Preparation method and application of canna edulis stem cells

Abstract

The invention discloses a preparation method and application of canna edulis stem cells, and belongs to the technical field of stem cells. The canna edulis stem cells are prepared by the following steps: A1, selecting canna edulis which is large in tuber, many in root, slightly red in bud end, good in growth vigor, free of breakage, germs and insect pests, cleaning, sterilizing, cutting a root tip meristematic region of the canna edulis into blocks, continuously sterilizing, infiltrating by using a sucrose solution, and washing by using an antioxidant solution; a2, inoculating the small blocks treated in the step A1 into a first culture medium for induction culture; a3, taking a cell population containing stem cells after induction culture in the step A2, and inoculating the cell population into a second culture medium for multiplication culture; and A4, inoculating the stem cells subjected to multiplication culture in the step 2 into a third culture medium for adaptive culture, and inoculating the stem cells into a fourth culture medium for suspension culture to obtain the canna edulis stem cells. The canna edulis stem cells can be applied to crop planting, and the crop yield can be obviously increased; the canna edulis stem cells can also be applied to health care products and cosmetics with anti-aging effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell technology, in particular to a preparation method and application of Canna indica stem cells. BACKGROUND

[0002] Canna indica, also known as Musa indica, Calamus orientalis or Zingiber musaefolium, is a perennial herbaceous plant of the Musaceae family, widely distributed in Southwest China such as Yunnan, Guizhou, Guangxi and Sichuan, as well as Southeast Asia and other tropical and subtropical regions. This plant has strong environmental adaptability, drought tolerance, poor soil tolerance, disease and pest resistance, rapid growth, and high biological yield. Its underground tubers are rich in starch, dietary fiber, polysaccharides, flavonoids, phenolic acids and various secondary metabolites with biological activity. In recent years, with the growing demand for high-value utilization of non-food crops and deep development of natural plant resources, Canna indica has not only been widely used as an important energy crop and feed resource, but its potential medicinal value and physiological function have also gradually attracted attention.

[0003] Studies have shown that Canna indica extract has significant antioxidant, anti-inflammatory, free radical scavenging and immune regulation biological activities, and is rich in flavonoids and polyphenols. These components can effectively inhibit oxidative stress, delay the aging process of cells, and protect cell membrane structure and DNA integrity, thus showing broad application prospects in anti-aging functional foods, health products and skin care products. In addition, Canna indica has strong regenerative ability and active meristematic tissue, making it easy to be cultured in vitro and an ideal material for plant cell engineering and stem cell research.

[0004] Plant stem cells are a type of undifferentiated cells present in the apical meristem or lateral meristem of plants, with the ability of continuous self-renewal and multi-directional differentiation potential. In recent years, plant stem cell culture technology, as an important direction of modern biotechnology, has been widely used in the sustainable production of high-value active ingredients. By selecting tissues rich in meristematic cells such as stem tips and axillary buds as explants under sterile conditions, surface disinfection, inoculation and induction of callus formation, and further screening, purification and amplification of cell populations with stem cell characteristics, large-scale cell proliferation can be achieved in an artificially controlled environment. By optimizing the medium formula, regulating light, temperature and gas environment, the synthesis and accumulation of specific secondary metabolites can also be directed to promote, thereby obtaining high-purity and high-activity functional extracts.

[0005] Currently, plant stem cell technology has been industrialized in various plants such as ginseng, grapes, apples, and rosemary. Its extracts are widely used in high-end skincare products, exerting anti-aging effects such as promoting skin cell regeneration, reducing wrinkles, brightening skin tone, and repairing the skin barrier. In the field of functional health products, it is used to enhance the body's antioxidant capacity and delay organ function decline. In agriculture, plant-derived active substances are also being explored for use as plant growth regulators or biostimulants.

