Rapid propagation method of Rubus chingii Hu by tissue culture

Abstract

This invention relates to a rapid tissue culture propagation method for *Rubus palmatus*, belonging to the field of plant biotechnology. The method includes five steps: explant collection, explant sterilization, induction culture, proliferation culture, and rooting culture. This invention can efficiently, rapidly, and in large quantities cultivate high-quality *Rubus palmatus* tissue culture seedlings, meeting the market demand for *Rubus palmatus* tissue culture seedlings, possessing promising market prospects, and being easy to promote and apply.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for rapid tissue culture propagation of Raspberry palmatum. Background Technology

[0002] *Rubus chingii* Hu, belonging to the genus *Rubus* in the family Rosaceae, is a vine-like shrub, 1.5-3 meters tall; its branches are slender, prickly, and glabrous. Leaves are simple, nearly orbicular, 4-9 cm in diameter, pubescent only along the veins on both surfaces or nearly glabrous, with a cordate base. Flowers are solitary and axillary, glabrous; the calyx tube is sparsely pubescent or nearly glabrous; sepals are ovate or ovate-oblong, with a pointed apex. Fruits are nearly spherical, red, 1.5-2 cm in diameter, densely covered with grayish-white pubescence. Flowering occurs from March to April, and fruiting from May to June.

[0003] The fruit of the palmate raspberry, when immature and green, can be used medicinally. It is listed in the Chinese Pharmacopoeia and has the effects of tonifying the kidneys, strengthening essence, reducing urination, nourishing the liver, and improving eyesight. When ripe and red, it can be eaten fresh. The fresh fruit is bright red, has a delicate texture, is sweet and sour, and is rich in nutrients, including amino acids and various vitamins. It has high economic, nutritional, and health benefits and is known as the "third-generation fruit," which is very popular among consumers.

[0004] The conventional propagation methods for Raspberry palmatum are mainly cuttings and division. These methods have drawbacks such as low propagation coefficient, slow speed, long cycle, easy degeneration of seed quality, susceptibility to natural environmental and climatic changes, and high demand for mother plants. Propagating Raspberry palmatum using plant tissue culture (TCM) offers numerous advantages, including a high propagation coefficient, fast speed, no geographical or seasonal limitations, consistent genetic background, short growth cycle, year-round production, uniform seedlings, and high-quality seedlings. TCM seedlings are well-received in the market.

[0005] Current research reports on tissue culture techniques for Raspberry palmatum mainly have the following problems:

[0006] 1. Current technology mainly uses current-year stem segments with buds, and the sampling range of these stem segments is relatively wide. Different sampled stem segments will affect the tissue culture effect.

[0007] 2. Existing technologies use the same sterilization method to sterilize stem segments, without specifying the size of the stem segments or reporting data on sterilization effectiveness. Furthermore, the thickness of the stem segments can affect the sterilization effect.

[0008] 3. The induction culture medium in the existing technology mainly uses MS basal medium supplemented with two of the following plant growth regulators: 6-benzylaminopurine, naphthaleneacetic acid, indoleacetic acid, and gibberellin. Only a few studies have reported data on the induction rate (68%, 80%, 93.3%, 95%), and the induction rate of induction culture needs to be improved.

[0009] 4. Existing proliferation media mainly use MS basal medium supplemented with two or three plant growth regulators such as 6-benzylaminopurine, naphthaleneacetic acid, gibberellin, indolebutyric acid, and kinetin. The reported proliferation coefficients are 3.6, 4.87, 6.0, and 15.1, etc. Only a few studies have reported qualitative and simple descriptions of the growth of proliferation materials, which are difficult to reflect the comprehensive quality of proliferation materials. There is a lack of research reports on vitrification, yellowing, callus formation, etc. of proliferation materials.

[0010] 5. Existing rooting media mainly use 1 / 2M basal medium supplemented with one or two plant growth regulators such as indolebutyric acid, naphthaleneacetic acid, and gibberellin. Rooting rates have been reported as 90.6%, 93.3%, and 96.49%, etc. Only a few studies have qualitatively and simply described plant growth, which is difficult to reflect the overall quality of the plant. Furthermore, the rooting rate needs improvement, and there is a lack of research reports on root development characteristics (where the roots originate and whether there is callus tissue) and a lack of comprehensive descriptions of plant growth.

[0011] Therefore, overcoming the shortcomings of existing technologies is an urgent problem to be solved in the field of plant biotechnology. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a rapid tissue culture propagation method for Raspberry palmatum. This method can efficiently, quickly, and in large quantities cultivate high-quality Raspberry palmatum tissue culture seedlings, meeting the market demand for Raspberry palmatum tissue culture seedlings and showing promising market prospects.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A rapid tissue culture propagation method for Raspberry palmatum includes the following steps:

[0015] A. Explant collection: During the growing season of Raspberry palmatum, select new, semi-lignified branches with undeveloped axillary buds, vigorous growth, and free from pests and diseases on a sunny midday. After cutting the branches, seal them for preservation and bring them back to the tissue culture room for processing within 2.5 to 3.0 hours.

[0016] B. Sterilization of explants: Cut the branches from step A into stem segments of 0.8-1.0 cm in length, with each stem segment having a petiole of 0.1-0.2 cm in length attached; soak the stem segments in a 1.5%-2.5% (v / v) detergent solution for 1.0-2.0 min, then rinse the stem segments with sterile water 2-3 times, and finally sterilize the stem segments;

[0017] C. Induction Culture: The stem segments sterilized in step B are inoculated into the induction culture medium and cultured. When strong buds are cultivated, the induction culture is completed.

[0018] The induction medium is: MS basal medium + 6-benzylaminopurine 0.5-0.8 mg / L + naphthaleneacetic acid 0.01-0.05 mg / L + citric acid 300-400 mg / L + white sugar 25-30 g / L + agar powder 5.5-5.8 g / L, pH 5.8-6.0;

[0019] Alternatively, the induction medium can be: basal medium WPM + 6-benzylaminopurine 0.5–0.8 mg / L + naphthaleneacetic acid 0.01–0.05 mg / L + citric acid 300–400 mg / L + granulated sugar 25–30 g / L + agar powder 5.5–5.8 g / L, pH 5.8–6.0;

[0020] D. Proliferation Culture: The buds obtained in step C are cut and separated into individual buds at their connection with the stem segment; the individual buds are inoculated into the proliferation medium to cultivate robust cluster buds, and then individual buds are separated from the cluster buds and inoculated into the proliferation medium again. This process of subculture and proliferation culture is repeated. The temperature of the proliferation culture room is 25℃±2℃, the relative humidity is 30%~45%, the light intensity is 2500Lx~3000Lx, and the light duration is 12~14h / d.

