A method for efficiently breeding saxifraga by creeping stem

Abstract

This invention provides a method for efficiently propagating Saxifraga stolonifera using stolons, comprising at least the following steps: using stolons of Saxifraga stolonifera with buds as explants, and subjecting the explants to sequential culture treatments including callus induction culture and adventitious bud induction culture to obtain Saxifraga stolonifera transplanted seedlings; wherein the induction culture medium used for callus induction culture includes one of MS+ZT+2,4-D+NAA, MS+2,4-D+NAA, MS+ZT+NAA, and MS+ZT+KT+2,4-D+NAA; and the adventitious bud induction culture medium used for adventitious bud induction culture includes one of MS+ZT+TDZ+IBA, MS+TDZ+IBA, MS+ZT+IBA, and MS+ZT+TDZ+IBA+GA3. This method does not harm the Saxifraga stolonifera parent plant and has a short propagation cycle and high genetic stability.

Description

Technical Field

[0001] This invention relates to a method for efficiently propagating Saxifraga stolonifera using stolons, belonging to the field of biotechnology. Background Technology

[0002] Saxifraga stolonifera, commonly known as tiger's ear grass, is a perennial herb belonging to the genus Saxifraga in the family Saxifragaceae. It is also known by other names such as Tong'er grass, Jinsi heye, Tianheye, Laohu'er, Jinxiandiao furong, Jinxianlian, Shidanyao, Fo'er grass, Tongqian grass, and Daochui lian. The plant grows to a height of 8-45 cm, with slender, slender stolons, red stems, and green leaves. The leaves are nearly heart-shaped, kidney-shaped, to flattened round, with white markings and glandular hairs on the surface, resembling the round ears of a tiger, hence the name tiger's ear grass. It flowers from May to August and fruits from July to November. It is mainly distributed in Hebei, Jiangsu, Anhui, Fujian, Hubei, Guangdong, and Taiwan provinces. It grows in forests, thickets, meadows, and shady, damp rock crevices at altitudes of 400-4500 meters. The optimal growth temperature is 15-25 degrees Celsius, and it can tolerate temperatures as low as 5 degrees Celsius. Saxifraga stolonifera is an important and distinctive traditional Chinese medicine. Its creeping stems contain bergenin, quercetin, gallic acid, protocatechuic acid, succinic acid, and methylfumaric acid; the stems contain catechins; and the roots contain volatile oils. Besides its anti-tumor and antibacterial effects, it is also used to treat lung heat cough, thick yellow phlegm, lung abscesses, itchy rashes, hemorrhoids, insect bites, blistering, necrosis, otitis media, urticaria, eczema, menorrhagia due to blood heat in women, hematemesis, external bleeding, burns, mumps, pharyngitis, and oral ulcers. Due to its significant medicinal value, dried Saxifraga stolonifera sells for around 40 yuan per kilogram, while stoxin can fetch as much as 700 yuan per kilogram. In addition to its medicinal value, Saxifraga stolonifera is also a popular choice for bonsai decoration and ground cover in gardens, with individual plants selling for 9 to 20 yuan, and new varieties reaching over 120 yuan. Therefore, exploring efficient methods for propagating tiger tail grass has significant social and economic value.

[0003] Traditional propagation methods for Saxifraga stolonifera mainly include division and layering. However, both methods suffer from low propagation coefficients and slow propagation speeds. To address these issues, researchers explored a series of rapid tissue culture propagation methods for Saxifraga stolonifera. The academic paper "Saxifraga stolonifera and its Tissue Culture Rapid Propagation Technology" describes the use of stem segments from mature Saxifraga stolonifera plants as explants for rapid tissue culture propagation.

[0004] However, the above-mentioned propagation methods have disadvantages such as damaging the parent plant of Saxifraga stolonifera, having a long propagation cycle, and low genetic stability. Summary of the Invention

[0005] This invention provides a method for efficiently propagating Saxifraga stolonifera using stolons. This method causes less damage to the Saxifraga stolonifera parent plant, has a shorter propagation cycle, and exhibits high genetic stability.

