A method for culturing orobanche embryonic stem cells

Abstract

This invention provides a method for culturing embryonic stem cells from Orobanchaceae plants. The steps include: obtaining explants from Orobanchaceae plants; inducing culture of the explants to obtain embryonic stem cells; and inoculating the embryonic stem cells into an expansion medium containing Artemisia annua extract for further culture. This invention provides, for the first time, a method for the isolation and rapid culture of embryonic stem cells from Orobanchaceae plants. This method is independent of host plants and can rapidly and massively obtain Orobanchaceae plant stem cells under artificial conditions, fundamentally solving the problem of ecological damage caused by traditional production methods. For the first time, Artemisia annua extract is applied to the culture of Orobanchaceae plant stem cells. Utilizing its natural antibacterial and bioactivity, it not only effectively inhibits microbial contamination during the culture process but also unexpectedly shows that it can significantly promote the growth of Orobanchaceae plant stem cells, shorten the culture cycle, and reduce production costs.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and specifically to a method for culturing embryonic stem cells from Orobanchaceae plants. Background Technology

[0002] Orobanchaceae plants are classified as holoparasitic or hemiparasitic, annual or perennial herbaceous plants, almost devoid of chlorophyll. Their stems are usually unbranched, or in a few species branched. Leaves are scale-like, spirally arranged, or densely arranged in a near-imbriated pattern at the base of the stem. Orobanchaceae comprises 15 genera and approximately 150 species, mainly distributed in the North Temperate Zone, with a few species found in Africa, Oceania, Asia, and the Americas. China has 9 genera, 40 species, and 3 varieties, mainly distributed in the west, with a few species distributed in the northeast, north, central, southwest, and south. Members of Orobanchaceae parasitize the roots of other plants. Some species have a wider host range, parasitizing various plants within a specific genus or family; for example, *Cistanche* parasitizes the roots of plants in the *Haloxylon* genus. Other species prefer to parasitize cultivated crops; for example, *Orobanchaceae var. branchi* often parasitizes the roots of plants such as cantaloupe and cucumber.

[0003] The Orobanchaceae family, as it is commonly known, is mainly composed of medicinal plants such as Cistanche deserticola, Cistanche salsa, wild rice, and yellow broomrape. Taking the common Cistanche deserticola as an example, it typically parasitizes the roots of plants like Haloxylon ammodendron, absorbing nutrients and water from them. Cistanche deserticola is used medicinally for its fleshy stems with scaly leaves, and it is often available in the market as a dried whole stem or sliced ​​products. Cistanche deserticola is believed to have effects such as tonifying the kidneys and replenishing essence, moistening dryness and promoting bowel movement, delaying aging, and enhancing immunity. Modern research shows that Cistanche deserticola contains phenylethanoid glycosides, iridoids, lignans, polysaccharides, more than a dozen amino acids, and various alkaloids, and is rich in trace elements needed by the human body. Among these, total phenylethanoid glycosides are the main active components of Cistanche deserticola, while phenylethanoid glycosides, including echinacoside and verbascoside, are indicator components for detecting Cistanche deserticola. Cistanche deserticola polysaccharides can enhance the body's immunity; betaine has anti-cancer and blood pressure-lowering functions. It is evident that medicinal plants of the Orobanchaceae family have extremely high value, but their growth characteristics should not be overlooked.

[0004] Because plants in the Orobanchaceae family are parasitic and require specific growing environments, often parasitizing the roots of other plants, indiscriminate harvesting severely damages the growth of their host plants, such as Haloxylon ammodendron, Salix matsudana, Salix babylonica, and Salix babylonica var. thunbergii. These host plants are often planted as pioneers in sand control and afforestation in barren and arid desert regions, and the harvesting of Orobanchaceae plants further exacerbates desertification. Meanwhile, my country's annual market demand for Cistanche deserticola alone is around 500 tons, while domestic production is only around 70 tons, far from meeting market demand.