[0021] The proliferation medium is: modified MS basal medium + 0.5-1.0 mg / L 6-benzylaminopurine + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0; the modified MS has KNO3, NH4NO3, and KH2PO4 contents of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, and the concentrations of other components are kept consistent with those of the basal MS medium.

[0022] Alternatively, the proliferation medium could be: MS basal medium + 0.5–1.0 mg / L 6-benzylaminopurine + 25–30 g / L sucrose + 5.5–5.8 g / L agar powder, pH 5.8–6.0;

[0023] E. Rooting culture: The cluster of buds obtained in step D is divided into individual buds. Individual buds with a length of 1.5 to 2.1 cm are selected and inoculated into rooting culture medium to obtain seedlings.

[0024] The rooting culture room temperature was 25℃±2℃, relative humidity was 30%~45%, light intensity was 2500Lx~3000Lx, light duration was 12~14h / d, and culture time was 35~50d;

[0025] The rooting medium is: 1 / 2 MS basal medium + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0;

[0026] Alternatively, the rooting medium can be: 1 / 2 MS basal medium + 0.1wt%–0.2wt% activated carbon + 25–30 g / L granulated sugar + 5.5–5.8 g / L agar powder, pH 5.8–6.0;

[0027] Alternatively, the rooting medium can be: MS basal medium + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0;

[0028] Alternatively, the rooting medium can be: MS basal medium + 0.1wt%–0.2wt% activated carbon + 25–30 g / L granulated sugar + 5.5–5.8 g / L agar powder, pH 5.8–6.0;

[0029] Alternatively, the rooting medium can be: basal medium WPM + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0;

[0030] Alternatively, the rooting medium can be: basal medium WPM + activated carbon 0.1wt%~0.2wt% + white sugar 25~30g / L + agar powder 5.5~5.8g / L, pH 5.8~6.0.

[0031] Furthermore, preferably, in step A, the explant collection targets are:

[0032] New shoots that are 45-60 days old on the main stem or lateral branches of Raspberry palmatum.

[0033] Furthermore, preferably, the specific method for sterilizing the stem segments in step B is as follows:

[0034] The stem segments are classified into three categories based on their stem thickness: thin stem segments, medium-thickness stem segments, and thick stem segments.

[0035] The thin stem segments have a stem diameter greater than or equal to 2.55 mm and less than or equal to 3.10 mm; the medium-thick stem segments have a stem diameter greater than 3.10 mm and less than or equal to 3.66 mm; and the thick stem segments have a stem diameter greater than 3.66 mm and less than or equal to 4.22 mm.

[0036] When sterilizing stem segments on a clean bench, first soak them in 75% alcohol for 30-40 seconds. Then, soak the thin stem segments in a 0.2% mercuric chloride solution containing Tween-20 for 4-5 minutes, the medium-thick stem segments for 6-7 minutes, and the thick stem segments for 8-9 minutes. Next, soak and rinse them in sterile water for 5-7 minutes. Finally, use sterile filter paper to absorb the surface moisture of the stem segments.

[0037] Add 1-2 drops of Tween-20 to 150-200 mL of a 0.2% mercuric chloride solution and mix well to form a 0.2% mercuric chloride solution containing Tween-20.

[0038] Furthermore, preferably, the specific method for stem segment culture in step C is as follows:

[0039] First, the buds were cultured in the dark for 8-10 days at a temperature of 25±2℃ and a relative humidity of 30%-45% to induce 1-2 buds with a length of 0.5-0.8cm. Then, they were cultured in an environment with a temperature of 25±2℃, a relative humidity of 30%-45%, a light intensity of 2500-3000Lx, and a light duration of 12-14h / d for 15-20 days to cultivate buds with a length of 1.5-2.0cm.

[0040] Furthermore, preferably, in step C, during dark culture, the induction medium is replaced daily from day 2 to day 5.

[0041] Furthermore, preferably, in step D, the number of subcultures is controlled to be 15-20; during subculture, proliferation media composed of different concentrations (0.5-1.0 mg / L) of 6-benzylaminopurine and different types of basal media (modified MS, MS) are used alternately. That is, in the proliferation media provided in this invention, each generation uses a proliferation medium composed of different concentrations of 6-benzylaminopurine and different types of basal media.

[0042] Furthermore, preferably, in step D, the culture time for proliferation culture is 15-20 days; first, all the callus tissue at the base of the bud cluster is removed, and then the bud cluster is separated into individual buds.

[0043] Furthermore, preferably, in step E, single buds shorter than 1.5 cm are cultured for proliferation according to step D.

[0044] Furthermore, preferably, the specific method for rooting culture in step E is as follows:

[0045] Remove 2-4 petioles from the stem at the base of each bud, leaving 2-3 leaves at the top of the stem; inoculate the bud into the rooting medium at a depth of 0.3-0.4 cm.

[0046] In step A of this invention, the explants are selected from newly grown branches of the main stem or lateral branches of Raspberry palmatum, which are 45 to 60 days old. At this time, the branches have vigorous growth and strong axillary bud sprouting ability, making them relatively easy to induce and culture.

[0047] In step C of this invention, the stem segments sterilized in step B are inoculated into an induction medium for culture, achieving an induction rate of 100%. The induced buds exhibit robust growth. During dark culture, the induction medium is changed daily from day 2 to day 5, using fresh, identical induction medium each time.

[0048] In step D of this invention, during the subculture and proliferation culture, the amount of callus tissue at the base of each bud cluster is very small, only 0.04 to 0.06 g, which has no effect on the differentiation quality of the bud cluster. At this time, the bud cluster has vigorous growth, no vitrification, no yellowing, and a proliferation coefficient of 5.52 to 6.14.

[0049] In step E of this invention, after rooting culture, the plant maturity rate is 100%, the rooting rate of the plants is 100%, the plant height is 2.41-2.58cm, the root quantity is 2.00-2.18cm, the root length is 4.92-5.55cm, the root diameter is 0.40-0.54mm, the roots emerge from the base, there is no callus tissue at the base, and the plants have good growth vigor: robust, green, uniform, without vitrification, and without yellowing.

[0050] In this invention, the strength of the agar powder is 1200 g / cm³. 2 .

