A culture method and culture medium for rapidly proliferating scoparia cell biomass

Abstract

The present invention provides a method for rapidly proliferating hyoscyamine cell biomass, comprising the following steps: S1, obtaining hyoscyamine callus; S2, proliferating and culturing the callus; S3, suspending and culturing primary hyoscyamine cell biomass; and S4, proliferating and culturing secondary hyoscyamine cell biomass. The present invention also provides hyoscyamine cells prepared by the aforementioned method for rapidly proliferating hyoscyamine cell biomass. The present invention utilizes bioengineering cell suspension culture technology, employing suspension culture of callus cells induced from the hypocotyl of the hyoscyamine plant as an explant, and establishes a suspension culture system for hyoscyamine cells, effectively addressing the current shortage of hyoscyamine plant resources. This method enables the rapid, large-scale production of hyoscyamine plant cultures with high levels of active ingredients, thereby meeting the current market demand for hyoscyamine medicinal materials.

Description

Technical Field

[0001] The invention belongs to the field of plant cell fermentation, and particularly relates to a culture method for rapidly proliferating scoparia cell biomass. Background Art

[0002] Scotswort is a medicinal plant endemic to my country's Qinghai-Tibet Plateau. It belongs to the Solanaceae family and is a perennial herb with a fleshy, thick taproot. The complex and often harsh habitat of scotswort plants results in a slow natural renewal rate and resource scarcity. However, with the deepening of modern research and the gradual expansion of its application, the domestic market for scotswort alkaloid drugs currently stands at approximately 8 billion yuan, with a future market potential exceeding 12 billion yuan, representing significant economic benefits. In stark contrast to its widespread use, scotswort medicinal resources are extremely limited. A rare plant endemic to the Qinghai-Tibet Plateau, scotswort grows in valleys at altitudes between 2,200 and 4,200 meters. As a traditional Tibetan medicine, scotswort is commonly used for analgesia, antispasmodic effects, blood circulation, blood stasis removal, hemostasis, and tissue regeneration. It has significant clinical value and broad market potential.

[0003] Bioengineering suspension culture technology is the only way to achieve industrial production of plant cells today. Compared with solid culture, suspension culture can achieve better mixing and fluidity between cultured cells and culture medium, as well as higher mass transfer efficiency, which is very conducive to the rapid growth of cultured cells. In addition, the suspension culture method can also pave the way for the subsequent development of precursor feeding technology, so as to purposefully add various precursor substances to the liquid culture medium and obtain more plant active ingredients through effective transformation and culture.

[0004] Currently, there are no reports on cell suspension culture of Hyoscyamus scoparia, only on callus induction. For example, Xu Wenhua et al., "Study on Tissue Culture Technology of the Tibetan Medicinal Herb Hyoscyamus scoparia," Biotechnology Bulletin, 2008 Supplement, reported rapid propagation through organ culture and bud differentiation using two-year-old field-cultivated Hyoscyamus scoparia buds (dormant buds) and young leaves with petioles as explants. Zeren Wangmo, "Tissue Culture of the Tibetan Medicinal Herb Anisodus," Tibet Science and Technology, Issue 1, 2010. This study used seeds of the Tibetan medicinal herb Anisodus as explants for induction and differentiation culture. MS medium was used for the experiment. Callus differentiation and adventitious bud induction were best achieved in a medium supplemented with 0.2 mg / L NAA and 1 mg / L 6-BA, while callus and adventitious bud induction were best achieved in a medium supplemented with 0.2 mg / L NAA and 2 mg / L 6-BA. After 15 days on MS medium supplemented with 0.5 mg / L IAA rooting medium, the rooting rate was 100%. Previous literature has focused on obtaining callus tissue and then conducting callus differentiation and rooting. These reported methods have resulted in low callus induction rates, high rates of browning, and slow callus growth. For industrial-scale production, it is urgently necessary to cultivate anisodus plant cells that can rapidly proliferate and contain a higher concentration of phytoactive ingredients. Summary of the Invention

[0005] In order to solve the technical problem in the above-mentioned background technology that a kind of scopolamine plant cell that can proliferate rapidly and contains more plant active ingredients is urgently needed to cultivate, the present invention provides a culture method with high callus induction rate, low callus growth browning rate, and rapid proliferation of scopolamine cell biomass.

