, dry weight showed an increasing trend with increasing concentration, and compared with the control group, increased by 39.35%, 69.67%, 89.89%, and 100.35%, respectively, and reached the maximum in K2 treatment ( Figure 3 (b)). Under different concentrations of brassinolide treatment, the dry weight showed a trend of first increasing and then decreasing with increasing concentration. Compared with the control group, it increased by 9.83%, 40.61%, 88.10%, and 34.97%, respectively, and reached the maximum at B3 ( Figure 3 (b)).

[0046] Depend on Figure 3 As shown in (c), chitosan and brassinolide treatments had a significant effect on the content of malondialdehyde in callus tissue (P<0.05). Under different concentrations of chitosan treatment, the content of malondialdehyde showed a decreasing trend with increasing concentration. Compared with the control group, the content of malondialdehyde decreased by 70.98%, 79.09%, 79.18%, and 96.76%, respectively, and reached the minimum value at K4 ( Figure 3 (c)). Under different concentrations of brassinolide treatment, the malondialdehyde content showed a trend of first decreasing and then increasing with the increase of concentration. Compared with the control group, it decreased by 77.87%, 86.03%, 95.52%, and 88.36%, respectively, and reached the minimum value in the B3 treatment ( Figure 3 (c)).

[0047] Depend on Figure 3 As shown in (d), chitosan and brassinolide treatments had a significant effect on the rate of superoxide anion production in callus tissue (P<0.05). Under different concentrations of chitosan treatment, O2 - The generation rate showed a trend of first decreasing and then increasing with the increase of concentration. Compared with the control group, it decreased by 13.86%, 64.71%, 69.33%, and 68.01%, respectively, and reached the minimum value at K3 ( Figure 3 (d)). Under different concentrations of brassinolide, O2 - The generation rate showed a trend of first decreasing and then increasing with the increase of concentration. Compared with the control group, it decreased by 10.33%, 26.62%, 61.63%, and 51.28%, respectively, and reached the minimum value at B3 ( Figure 3 (d)).

[0048] Depend on Figure 3As shown in (e), chitosan and brassinolide treatments had a significant effect on the proline content in callus (P<0.05). Under different concentrations of chitosan treatment, the proline content showed a trend of first increasing and then decreasing with increasing concentration. Compared with the control group, the proline content increased by 1.56 times, 1.73 times, 0.69 times, and 0.83 times, respectively, and reached the maximum at K2 ( Figure 3 (e)). Under different concentrations of brassinolide treatment, the proline content showed a trend of first increasing and then decreasing with increasing concentration. Compared with the control group, it increased by 0.07 times, 0.79 times, 2.50 times, and 0.93 times, respectively, and reached the maximum at B3 ( Figure 3 (e)).

[0049] Depend on Figure 3 As shown in (f), chitosan and brassinolide treatments had a significant effect on the glutathione content in callus tissue (P<0.05). Under different concentrations of chitosan treatment, the glutathione content of all treatments was higher than that of the control group. The glutathione content reached a peak at K4, increasing by 1.05 times. K1, K2, and K3 increased by 0.75 times, 1.03 times, and 1.01 times, respectively. Figure 3 (f)). Under different concentrations of brassinolide treatment, the glutathione content showed a trend of increasing first and then decreasing with increasing concentration. Compared with the control group, it increased by 0.47 times, 0.98 times, 1.51 times, and 0.47 times, respectively, and reached the maximum at B3 ( Figure 3 (f)).

[0050] Depend on Figure 3 As shown in (g), chitosan and brassinolide treatments had a significant effect on callus peroxidase activity (P<0.05). Under different concentrations of chitosan treatment, compared with the control group, the peroxidase activities of K2, K3, and K4 increased by 48.03%, 93.88%, and 46.61%, respectively, and reached the maximum in K3, while the peroxidase activity of K1 did not show a significant increase ( Figure 3 (g)). Under different concentrations of brassinolide treatment, the peroxidase activity first increased and then decreased with the increase of concentration. Compared with the control group, the peroxidase activity increased by 30.60%, 65.27%, 22.54%, and 15.49%, respectively, and reached the maximum at B2 ( Figure 3 (g)).

[0051] Depend on Figure 3As shown in (h), different concentrations of chitosan and brassinolide had significant effects on superoxide dismutase activity (P<0.05). Under different concentrations of chitosan treatment, superoxide dismutase activity showed an increasing trend with increasing concentrations. Compared with the control group, the superoxide dismutase activity increased by 27.66%, 37.63%, 53.33%, and 108.33%, respectively, and reached the maximum at K4 ( Figure 3 (h)). Under different concentrations of brassinolide treatment, superoxide dismutase activity showed a trend of first increasing and then decreasing with increasing concentration. Compared with the control group, it increased by 152.66%, 145.33%, 216.67%, and 139.33%, respectively, and reached the maximum at B3 ( Figure 3 (h)).