[0051] Compared with the prior art, the beneficial effects of this invention are as follows:

[0052] (1) The present invention selects new branches of 45-60 days as explants, which have a good induction culture effect: they are relatively easy to induce, with an induction rate of 100%, and 1-2 buds of 0.5-0.8 cm in length are induced in 8-10 days, and buds of 1.5-2.0 cm in length can be cultured within 23-30 days;

[0053] (2) According to the thickness of the stem segments, the present invention divides the stem segments into three categories: thin stem segments, medium-thick stem segments, and thick stem segments, and adopts three different suitable sterilization methods accordingly, all of which have achieved good sterilization results;

[0054] (3) In the induction culture, the present invention uses a suitable induction culture medium and induction culture method, which can greatly reduce the influence of stem browning, improve the induction culture effect, and achieve an induction rate of 100%. The buds induced by the culture show strong growth.

[0055] (4) The present invention uses a suitable proliferation culture medium and proliferation culture method to achieve efficient and high-quality proliferation and propagation of buds: the bud clusters grow vigorously, without vitrification or yellowing, and the proliferation coefficient reaches 5.52 to 6.14;

[0056] (5) The present invention uses a suitable rooting culture medium and rooting culture method to cultivate high-quality seedlings: the maturity rate is 100%, the rooting rate of the plants is 100%, the plant height is 2.41-2.58cm, the root amount is 2.00-2.18cm, the root length is 4.92-5.55cm, the root diameter is 0.40-0.54mm, the roots emerge from the base, there is no callus tissue at the base, and the plants have good growth vigor (robust, green, uniform, no vitrification, no yellowing); at the same time, the small buds are returned to the proliferation culture stage for recycling, which is conducive to increasing the number of proliferation mother bottles and expanding the base number of mother bottles;

[0057] (6) The culture medium used in this invention has a simple composition (two plant growth regulators are used in the induction culture stage, only one plant growth regulator is used in the proliferation culture stage, and zero plant growth regulators are used in the rooting culture stage). The culture method is easy to operate, which can not only reduce costs, but also efficiently, quickly and in large quantities cultivate high-quality Raspberry palmatum tissue culture seedlings, realize the rapid propagation of Raspberry palmatum tissue culture, meet the market demand for Raspberry palmatum tissue culture seedlings, and has a good market prospect. This invention is easy to implement and promote. Attached Figure Description

[0058] Figure 1 The images show the plant conditions of the Raspberry palmatum explants in this embodiment of the invention. Image A shows the plant growth, indicating that the plant is growing vigorously. Image B shows the fruit, indicating that the fruit is red.

[0059] Figure 2 The diagram shows the induction culture of Raspberry palmatum in this embodiment of the invention; wherein, diagram A shows a 0.7 cm long bud induced after 8 days of induction culture, and diagram B shows a 1.6 cm long bud formed after 25 days of induction culture, showing a good induction culture effect;

[0060] Figure 3 This is a propagation culture diagram of Raspberry palmatum in an embodiment of the present invention. The cluster buds in the diagram are growing vigorously, without vitrification or yellowing, showing a good propagation culture effect.

[0061] Figure 4 The following are rooting culture diagrams of Raspberry palmatum in an embodiment of the present invention; wherein, diagram A is a rooting culture diagram in a rooting medium without activated carbon, and diagram B is a rooting culture diagram in a rooting medium with activated carbon; the diagrams show that the rooted plants have good growth vigor: robust, green, uniform, without vitrification or yellowing, and good root quality, demonstrating a good rooting culture effect.

[0062] Figure 5This is a time dynamic diagram of the proliferation culture of Raspberry palmatum in an embodiment of the present invention. The diagram shows that the suitable proliferation culture time is 15 to 20 days. At this time, the amount of callus tissue at the base of each bud cluster is very small, only 0.04 to 0.06 g, which has no effect on the differentiation quality of the bud cluster. The bud cluster is robust, without vitrification or yellowing, and the proliferation coefficient is 5.52 to 6.14, achieving good proliferation. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to the embodiments.

[0064] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0065] Example 1

[0066] A rapid tissue culture propagation method for Raspberry palmatum includes the following steps:

[0067] A. Explant collection: During the growing season of Raspberry palmatum, select new, semi-lignified branches with undeveloped axillary buds, vigorous growth, and free from pests and diseases on a sunny midday. After cutting the branches, seal them for preservation and bring them back to the tissue culture room for processing within 2.5 to 3.0 hours.

[0068] B. Sterilization of explants: Cut the branches from step A into stem segments of 0.8-1.0 cm in length, with each stem segment having a petiole of 0.1-0.2 cm in length attached; soak the stem segments in a 2% (v / v) detergent solution for 1.5 min, then rinse the stem segments twice with sterile water, and then sterilize the stem segments.

[0069] C. Induction Culture: The stem segments sterilized in step B are inoculated into the induction culture medium and cultured. When strong buds are cultivated, the induction culture is completed.

[0070] The induction medium was: MS basal medium + 0.6 mg / L 6-benzylaminopurine + 0.03 mg / L naphthaleneacetic acid + 350 mg / L citric acid + 28 g / L granulated sugar + 5.7 g / L agar powder, pH 5.9;

[0071] Alternatively, the induction medium could be: basal medium WPM + 6-benzylaminopurine 0.6 mg / L + naphthaleneacetic acid 0.03 mg / L + citric acid 350 mg / L + granulated sugar 28 g / L + agar powder 5.7 g / L, pH 5.9;

[0072] D. Proliferation Culture: The buds obtained in step C are cut and separated into individual buds at their connection with the stem segment; the individual buds are inoculated into the proliferation medium to cultivate robust cluster buds, and then individual buds are separated from the cluster buds and inoculated into the proliferation medium again. This process of subculture and proliferation culture is repeated. The temperature of the proliferation culture room is 25℃±2℃, the relative humidity is 30%~45%, the light intensity is 2500Lx~3000Lx, and the light duration is 12~14h / d.

[0073] The proliferation medium is: modified MS basal medium + 0.8 mg / L 6-benzylaminopurine + 27 g / L granulated sugar + 5.7 g / L agar powder, pH 5.9; the modified MS has KNO3, NH4NO3, and KH2PO4 contents of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, and the concentrations of other components are kept consistent with those of the basal MS medium;

[0074] Alternatively, the proliferation medium could be: MS basal medium + 0.8 mg / L 6-benzylaminopurine + 26 g / L sucrose + 5.5–5.8 g / L agar powder, pH 5.8–6.0;

[0075] E. Rooting culture: The cluster of buds obtained in step D is divided into individual buds. Individual buds with a length of 1.5 to 2.1 cm are selected and inoculated into rooting culture medium to obtain seedlings.