[0006] The present invention provides a culture method for rapidly proliferating scoparia cell biomass, comprising the following steps:

[0007] S1. Obtaining scopolamine callus: sterilizing scopolamine seeds and inoculating them into MS culture medium for germination to obtain sterile seedlings, taking the hypocotyls of the sterile seedlings and inoculating them into scopolamine callus induction medium, and obtaining the scopolamine callus through induction culture;

[0008] S2, callus proliferation culture: selecting the light yellow portion of the scopolamine callus obtained in step S1 and inoculating it into a scopolamine callus proliferation medium, and culturing it to obtain a proliferated callus;

[0009] S3, primary generation scopolamine cell suspension culture: selecting the light yellow portion of the proliferated callus obtained in step S2 and inoculating it into a primary generation scopolamine cell liquid culture medium, and culturing to obtain primary generation scopolamine cells;

[0010] S4. Subculture of scopolamine cells for proliferation: The primary scopolamine cells described in step S3, which have been cultured for 24 to 28 days, are inoculated into the secondary scopolamine cell proliferation culture liquid medium to obtain scopolamine cells after culture.

[0011] The scopolamine callus induction medium described in step S1 is based on a modified MS medium, wherein each liter of the modified MS medium contains the following components:

[0012] 0.4-0.8 mg of naphthaleneacetic acid, 0.5-1.5 mg of 6-benzylaminopurine, 1-2 mg of 2,4-dichlorophenoxyacetic acid, 0.1-0.3 mg of zeatin, 20-40 mg of L-cysteine, and 20-40 g of sucrose; the MS medium does not contain ammonium nitrate, and the concentration of potassium nitrate is 2850 mg / L;

[0013] The scopolamine callus proliferation culture medium described in step S2 is based on MS culture medium, and each liter of the MS culture medium contains the following components:

[0014] Naphthaleneacetic acid 0.6-1.0 mg, 6-benzylaminopurine 0.1-0.3 mg, 2,4-dichlorophenoxyacetic acid 1-3 mg, arginine 2-4 mg, ornithine 2-4 mg, sucrose 25-45 g;

[0015] The primary scopolamine cell liquid culture medium described in step S3 is based on a modified MS medium, wherein each liter of the modified MS medium contains the following components:

[0016] 6-benzylaminopurine 0.1-0.2 mg, 2,4-dichlorophenoxyacetic acid 1.5-2.5 mg, kinetin 0.1-0.3 mg, yeast polysan 40-80 mg, glutamine 60-100 mg, sucrose 15-35 g;

[0017] The concentration of folic acid in the modified MS medium is 1 mg / L, the concentration of vitamin B6 is 10 mg / L, and the concentration of vitamin B1 is 1 mg / L;

[0018] The subcultured scopolamine cell proliferation liquid culture medium in step S4 is based on a modified MS medium, wherein each liter of the modified MS medium contains the following components:

[0019] 6-benzylaminopurine 0.3-0.5 mg, 2,4-dichlorophenoxyacetic acid 1.0-3 mg, kinetin 0.1-0.3 mg, yeast polysaccharide 100-200 mg, phenylalanine 60-80 mg, sucrose 20-40 g;

[0020] The improved MS medium refers to an MS medium in which the KI concentration is 0.75 mg / L, the H3BO3 concentration is 3 mg / L, and the MnSO4·H2O concentration is 10 mg / L.

[0021] Further preferably, the MS culture medium in step S1 contains the following ingredients per liter: 0.6 mg of naphthaleneacetic acid, 1 mg of 6-benzylaminoadenine, 1.5 mg of 2,4-dichlorophenoxyacetic acid, 0.2 mg of zeatin, 30 mg of L-cysteine, and 30 g of sucrose;

[0022] The MS medium in step S2 contains the following ingredients per liter: 0.8 mg of naphthaleneacetic acid, 0.2 mg of 6-benzylaminoadenine, 2 mg of 2,4-dichlorophenoxyacetic acid, 3 mg of arginine, 3 mg of ornithine, and 35 g of sucrose;

[0023] The primary culture medium for scopolamine cells in step S3 is based on a modified MS medium containing the following ingredients per liter: 0.15 mg of 6-benzylaminoadenine, 2 mg of 2,4-dichlorophenoxyacetic acid, 0.2 mg of kinetin, 60 mg of zymosan, 80 mg of glutamine, and 25 g of sucrose. The secondary culture medium for scopolamine cell proliferation in step S4 contains the following ingredients per liter: 0.4 mg of 6-benzylaminoadenine, 2 mg of 2,4-dichlorophenoxyacetic acid, 0.2 mg of kinetin, 150 mg of zymosan, 70 mg of phenylalanine, and 35 g of sucrose.

[0024] The induction culture conditions in step S1 are: culture at 20-28° C. in the dark.

[0025] The proliferation culture conditions in step S2 are: culture at 25-29° C. in the dark.

[0026] The inoculation amount in step S3 is 20-40 g / L, and the culture conditions are: a gyroscopic shaker with a rotation speed of 90-130 r / min and culture at a temperature of 22-28° C. in the dark.

