Strawberry tissue culture rapid propagation method based on natural extract

Abstract

The application discloses a strawberry tissue culture and rapid propagation method based on natural extracts and belongs to the technical field of strawberry tissue culture and rapid propagation. The application takes MS culture medium as a basis, uniformly adds sucrose as a carbon source in the culture medium, and uniformly adds ascorbic acid (VC) and thiamine (VB1) as coenzyme vitamins to provide a uniform nutrient environment, and takes eleutherococcus senticosus extract as a growth regulator to replace traditional 6-BA, so that the browning rate and the malformation rate of the strawberry tissue culture and rapid propagation can be effectively reduced, and the proliferation rate, the survival rate and the morphological performance are equivalent to those of conventional culture (6-BA is added in the culture medium). The application solves the problems of synthetic hormone dependence and residues in the prior art, reduces the malformation rate and the browning rate of the tissue culture seedlings, and has a good popularization and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of strawberry tissue culture and rapid propagation technology, and particularly relates to a strawberry tissue culture and rapid propagation method based on natural extracts. BACKGROUND

[0002] The commonly used cell division factor in the process of strawberry tissue culture and rapid propagation is 6-benzylaminopurine (6-BA). As the mainstream hormone for inducing and promoting buds, 6-BA has achieved remarkable proliferation effect in the tissue culture of many strawberry varieties. However, the existing 6-BA-based tissue culture and rapid propagation system also has many drawbacks: (1) synthetic hormone residue problem: since 6-BA is an artificially synthesized plant growth regulator, a certain amount of residual hormone is often accumulated in the tissue culture seedlings. Studies have shown that the residual 6-BA in the tissue culture process may affect the subsequent growth and tillering of the plants, and some test tube seedlings may exhibit the effects of residual hormones after rooting and transplanting. Such chemical hormone residues not only may affect the growth stability of the tissue culture seedlings, but also cause concerns about food quality and ecological safety; (2) strong dependence on hormones: some strawberry varieties show strong dependence on 6-BA and other exogenous hormones, i.e., only in the presence of a high concentration of hormones can ideal bud proliferation effect be achieved. Once the hormone dosage is reduced, the proliferation coefficient and growth performance of these varieties will significantly decrease, making it difficult to maintain good propagation under low-hormone or hormone-free conditions. This hormone-dependent phenomenon makes it difficult to eliminate the long-term use of synthetic hormones in production; (3) high rate of abnormality: although the use of 6-BA to promote cluster bud proliferation can increase the number of buds, it often accompanies a high rate of abnormal cluster buds and abnormal seedlings. Especially when the concentration of 6-BA is relatively high, it is more likely to induce abnormal differentiation of growth points and other adverse phenomena such as dwarfing and clustering of the tissue culture seedlings. Studies have shown that high concentrations of 6-BA can inhibit the normal germination and continuous proliferation of strawberry stem tips, leading to severe clustering and dwarfing and abnormality. Abnormal seedlings not only reduce the quality of tissue culture seedlings, but also increase the elimination rate after seedling raising and transplanting; (4) poor sustainability: long-term reliance on synthetic hormones for tissue culture and rapid propagation raises environmental and economic concerns. On the one hand, the large-scale use of synthetic hormones may cause problems in the treatment of culture medium waste liquid and environmental safety; on the other hand, the cost of hormone input and the possible resistance problems make the existing technology difficult to be sustainable in large-scale industrialized seedling raising. In general, although the traditional 6-BA system is efficient, it has limitations in terms of green safety and long-term application.

[0003] In recent years, there has been a trend in the field of tissue culture to replace synthetic growth regulators with natural plant active substances. Natural plant extracts are rich in secondary metabolites that have growth-regulating effects, such as plant hormone analogues and biological stimulants, making them a more environmentally friendly and safe choice for tissue culture additives. Among them, Eleutherococcus senticosus has attracted attention due to its rich natural active ingredients. Eleutherococcus senticosus belongs to the family Araliaceae, and its roots, stems, and leaves contain various natural compounds such as saponins, flavonoids, and polysaccharides. Studies have shown that components such as total ginsenosides, total flavonoids, and polysaccharides have significant biological activity in plant pharmacology. These substances have outstanding effects in improving stress resistance, promoting metabolism, antioxidant, and immune regulation. Compared with synthetic hormones with single components, natural extracts are often a combination of multiple active factors, which are expected to have a mild and comprehensive regulating effect on plant growth. Therefore, the application of natural plant active substances such as Eleutherococcus senticosus in plant tissue culture has important significance for establishing a green and safe tissue culture and rapid propagation method. SUMMARY