[0006] This invention provides a method for efficiently propagating Saxifraga stolonifera using stolons, comprising at least the following steps:

[0007] Saxifraga stolons with buds were used as explants, and the explants were subjected to culture treatments including callus induction culture and adventitious bud induction culture in sequence to obtain Saxifraga stolon transplant seedlings.

[0008] The induction medium used for the callus induction culture includes one of the following: MS + 0.2-0.6 mg / L ZT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA, MS + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA, MS + 1.1-1.5 mg / L ZT + 0.5-1.0 mg / L NAA, or MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA.

[0009] The adventitious bud induction culture medium used includes one of the following: MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA, MS + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA, MS + 0.05-0.1 mg / L ZT + 1.5-3.0 mg / L IBA, MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

[0010] As described above, the induction medium comprises MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA.

[0011] As described above, the adventitious bud induction medium comprises MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

[0012] As described above, the explant has one bud point.

[0013] The method described above further includes a disinfection process before the callus induction culture, which includes the following steps:

[0014] The explants were soaked in a laundry detergent solution for 5 minutes, then rinsed with clean water for 45-60 minutes. After that, they were soaked in 75% medical alcohol for 30 seconds, rinsed with sterile water, and then soaked in 0.1% mercuric chloride solution with 3-5 drops of Tween 80 for 5-10 minutes. The resulting explants were then rinsed to obtain sterilized explants.

[0015] The callus induction culture, as described above, includes the following steps:

[0016] The sterilized explants were inoculated onto the induction medium. The induction medium was placed in the dark for cultivation from day 1 to day 7. From day 8 onwards, the explants were cultured under fluorescent lamps with a light intensity of 2500-3000 lx and a light duration of 12 h / d to obtain callus tissue with a length of 0.5-1.0 cm and a width of 0.5-1.0 cm.

[0017] The culture temperature for inducing callus tissue is 23-27℃.

[0018] The method described above further includes callus proliferation culture between the callus induction culture and the adventitious shoot induction culture, and adventitious shoot proliferation culture is also included after the adventitious shoot induction culture.

[0019] The proliferation medium used for the callus proliferation culture includes MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA;

[0020] The adventitious bud proliferation culture medium used in the adventitious bud proliferation culture includes MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

[0021] The method described above further includes seedling rooting culture after the adventitious bud proliferation culture.

[0022] The seedling rooting culture medium used in the seedling rooting culture includes MS + 0.5 mg / L IBA + 1 g / L Ac.

[0023] The method described above further includes a hardening-off culture after the seedling rooting culture, which includes the following steps:

[0024] After the seedling rooting culture, the seedlings with no less than 6 leaves and more than 20 roots obtained are transferred to the hardening environment for 3 days. Then the culture device is opened and the seedlings are cultured in the hardening environment for another 2 days.

[0025] The seedling hardening environment is characterized by a daytime temperature of 21℃~25℃, a nighttime temperature of 15℃~18℃, and an ambient humidity of 60%~80%.

[0026] The method described above further includes transplanting culture after the seedling hardening-off culture, and the transplanting culture includes:

[0027] After hardening off, the rooted seedlings were washed and sterilized in sequence, and then the rooted seedlings were transferred to seedling pots containing substrate to cultivate the Saxifraga stolonifera transplant seedlings with more than 9 leaves.

[0028] The matrix is ​​prepared by mixing perlite, peat, and vermiculite in a volume ratio of 1:2:1.

[0029] This invention provides a method for the efficient propagation of Saxifraga stolonifera using its runners. Using runners as explants, the explants undergo sequential culture treatments, including callus induction culture and adventitious bud induction culture, in a specific culture medium to obtain transplanted Saxifraga stolonifera seedlings. This method not only significantly reduces damage to the Saxifraga stolonifera parent plant but also significantly shortens the propagation cycle and improves genetic stability. Attached Figure Description

[0030] Figure 1 Image of the parent plant of Saxifraga stolonifera used in this invention;

[0031] Figure 2 Images of callus tissue induced in Example 1 of this invention;

[0032] Figure 3 Images of adventitious bud clusters induced in Example 1 of this invention;

[0033] Figure 4 This is an image of the proliferated adventitious bud clusters obtained through adventitious bud proliferation culture in Example 1 of the present invention;