[0076] The rooting culture room temperature was 25℃±2℃, relative humidity was 30%~45%, light intensity was 2500Lx~3000Lx, light duration was 12~14h / d, and culture time was 35d;

[0077] The rooting medium is: 1 / 2 MS basal medium + 26 g / L granulated sugar + 5.7 g / L agar powder, pH 5.8;

[0078] Alternatively, the rooting medium could be: 1 / 2 MS basal medium + 0.15 wt% activated carbon + 28 g / L granulated sugar + 5.6 g / L agar powder, pH 5.9;

[0079] Alternatively, the rooting medium can be: MS basal medium + 27 g / L granulated sugar + 5.7 g / L agar powder, pH 5.9;

[0080] Alternatively, the rooting medium could be: MS basal medium + 0.16wt% activated carbon + 27g / L granulated sugar + 5.7g / L agar powder, pH 5.9;

[0081] Alternatively, the rooting medium can be: basal medium WPM + 28 g / L granulated sugar + 5.7 g / L agar powder, pH 5.9;

[0082] Alternatively, the rooting medium can be: basal medium WPM + activated carbon 0.15wt% + white sugar 26g / L + agar powder 5.7g / L, pH 5.9.

[0083] Example 2

[0084] A rapid tissue culture propagation method for Raspberry palmatum includes the following steps:

[0085] A. Explant collection: During the growing season of Raspberry palmatum, select new, semi-lignified branches with undeveloped axillary buds, vigorous growth, and free from pests and diseases on a sunny midday. After cutting the branches, seal them for preservation and bring them back to the tissue culture room for processing within 2.5 to 3.0 hours.

[0086] B. Sterilization of explants: Cut the branches from step A into stem segments of 0.8-1.0 cm in length, with each stem segment having a petiole of 0.1-0.2 cm in length attached; soak the stem segments in a 1.5% (v / v) detergent solution for 1.0 min, then rinse the stem segments twice with sterile water, and finally sterilize the stem segments.

[0087] C. Induction Culture: The stem segments sterilized in step B are inoculated into the induction culture medium and cultured. When strong buds are cultivated, the induction culture is completed.

[0088] The induction medium is: MS basal medium + 0.5 mg / L 6-benzylaminopurine + 0.01 mg / L naphthaleneacetic acid + 300 mg / L citric acid + 25 g / L granulated sugar + 5.5 g / L agar powder, pH 5.8;

[0089] Alternatively, the induction medium could be: basal medium WPM + 6-benzylaminopurine 0.5 mg / L + naphthaleneacetic acid 0.01 mg / L + citric acid 300 mg / L + granulated sugar 25 g / L + agar powder 5.5 g / L, pH 5.8;

[0090] D. Proliferation Culture: The buds obtained in step C are cut and separated into individual buds at their connection with the stem segment; the individual buds are inoculated into the proliferation medium to cultivate robust cluster buds, and then individual buds are separated from the cluster buds and inoculated into the proliferation medium again. This process of subculture and proliferation culture is repeated. The temperature of the proliferation culture room is 25℃±2℃, the relative humidity is 30%~45%, the light intensity is 2500Lx~3000Lx, and the light duration is 12~14h / d.

[0091] The proliferation medium is: modified MS basal medium + 0.5 mg / L 6-benzylaminopurine + 25 g / L granulated sugar + 5.5 g / L agar powder, pH 5.8; the modified MS has KNO3, NH4NO3, and KH2PO4 contents of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, and the concentrations of other components are kept consistent with those of the basal MS medium;

[0092] Alternatively, the proliferation medium could be: MS basal medium + 0.5 mg / L 6-benzylaminopurine + 25 g / L sucrose + 5.5 g / L agar powder, pH 5.8;

[0093] E. Rooting culture: The cluster of buds obtained in step D is divided into individual buds. Individual buds with a length of 1.5 to 2.1 cm are selected and inoculated into rooting culture medium to obtain seedlings.

[0094] The rooting culture room temperature was 25℃±2℃, relative humidity was 30%~45%, light intensity was 2500Lx~3000Lx, light duration was 12~14h / d, and culture time was 35d;

[0095] The rooting medium is: 1 / 2 MS basal medium + 25 g / L granulated sugar + 5.5 g / L agar powder, pH 5.8;

[0096] Alternatively, the rooting medium can be: 1 / 2 MS basal medium + 0.1wt%~0.2wt% activated carbon + 25g / L white sugar + 5.5g / L agar powder, pH 5.8;

[0097] Alternatively, the rooting medium can be: MS basal medium + 25 g / L granulated sugar + 5.5 g / L agar powder, pH 5.8;

[0098] Alternatively, the rooting medium could be: MS basal medium + 0.1 wt% activated carbon + 25 g / L granulated sugar + 5.5 g / L agar powder, pH 5.8;

[0099] Alternatively, the rooting medium can be: basal medium WPM + 25 g / L granulated sugar + 5.5 g / L agar powder, pH 5.8;

[0100] Alternatively, the rooting medium can be: basal medium WPM + activated carbon 0.1wt% + white sugar 25g / L + agar powder 5.5g / L, pH 5.8.

[0101] In step A, the explant collection targets are:

[0102] New shoots that are 45-50 days old on the main stem or lateral branches of Raspberry palmatum.

[0103] In step B, the specific method for sterilizing the stem segments is as follows:

[0104] The stem segments are classified into three categories based on their stem thickness: thin stem segments, medium-thickness stem segments, and thick stem segments.

[0105] The thin stem segments have a stem diameter greater than or equal to 2.55 mm and less than or equal to 3.10 mm; the medium-thick stem segments have a stem diameter greater than 3.10 mm and less than or equal to 3.66 mm; and the thick stem segments have a stem diameter greater than 3.66 mm and less than or equal to 4.22 mm.

[0106] When sterilizing stem segments on a clean bench, first soak them in 75% alcohol for 30 seconds, then soak them in a 0.2% mercuric chloride solution containing Tween-20 for 4 minutes for fine stem segments, 6 minutes for medium-thick stem segments, and 8 minutes for thick stem segments, respectively. Then soak them in sterile water for 5 minutes, and finally use sterile filter paper to absorb the surface moisture of the stem segments.

[0107] Add 1 drop of Tween-20 to 150 mL of 0.2% mercuric chloride solution and mix well to form a 0.2% mercuric chloride solution containing Tween-20.

[0108] In step C, the specific method for stem segment culture is as follows:

[0109] First, the buds were cultured in the dark for 8-10 days at a temperature of 25±2℃ and a relative humidity of 30%-45% to induce 1-2 buds with a length of 0.5-0.8cm. Then, they were cultured in an environment with a temperature of 25±2℃, a relative humidity of 30%-45%, a light intensity of 2500-3000Lx, and a light duration of 12-14h / d for 15 days to cultivate buds with a length of 1.5-2.0cm.