[0005] Currently, the main method for increasing production of Orobanchaeidae plants is artificial cultivation. However, due to the limitations of their growth characteristics, they primarily grow via parasitic roots, failing to fundamentally address the environmental damage issue. While cultivating Orobanchaeidae plants independently of their host plants presents significant challenges, liquid cell culture is relatively easier. Establishing a more universal cell culture system for Orobanchaeidae plants would allow for large-scale, industrialized production of the required plant cells under controlled conditions. By adding specific components, the cell culture cycle can be shortened, saving costs while simultaneously enabling resource reuse. This approach not only protects existing wild resources of Orobanchaeidae plants but also fundamentally avoids damage to host plants. Therefore, the current technology warrants further development. Summary of the Invention

[0006] To address the shortcomings of existing technologies and solve the aforementioned problems, a method for culturing embryonic stem cells from Orobanchaceae plants is proposed, and the following technical solution is provided: A method for culturing embryonic stem cells from Orobanchaceae plants includes the following steps: obtaining explants from Orobanchaceae plants, inducing and culturing the explants to obtain embryonic stem cells, and inoculating the embryonic stem cells into an expansion medium containing Artemisia annua extract for further culture.

[0007] Furthermore, the Artemisia annua extract is at least one of Artemisia annua stem cell extract, Artemisia annua water extract, or Artemisia annua alcohol extract.

[0008] Furthermore, the amount of Artemisia annua extract added is 0.5-2 mg / L, calculated based on its artemisinin content.

[0009] Furthermore, the Artemisia annua extract was obtained by using Artemisia annua stem cell powder as raw material, followed by ultrasonication and centrifugation, and then taking the supernatant.

[0010] Furthermore, embryonic stem cells were seeded into an expansion medium containing Artemisia annua extract and then cultured in the dark for 20-22 days at 22-28°C and 100-120 rpm.

[0011] Furthermore, the explant is a scale from a plant of the Orobanchaceae family.

[0012] Furthermore, the explants are cultured in an induction medium to obtain embryonic stem cells. The induction medium contains a basal medium, a first carbon source, a first plant growth regulator, and a first anti-browning agent.

[0013] Furthermore, the first carbon source is sucrose with a concentration of 15-30 g / L, the first plant growth regulator is selected from one or more of 2,4-dichloronaphthyloxyacetic acid, kinetin, and indoleacetic acid, and the first anti-browning agent is selected from one or two of ascorbic acid and citric acid.

[0014] Furthermore, the amplification medium also includes a basal medium, a second carbon source, a second organic additive, a second plant growth regulator, and a second anti-browning agent.

[0015] Furthermore, the second carbon source is sucrose at a concentration of 30-50 g / L, the second organic additive includes inositol and hydrolyzed casein, the second plant growth regulator includes 2,4-dichloronaphthyloxyacetic acid and indoleacetic acid, and the second anti-browning agent includes one or more of ascorbic acid, citric acid, and polyvinylpyrrolidone.

[0016] Beneficial effects: 1. This invention is the first to apply Artemisia annua extract to the culture of Orobanchaeaceae plant stem cells. Utilizing its natural antibacterial and bioactivity, it not only effectively inhibits microbial contamination during the culture process, but also unexpectedly discovers that it can significantly promote the growth of Orobanchaeaceae plant stem cells, shorten the culture cycle, and reduce production costs.

[0017] 2. This invention provides for the first time a method for isolating and rapidly culturing embryonic stem cells of Orobanchaeaceae plants. This method does not rely on host plants and can rapidly and massively obtain Orobanchaeaceae plant stem cells under artificial conditions, fundamentally solving the problem of ecological damage caused by traditional production methods.

[0018] 3. The present invention, through an optimized induction culture medium and an expansion culture system supplemented with Artemisia annua extract, yields embryonic stem cells with high purity, strong vitality, and rapid proliferation rate, laying the foundation for their industrial-scale production. Attached Figure Description

[0019] Figure 1 This is a growth curve of Cistanche deserticola stem cells after adding different concentrations of Artemisia annua extract in Example 1 of the present invention; Figure 2 This is a growth curve of Cistanche stem cells after adding different concentrations of Artemisia annua extract in Example 2 of the present invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Based on the embodiments in this application, other similar embodiments obtained by those skilled in the art without creative effort should all fall within the scope of protection of this application.

[0021] According to embodiments of the present invention, a method for culturing embryonic stem cells from Orobanchaceae plants is provided. The steps include: obtaining explants from Orobanchaceae plants; inducing culture of the explants to obtain embryonic stem cells; and inoculating the embryonic stem cells into an expansion medium containing Artemisia annua extract for culture. This invention provides, for the first time, a method for the isolation and rapid culture of embryonic stem cells from Orobanchaceae plants. This method is independent of host plants and can rapidly and massively obtain Orobanchaceae plant stem cells under artificial conditions, fundamentally solving the problem of ecological damage caused by traditional production methods. It is also the first time that Artemisia annua extract has been applied to the culture of Orobanchaceae plant stem cells. The extract contains artemisinin, which, utilizing its natural antibacterial and bioactive properties, not only effectively inhibits microbial contamination during the culture process but also unexpectedly shows that it can significantly promote the growth of Orobanchaceae plant stem cells, shorten the culture cycle, and reduce production costs.