[0052] Depend on Figure 4 As shown in (a), different concentrations of chitosan and brassinolide had significant effects on the soluble sugar content of callus (P<0.05). Under different concentrations of chitosan treatment, the soluble sugar content showed an increasing trend with the increase of concentration. Compared with the control group, it increased by 3.54%, 22.69%, 73.04%, and 202.12%, respectively, and reached the maximum at K4 ( Figure 4 (a)). Under different concentrations of brassinolide treatment, the soluble sugar content showed a trend of increasing first and then decreasing with increasing concentration. Compared with the control group, it increased by 102.12%, 145.03%, 168.79%, and 92.91%, respectively, and reached the maximum value at B3 ( Figure 4 (a)).

[0053] Depend on Figure 4 As shown in (b), different concentrations of chitosan and brassinolide had a significant effect on the soluble protein content of callus (P<0.05). Under different concentrations of chitosan treatment, the soluble protein content first decreased and then increased with the increase of concentration. Compared with the control group, it increased by 28.63%, 16.32%, 43.31%, and 153.32%, respectively, and reached the maximum at K4 ( Figure 4 (b)). Under different concentrations of brassinolide treatment, compared with the control group, the soluble protein content of B1, B2, and B3 increased by 13.04%, 14.44%, and 18.95%, respectively, and reached the maximum in B3, while the soluble protein content of B4 showed no significant increase trend ( Figure 4 (b)).

[0054] Depend on Figure 4As shown in (c), different concentrations of chitosan and brassinolide had significant effects on the vitamin C content of callus (P<0.05). Under different concentrations of chitosan treatment, the vitamin C content showed an increasing trend with the increase of concentration. Compared with the control group, it increased by 44.38%, 58.19%, 98.74%, and 269.86%, respectively, and reached the maximum value at K4 ( Figure 4 (c)). Under different concentrations of brassinolide treatment, the vitamin C content showed a trend of increasing first and then decreasing with increasing concentration. Compared with the control group, it increased by 57.75%, 252.93%, 555.06%, and 334.03%, respectively, and reached the maximum value at B3 ( Figure 4 (c)).

[0055] Depend on Figure 4 As shown in (d), different concentrations of chitosan and brassinolide had significant effects on the alkaloid content of callus (P<0.05). Under different concentrations of chitosan treatment, the alkaloid content increased with the increase of concentration. Compared with the control group, it increased by 1.16 times, 4.82 times, 5.09 times, and 6.39 times, respectively, and reached the maximum at K4 ( Figure 4 (d)). Under different concentrations of brassinolide treatment, the alkaloid content increased with the increase of concentration. Compared with the control group, it increased by 1.08 times, 1.32 times, 4.08 times, and 4.77 times, respectively, and reached the maximum value in B4 ( Figure 4 (d)).

[0056] Depend on Figure 4 As shown in (e), different concentrations of chitosan and brassinolide had significant effects on the polysaccharide content of callus (P<0.05). Under different concentrations of chitosan treatment, compared with the control group, the polysaccharide content of K1 and K4 increased by 50.97% and 39.37%, respectively, and reached the maximum in K1, while the polysaccharide content of K2 and K3 showed no obvious increasing trend ( Figure 4 (e)). Under different concentrations of brassinolide treatment, compared with the control group, the polysaccharide contents of B1, B2, and B3 increased by 51.19%, 6.34%, and 12.68%, respectively, and reached the maximum in B1, while the polysaccharide content of B4 showed no obvious increasing trend ( Figure 4 (e)).

[0057] Depend on Figure 4As shown in (f), chitosan and brassinolide at different concentrations significantly affected the total flavonoid content of Astragalus membranaceus callus (P<0.05). Under different concentrations of chitosan treatment, the total flavonoid content of K2, K3, and K4 increased by 1.939 times, 2.507 times, and 2.995 times, respectively, compared with the control group, and reached the maximum in K4, while the total flavonoid content of K1 showed no significant increase ( Figure 4 (f)). Under different concentrations of brassinolide treatment, the total flavonoid content showed a trend of increasing first and then decreasing with increasing concentration. Compared with the control group, it increased by 0.77 times, 1.09 times, 3.55 times, and 0.89 times, respectively, and reached the maximum value at B3 ( Figure 4 (f)).