[0006] However, despite the excellent stress resistance, high biomass and rich active ingredients of the Chinese lotus, its development and utilization are currently mainly focused on tuber starch extraction and crude processing. A systematic technical system for the isolation, culture and preparation of functional products of Chinese lotus stem cells has not yet been established.

[0007] In summary, there is currently no systematic preparation method for lotus root stem cells. Therefore, it is urgent to develop an efficient, stable, and scalable process for the extraction and culture of lotus root stem cells. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing and applying lotus root stem cells, so as to solve the technical problems mentioned in the background art.

[0009] The technical solution to achieve the objective of this invention is:

[0010] In a first aspect, the present invention provides a method for preparing lotus root stem cells, comprising the following preparation steps:

[0011] A1. Select rhizomes with large tubers, many roots, slightly reddish bud tips, good growth, no rot, no pathogens, and no pests. This indicates that the plant is in a vigorous growth period, with active cell division in the meristematic tissue and rich in undifferentiated cells, which is conducive to the subsequent induction and separation of stem cells. After cleaning, sterilize the rhizomes. Cut the root tip meristem into small pieces, sterilize again, and then soak them in a sucrose solution. Finally, rinse them with an antioxidant solution.

[0012] A2. Inoculate the small pieces processed in step A1 into the first culture medium for induction culture;

[0013] A3. Take the cell population containing stem cells after the induction culture in step A2 and inoculate it into the second culture medium for proliferation culture;

[0014] A4. After the stem cells in step 2 have been proliferated and cultured, they are inoculated into the third culture medium for adaptive culture, and then into the fourth culture medium for suspension culture to obtain lotus root stem cells.

[0015] Furthermore, the antioxidant solution is a 150 mg / L citric acid solution.

[0016] Furthermore, steps A2 to A4 are all performed under dark conditions.

[0017] Furthermore, the first culture medium is B5 medium, which contains 6 mg / L LIAAA, 6 mg / L LIABA, 100 mg / L L-ascorbic acid, 150 mg / L citric acid, 7 mg / L agar, and 20 mg / L sucrose.

[0018] Furthermore, the second culture medium is B5 medium, which contains 2.9 mg / L LIAAA, 2.6 mg / L LIABA, 5.9 mg / L NAA, 500-1000 mg / L buckwheat pollen water extract, 7 mg / L agar, and 20 mg / L sucrose.

[0019] Furthermore, the third culture medium is B5 medium, supplemented with 2.9 mg / L LAA, 2.6 mg / L LBA, 5.9 mg / L NAA, 500–1000 mg / L buckwheat pollen water extract, and 20 mg / L sucrose.

[0020] Furthermore, the fourth culture medium is B5 medium, supplemented with 5 mg / L LIAAA, 5 mg / L LIABA, 5 mg / L NAA, 500–1000 mg / L buckwheat pollen water extract, and 20 mg / L sucrose. The pH is 5.8–6.6.

[0021] Further, the preparation steps of the buckwheat pollen water extract are as follows: the buckwheat pollen is dried, pulverized, sieved, and then stored at -20℃ for later use; 4-6 parts by weight of pollen are mixed with 100 parts by weight of distilled water and vortexed, then placed in a 4℃ refrigerator for 4-6 hours, then taken out and sonicated at 38-42℃ for 80-100 minutes, with shaking once every 30 minutes, then placed in a 4℃ refrigerator overnight, then filtered, diluted to volume, centrifuged for 10 minutes, and then deionized water is added to obtain a buckwheat pollen water extract of 500-1000 mg / L.

[0022] Secondly, an application of *Rhizophora stylosa* stem cells in crops involves adding the *Rhizophora stylosa* stem cells prepared by the method described in the first aspect to crop fertilizers for fertilization, specifically to fertilizers used during the seedling, growth, and flowering / fruiting stages of crops.