[0034] Figure 5 This is a scene diagram of seedling rooting culture in Embodiment 1 of the present invention;

[0035] Figure 6 This is a picture of the rooted seedlings obtained after 15 days of robust seedling rooting culture in Example 1 of the present invention;

[0036] Figure 7 This is a picture of the rooted seedlings obtained after 30 days of robust seedling rooting culture in Example 1 of the present invention;

[0037] Figure 8 This is a diagram of the seedling hardening and cultivation scenario in Embodiment 1 of the present invention;

[0038] Figure 9 This is a scene diagram illustrating the cleaning process of rooted seedlings after hardening-off cultivation in Embodiment 1 of the present invention;

[0039] Figure 10 This is a grading diagram of rooted seedlings in Embodiment 1 of the present invention;

[0040] Figure 11 These are growth diagrams of rooted seedlings of different grades after 7 days of transplanting and cultivation in Example 1 of this invention;

[0041] Figure 12 These are growth diagrams of rooted seedlings of different grades after 21 days of transplanting and cultivation in Example 1 of this invention;

[0042] Figure 13 These are growth diagrams of rooted seedlings of different grades after 45 days of transplanting and cultivation in Example 1 of this invention;

[0043] Figure 14 This is a landscape application diagram of the Saxifraga stolonifera transplanted seedlings obtained in Example 1 of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0045] This invention provides a method for efficiently propagating Saxifraga stolonifera using stolons, comprising at least the following steps:

[0046] Saxifraga stolons with buds were used as explants, and the explants were subjected to culture treatments including callus induction culture and adventitious bud induction culture in sequence to obtain Saxifraga stolon transplant seedlings.

[0047] The induction medium used for callus induction culture includes one of the following: MS + 0.2-0.6 mg / L ZT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA, MS + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA, MS + 1.1-1.5 mg / L ZT + 0.5-1.0 mg / L NAA, or MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA.

[0048] The adventitious bud induction culture medium used includes one of the following: MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA, MS + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA, MS + 0.05-0.1 mg / L ZT + 1.5-3.0 mg / L IBA, MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

[0049] The method provided by this invention establishes a highly efficient propagation system that uses the stolons of Saxifraga stolonifera as explants, induces callus formation from the explants, and then induces adventitious buds from the callus. In this system, Saxifraga stolonifera is rapidly propagated through a specific culture medium, which shortens the propagation cycle and improves the genetic stability of the efficient propagation of Saxifraga stolonifera.

[0050] In detail, the creeping stems of Saxifraga stolonifera with buds were cut using sterile instruments as explants. The explants were then inoculated onto an induction medium for induction culture to obtain callus tissue. Subsequently, the callus tissue was inoculated onto an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious bud clusters.

[0051] This invention does not limit the specific growth time of Saxifraga stolons. To reduce the contamination rate of the stolons used as explants, relatively young stolons can be selected as explants.

[0052] In one specific embodiment, the stolons of 1-2 month old Saxifraga stolonifera were selected as explants.

[0053] This invention does not limit the specific length of the stolons; a suitable length can be selected based on the actual cultivation process.

[0054] In one specific embodiment, 2-4 cm stolons are selected as explants.

[0055] It is understood that the cultivation treatment also includes seedling strengthening, rooting, and transplanting, until the *Saxifraga stolonifera* transplanted seedlings are obtained. This invention does not limit the specific steps of seedling strengthening, rooting, and transplanting; methods commonly used in the art can be selected to perform these treatments on the adventitious bud clusters.

[0056] The MS medium formulation of the present invention is disclosed. The MS medium includes inorganic salts, organic substances, vitamins, etc., which can effectively promote the division and growth of plant cells. In the present invention, ZT refers to zeatin, KT refers to kinetin, NAA refers to naphthaleneacetic acid, 2,4-D refers to dichlorophenoxyacetic acid, TDZ refers to phenylthiazolylurea, IBA refers to indolebutyric acid, and GA3 refers to gibberellin.

[0057] To ensure sufficient carbon source for each culture medium and improve its support, the culture medium of the present invention may also include sucrose and agar. The present invention does not limit the specific content of sucrose and agar, and the content of sucrose and agar can be selected according to actual needs.