[0027] Among them, in step S4, the inoculation amount is 35-55 g / L, and the culture conditions are to select a gyratory shaking shaker with a rotation speed of 100-140 r / min, a temperature of 24-28° C., 8-12 hours of light per day, and a light intensity of 800-1200 lx.

[0028] The present invention also provides a scopolamine cell, which is prepared by the culturing method for rapidly proliferating scopolamine cell biomass.

[0029] After obtaining the callus tissue, the present invention performs suspension culture of the callus tissue. The suspension cultured scopolamine cells grow faster and increase their biomass faster. Currently, there is no relevant report on suspension culture of scopolamine cells in Chinese literature.

[0030] The present invention optimizes the components and composition of the culture medium to increase the callus induction rate and proliferation rate, thereby achieving rapid proliferation. Furthermore, the present invention utilizes a suspension culture method to grow anisodactyl cells in a liquid culture medium, avoiding the problem of uneven nutrient distribution on solid culture media. This results in faster growth and higher biomass, helping to improve the culture efficiency and yield of anisodactyl cells.

[0031] The present invention has the beneficial effect of specifically proposing a cell suspension culture method using biotechnology to achieve rapid production of hyoscyamus plant cells under artificially controlled conditions, thereby alleviating the current severe shortage of hyoscyamus medicinal material resources and ensuring the sustainable utilization of hyoscyamus plant resources. The present invention utilizes bioengineering cell suspension culture technology to induce callus cells produced using the hypocotyl of the hyoscyamus plant as an explant for suspension culture, and establishes a suspension culture system for hyoscyamus cells, effectively resolving the current shortage of hyoscyamus plant resources. This method can achieve large-scale and rapid production of cultures with high content of hyoscyamus plant active ingredients, thereby meeting the current large market demand for hyoscyamus medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison of the growth conditions of cotyledon and hypocotyl callus of the present invention (Figure A is cotyledon callus; Figure B is hypocotyl callus) DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] Example 1 Cultivation method for rapidly proliferating scoparia cell biomass of the present invention

[0035] S1. Obtaining callus of hyacinthus angustifolius: sterilized hyacinthus angustifolius seeds were inoculated into sterile seedlings germinated and grown on MS culture medium for 14 days, and the hypocotyls were selected as explants. The hypocotyls were placed flat on the surface of the culture medium and inoculated into a solid culture medium of modified MS + IAA 0.6 mg / L + 6-BA 1 mg / L + 2,4-D 1.5 mg / L + zeatin 0.2 mg / L + L-cysteine 30 mg / L + sucrose 30 g / L + agar 6.5 g / L, and callus induction culture was carried out at a temperature of 24 ° C and no light. The modified MS culture medium refers to removing the ammonium nitrate in the culture medium and replacing it with 2850 mg / L of potassium nitrate. After 28 days of culture, the callus induction rate was 100% and the browning rate was 0%.

[0036] S2. Proliferation culture of callus tissue: Select the light yellow callus tissue induced in step S1 and evenly spread it on MS + NAA 0.8 mg / L + 6-BA 0.2 mg / L + 2,4-D 2 mg / L + arginine 3 mg / L + ornithine 3 mg / L + activated carbon 1.5 mg / L -1 The culture medium was filled with 35 g / L sucrose and 4.5 g / L agar, and cultured at 27°C in the absence of light for 30 days. The callus proliferation was 9.13 times, and the browning rate was 3.18%.

[0037] S3. Primary cell suspension culture of Hyoscyamus scoparia: Select Hyoscyamus scoparia callus with light yellow-white color and loose texture produced by proliferation culture, inoculate it into modified MS+6-BA0.15mg / L+2,4-D2mg / L+KT0.2mg / L+zymosan 60mg / L+glutamine 80mg / L+sucrose 25g / L liquid culture medium with an inoculum size of 30g / L, select an orbital shaker with a speed of 110r / min, and culture at a temperature of 25℃ and no light. It was measured that the growth cycle of the primary cultured scopolamine cells was 35 days, including a stagnation period of 0 to 6 days, a delay period of 7 to 15 days, a logarithmic growth period of 16 to 28 days, and a stable growth period of 29 to 35 days; the folic acid concentration of the modified MS culture medium was changed to 1 mg / L, vitamin B6 was changed to 1 mg / L, and vitamin B1 was changed to 0.1 mg / L; the proliferation multiple of the scopolamine cells was counted to be 9.76 times after 35 days of primary culture.