[0023] Thirdly, the application of lotus root stem cells in health products and cosmetics with anti-aging effects, wherein the lotus root stem cells are prepared by the method for preparing lotus root stem cells described in the first aspect.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects:

[0025] (1) This invention uses lotus root as raw material to extract lotus root stem cells. Its process design is scientific and reasonable, the steps are clear and easy to operate, and it has high efficiency, stability and good industrial scale-up potential. First, in the explant selection and pretreatment stage, lotus roots with large tubers, many roots, slightly red bud tips and good growth without pests and diseases are selected for cleaning and sterilization to ensure the high viability and sterility of the starting material. Then, the meristematic zone of the root tip of the lotus root is cut into small pieces, sterilized again, and then permeated with sucrose solution and rinsed with antioxidant solution to reduce the damage of oxidative stress to the cells, thus laying the foundation for subsequent efficient induction. In the first culture medium, an appropriate amount of auxin and cytokinin are added to induce the dedifferentiation of explant cells to form callus tissue. This process activates the expression of key stem cell regulatory genes and promotes the reconstruction of meristematic characteristics. Then, the cell population containing stem cells is transferred to the second culture medium. The first culture medium, through optimized hormone ratios and the introduction of buckwheat pollen water extract, rich in various amino acids and monosaccharides, synergistically promotes cell cycle progression, enhances mitochondrial function, and improves cell viability and division rate, significantly increasing stem cell proliferation efficiency and stability. The second culture medium is used for adaptive culture, helping stem cells gradually adapt to the in vitro environment, enhancing their tolerance to environmental stresses such as pH fluctuations and dissolved oxygen changes, and maintaining stem gene expression. Finally, the third culture medium uses suspension culture, facilitating large-scale expansion and subsequent collection, while continuing to add buckwheat pollen water extract to continuously supply amino acids and sugars, maintaining cell activity and product synthesis capacity under high-density culture. This multi-stage culture strategy achieves precise control of the entire process from "induction → expansion → adaptation → large-scale production," ensuring that the final obtained lotus root stem cells are numerous, highly pure, and physiologically stable.

[0026] (2) The dry lotus stem cells prepared by this invention show broad application prospects in agriculture, anti-aging health products and cosmetics. In agriculture, adding dry lotus stem cells or their extracts to fertilizers can promote seed germination, root development, improve photosynthetic efficiency and crop resistance, thereby increasing yield.

[0027] (3) In the field of anti-aging health products, lotus root stem cells are rich in antioxidant active ingredients and can be used to prepare oral capsules, tablets or functional drinks. They can delay aging by scavenging free radicals, activating longevity pathways and regulating intestinal flora. Long-term use can help improve fatigue, enhance immunity and delay organ function decline, and is suitable for health management of middle-aged and elderly people.

[0028] (4) In the cosmetics field, lotus root stem cell extract can promote fibroblast proliferation, increase collagen and elastin synthesis, brighten skin tone, repair the skin barrier, and relieve skin inflammation caused by ultraviolet rays. It can be used as a high-end skin care ingredient. Detailed Implementation

[0029] To better understand the above technical solution, the following will provide a detailed explanation of the technical solution in conjunction with specific implementation methods.

[0030] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0031] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0032] Example

[0033] A method for preparing lotus root stem cells includes the following preparation steps:

[0034] A1. Select rhizomes with large tubers, well-developed root systems, slightly reddish bud tips, robust growth, no damage, no pathogen infection, and no pests. This indicates that the plant is in a vigorous growth period, with active cell division in its meristematic tissues, rich in undifferentiated cells, which is beneficial for subsequent stem cell induction and separation. After cleaning the selected rhizomes, soak them in 70% ethanol for 5 minutes, then rinse them three times with sterile water. Next, soak them in 7.5% sodium hypochlorite solution for 5 minutes, and rinse them three times again with sterile water. Subsequently... Take the root tip meristem of the dry lotus and cut it into 30 small pieces, each 3mm long, 3mm wide, and 0.3mm thick. Soak the cut pieces again in 70% ethanol for 5 minutes, rinse three times with sterile water, then soak in 1% sodium hypochlorite solution for 15 minutes, rinse with sterile water for 5 minutes, then soak in 0.6mol / L sucrose solution for 24 hours, then transfer to 0.3mol / L sucrose solution for 15 minutes, and finally rinse with 150mg / L citric acid solution.