[0058] Depend on Figure 4 As shown in (g), different concentrations of chitosan and brassinolide had a significant effect on the total saponin content of Astragalus membranaceus callus (P<0.05). Under different concentrations of chitosan treatment, the total saponin content of Astragalus membranaceus callus showed an increasing trend with the increase of concentration. Compared with the control group, it increased by 0.94 times, 1.45 times, 1.86 times, and 2.63 times, respectively, and reached the maximum at K4 ( Figure 4 (g). Under different concentrations of brassinolide treatment, the content of total saponins showed a trend of increasing first and then decreasing with increasing concentration. Compared with the control group, the content increased by 0.81 times, 1.14 times, 1.97 times, and 0.28 times, respectively, and reached the maximum value at B3 ( Figure 4 (g)).

[0059] Depend on Figure 4 As shown in (h), different concentrations of chitosan and brassinolide had a significant effect on the activity of phenylalanine ammonia lyase in callus tissue (P<0.05). Under different concentrations of chitosan treatment, the activity of phenylalanine ammonia lyase increased with the increase of concentration. Compared with the control group, the activity increased by 23.51%, 50.64%, 51.83%, and 76.93%, respectively, and reached the maximum at K4 ( Figure 4 (h)). Under different concentrations of brassinolide treatment, phenylalanine ammonia lyase activity increased first and then decreased with increasing concentration. Compared with the control group, it increased by 29.78%, 76.11%, 100.34%, and 53.99%, respectively, and reached the maximum at B3 ( Figure 4 (h)).

[0060] As shown in the results, chitosan treatment at a concentration of 200 mg / L resulted in the highest soluble sugar, soluble protein, and vitamin C contents in Astragalus membranaceus calli, as well as high secondary metabolite (alkaloids, flavonoids, and saponins) content and secondary metabolic enzyme activity (PAL). Polysaccharide content reached a high level at the low chitosan concentration (100 mg / L). Brassinolide treatment at a concentration of 0.1 mg / L resulted in the highest soluble sugar, soluble protein, and vitamin C contents in Astragalus membranaceus calli, as well as high secondary metabolite (flavonoids and saponins) content and secondary metabolic enzyme activity (PAL). Polysaccharide content reached its highest level at the low brassinolide concentration (0.05 mg / L), while alkaloid content reached its highest level at the high brassinolide concentration (0.15 mg / L).

[0061] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for increasing the growth efficiency and medicinal value of Astragalus callus, characterized in that: The following steps are involved: S1: Before sowing, wash the Astragalus seeds and soak them in pure water for 24 hours; then soak them in 0.5% sodium hypochlorite solution for 10-15 minutes, rinse with sterile water and dry them, and inoculate the seeds onto pre-sterilized MS medium supplemented with 30g / L sucrose and 7.5g / L agar, pH 5.8; and culture them in a tissue culture room. S2: Using sterile seedlings that have been cultured for one month and are in good growth condition, explant stem segments were cut and placed flat on MS medium containing 0.5-1.5 mg / L 6-BA, 0.5-1.5 mg / L NAA, 30 g / L sucrose, and 7.5 g / L agar; and cultured in a tissue culture room. S3: When the callus tissue grows to 35 days, the callus tissue with good growth condition is inoculated on MS medium containing 0.5-1.5 mg / L 6-BA, 0.5-1.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar for subculture. Subculture is performed every 35 days for a total of three subcultures. S4: Select callus tissue with good growth condition, cut it, and inoculate it on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 0.01-0.15 mg / L brassinolide, 30 g / L sucrose and 7.5 g / L agar for culture; alternatively, inoculate it on MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 50-200 mg / L chitosan, 30 g / L sucrose and 7.5 g / L agar for culture.

2. The culture method according to claim 1, wherein In step S1, the sample is soaked in a 0.5% sodium hypochlorite solution for 10-15 minutes, and then rinsed with sterile water 7-10 times.

3. The culture method according to claim 1, wherein The culture medium used in steps S2 and S3 is: MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar.

4. The culture method according to claim 1, wherein The culture medium used in step S4 is: MS medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 0.01 mg / L brassinolide, 30 g / L sucrose and 7.5 g / L agar.

5. The culture method according to claim 1, wherein The culture medium used in step S4 is: MS culture medium containing 1.0 mg / L 6-BA, 0.5 mg / L NAA, 100 mg / L chitosan, 30 g / L sucrose and 7.5 g / L agar.

6. The culture method according to claim 1, wherein The culture conditions of steps S1 to S4 are: temperature (25±1)°C, humidity 60%, photoperiod 16 h / d, and light intensity 3000 lx.

7. The culture method according to any one of claims 1 to 6, characterized in that The astragalus is Astragalus membranaceus.

8. Application of chitosan or brassinolide in Astragalus tissue culture technology.

9. The use according to claim 8, characterized in that The astragalus is Astragalus membranaceus.

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