[0004] The purpose of the present application is to provide a strawberry tissue culture and rapid propagation method based on natural extracts to solve the problems existing in the prior art. The present application uses Eleutherococcus senticosus natural extract to replace traditional 6-BA for strawberry tissue culture and rapid propagation, which can effectively reduce the browning rate and malformation rate of strawberry tissue culture and rapid propagation, and maintain the proliferation rate, survival rate and morphological performance comparable to conventional culture (add 6-BA in the medium), while solving the problem of synthetic hormone dependence and residue in the prior art, and has good popularization and application prospect.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The present application provides the application of Eleutherococcus senticosus extract in reducing the browning rate and malformation rate of strawberry tissue culture and rapid propagation, wherein the Eleutherococcus senticosus extract is an ethanol extract of dried roots of Eleutherococcus senticosus.

[0007] Further, the preparation method of the Eleutherococcus senticosus extract comprises the following steps:

[0008] After the dried roots of Eleutherococcus senticosus are crushed, an ethanol solution with a volume fraction of 75% is used as the extraction agent, the solid-liquid ratio is 1g:10mL, and the extraction is carried out twice at 60℃ in a water bath under reflux for 2h each time. After combining the two extraction solutions, filtering and reducing pressure concentration, the Eleutherococcus senticosus extract is obtained.

[0009] The present application also provides a culture medium for strawberry tissue culture and rapid propagation based on natural extracts, wherein the culture medium uses Eleutherococcus senticosus extract as a growth regulator and does not contain other hormones.

[0010] The acanthopanax extract is an ethanol extract of dried roots of acanthopanax.

[0011] Further, the preparation method of the acanthopanax extract comprises the following steps:

[0012] After the dried roots of acanthopanax are crushed, an ethanol solution with a volume fraction of 75% is used as an extraction agent, the solid-liquid ratio is 1g:10mL, and the extraction is carried out twice at 60 DEG C in a water bath reflux for 2h each time; after the two extraction solutions are combined, filtration and concentration under reduced pressure are carried out to obtain the acanthopanax extract.

[0013] Further, the volume concentration of the acanthopanax extract in the culture medium is 10% to 15%.

[0014] Further, the culture medium is a MS culture medium as a basic culture medium, and the components further comprise sucrose 30g / L, ascorbic acid 1mg / L and thiamine 10mg / L.

[0015] The application further provides a strawberry tissue culture and rapid propagation method based on a natural extract, which comprises the step of using the culture medium to carry out tissue culture on a strawberry explant.

[0016] Further, the temperature of the tissue culture is 25 DEG C, the humidity is 70%, the photoperiod is 16h light / 8h dark, and the light intensity is 3000lx.

[0017] Further, the culture medium is not replaced and subculture is not carried out during the tissue culture process.

[0018] Further, the strawberry explant comprises callus buds or healthy axillary buds of strawberries.

[0019] The application discloses the following technical effects:

[0020] The application finds through experiments that the use of acanthopanax natural extract instead of traditional 6-BA for strawberry tissue culture and rapid propagation can effectively reduce the browning rate and deformity rate of strawberry tissue culture and rapid propagation, and keep the proliferation rate, survival rate and morphological performance equivalent to conventional culture (addition of 6-BA in the culture medium).

[0021] The application uses MS culture medium as a basis, uniformly adds sucrose as a carbon source, ascorbic acid (VC) and thiamine (VB1) as coenzyme vitamins in the culture medium to provide a uniform nutrient environment, uses acanthopanax extract as a growth regulator, is safe in source and has no toxic side effects, and can significantly reduce the chemical hormone residues of tissue culture seedlings after replacing 6-BA, thereby meeting the production concept of "green safety".