[0022] Example 1 Methods for Isolation and Rapid Culture of Cistanche deserticola Stem Cells The specific steps are as follows: (1) Obtaining Cistanche deserticola stem cells Select scales from healthy Cistanche deserticola plants, rinse with tap water for 10 min, soak in 75% ethanol for 5 min, rinse with sterile water for 2 min, soak in 1% sodium hypochlorite for 10 min, rinse with sterile water for 10 min, and blot dry the explant surface with sterile filter paper. Under a dissecting microscope, cut tissue near the stem, approximately 0.2-0.5 mm in size. Cut the material as small as possible and place it in an Erlenmeyer flask containing induction medium for culture.

[0023] (2) Induction culture The embryonic stem cell induction medium consisted of: 20 g / L sucrose, 90 mg / L inositol, 1.8 mg / L 2,4-dichloronaphthylacetic acid, 0.6 mg / L kinetin, 3 mg / L indoleacetic acid, 30 mg / L ascorbic acid, 100 mg / L citric acid, and 2 g / L 1 / 2 MS medium, pH 5.8. The cells were incubated in the dark at 25℃ and 100 rpm for 24 h.

[0024] 1 / 2 MS medium Ingredients: Ammonium nitrate 750 mg / L, potassium nitrate 1000 mg / L, calcium chloride dihydrate 250 mg / L, magnesium sulfate heptahydrate 160 mg / L, potassium dihydrogen phosphate 75 mg / L, boric acid 3 mg / L, sodium molybdate heptahydrate 0.125 mg / L, manganese sulfate tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.0125 mg / L, zinc sulfate heptahydrate 5 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, potassium iodide 0.5 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate 28.7 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, glycine 2 mg / L, pyridoxine hydrochloride 0.5 mg / L, ammonium sulfate hydrochloride 0.1 mg / L.

[0025] (3) Microscopic examination The culture was passed through a 40 μm sieve to separate larger tissue slices and cell clusters, while retaining single cells.

[0026] Take 1 mL of culture, centrifuge at 2500 rpm for 5 min, and remove the supernatant. Add pH 7.0 PBS buffer and wash the cell pellet twice. Finally, add 1 mL of PBS buffer to fully resuspend the cells, perform serial dilutions, and spread 10 μL of each solution evenly onto a glass slide. Fix the slide, add 1 drop of 0.1% neutral red staining solution, fully cover the slide, and incubate at room temperature for 20 min. Wash off the staining solution with water, blot dry, cover with a coverslip, and examine under a microscope.

[0027] Plant stem cells are characterized by multiple small vacuoles, high mitochondrial activity, and cell walls that do not contain lignin. In the identification of stem cells, the main approach is to observe the staining of certain organelles based on their characteristics to distinguish them from non-stem cell lines such as callus tissue.

[0028] (4) Preparation of Artemisia annua extract Using Artemisia annua stem cell powder as raw material, 100 mg was weighed, added to 1 mL of distilled water, sonicated for 30 min, and centrifuged at 12000 rpm for 2 min. The supernatant was the Artemisia annua stock solution (Artemisia annua mass concentration of 100 mg / L). The artemisinin content was determined by mass spectrometry, and the solution was diluted to 0, 0.5, 1, 1.5, and 2 mg / L for later use.

[0029] (5) Add Artemisia annua extract Embryonic stem cells were used as seeds and inoculated at a rate of 1‰ in 1 L of liquid amplification medium. They were then cultured in the dark at 25°C and 110 rpm for 20 days.

[0030] The liquid amplification medium consisted of 30 g / L sucrose, 90 mg / L inositol, 200 mg / L hydrolyzed casein, 2 mg / L 2,4-dichloronaphthylacetic acid, 3 mg / L indoleacetic acid, 30 mg / L ascorbic acid, 100 mg / L citric acid, 0.18% PVP, and 2.8 g / L 1 / 2 MS medium at pH 5.8.