[0110] In step C, during dark incubation, the induction medium should be changed daily from day 2 to day 5.

[0111] In step D, the number of subcultures is controlled at 15. During subculture, different proliferation media composed of different concentrations (0.5–1.0 mg / L) of 6-benzylaminopurine and different types of basal media (modified MS, MS) are used alternately. That is, in the proliferation media given in this invention, each generation uses a proliferation medium composed of different concentrations of 6-benzylaminopurine and different types of basal media.

[0112] In step D, the culture time for proliferation culture is 15 days; first, all the callus tissue at the base of the bud cluster is removed, and then the bud cluster is separated into individual buds.

[0113] In step E, single buds shorter than 1.5 cm are cultured for proliferation according to step D.

[0114] In step E, the specific method for rooting culture is as follows:

[0115] Remove the two petioles from the base of the stem of each bud, leaving the top of the stem with two leaves; inoculate the bud into the rooting medium at a depth of 0.3 cm.

[0116] Example 3

[0117] A rapid tissue culture propagation method for Raspberry palmatum includes the following steps:

[0118] A. Explant collection: During the growing season of Raspberry palmatum, select new, semi-lignified branches with undeveloped axillary buds, vigorous growth, and free from pests and diseases on a sunny midday. After cutting the branches, seal them for preservation and bring them back to the tissue culture room for processing within 2.5 to 3.0 hours.

[0119] B. Sterilization of explants: Cut the branches from step A into stem segments of 0.8-1.0 cm in length, with each stem segment having a petiole of 0.1-0.2 cm in length attached; soak the stem segments in a 2.5% detergent solution for 2.0 min, then rinse the stem segments three times with sterile water, and then sterilize the stem segments.

[0120] C. Induction Culture: The stem segments sterilized in step B are inoculated into the induction culture medium and cultured. When strong buds are cultivated, the induction culture is completed.

[0121] The induction medium was: MS basal medium + 0.8 mg / L 6-benzylaminopurine + 0.05 mg / L naphthaleneacetic acid + 400 mg / L citric acid + 30 g / L granulated sugar + 5.8 g / L agar powder, pH 6.0;

[0122] Alternatively, the induction medium could be: basal medium WPM + 6-benzylaminopurine 0.8 mg / L + naphthaleneacetic acid 0.05 mg / L + citric acid 400 mg / L + granulated sugar 30 g / L + agar powder 5.8 g / L, pH 6.0;

[0123] D. Proliferation Culture: The buds obtained in step C are cut and separated into individual buds at their connection with the stem segment; the individual buds are inoculated into the proliferation medium to cultivate robust cluster buds, and then individual buds are separated from the cluster buds and inoculated into the proliferation medium again. This process of subculture and proliferation culture is repeated. The temperature of the proliferation culture room is 25℃±2℃, the relative humidity is 30%~45%, the light intensity is 2500Lx~3000Lx, and the light duration is 12~14h / d.

[0124] The proliferation medium is: modified MS basal medium + 1.0 mg / L 6-benzylaminopurine + 30 g / L granulated sugar + 5.8 g / L agar powder, pH 6.0; the modified MS has KNO3, NH4NO3, and KH2PO4 contents of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, and the concentrations of other components are kept consistent with those of the basal MS medium.

[0125] Alternatively, the proliferation medium could be: MS basal medium + 1.0 mg / L 6-benzylaminopurine + 30 g / L sucrose + 5.8 g / L agar powder, pH 6.0;

[0126] E. Rooting culture: The cluster of buds obtained in step D is divided into individual buds. Individual buds with a length of 1.5 to 2.1 cm are selected and inoculated into rooting culture medium to obtain seedlings.

[0127] The rooting culture room temperature was 25℃±2℃, relative humidity was 30%~45%, light intensity was 2500Lx~3000Lx, light duration was 12~14h / d, and culture time was 50d;

[0128] The rooting medium is: 1 / 2 MS basal medium + 30 g / L granulated sugar + 5.8 g / L agar powder, pH 6.0;

[0129] Alternatively, the rooting medium can be: 1 / 2 MS basal medium + 0.2 wt% activated carbon + 30 g / L granulated sugar + 5.8 g / L agar powder, pH 6.0;

[0130] Alternatively, the rooting medium can be: MS basal medium + 30 g / L granulated sugar + 5.8 g / L agar powder, pH 6.0;

[0131] Alternatively, the rooting medium could be: MS basal medium + 0.2wt% activated carbon + 30g / L granulated sugar + 5.8g / L agar powder, pH 6.0;

[0132] Alternatively, the rooting medium can be: basal medium WPM + 30 g / L granulated sugar + 5.8 g / L agar powder, pH 6.0;

[0133] Alternatively, the rooting medium can be: basal medium WPM + activated carbon 0.2wt% + white sugar 30g / L + agar powder 5.8g / L, pH 6.0.

[0134] In step A, the explant collection targets are:

[0135] New shoots that are 53-60 days old on the main stem or lateral branches of Raspberry palmatum.

[0136] In step B, the specific method for sterilizing the stem segments is as follows:

[0137] The stem segments are classified into three categories based on their stem thickness: thin stem segments, medium-thickness stem segments, and thick stem segments.

[0138] The thin stem segments have a stem diameter greater than or equal to 2.55 mm and less than or equal to 3.10 mm; the medium-thick stem segments have a stem diameter greater than 3.10 mm and less than or equal to 3.66 mm; and the thick stem segments have a stem diameter greater than 3.66 mm and less than or equal to 4.22 mm.

[0139] When sterilizing stem segments on a clean bench, first soak them in 75% alcohol for 40 seconds, then soak them in a 0.2% mercuric chloride solution containing Tween-20 for 5 minutes, medium-thick stem segments for 7 minutes, and thick stem segments for 9 minutes, respectively. Then soak them in sterile water for 7 minutes, and finally use sterile filter paper to absorb the surface moisture of the stem segments.

[0140] Add 2 drops of Tween-20 to 200 mL of 0.2% mercuric chloride solution and mix well to form a 0.2% mercuric chloride solution containing Tween-20.

[0141] In step C, the specific method for stem segment culture is as follows:

[0142] First, the buds were cultured in the dark for 8-10 days at a temperature of 25±2℃ and a relative humidity of 30%-45% to induce 1-2 buds with a length of 0.5-0.8cm. Then, they were cultured in an environment with a temperature of 25±2℃, a relative humidity of 30%-45%, a light intensity of 2500-3000Lx, and a light duration of 12-14h / d for 15-20 days to cultivate buds with a length of 1.5-2.0cm.