[0022] The application is a strawberry tissue culture rapid propagation method with natural acanthopanax senticosus extract as the core, the whole rapid propagation culture cycle is 28 days, during which no subculture is performed (i.e., one-step culture completes proliferation), so as to reduce the stress caused by repeated subculture on test-tube seedlings, improve the propagation efficiency and simplify the operation process, solve the problems of hormone dependence and residue in the prior art, and reduce the malformation rate and browning rate of tissue culture seedlings, and has a good popularization and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0024] Figure 1 Effect of different acanthopanax senticosus extract concentrations on strawberry tissue culture rapid propagation malformation rate;

[0025] Figure 2 Effect of different acanthopanax senticosus extract concentrations on strawberry tissue culture rapid propagation browning rate;

[0026] Figure 3 Effect of different acanthopanax senticosus extract concentrations on strawberry tissue culture rapid propagation proliferation rate;

[0027] Figure 4 Effect of different acanthopanax senticosus extract concentrations on strawberry tissue culture rapid propagation survival rate. DETAILED DESCRIPTION

[0028] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0029] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range, and any other stated value or intermediate value in the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example

[0034] I. Experimental Methods

[0035] 1. Preparation of Acanthopanax senticosus extract

[0036] The dried roots of the wild medicinal plant Eleutherococcus senticosus were used as raw material. After pulverizing the roots, a crude extract was obtained using an ethanol-water solvent extraction method. The specific steps are as follows:

[0037] Using 75% ethanol as the extraction solvent, the Acanthopanax senticosus root powder and ethanol extractant were mixed at a material-to-liquid ratio of 1:10 (1g of Acanthopanax senticosus root powder corresponds to 10mL of ethanol solution). The mixture was refluxed twice in a 60℃ water bath, 2 hours each time. The extracts were combined, filtered through filter paper to remove residue, and then concentrated under reduced pressure to remove ethanol, yielding a crude Acanthopanax senticosus extract mother liquor. To ensure sterility in the culture medium, the extract was sterilized by filtration through a 0.22μm microporous membrane for later use.

[0038] 2. Selection and inoculation of explants

[0039] The strawberry "Red Beauty" variety was pre-cultured to produce callus buds or healthy axillary buds as explant material, and the length of the bud body was about 0.2-0.5 cm (with a little callus base or inter-nodal tissue). To ensure uniformity of culture, the number of bud bodies inoculated in each culture bottle was 5, with appropriate spacing and competition conditions between the bud seedlings. Strict aseptic operation was followed during inoculation, and the explants were surface sterilized with 75% alcohol and 0.1% mercury (or sodium hypochlorite solution), then rinsed with sterile water, and quickly inoculated on culture medium with different treatment substances.

[0040] 3. Tissue culture

[0041] The formula of the culture medium is as follows: MS medium is used as the basis, and 30 g / L sucrose is uniformly added as a carbon source, 1 mg / L ascorbic acid (VC) and 10 mg / L thiamine (VB1) are added as coenzyme vitamins to provide a uniform nutritional environment. On this basis, different concentration gradients of acanthopanax extract are added to form four groups of treatments:

[0042] (1) The final concentration of acanthopanax extract is 5% (volume fraction, the same below), denoted as T1;

[0043] (2) The final concentration of acanthopanax extract is 10%, denoted as T2;

[0044] (3) The final concentration of acanthopanax extract is 15%, denoted as T3;

[0045] (4) The final concentration of acanthopanax extract is 20%, denoted as T4.

[0046] To facilitate comparison of effects, two groups of controls were set up: one was a blank control group without the addition of any bud-promoting hormones (i.e. no 6-BA and no acanthopanax extract in the culture medium), used to evaluate the basic effect of the culture medium on the growth of explants, denoted as CK1; the other was a conventional control group with the addition of standard concentration 6-BA, with an addition concentration of 1.0 mg / L, as a control treatment to represent the traditional bud-promoting proliferation level, denoted as CK2. The above treatment groups and control groups were consistent in other culture conditions, so that the differences in the effects of different growth-promoting substances on strawberry tissue culture and rapid propagation could be compared.