[0031] Artemisinin was added at concentrations of 0, 0.5, 1, 1.5, and 2 mg / L. One mL of culture was collected daily, and OD600 was measured to plot growth curves. The control group received 0 mg / L artemisinin. Results showed that cells in all groups supplemented with Artemisia annua extract entered the logarithmic growth phase earlier than the control group, and had higher cell density at the plateau phase. The experimental groups with concentrations of 1.0 mg / L and 1.5 mg / L exhibited the most vigorous cell growth, reaching the plateau phase approximately 3-5 days earlier than the control group, and ultimately achieving significantly higher biomass. This indicates that Artemisia annua extract can effectively promote the growth of Cistanche deserticola embryonic stem cells and shorten the culture period.

[0032] Example 2 Cistanche stem cell isolation and rapid culture method The specific steps are as follows: (1) Obtaining Cistanche stem cells Select scales from healthy Cistanche deserticola plants, rinse with tap water for 10 min, soak in 75% ethanol for 5 min, rinse with sterile water for 2 min, soak in 1% sodium hypochlorite for 10 min, rinse with sterile water for 10 min, and blot dry the explant surface with sterile filter paper. Under a dissecting microscope, cut tissue near the stem, approximately 0.2-0.5 mm in size. Cut the material as small as possible and place it in an Erlenmeyer flask containing induction medium for culture.

[0033] (2) Induction culture The embryonic stem cell induction medium consisted of 20 g / L sucrose, 90 mg / L inositol, 1.8 mg / L 2,4-dichloronaphthylacetic acid, 0.6 mg / L kinetin, 3 mg / L indoleacetic acid, 30 mg / L ascorbic acid, 100 mg / L citric acid, and 2 g / L 1 / 2 MS medium, pH 5.8. The cells were incubated in the dark at 25°C and 100 rpm for 24 h.

[0034] 1 / 2 MS medium Ingredients: Ammonium nitrate 750 mg / L, potassium nitrate 1000 mg / L, calcium chloride dihydrate 250 mg / L, magnesium sulfate heptahydrate 160 mg / L, potassium dihydrogen phosphate 75 mg / L, boric acid 3 mg / L, sodium molybdate heptahydrate 0.125 mg / L, manganese sulfate tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.0125 mg / L, zinc sulfate heptahydrate 5 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, potassium iodide 0.5 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate 28.7 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, glycine 2 mg / L, pyridoxine hydrochloride 0.5 mg / L, ammonium sulfate hydrochloride 0.1 mg / L.

[0035] (3) Microscopic examination: The culture was passed through a 40 μm sieve to separate larger tissue slices and cell clusters, while retaining single cells.

[0036] Take 1 mL of culture, centrifuge at 2500 rpm for 5 min, and remove the supernatant. Add pH 7.0 PBS buffer and wash the cell pellet twice. Finally, add 1 mL of PBS buffer to fully resuspend the cells, perform serial dilutions, and spread 10 μL of each solution evenly onto a glass slide. Fix the slide, add 1 drop of 0.1% neutral red staining solution, fully cover the slide, and incubate at room temperature for 20 min. Wash off the staining solution with water, blot dry, cover with a coverslip, and examine under a microscope.

[0037] Plant stem cells are characterized by multiple small vacuoles, high mitochondrial activity, and cell walls that do not contain lignin. In the identification of stem cells, the main approach is to observe the staining of certain organelles based on their characteristics to distinguish them from non-stem cell lines such as callus tissue.

[0038] (4) Preparation of Artemisia annua extract Using Artemisia annua stem cell powder as raw material, 100 mg was weighed and added to 1 mL of distilled water. The mixture was sonicated for 30 min and centrifuged at 12000 rpm for 2 min. The supernatant was the Artemisia annua stock solution (Artemisia annua mass concentration of 100 mg / L). After centrifugation and cell culture, the culture medium was centrifuged again and filtered to obtain Artemisia annua cell-free cell culture. The artemisinin content in the culture medium was determined using mass spectrometry, and the culture was diluted to 0, 0.5, 1, 1.5, and 2 mg / L for later use.

[0039] (5) Add Artemisia annua extract Embryonic stem cells were used as seeds and inoculated at a rate of 1‰ in 1 L of liquid culture medium. They were then cultured in the dark at 25°C and 110 rpm for 20 days.

[0040] The liquid culture medium consisted of 30 g / L sucrose, 90 mg / L inositol, 200 mg / L hydrolyzed casein, 2 mg / L 2,4-dichloronaphthylacetic acid, 3 mg / L indoleacetic acid, 30 mg / L ascorbic acid, 100 mg / L citric acid, 0.18% PVP, and 2.8 g / L 1 / 2 MS medium at pH 5.8.