[0035] A2. The small pieces treated in step A1 were inoculated into the first culture medium and induced under dark conditions at a temperature of 25°C for 30 days. The first culture medium was B5 medium, with 6 mg / L IAA, 6 mg / L IBA, 100 mg / L L-ascorbic acid, 150 mg / L citric acid, 7 mg / L agar and 20 g / L sucrose added. The induction rate was 52.41%, where the induction rate (%) = 100% × number of small pieces that grew stem cells / total number of small pieces.

[0036] A3. In the culture medium after induction culture in step A2, select the cell population containing stem cells and inoculate it into the second culture medium. Proliferate and culture at 25℃ in the dark, subculturing every 2 weeks for 30 days. The second culture medium is B5 medium, supplemented with 2.9 mg / L IAA, 2.6 mg / L IBA, 5.9 mg / L NAA, 1000 mg / L buckwheat pollen water extract, 7 mg / L agar, and 20 g / L sucrose. Proliferation amount = weight of stem cells after proliferation culture - mass of the cell population containing stem cells; the proliferation amount is 1.34 g.

[0037] A4. The stem cells proliferated and cultured in step 2 were seeded into the third culture medium and subjected to adaptive culture at 25°C, in the dark, and with shaking at a speed of 100 rpm. After 14 days of culture, the suspension was filtered through a 100 μm cell sieve, and the filtrate was centrifuged at 1000 rpm. The precipitate was then transferred to the fourth culture medium for further culture. The suspension was cultured at 25°C, in the dark, and with shaking at a speed of 100 rpm, with culture medium replenished weekly and subcultured every two weeks for 30 days. After culture, the cell suspension was centrifuged, freeze-dried, pulverized, and sieved to obtain *Rhizophora stylosa* stem cells. The third culture medium was B5 medium supplemented with 2.9 mg / L IAA, 2.6 mg / L IBA, 5.9 mg / L NAA, 1000 mg / L buckwheat pollen water extract, and 20 g / L sucrose. The fourth culture medium was also B5 medium supplemented with 5 mg / L IAA, 5 mg / L... IBA, 5 mg / L NAA, 1000 mg / L buckwheat pollen water extract and 20 g / L sucrose, pH adjusted to 6.2; proliferation amount = weight of stem cells after suspension culture - mass of cell population containing stem cells; proliferation amount was 3.68 g.

[0038] Comparative Example

[0039] A method for preparing lotus root stem cells includes the following preparation steps:

[0040] A1. Select rhizomes with large tubers, well-developed root systems, slightly reddish bud tips, robust growth, no damage, no pathogen infection, and no pests. This indicates that the plant is in a vigorous growth period, with active cell division in its meristematic tissues, rich in undifferentiated cells, which is beneficial for subsequent stem cell induction and separation. After cleaning the selected rhizomes, soak them in 70% ethanol for 5 minutes, then rinse them three times with sterile water. Next, soak them in 7.5% sodium hypochlorite solution for 5 minutes, and rinse them three times again with sterile water. Subsequently... Take the root tip meristem of the dry lotus and cut it into 30 small pieces, each 3mm long, 3mm wide, and 0.3mm thick. Soak the cut pieces again in 70% ethanol for 5 minutes, rinse three times with sterile water, then soak in 1% sodium hypochlorite solution for 15 minutes, rinse with sterile water for 5 minutes, then soak in 0.6mol / L sucrose solution for 24 hours, then transfer to 0.3mol / L sucrose solution for 15 minutes, and finally rinse with 150mg / L citric acid solution.