[0047] All groups were cultured at approximately 25℃, with a photoperiod of 16 hours light / 8 hours dark (16h / 8h), a light intensity of approximately 3000 lx (using LED plant growth lights to provide uniform illumination), and a relative humidity of approximately 70%. Culture containers were placed on culture racks in the culture room, using well-ventilated caps to facilitate gas exchange. The culture period was 28 days. During this entire 28-day period, no opening or transfer of the containers was performed, and the culture medium was not changed, allowing the test-tube seedlings to grow continuously in the same medium for four weeks. Throughout this process, the bud proliferation, growth, and rooting of each group of test-tube seedlings were observed and recorded daily without disturbing the culture environment.

[0048] 4. Evaluation Indicators

[0049] This invention uses malformation rate, browning rate, survival rate, proliferation rate, plant height, number of leaves, rooting status, and dry matter weight as evaluation indicators. These indicators are obtained through regular observation and statistics to form a complete data record, in order to measure the effectiveness of Acanthopanax senticosus extract as a hormone substitute from multiple perspectives.

[0050] II. Experimental Results

[0051] 1. Effect of Acanthopanax senticosus extract concentration on the rate of deformed strawberry in rapid propagation

[0052] like Figure 1 As shown, after 28 days of continuous culture, compared with the blank control (CK1), all other treatment groups significantly increased the probability of deformed seedlings in strawberry tissue culture propagation. The deformed seedling rate was as high as 20.93% in the T4 treatment, while it remained in the range of 7.57%–11.92% in the T1–T3 treatments. In the treatment group using conventional bud-promoting hormones (CK2), the deformed seedling rate also reached 15.63%. This indicates that the addition of exogenous growth-promoting substances to the culture medium during tissue culture propagation increases the probability of deformed seedlings to some extent. The addition of 20% Acanthopanax senticosus extract resulted in a significantly higher deformed seedling rate than conventional bud-promoting culture.

[0053] 2. Effect of Acanthopanax senticosus extract concentration on the browning rate of strawberry rapid propagation

[0054] like Figure 2 As shown, compared with the blank control CK1, the treatment groups T1, T2 and the control group CK2 significantly reduced the browning rate of strawberry tissue culture rapid propagation. The treatment group T3 showed no significant difference from the blank control CK1, while the treatment group T4 significantly increased the browning rate of strawberry tissue culture rapid propagation. This indicates that adding certain concentrations of growth-promoting substances does not significantly increase the browning rate of strawberry tissue culture rapid propagation, and may even inhibit it, such as low concentrations of Acanthopanax senticosus extract (5%, 10%) and 1.0 mg / L 6-BA.

[0055] 3. Effect of Acanthopanax senticosus extract concentration on the rapid propagation rate of strawberry

[0056] like Figure 3 As shown, compared to the blank control CK1, all other treatment groups significantly increased the proliferation rate of strawberry tissue culture rapid propagation, suggesting that adding exogenous growth-promoting substances to the culture medium can effectively improve the proliferation rate of strawberry tissue culture rapid propagation. Compared to the CK2 group, the proliferation rate under the T3 treatment was significantly increased, while the T2 and T4 treatments showed no significant difference from CK2, and the T1 treatment was significantly lower than CK2. This indicates that compared to the standard concentration of 6-BA commonly used in production, adding 15% Acanthopanax senticosus extract to the culture medium can effectively improve the proliferation rate of strawberry tissue culture rapid propagation and can effectively replace 6-BA. However, too high or too low a concentration of Acanthopanax senticosus extract will not achieve a significant promoting effect.

[0057] 4. Effect of Acanthopanax senticosus extract concentration on the survival rate of strawberry rapid propagation

[0058] like Figure 4 As shown, compared with the blank control CK1, all other treatment groups reduced the survival rate of strawberry tissue culture propagation, and the survival rate gradually decreased with the increase of Acanthopanax senticosus concentration, with the survival rate of T4 treatment being only 86.67%. Compared with CK2, the survival rate of strawberry tissue culture propagation was significantly reduced under T3 and T4 treatments, while there was no significant difference between T1 and T2 treatments and CK2.