[0038] S4. Subculture of hyoscyamine cell proliferation: Select hyoscyamine cells that have been cultured for 26 days. Ensure that the newly added culture medium accounts for 8 / 10 of the total culture medium volume for subculture, and the cell inoculation volume is 45g / L. The subculture growth medium is a liquid culture medium composed of modified MS+6-BA0.4mg / L+2,4-D2mg / L+KT0.2mg / L+zymosan 150mg / L+phenylalanine 70mg / L+sucrose 35g / L. Select a gyroscopic shaker with a speed of 120r / min. Incubate at a temperature of 26℃ and with daily light. The cells were cultured under conditions of 10 hours of illumination and a light intensity of 1000 lx; the growth cycle of the subcultured scopolamine cells was measured to be 30 days, including a stagnation period of 0 to 4 days, a delay period of 5 to 12 days, a logarithmic growth period of 13 to 25 days, and a stable growth period of 25 to 30 days; KI in the modified MS culture medium was changed to 0.75 mg / L, H3BO3 was changed to 3 mg / L, MnSO4·4H2O was changed to 0 mg / L, and MnSO4·4H2O was changed to 10 mg / L, and the proliferation multiples of the scopolamine cells were statistically calculated to be 14.96 times after 30 days of subculture.

[0039] Calculation of cell proliferation multiples: Take the scopolamine cells cultured in step S4 and centrifuge them at 4500 rpm for 10 min. Discard the supernatant and weigh the cell to obtain the fresh weight.

[0040] Cell proliferation multiple = (harvest fresh weight - inoculum fresh weight) / inoculum fresh weight;

[0041] The pH value of the above culture medium is 6.

[0042] Example 2 Cultivation method for rapidly proliferating scoparia cell biomass of the present invention

[0043] S1. Obtaining callus tissue of hyoscyamine: sterilized hyoscyamine seeds were inoculated into sterile seedlings germinated and grown on MS culture medium for 14 days, and the hypocotyls were selected as explants. They were placed flat on the surface of the culture medium and inoculated into a solid culture medium of modified MS+IAA0.4mg / L+6-BA0.5mg / L+2,4-D1mg / L+KT0.1mg / L+L-cysteine20mg / L+sucrose20g / L+agar6g / L, and callus induction culture was carried out at a temperature of 20°C and in the absence of light. The modified MS culture medium refers to removing the ammonium nitrate in the culture medium and changing the potassium nitrate content to 2850mg / L. After 28 days of culture, the callus induction rate was 91.13% and the browning rate was 2.67%.

[0044] S2. Proliferation culture of callus tissue: Select the light yellow callus tissue induced in step S2 and evenly spread it on MS + NAA 0.6 mg / L + 6-BA 0.1 mg / L + 2,4-D 1 mg / L + arginine 2 mg / L + ornithine 2 mg / L + activated carbon 1 mg·L -1 The culture medium was filled with 25 g / L sucrose and 4 g / L agar, and cultured at 25°C in the absence of light for 30 days. The callus proliferation was 8.04 times, and the browning rate was 2.98%.

[0045] S3. Primary generation of scopolamine cell suspension culture: Select scopolamine callus tissue with light yellow-white color and loose texture produced by proliferation culture, and use 20g·L -1 The cells were inoculated with a modified MS medium containing 0.1 mg / L 6-BA, 1.5 mg / L 2,4-D, 0.1 mg / L KT, 40 mg / L yeast polysaccharide, 60 mg / L glutamine, and 15 g / L sucrose. Culture was performed on an orbital shaker at 90 rpm at 22°C in the absence of light. The growth cycle of primary cultures of Hyoscyamus scoparia cells was measured to be 35 days, with a lag phase of 0 to 6 days, a delay phase of 7 to 15 days, a logarithmic growth phase of 16 to 28 days, and a stationary growth phase of 29 to 35 days. The modified MS medium was modified with 1 mg / L folic acid, 1 mg / L vitamin B6, and 10 mg / L vitamin B1. After 35 days of primary culture, the proliferation rate of Hyoscyamus scoparia cells was 8.24-fold.

[0046] S4. Subculture of hyoscyamine cell proliferation: Select hyoscyamine cells that have been cultured for 24 days. Subculture to ensure that the newly added culture medium accounts for 7 / 10 of the total culture medium volume. The cell inoculation volume is 35 g·L -1The subculture growth medium was a liquid culture medium consisting of modified MS+6-BA0.3mg / L+2,4-D1.0mg / L+KT0.1mg / L+zymosan 100mg / L+phenylalanine 60mg / L+sucrose 30g / L, and cultured on a gyratory shaker at a speed of 100r / min at a temperature of 24°C, 8 hours of light per day, and a light intensity of 800lx. The results showed that the subcultured scopolamine The cell growth cycle is 30 days, including a stagnation phase of 0 to 4 days, a delay phase of 5 to 12 days, a logarithmic growth phase of 13 to 25 days, and a stable growth phase of 25 to 30 days. In the improved MS culture medium, KI is changed to 0.75 mg / L, H3BO3 is changed to 3 mg / L, MnSO4·4H2O is changed to 0 mg / L, and MnSO4·4H2O is changed to 10 mg / L. After subculture for 30 days, the proliferation multiple of the scopolamine cells is statistically 13.67 times.