[0143] In step C, during dark incubation, the induction medium should be changed daily from day 2 to day 5.

[0144] In step D, the number of subcultures is controlled at 20. During subculture, different proliferation media composed of different concentrations (0.5–1.0 mg / L) of 6-benzylaminopurine and different types of basal media (modified MS, MS) are used alternately. That is, in the proliferation media given in this invention, each generation uses a proliferation medium composed of different concentrations of 6-benzylaminopurine and different types of basal media.

[0145] In step D, the culture time for proliferation culture is 20 days; first, all the callus tissue at the base of the bud cluster is removed, and then the bud cluster is separated into individual buds.

[0146] In step E, single buds shorter than 1.5 cm are cultured for proliferation according to step D.

[0147] In step E, the specific method for rooting culture is as follows:

[0148] Remove the four petioles from the base of the stem of each bud, leaving the top of the stem with three leaves; inoculate the bud into the rooting medium at a depth of 0.4 cm.

[0149] Example 4

[0150] A rapid tissue culture propagation method for Raspberry palmatum includes the following steps:

[0151] A. Explant collection: During the growing season of Raspberry palmatum, select new, semi-lignified branches with undeveloped axillary buds, vigorous growth, and free from pests and diseases on a sunny midday. After cutting the branches, seal them for preservation and bring them back to the tissue culture room for processing within 2.5 to 3.0 hours.

[0152] B. Sterilization of explants: Cut the branches from step A into stem segments of 0.8-1.0 cm in length, with each stem segment having a petiole of 0.1-0.2 cm in length attached; soak the stem segments in a 2% (v / v) detergent solution for 1.5 min, then rinse the stem segments twice with sterile water, and then sterilize the stem segments.

[0153] C. Induction Culture: The stem segments sterilized in step B are inoculated into the induction culture medium and cultured. When strong buds are cultivated, the induction culture is completed.

[0154] The induction medium was: MS basal medium + 0.7 mg / L 6-benzylaminopurine + 0.03 mg / L naphthaleneacetic acid + 350 mg / L citric acid + 28 g / L granulated sugar + 5.7 g / L agar powder, pH 5.9;

[0155] Alternatively, the induction medium could be: basal medium WPM + 6-benzylaminopurine 0.7 mg / L + naphthaleneacetic acid 0.03 mg / L + citric acid 370 mg / L + granulated sugar 28 g / L + agar powder 5.7 g / L, pH 5.9;

[0156] D. Proliferation Culture: The buds obtained in step C are cut and separated into individual buds at their connection with the stem segment; the individual buds are inoculated into the proliferation medium to cultivate robust cluster buds, and then individual buds are separated from the cluster buds and inoculated into the proliferation medium again. This process of subculture and proliferation culture is repeated. The temperature of the proliferation culture room is 25℃±2℃, the relative humidity is 30%~45%, the light intensity is 2500Lx~3000Lx, and the light duration is 12~14h / d.

[0157] The proliferation medium is: modified MS basal medium + 0.8 mg / L 6-benzylaminopurine + 27 g / L granulated sugar + 5.6 g / L agar powder, pH 5.9; the modified MS has KNO3, NH4NO3, and KH2PO4 contents of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, and the concentrations of other components are kept consistent with those of the basal MS medium.

[0158] Alternatively, the proliferation medium could be: MS basal medium + 0.8 mg / L 6-benzylaminopurine + 28 g / L sucrose + 5.7 g / L agar powder, pH 5.9;

[0159] E. Rooting culture: The cluster of buds obtained in step D is divided into individual buds. Individual buds with a length of 1.5 to 2.1 cm are selected and inoculated into rooting culture medium to obtain seedlings.

[0160] The rooting culture room temperature was 25℃±2℃, relative humidity was 30%~45%, light intensity was 2500Lx~3000Lx, light duration was 12~14h / d, and culture time was 45d;

[0161] The rooting medium is: 1 / 2 MS basal medium + 28 g / L granulated sugar + 5.6 g / L agar powder, pH 5.9;

[0162] Alternatively, the rooting medium could be: 1 / 2 MS basal medium + 0.12 wt% activated carbon + 28 g / L granulated sugar + 5.6 g / L agar powder, pH 5.9;

[0163] Alternatively, the rooting medium can be: MS basal medium + 29 g / L granulated sugar + 5.7 g / L agar powder, pH 5.9;

[0164] Alternatively, the rooting medium could be: MS basal medium + 0.18wt% activated carbon + 28g / L granulated sugar + 5.7g / L agar powder, pH 5.9;

[0165] Alternatively, the rooting medium could be: basal medium WPM + 27 g / L granulated sugar + 5.6 g / L agar powder, pH 5.9;

[0166] Alternatively, the rooting medium can be: basal medium WPM + activated carbon 0.15wt% + white sugar 26g / L + agar powder 5.7g / L, pH 5.9.

[0167] In step A, the explant collection targets are:

[0168] New shoots that are 47-55 days old on the main stem or lateral branches of Raspberry palmatum.

[0169] In step B, the specific method for sterilizing the stem segments is as follows:

[0170] The stem segments are classified into three categories based on their stem thickness: thin stem segments, medium-thickness stem segments, and thick stem segments.

[0171] The thin stem segments have a stem diameter greater than or equal to 2.55 mm and less than or equal to 3.10 mm; the medium-thick stem segments have a stem diameter greater than 3.10 mm and less than or equal to 3.66 mm; and the thick stem segments have a stem diameter greater than 3.66 mm and less than or equal to 4.22 mm.

[0172] When sterilizing stem segments on a clean bench, first immerse them in 75% alcohol for 35 seconds, then immerse them in a 0.2% mercuric chloride solution containing Tween-20 for 4.5 minutes for fine stem segments, 6.5 minutes for medium-thick stem segments, and 8.5 minutes for thick stem segments, respectively. Then, immerse them in sterile water for 6 minutes and finally use sterile filter paper to absorb the surface moisture of the stem segments.

[0173] Add 1 drop of Tween-20 to 180 mL of 0.2% mercuric chloride solution and mix well to form a 0.2% mercuric chloride solution containing Tween-20.

[0174] In step C, the specific method for stem segment culture is as follows:

[0175] First, the buds were cultured in the dark for 8-10 days at a temperature of 25±2℃ and a relative humidity of 30%-45% to induce 1-2 buds with a length of 0.5-0.8cm. Then, they were cultured in an environment with a temperature of 25±2℃, a relative humidity of 30%-45%, a light intensity of 2500-3000Lx, and a light duration of 12-14h / d for 18 days to cultivate buds with a length of 1.5-2.0cm.