[0059] 5. Effects of Acanthopanax senticosus extract concentration on rapid propagation and growth of strawberries

[0060] As shown in Table 1, compared with CK1, the addition of exogenous growth promoters to the culture medium inhibited the plant height, leaf number, rooting rate, and dry weight of strawberry tissue culture seedlings. This is because the hormone group (such as 6-BA or Acanthopanax senticosus extract) mainly stimulates cell proliferation, and the seedlings quickly form a large number of tender new tissues. These tissues have high water content, loose structure, and thin cell walls, and have not yet completed sufficient lignification and dry matter accumulation. As a result, the fresh weight of the seedlings increases rapidly, but the dry weight increases slowly, and the dry matter content per unit area is relatively low. In addition, the control group CK1 has no exogenous hormones, the plant proliferation is slow, the number of leaves is small, and the growth rhythm is natural. The tissues have enough time to accumulate assimilates (such as starch, cellulose, and structural proteins) under light. At the same time, the cell water content is lower, the tissues are more compact, and the dry matter content is naturally higher.

[0061] In addition, compared with CK2, the plant height of strawberry tissue culture seedlings under T1 and T2 treatments had no significant difference with CK2, while T3 and T4 treatments were significantly smaller than CK2; the leaf number of strawberry tissue culture seedlings under T1, T2, T3 and T4 treatments had no significant difference with CK2; the rooting rate under T1 treatment was significantly higher than CK2, the rooting rate under T2 and T3 treatments had no significant difference with CK2, while the rooting rate under T4 treatment was 0 and significantly smaller than CK2; in addition, the dry weight of strawberry tissue culture seedlings under T1 treatment was significantly higher than CK2, T2 and T3 treatments had no significant difference with CK2, while T4 treatment was significantly smaller than CK2. From the morphology of strawberry tissue culture seedlings, with the increase of the concentration of acanthopanax extract, the morphology of the plant gradually deteriorated, among which the morphology of the tissue culture seedlings treated with 10% and 15% acanthopanax extract was equivalent to the treatment of adding 1.0 mg / L 6-BA, while the treatment group of adding 5% acanthopanax extract was better than the treatment group of adding 1.0 mg / L 6-BA.

[0062] Table 1 Morphological performance of strawberry tissue culture seedlings under different concentrations of acanthopanax extract

[0063]

[0064] Note: According to Duncan test, different letters under the same index represent significant difference at 0.05 level.

[0065] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

1. Application of acanthopanax extract in reducing browning rate of strawberry tissue culture rapid propagation, characterized in that, The acanthopanax extract is an ethanol extract of dried roots of acanthopanax senticosus; The preparation method of the acanthopanax extract comprises the following steps: The dried roots of acanthopanax senticosus are crushed, and then extracted with an ethanol solution with a volume fraction of 75% as an extracting agent at a solid-liquid ratio of 1 g:10 mL under water bath reflux at 60 ℃ for 2 hours for two times, and then the two times of extracting solutions are combined, filtered and concentrated under reduced pressure to obtain the acanthopanax extract; The acanthopanax extract has a volume concentration of 5% or 10% in the culture medium.

2. A medium for strawberry tissue culture rapid propagation based on natural extract, characterized in that, The active ingredient in the culture medium is the acanthopanax extract, and no other hormone is contained. The acanthopanax extract is an ethanol extract of dried roots of acanthopanax senticosus; The preparation method of the acanthopanax extract comprises the following steps: The dried roots of acanthopanax senticosus are crushed, and then extracted with an ethanol solution with a volume fraction of 75% as an extracting agent at a solid-liquid ratio of 1 g:10 mL under water bath reflux at 60 ℃ for 2 hours for two times, and then the two times of extracting solutions are combined, filtered and concentrated under reduced pressure to obtain the acanthopanax extract; The acanthopanax extract has a volume concentration of 5% or 10% in the culture medium.

3. The medium of claim 2, wherein, The culture medium is a MS culture medium, and the non-active ingredients are sucrose 30 g / L, ascorbic acid 1 mg / L and thiamine 10 mg / L.

4. A method for in vitro clonal propagation of strawberry based on natural extract, characterized in that, The method comprises the step of culturing strawberry explants by using the culture medium according to claim 2 or 3. The strawberry explants comprise callus buds or healthy axillary buds of strawberries.

5. The method of claim 4, wherein, The temperature of the tissue culture is 25 ℃, the humidity is 70%, the light period is 16 hours of light / 8 hours of darkness, and the light intensity is 3000 lx.

6. The method of claim 4, wherein, The culture medium is not replaced during the tissue culture, and no subculture is performed.

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