[0047] Calculation of cell proliferation multiples: Take the cultured scopolamine cells and centrifuge them at 4500 rpm for 10 min. Discard the supernatant and weigh the cell to obtain the fresh weight.

[0048] Cell proliferation multiple = (harvest fresh weight - inoculum fresh weight) / inoculum fresh weight;

[0049] The pH value of the above culture medium is 5.8.

[0050] Example 3 Cultivation method for rapidly proliferating scoparia cell biomass of the present invention

[0051] S1. Obtaining callus tissue of hyoscyamine: sterilized hyoscyamine seeds were inoculated into sterile seedlings germinated and grown on MS culture medium for 14 days, and the hypocotyls were selected as explants. They were placed flat on the surface of the culture medium and inoculated into a solid culture medium of modified MS+IAA0.8mg / L+6-BA1.5mg / L+2,4-D2mg / L+KT0.3mg / L+L-cysteine40mg / L+sucrose40g / L+agar7g / L, and callus induction culture was carried out at a temperature of 28°C and in the absence of light. The modified MS culture medium refers to removing the ammonium nitrate in the culture medium and changing the potassium nitrate content to 2850mg / L. After 28 days of culture, the callus induction rate was 94.21% and the browning rate was 10.62%.

[0052] S2. Proliferation culture of callus tissue: Select the light yellow callus tissue induced in step S1 and spread it evenly on MS + NAA 1.0 mg / L + 6-BA 0.3 mg / L + 2,4-D 3 mg / L + arginine 4 mg / L + ornithine 4 mg / L + activated carbon 2.0 mg / L -1On the surface of the culture medium containing 45 g / L sucrose and 5 g / L agar, the callus tissue was cultured at 29°C in the absence of light for 30 days. The proliferation rate was 8.27 times and the browning rate was 11.58%.

[0053] S3. Primary generation of scopolamine cell suspension culture: Select scopolamine callus tissue with light yellow-white color and loose texture produced by proliferation culture, and use 40g·L -1 The cells were inoculated with a modified MS medium containing 0.2 mg / L 6-BA, 2.5 mg / L 2,4-D, 0.3 mg / L KT, 80 mg / L yeast polysaccharide, 100 mg / L glutamine, and 35 g / L sucrose. Culture was performed on an orbital shaker at 120 rpm at 28°C in the absence of light. The growth cycle of primary cultures of Hyoscyamus scoparia cells was measured to be 35 days, with a lag phase of 0 to 6 days, a delay phase of 7 to 15 days, a logarithmic growth phase of 16 to 28 days, and a stationary growth phase of 29 to 35 days. The modified MS medium was modified with 1 mg / L folic acid, 1 mg / L vitamin B6, and 10 mg / L vitamin B1. After 35 days of primary culture, the proliferation rate of Hyoscyamus scoparia cells was 8.56-fold.

[0054] S4. Subculture of scopolamine cells: Select scopolamine cells that have been cultured for 28 days. Subculture to ensure that the newly added culture medium accounts for 9 / 10 of the total culture medium volume. The cell inoculation volume is 55 g·L -1 The subculture growth medium was a liquid medium consisting of modified MS+6-BA0.5mg / L+2,4-D3mg / L+KT0.3mg / L+zymosan 200mg / L+phenylalanine 80mg / L+sucrose 40g / L, and cultured on a gyratory shaker at a speed of 140r / min at a temperature of 28°C, 12 hours of light per day, and a light intensity of 1200lx. The cell growth cycle is 30 days, including a stagnation phase of 0 to 4 days, a delay phase of 5 to 12 days, a logarithmic growth phase of 13 to 25 days, and a stable growth phase of 25 to 30 days. In the improved MS culture medium, KI is changed to 0.75 mg / L, H3BO3 is changed to 3 mg / L, MnSO4·4H2O is changed to 0 mg / L, and MnSO4·4H2O is changed to 10 mg / L. After subculture for 30 days, the proliferation multiple of the scopolamine cells is statistically 14.13 times.

[0055] Calculation of cell proliferation multiples: Take the scopolamine cells cultured in step S4 and centrifuge them at 4500 rpm for 10 min. Discard the supernatant and weigh the cell to obtain the fresh weight.

[0056] Cell proliferation multiple = (harvest fresh weight - inoculum fresh weight) / inoculum fresh weight;

[0057] The pH value of the above culture medium is 6.2.