[0176] In step C, during dark incubation, the induction medium should be changed daily from day 2 to day 5.

[0177] In step D, the number of subcultures is controlled at 18. During subculture, different proliferation media composed of different concentrations (0.5–1.0 mg / L) of 6-benzylaminopurine and different types of basal media (modified MS, MS) are used alternately. That is, in the proliferation media given in this invention, different concentrations of 6-benzylaminopurine and different types of basal media are used in each generation.

[0178] In step D, the culture time for proliferation culture is 17 days; first, all the callus tissue at the base of the bud cluster is removed, and then the bud cluster is separated into individual buds.

[0179] In step E, single buds shorter than 1.5 cm are cultured for proliferation according to step D.

[0180] In step E, the specific method for rooting culture is as follows:

[0181] Remove the three petioles from the stem at the base of each bud, leaving the top of the stem with two leaves; inoculate the bud into the rooting medium at a depth of 0.35 cm.

[0182] Experimental research:

[0183] A systematic study on the rapid tissue culture propagation experiment of *Rubus palmatus* was conducted at the tissue culture laboratory of Linhan Nursery in Hongta District, Yuxi City, Yunnan Province from February 2023 to June 2024. Explants were collected from Shengfeng Raspberry Seedling Harvesting Base in Jiangchuan District, Yuxi City, Yunnan Province. The main experimental results are as follows:

[0184] 1. Explant sterilization test

[0185] Three types of stem segments—thin, medium, and thick—were sterilized using a 0.2% mercuric chloride solution containing Tween-20 (1 drop of Tween-20 added to 200 mL of 0.2% mercuric chloride solution). The results (Table 1) showed that for thin stem segments, the contamination rate was less than 27% and the mortality rate was less than 21% after 4–5 minutes of sterilization, indicating a good sterilization effect. For medium-thick stem segments, the contamination rate was less than 24% and the mortality rate was less than 25% after 6–7 minutes of sterilization, indicating a good sterilization effect. For thick stem segments, the contamination rate was less than 21% and the mortality rate was less than 29% after 8–9 minutes of sterilization, indicating a good sterilization effect.

[0186] In existing technologies, stem segments are sterilized using the same sterilization method (regardless of thickness). The problem is that the sterilization effect is affected by variations in stem segment thickness. In contrast, this invention classifies stem segments into three categories based on their thickness: bacterial segments, medium-thickness segments, and thick segments, and employs three different suitable sterilization methods for each category. Experiments show that each sterilization method for the three types of stem segments has a good sterilization effect (Table 1).

[0187] Table 1. Sterilization effect test of Raspberry stem segments

[0188]

[0189] 2. Induction culture experiment

[0190] The four induction culture media of the present invention were used to carry out the induction culture experiment of Raspberry palmatum stem segments. The results (Table 2) show that the induction rate of Raspberry palmatum stem segments by the present invention is 100%, the browning rate is low, which is only 1.15% to 2.01%, and the buds have strong growth vigor, showing a good induction culture effect.

[0191] Table 2. Induction culture effect of Raspberry stem segments

[0192]

[0193] Note: In the table, 6-BA, NAA, and CA are abbreviations for 6-benzylaminopurine, naphthaleneacetic acid, and citric acid, respectively, and the same applies below.

[0194] 3. Proliferation Culture Experiment

[0195] The four proliferation culture media of the present invention were used to carry out the proliferation culture experiment of Raspberry palmatum. The results (Table 3) show that after proliferation culture, the buds formed cluster buds, which grew vigorously, without vitrification or yellowing, and with small callus tissue attached to the base. The proliferation coefficient was 5.52 to 6.14, showing good proliferation culture effect.

[0196] Table 3. Propagation Culture Effect Experiment of Raspberry Palmarosa

[0197]

[0198] Note: The modified MS in the table has KNO3, NH4NO3, and KH2PO4 concentrations of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, while the concentrations of other components remain consistent with those in the basal medium MS.

[0199] In this invention, the propagation culture time has a significant impact on the propagation culture effect of *Rubus palmatus* (see attached). Figure 5 As the propagation culture time increased, the propagation coefficient of the shoot clusters gradually increased, and the fresh weight of callus tissue at the base of the shoot clusters also gradually increased. However, the quality of the shoot clusters gradually weakened, manifested as a decrease in growth vigor and an increase in the proportion of vitrification and yellowing. When the propagation culture time was 15–20 days, the amount of callus tissue at the base of each shoot cluster was minimal, only 0.04–0.06 g, which had no effect on the differentiation quality of the shoot clusters. At this time, the shoot clusters were robust, without vitrification or yellowing, and the propagation coefficient was 5.52–6.14, achieving good propagation. This indicates that the propagation culture of *Raspberry palmatum* has strict requirements on the culture time, and the suitable propagation culture time is 15–20 days.

[0200] Compared with existing technologies, the proliferation culture medium of the present invention has a simple composition, using only one plant growth regulator. Moreover, the shoots cultured by the present invention grow vigorously, without vitrification or yellowing, and with tiny callus tissue attached to the base, showing better proliferation culture effect.

[0201] 4. Rooting Culture Experiment

[0202] Nine rooting culture media of the present invention were used to conduct rooting culture experiments on Raspberry palmatum. The results (Table 4) show that after rooting culture, the buds reached 100% maturity and 100% rooting rate. The plant height was 2.41-2.58 cm, the root mass was 2.00-2.18 cm, the root length was 4.92-5.55 cm, and the root diameter was 0.40-0.54 mm. The roots emerged from the base without callus tissue. The plants showed good growth (robust, green, uniform, without vitrification or yellowing), demonstrating good rooting culture effect.

[0203] Compared with the prior art, the rooting culture medium of the present invention has a simple composition and does not use any plant growth regulators. Moreover, the plants cultivated by the technology of the present invention show better overall growth in terms of maturity rate, rooting rate, plant height, root quantity, root length, root thickness, and growth vigor. In addition, the quality of root development is better: roots emerge from the base and there is no callus tissue at the base.

[0204] Table 4. Rooting Culture Effect Experiment of Raspberry Palmarosa

[0205]

[0206] Note: AC in the table is short for activated carbon.