[0041] A2. The small pieces treated in step A1 were inoculated into the first culture medium and induced under dark conditions at a temperature of 25°C for 30 days. The first culture medium was B5 medium, supplemented with 6 mg / L IAA, 6 mg / L IBA, 100 mg / L L-ascorbic acid, 150 mg / L citric acid, 7 mg / L agar, and 20 g / L sucrose; the induction rate was 52.38%.

[0042] A3. In the culture medium after induction culture in step A2, select the cell population containing stem cells and inoculate it into the second culture medium. Proliferate and culture it at 25°C in the dark, subculturing it every 2 weeks for 30 days. The second culture medium is B5 medium, supplemented with 2.9 mg / L IAA, 2.6 mg / L IBA, 5.9 mg / L NAA, 1000 mg / L buckwheat pollen water extract, 7 mg / L agar and 20 g / L sucrose; the proliferation amount is 0.89 g.

[0043] A4. The stem cells after proliferation culture in step 2 were seeded into the third culture medium and subjected to adaptive culture at 25°C, in the dark, and under shaking conditions, with a shaking speed of 100 r / min. After 14 days of culture, the suspension was filtered through a 100 μm cell sieve, and the filtrate was centrifuged at 1000 r / min. The precipitate was then transferred to the fourth culture medium for further culture. The cells were cultured in suspension at 25°C in the dark with shaking, at a speed of 100 r / min. The culture medium was replenished weekly, and the cells were subcultured every two weeks for 30 days. After culture, the cell suspension was centrifuged, freeze-dried, pulverized, and sieved to obtain *Rhizophora stylosa* stem cells. The third culture medium was B5 medium, supplemented with 2.9 mg / L IAA, 2.6 mg / L IBA, 5.9 mg / L NAA, 1000 mg / L buckwheat pollen water extract, and 20 g / L sucrose. The fourth culture medium was also B5 medium, supplemented with 5 mg / L IAA, 5 mg / L IBA, 5 mg / L NAA, 1000 mg / L buckwheat pollen water extract, and 20 g / L sucrose, with the pH adjusted to 6.2. The proliferation rate was 2.36 g.

[0044] Compared with the comparative example, the stem cells of lotus root cultured with buckwheat pollen water extract in the example showed a larger proliferation rate.

[0045] Table 1 below shows the EC50 test results of the ABTS free radical scavenging ability of the *Rhizophora stylosa* stem cells obtained in the examples and comparative examples:

[0046] Table 1

[0047] ABTS radical scavenging capacity EC50(mg / mL) Examples 1.98 Comparative examples 5.64

[0048] The smaller the EC50, the stronger its ability to scavenge free radicals. As shown in Table 1, the lotus root stem cells obtained in the embodiments and comparative examples of the present invention all have good anti-aging properties and are suitable for health products and cosmetics with anti-aging functions. Among them, the lotus root stem cells prepared by this embodiment have better anti-aging properties.

[0049] Application Example 1

[0050] During rice cultivation in a certain area of ​​Guangxi, in addition to normal fertilization and cultivation management, the *Rhizophora stylosa* stem cells obtained in Example 1 were mixed with compound fertilizer, with the *Rhizophora stylosa* stem cells added at a rate of 0.3 wt%. The mixture was applied at four different times: during the rice jointing stage (June 12, 2024), booting stage (July 8, 2024), grain-filling stage (August 12, 2024), and milk stage (August 21, 2024). The mixture was then sprayed evenly on the leaves, with a dosage of 40 kg / mu each time, for a total of four applications. Each application was carried out on cloudy days or in the morning after the dew had dried on sunny days, or after 4:00 PM, avoiding the midday heat and strong sunlight to prevent rapid evaporation of the fertilizer solution. Spraying was avoided in windy conditions. The amount of fertilizer applied was such that the leaves were just about to drip, fully moistened but not running, while keeping the soil surface slightly moist to ensure effective absorption.