[0058] Comparative Example 1:

[0059] In step S1 of Example 1, the explant was inserted into the culture medium. The other steps were the same as those of Example 1. After culturing for 28 days, the callus induction rate was 77% and the browning rate was 29.18%.

[0060] Comparative Example 2:

[0061] In step S1 of Example 1, the basic culture medium was changed to MS and the L-cysteine component in the culture medium was removed. The other steps were the same as in Example 1. After culturing for 28 days, the callus induction rate was 76.93% and the browning rate was 37.98%.

[0062] Comparative Example 3:

[0063] In step S2 of Example 1, callus tissue with brown or dark yellow color was selected, and the other steps were the same as those of Example 1. After culturing for 30 days, the callus tissue proliferation rate was 3.25 times, and the browning rate was higher than 63.15%.

[0064] Comparative Example 4:

[0065] In step S2 of Example 1, arginine and ornithine were removed from the culture medium. Other steps were the same as in Example 1. After culturing for 30 days, the callus proliferation rate was 6.17 times, and the browning rate was 12.18%.

[0066] Comparative Example 5

[0067] In step S2 of Example 1, the agar in the culture medium was changed to 6.5 g / L. The other steps were the same as in Example 1. After 30 days of culture, the callus proliferation rate was 7.62 times, and the browning rate was 23.68%. (Agar had no effect on the growth results.)

[0068] Comparative Example 6

[0069] In step S2 of Example 1, the culture conditions were changed to a temperature of 30° C., 12 hours of light per day, and a light intensity of 1400 lx. The other steps were the same as in Example 1. After 30 days of culture, the callus proliferation was 5.97 times, and the browning rate was 34.63%.

[0070] Comparative Example 7

[0071] In step S3 of Example 1, callus tissue with yellow color and compact texture was selected, and the other steps were the same as in Example 1. The callus tissue proliferation rate was statistically calculated to be 5.38 times after 35 days of primary culture.

[0072] Comparative Example 8

[0073] In step S3 of Example 1, the yeast polysaccharide and glutamine components in the culture medium were removed, and the other steps were the same as in Example 1. The callus proliferation rate after 35 days of primary culture was 6.41 times.

[0074] Comparative Example 9

[0075] In step S3 of Example 1, a reciprocating shaking table was used instead, and the other steps were the same as in Example 1. The callus proliferation rate was statistically calculated to be 7.62 times after 35 days of primary culture.

[0076] Comparative Example 10

[0077] In step S3 of Example 1, the basic culture medium was changed to MS, and the other steps were the same as in Example 1. The callus proliferation rate was statistically calculated to be 6.73 times after 35 days of primary culture.

[0078] Comparative Example 11

[0079] In step S4 of Example 1, the 40-day-primary cultured scoparia cells were selected for subculture. The other steps were the same as those of Example 1. The proliferation multiple of the callus tissue after 30 days of subculture was 8.03 times.

[0080] Comparative Example 12

[0081] In step S4 of Example 1, the cell inoculation volume was changed to 25 g·L -1 The other steps were the same as those in Example 1. The callus proliferation rate after 30 days of subculture was 7.34 times.

[0082] Comparative Example 13

[0083] In step S4 of Example 1, the basic culture medium was changed to MS, and the yeast polysaccharide and phenylalanine components in the culture medium were removed. The other steps were the same as in Example 1. After subculture for 30 days, the callus proliferation rate was statistically 8.54 times.

[0084] In order to further illustrate the advantages of the technical solution of the present invention, the following experimental verification is carried out:

[0085] Test Example 1 Parameter selection test of the culture method of the present invention

[0086] 1. Screening results of the best explants for callus induction

[0087] Different explants have different degrees of differentiation and different abilities to form callus tissue. This experiment used 4 types of explants. After 28 days of culture, the callus tissue induction growth status was statistically analyzed and shown in Table 1. Figure 1 shown.

[0088] Table 1 Effects of different explants on callus induction of Cistanche deserticola

[0089]

[0090] Summary: The results showed that callus could be induced from all four explants of Hyoscyamus scoparia: cotyledons, true leaves, hypocotyls, and petioles. However, the callus induction rate and browning rate varied significantly between the explants. Specific analysis of the experimental results revealed that hypocotyl explants had a callus induction rate as high as 83.3%, with a browning rate of only 6.8%. Cotyledon explants had the lowest callus induction rate, at only 33.1%, and the callus growth rate was slow. True leaf and petiole explants had callus induction rates of 51.7% and 42.4%, respectively. However, true leaf explants had the highest browning rate of 32.4%, while radicle explants had a browning rate of 6.8%.

[0091] Therefore, considering the callus induction and browning of the four explants of Scots dasyphylla, it was finally determined that the hypocotyl explant was the best explant for callus induction of Scots dasyphylla.