[0207] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid tissue culture propagation of Raspberry palmatum, characterized in that, Includes the following steps: A. Explant collection: During the growing season of Raspberry palmatum, select new, semi-lignified branches with undeveloped axillary buds, vigorous growth, and free from pests and diseases on a sunny midday. After cutting the branches, seal them for preservation and bring them back to the tissue culture room for processing within 2.5 to 3.0 hours. B. Sterilization of explants: Cut the branches from step A into stem segments of 0.8-1.0 cm in length, with each stem segment having a petiole of 0.1-0.2 cm in length attached; soak the stem segments in a 1.5%-2.5% (v / v) detergent solution for 1.0-2.0 min, then rinse the stem segments with sterile water 2-3 times, and finally sterilize the stem segments; C. Induction Culture: The stem segments sterilized in step B are inoculated into the induction culture medium and cultured. When strong buds are cultivated, the induction culture is completed. The induction medium is: MS basal medium + 6-benzylaminopurine 0.5-0.8 mg / L + naphthaleneacetic acid 0.01-0.05 mg / L + citric acid 300-400 mg / L + white sugar 25-30 g / L + agar powder 5.5-5.8 g / L, pH 5.8-6.0; Alternatively, the induction medium can be: basal medium WPM + 6-benzylaminopurine 0.5–0.8 mg / L + naphthaleneacetic acid 0.01–0.05 mg / L + citric acid 300–400 mg / L + granulated sugar 25–30 g / L + agar powder 5.5–5.8 g / L, pH 5.8–6.0; D. Proliferation Culture: The buds obtained in step C are cut and separated into individual buds at their connection with the stem segment; the individual buds are inoculated into the proliferation medium to cultivate robust cluster buds, and then individual buds are separated from the cluster buds and inoculated into the proliferation medium again. This process of subculture and proliferation culture is repeated. The temperature of the proliferation culture room is 25℃±2℃, the relative humidity is 30%~45%, the light intensity is 2500Lx~3000Lx, and the light duration is 12~14h / d. The proliferation medium is: modified MS basal medium + 0.5-1.0 mg / L 6-benzylaminopurine + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0; the modified MS has KNO3, NH4NO3, and KH2PO4 contents of 2200 mg / L, 1400 mg / L, and 380 mg / L, respectively, and the concentrations of other components are kept consistent with those of the basal MS medium. Alternatively, the proliferation medium could be: MS basal medium + 0.5–1.0 mg / L 6-benzylaminopurine + 25–30 g / L sucrose + 5.5–5.8 g / L agar powder, pH 5.8–6.0; E. Rooting culture: The cluster of buds obtained in step D is divided into individual buds. Individual buds with a length of 1.5 to 2.1 cm are selected and inoculated into rooting culture medium to obtain seedlings. The rooting culture room temperature was 25℃±2℃, relative humidity was 30%~45%, light intensity was 2500Lx~3000Lx, light duration was 12~14h / d, and culture time was 35~50d; The rooting medium is: 1 / 2 MS basal medium + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0; Alternatively, the rooting medium can be: 1 / 2 MS basal medium + 0.1wt%–0.2wt% activated carbon + 25–30 g / L granulated sugar + 5.5–5.8 g / L agar powder, pH 5.8–6.0; Alternatively, the rooting medium can be: MS basal medium + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0; Alternatively, the rooting medium can be: MS basal medium + 0.1wt%–0.2wt% activated carbon + 25–30 g / L granulated sugar + 5.5–5.8 g / L agar powder, pH 5.8–6.0; Alternatively, the rooting medium can be: basal medium WPM + 25-30 g / L granulated sugar + 5.5-5.8 g / L agar powder, pH 5.8-6.0; Alternatively, the rooting medium can be: basal medium WPM + activated carbon 0.1wt%~0.2wt% + white sugar 25~30g / L + agar powder 5.5~5.8g / L, pH 5.8~6.0; In step A, the explant collection targets are: New shoots that are 45-60 days old on the main stem or lateral branches of Raspberry palmatum; In step C, the specific method for stem segment culture is as follows: First, the buds were cultured in the dark for 8-10 days at a temperature of 25±2℃ and a relative humidity of 30%-45% to induce 1-2 buds with a length of 0.5-0.8cm. Then, they were cultured in an environment with a temperature of 25±2℃, a relative humidity of 30%-45%, a light intensity of 2500-3000Lx, and a light duration of 12-14h / d for 15-20 days to cultivate buds with a length of 1.5-2.0cm.

2. The method for rapid tissue culture propagation of Raspberry palmatum according to claim 1, characterized in that, In step B, the specific method for sterilizing the stem segments is as follows: The stem segments are classified into three categories based on their stem thickness: thin stem segments, medium-thickness stem segments, and thick stem segments. The thin stem segments have a stem diameter greater than or equal to 2.55 mm and less than or equal to 3.10 mm; the medium-thick stem segments have a stem diameter greater than 3.10 mm and less than or equal to 3.66 mm; and the thick stem segments have a stem diameter greater than 3.66 mm and less than or equal to 4.22 mm. When sterilizing stem segments on a clean bench, first soak them in 75% alcohol for 30-40 seconds. Then, soak the thin stem segments in a 0.2% mercuric chloride solution containing Tween-20 for 4-5 minutes, the medium-thick stem segments for 6-7 minutes, and the thick stem segments for 8-9 minutes. Next, soak and rinse them in sterile water for 5-7 minutes. Finally, use sterile filter paper to absorb the surface moisture of the stem segments. Add 1-2 drops of Tween-20 to 150-200 mL of a 0.2% mercuric chloride solution and mix well to form a 0.2% mercuric chloride solution containing Tween-20.

3. The method for rapid tissue culture propagation of *Raspberry palmatum* according to claim 1, characterized in that, In step C, during dark incubation, the induction medium should be changed daily from day 2 to day 5.

4. The method for rapid tissue culture propagation of *Raspberry palmatum* according to claim 1, characterized in that, In step D, the number of subcultures is controlled between 15 and 20; during subculture, proliferation media composed of different concentrations of 6-benzylaminopurine and different types of basal media are used alternately.

5. The method for rapid tissue culture propagation of *Raspberry palmatum* according to claim 1, characterized in that, In step D, the culture time for proliferation culture is 15-20 days; first, all the callus tissue at the base of the bud cluster is removed, and then the bud cluster is separated into individual buds.

6. The method for rapid tissue culture propagation of *Raspberry palmatum* according to claim 1, characterized in that, In step E, single buds shorter than 1.5 cm are cultured for proliferation according to step D.

7. The method for rapid tissue culture propagation of Raspberry palmatum according to claim 1, characterized in that, In step E, the specific method for rooting culture is as follows: Remove 2-4 petioles from the stem at the base of each bud, leaving 2-3 leaves at the top of the stem; inoculate the bud into the rooting medium at a depth of 0.3-0.4 cm.

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