[0041] Artemisia annua extract was added at concentrations of 0, 0.5, 1, 1.5, and 2 mg / L of artemisinin. 1 mL of culture was collected daily, and 0D600 was measured. Growth curves were plotted as shown below. Figure 2 As shown in the figure, similar to the results for Cistanche deserticola, the addition of 0.5-2 mg / L (calculated as artemisinin) of Artemisia annua extract significantly promoted the proliferation of Cistanche deserticola embryonic stem cells, with the optimal addition level of 1.0-1.5 mg / L showing the best effect, effectively shortening the culture period and increasing cell yield. This result demonstrates the good universality of the method of this invention for different Orobanchaceae plants. Without the addition of Artemisia annua extract, cell growth was slow, the entry into the logarithmic growth phase was delayed, and the final biomass was significantly lower than that of the groups in Example 1 where Artemisia annua extract was added. Simultaneously, varying degrees of microbial contamination were observed in some culture flasks during the culture process, requiring frequent inspection and treatment.

[0042] The results of Examples 1-2 demonstrate that the method for culturing embryonic stem cells of Orobanchaceae plants provided by this invention, through optimization of the induction medium, yields highly viable embryonic stem cells. Furthermore, the innovative addition of a specific concentration of Artemisia annua extract to the expansion medium significantly promotes cell growth, shortens the culture cycle, and may reduce the risk of contamination due to its natural antibacterial activity. This method is efficient, stable, and independent of the host plant, providing a novel solution for the sustainable utilization of Orobanchaceae medicinal plant resources.

[0043] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for culturing embryonic stem cells from Orobanchaceae plants, characterized by the following steps: include: Explants were obtained from plants of the Orobanchaceae family. The explants were induced and cultured to obtain embryonic stem cells. The embryonic stem cells were then seeded into an expansion medium containing Artemisia annua extract for further culture.

2. The method for culturing embryonic stem cells of Orobanchaceae plants according to claim 1, characterized in that, The Artemisia annua extract is at least one of Artemisia annua stem cell extract, Artemisia annua water extract, or Artemisia annua alcohol extract.

3. The method for culturing embryonic stem cells of Orobanchaceae plants according to claim 1, characterized in that, The amount of Artemisia annua extract added is 0.5-2 mg / L, calculated based on its artemisinin content.

4. The method for culturing embryonic stem cells of Orobanchaceae plants according to claim 1, characterized in that, Artemisia annua extract is obtained by using Artemisia annua stem cell powder as raw material, followed by ultrasonication and centrifugation, and then taking the supernatant.

5. A method for culturing embryonic stem cells of a Broomraceae plant according to claim 1, characterized in that, Embryonic stem cells were seeded into an expansion medium containing Artemisia annua extract and then cultured in the dark at 22-28℃ and 100-120 rpm for 20-22 days.

6. The method for culturing embryonic stem cells of Orobanchaceae plants according to claim 1, characterized in that, The explants are scales of plants from the Orobanchaceae family.

7. The method for culturing embryonic stem cells of Orobanchaceae plants according to claim 1, characterized in that, Explants were cultured in an induction medium to obtain embryonic stem cells. The induction medium contained a basal medium, a first carbon source, a first plant growth regulator, and a first anti-browning agent.

8. A method for culturing embryonic stem cells of a Broomraceae plant according to claim 7, characterized in that, The first carbon source is sucrose with a concentration of 15-30 g / L, the first plant growth regulator is selected from one or more of 2,4-dichloronaphthyloxyacetic acid, kinetin, and indoleacetic acid, and the first anti-browning agent is selected from one or two of ascorbic acid and citric acid.

9. A method for culturing embryonic stem cells of a Broomraceae plant according to claim 1, characterized in that, The amplification medium also contains a basal medium, a second carbon source, a second organic additive, a second plant growth regulator, and a second anti-browning agent.

10. A method for culturing embryonic stem cells of a Broomraceae plant according to claim 9, characterized in that, The second carbon source is sucrose at a concentration of 30-50 g / L; the second organic additive includes inositol and hydrolyzed casein; the second plant growth regulator includes 2,4-dichloronaphthoxyacetic acid and indoleacetic acid; and the second anti-browning agent includes one or more of ascorbic acid, citric acid, and polyvinylpyrrolidone.

Images