[0092] 2. Effect of sucrose concentration on the growth of scoparia cells

[0093] Sucrose was used as the carbon source in the culture medium at concentrations of 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L. After inoculation with scopolamine cells and culturing for 18 days, the experimental results are shown in Table 2.

[0094] Table 2 Effect of sucrose concentration on the growth of Anisodactyla suspension cells

[0095]

[0096]

[0097] Summary: As the sucrose concentration increased from 10 g / L to 60 g / L, the proliferation multiple and growth rate of scopolamine cells showed a trend of first increasing and then decreasing. At a sucrose concentration of 20 g / L, the proliferation multiple reached a maximum of 9.63 times, and the growth rate was the fastest at 1.77 g / (L·d). Under these conditions, the cells grew rapidly, the particles were uniform, there was no browning, and the color was light yellow or light cyan. When the sucrose concentration was 60 g / L, the proliferation multiple was the lowest at only 2.3 times, the growth rate was the slowest at 0.54 g / (L·d), the cells appeared light gray, and browning was severe. This is because the high concentration of sucrose affects the cell osmotic pressure, which seriously affects cell growth. The results show that a sucrose concentration of 20 g / L in the culture medium is most conducive to the growth of scopolamine plant cells.

[0098] 3. Effect of inoculum size on the growth of hyoscyamus cells

[0099] To screen the optimal inoculum size for suspension culture of scopolamine cells, 2.0 g, 3.0 g, 4.0 g, 5.0 g, and 6.0 g of scopolamine cells were inoculated per 100 mL of culture medium, respectively. The statistical results after 18 days of culture are shown in Table 3.

[0100] Table 3 Effect of inoculum size on the growth of suspension culture cells of Hyoscyamus scoparia

[0101]

[0102] When the cell inoculum size ranged from 2.0 to 12.0 g, the proliferation multiple of the hyoscyamus plant cells showed a trend of first increasing and then decreasing with increasing inoculum size. When the inoculum size was 4.0 g, the cell proliferation multiple reached a maximum of 11.70 times. Thereafter, as the inoculum size increased, the cell proliferation multiple decreased, reaching only 2.50 times when the inoculum size reached 12.0 g. The cell growth rate of the hyoscyamus plant cells also showed a trend of first increasing and then decreasing with increasing inoculum size. When the inoculum size was 8.0 g, the cell growth rate was the fastest at 3.60 g / (L·d); when the inoculum size was 2.0 g, the cell growth rate was the slowest at 1.15 g / (L·d). The results showed that to maximize the cell proliferation multiple, the optimal inoculum size per 100 mL of culture medium was 4.0 g; and to rapidly increase the cell biomass, the optimal inoculum size per 100 mL of culture medium was 8.0 g.

[0103] 4. Effect of shaker speed on the growth of scoparia cells

[0104] The inoculated scopolamine cells were placed on a shaker at rotation speeds of 100, 110, 120, and 130 r / min, respectively. After 18 days, the proliferation times of the scopolamine plant cells were counted. The results are shown in Table 4.

[0105] Table 4 Effect of shaker speed on the growth of Anisodactyla suspension culture cells

[0106]

[0107] The shaker speed significantly affected the proliferation of scopolamine cells. Within the speed range of 100 to 130 r / min, the proliferation fold and growth rate of scopolamine cells first increased and then decreased. When the shaker speed was 100 r / min, the proliferation fold of scopolamine cells was the lowest at 7.07, and the cell growth rate was the slowest at 1.79 g / (L·d). When the shaker speed increased to 120 r / min, the proliferation fold of scopolamine cells reached a maximum of 12.33, and the cell growth rate reached a maximum of 2.96 g / (L·d). When the speed continued to increase to 130 r / min, the proliferation fold of scopolamine cells began to decrease to 11.30, and the cell growth rate decreased to 2.73 g / (L·d). These results indicate that a shaker speed of 120 r / min is the optimal condition for the growth of scopolamine cells.

[0108] 5. Effect of light duration on the proliferation of callus tissue of Ranunculaceae

[0109] The effects of different light exposure times on the proliferation of callus tissue of Ranunculaceae are shown in Table 5.