[0051] Table 2 below shows the yield data of rice fields with no application of compound fertilizer containing lotus stem cells as a blank example and rice fields with application example 1:

[0052] Table 2

[0053] Yield per acre / kg Application example 1 378.70 Blank example 329.31

[0054] As shown in Table 2 above, the stem cell-containing fertilizer obtained in the examples can effectively increase crop yield when added to crop fertilizer.

[0055] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are 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 within the protection scope of the present invention.

Claims

1. A method for preparing lotus root stem cells, characterized in that, The preparation steps include the following: A1. Select dry lotus roots with large tubers, many roots, slightly reddish bud tips, good growth, no rot, no pathogens, and no pests. After cleaning, sterilize them. Cut the root tip meristem of the dry lotus root into small pieces, continue sterilizing, then penetrate with sucrose solution, and then rinse with an antioxidant solution. A2. Inoculate the small pieces processed in step A1 into the first culture medium for induction culture; A3. Take the cell population containing stem cells after the induction culture in step A2 and inoculate it into the second culture medium for proliferation culture; A4. After the stem cells in step 2 have been proliferated and cultured, they are inoculated into the third culture medium for adaptive culture, and then into the fourth culture medium for suspension culture to obtain lotus root stem cells.

2. The method for preparing lotus root stem cells according to claim 1, characterized in that, The antioxidant solution used is a 150 mg / L citric acid solution.

3. The method for preparing lotus root stem cells according to claim 1, characterized in that, All steps A2 to A4 were performed under dark conditions.

4. The method for preparing lotus root stem cells according to claim 1, characterized in that, The first culture medium is B5 medium, which contains 6 mg / L LIAAA, 6 mg / L LIABA, 100 mg / L L-ascorbic acid, 150 mg / L citric acid, 7 mg / L agar, and 20 mg / L sucrose.

5. The method for preparing lotus root stem cells according to claim 1, characterized in that, The second culture medium is B5 medium, which contains 2.9 mg / L LAA, 2.6 mg / L LBA, 5.9 mg / L NAA, 500-1000 mg / L buckwheat pollen water extract, 7 mg / L agar, and 20 mg / L sucrose.

6. The method for preparing lotus root stem cells according to claim 1, characterized in that, The third culture medium is B5 medium, supplemented with 2.9 mg / L LAA, 2.6 mg / L LBA, 5.9 mg / L NAA, 500–1000 mg / L buckwheat pollen water extract, and 20 mg / L sucrose.

7. The method for preparing lotus root stem cells according to claim 1, characterized in that, The fourth culture medium is B5 medium, supplemented with 5 mg / L LIAAA, 5 mg / L LIABA, 5 mg / L NAA, 500–1000 mg / L buckwheat pollen water extract, and 20 mg / L sucrose. The pH is 5.8–6.

6.

8. The method for preparing lotus root stem cells according to claims 5-7, characterized in that, The preparation steps of the buckwheat pollen water extract are as follows: Dry the buckwheat pollen, pulverize it, sieve it, and then store it at -20℃ for later use; take 4-6 parts by weight of pollen and mix it with 100 parts by weight of distilled water, vortex and shake it, put it in a 4℃ refrigerator for 4-6 hours, take it out and sonicate it at 38-42℃ for 80-100 minutes, shaking it once every 30 minutes, then put it in a 4℃ refrigerator overnight, take it out and filter it, make up the volume, centrifuge it for 10 minutes, and then add deionized water to obtain a buckwheat pollen water extract of 500-1000 mg / L.

9. An application of lotus root stem cells in agricultural crops, characterized in that, The dried lotus stem cells prepared by the method described in claim 8 are added to crop fertilizers for fertilization, specifically to fertilizers used during the seedling, growth, and flowering / fruiting stages of crops.

10. The application of lotus root stem cells in health products and cosmetics with anti-aging effects, characterized in that, The *Rhizophora stylosa* stem cells are prepared by the method for preparing *Rhizophora stylosa* stem cells as described in claim 8.