[0110] Table 5 Effects of different light exposure times on the proliferation of callus tissue of Ranunculaceae

[0111]

[0112] Throughout this specification, terms such as "one embodiment," "some embodiments," and "specific embodiments" mean that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapidly proliferating the biomass of hyssop cells, characterized in that: The following steps are involved: S1. Obtaining scopolamine callus: sterilizing scopolamine seeds and inoculating them into MS culture medium for germination to obtain sterile seedlings, taking the hypocotyls of the sterile seedlings and inoculating them into scopolamine callus induction medium, and obtaining the scopolamine callus through induction culture; S2, callus proliferation culture: selecting the light yellow portion of the scopolamine callus obtained in step S1 and inoculating it into a scopolamine callus proliferation medium, and culturing it to obtain a proliferated callus; S3, primary generation scopolamine cell suspension culture: selecting the light yellow portion of the proliferated callus obtained in step S2 and inoculating it into a primary generation scopolamine cell liquid culture medium, and culturing to obtain primary generation scopolamine cells; S4. Subculture of scopolamine cells for proliferation: inoculating the primary scopolamine cells described in step S3 after culturing for 24 to 28 days into a liquid culture medium for secondary scopolamine cell proliferation to obtain scopolamine cells; The scopolamine callus induction medium described in step S1 is based on a modified MS medium, wherein each liter of the modified MS medium contains the following components: 0.4-0.8 mg of naphthaleneacetic acid, 0.5-1.5 mg of 6-benzylaminopurine, 1-2 mg of 2,4-dichlorophenoxyacetic acid, 0.1-0.3 mg of zeatin, 20-40 mg of L-cysteine, and 20-40 g of sucrose; the MS medium does not contain ammonium nitrate, and the concentration of potassium nitrate is 2850 mg / L; The scopolamine callus proliferation culture medium described in step S2 is based on MS culture medium, and each liter of the MS culture medium contains the following components: Naphthaleneacetic acid 0.6-1.0 mg, 6-benzylaminopurine 0.1-0.3 mg, 2,4-dichlorophenoxyacetic acid 1-3 mg, arginine 2-4 mg, ornithine 2-4 mg, sucrose 25-45 g; The primary scopolamine cell liquid culture medium described in step S3 is based on a modified MS medium, wherein each liter of the modified MS medium contains the following components: 6-benzylaminopurine 0.1-0.2 mg, 2,4-dichlorophenoxyacetic acid 1.5-2.5 mg, kinetin 0.1-0.3 mg, yeast polysan 40-80 mg, glutamine 60-100 mg, sucrose 15-35 g; The modified MS medium contains 1 mg / L of folic acid, 10 mg / L of vitamin B6, and 1 mg / L of vitamin B1. The subcultured scopolamine cell proliferation liquid culture medium in step S4 is based on a modified MS medium, wherein each liter of the modified MS medium contains the following components: 6-benzylaminopurine 0.3-0.5 mg, 2,4-dichlorophenoxyacetic acid 1.0-3 mg, kinetin 0.1-0.3 mg, yeast polysaccharide 100-200 mg, phenylalanine 60-80 mg, sucrose 20-40 g; The modified MS medium refers to the MS medium in which the KI concentration is 0.75 mg / L, the H3BO3 concentration is 3 mg / L, and the MnSO4·H2O concentration is 10 mg / L; Among them, the conditions for the induction culture in step S1 are: culture in the dark at 20-28°C; the conditions for the proliferation culture in step S2 are: culture in the dark at 25-29°C; the inoculation amount in step S3 is 20-40 g / L, and the culture conditions are selecting a gyroscopic shaker, a speed of 90-130 r / min, and culture at a temperature of 22-28°C in the dark; in step S4, the inoculation amount is 35-55 g / L, and the culture conditions are selecting a gyroscopic shaker, a speed of 100-140 r / min, and a light exposure of 8-12 hours per day at a temperature of 24-28°C, with a light intensity of 800-1200 lx.

2. The culture method according to claim 1, wherein: The MS medium in step S1 contains the following ingredients per liter: 0.6 mg of naphthaleneacetic acid, 1 mg of 6-benzylaminoadenine, 1.5 mg of 2,4-dichlorophenoxyacetic acid, 0.2 mg of zeatin, 30 mg of L-cysteine, and 30 g of sucrose; The MS medium in step S2 contains the following ingredients per liter: 0.8 mg of naphthaleneacetic acid, 0.2 mg of 6-benzylaminoadenine, 2 mg of 2,4-dichlorophenoxyacetic acid, 3 mg of arginine, 3 mg of ornithine, and 35 g of sucrose; The primary scopolamine cell liquid culture medium in step S3 is based on a modified MS medium containing the following ingredients per liter: 0.15 mg of 6-benzylaminopurine, 2 mg of 2,4-dichlorophenoxyacetic acid, 0.2 mg of kinetin, 60 mg of zymosan, 80 mg of glutamine, and 25 g of sucrose; The subcultured scoparia cell proliferation culture liquid medium in step S4 contains the following ingredients per liter: 0.4 mg of 6-benzylaminopurine, 2 mg of 2,4-dichlorophenoxyacetic acid, 0.2 mg of kinetin, 150 mg of zymosan, 70 mg of phenylalanine, and 35 g of sucrose.

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