[0058] In one specific embodiment, the culture medium contains 30g of sucrose and 7g of agar.

[0059] This invention does not limit the pH value of the culture medium; a suitable pH range can be selected according to the actual situation, such as pH 5.5-6.0.

[0060] The present invention does not limit the method of adjusting the pH of the culture medium. For example, before the culture medium is completely dissolved and dispensed, the initial pH value of the culture medium is first measured with pH test paper or pH meter, and then 1 mol / L HCl or NaOH is added dropwise to the culture medium according to the test value to adjust the pH value of the solution to 5.5-6.0.

[0061] This invention does not limit the culture conditions for adventitious bud induction culture; appropriate culture conditions can be selected according to actual conditions.

[0062] In one specific embodiment, during the adventitious bud induction culture, the light intensity was 2500-3000 lx, the culture temperature was 23-27℃, and the light duration was 12h / d.

[0063] The method provided by this invention uses the stolons of Saxifraga stolonifera as explants. The explants are subjected to culture treatments including callus induction culture and adventitious bud induction culture in a specific culture medium. Under the synergistic effect of the various components in the culture medium, adventitious buds are induced by callus to achieve efficient propagation of Saxifraga stolonifera, shortening the propagation cycle of Saxifraga stolonifera and improving the genetic stability of efficient propagation of Saxifraga stolonifera.

[0064] Furthermore, in a specific embodiment of the present invention, the induction culture medium comprises MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA.

[0065] In detail, the induction medium includes, but is not limited to, MS + 0.01 g / L ZT + 0.6 mg / L KT + 3.0 mg / L 2,4-D + 0.5 mg / L NAA, MS + 0.2 g / L ZT + 0.7 mg / L KT + 4.0 mg / L 2,4-D + 0.8 mg / L NAA, MS + 0.03 g / L ZT + 0.8 mg / L KT + 5.0 mg / L 2,4-D + 1.0 mg / L NAA, or other induction media that meet the above component content ranges.

[0066] Through extensive and innovative experiments, the inventors discovered that when the composition of each component of the induction medium meets the above-mentioned range, the callus induction rate can be significantly improved, thereby increasing the propagation coefficient during the breeding process. This is because, in addition to MS as the basal medium, the specific amounts of ZT, KT, NAA, and 2,4-D in the above-mentioned induction medium work synergistically to efficiently induce callus formation at the bud point from explants.

[0067] Furthermore, in a specific embodiment of the present invention, the adventitious bud induction culture medium comprises MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

[0068] In detail, the adventitious bud induction medium includes, but is not limited to, MS + 0.05 mg / L ZT + 0.1 mg / L TDZ + 1.5 mg / L IBA + 0.2 g / L GA3, MS + 0.06 mg / L ZT + 0.2 mg / L TDZ + 2.0 mg / L IBA + 0.3 g / L GA3, MS + 0.08 mg / L ZT + 0.3 mg / L TDZ + 2.5 mg / L IBA + 0.4 g / L GA3, MS + 0.9 mg / L ZT + 0.4 mg / L TDZ + 3.0 mg / L IBA + 0.4 g / L GA3, or any other adventitious bud induction medium within the above range.

[0069] When the composition of each component of the adventitious bud induction medium meets the above range, the adventitious bud induction medium can accurately simulate the plant auxin signals in the plant growth process, promote the differentiation and regeneration of callus tissue, induce the growth of adventitious buds from callus tissue, and lay the foundation for the efficient propagation of Saxifraga stolonifera.

[0070] Furthermore, in one specific embodiment of the present invention, the explant has one bud point.

[0071] Understandably, if the collected Saxifraga stolon has multiple buds, sterile instruments can be used to cut the Saxifraga stolon into explants of appropriate length with one bud.

[0072] Through extensive and innovative experiments, the inventors discovered that when an explant has one bud, the induction rate is no less than 97.8%. This is because explants with a single bud have a more concentrated nutrient supply than those with multiple buds, resulting in a greater quantity and higher quality of induced callus. In contrast, explants with multiple buds experience an uneven nutrient supply, leading to inconsistent quantity and quality of induced callus.

[0073] Furthermore, in one specific embodiment of the present invention, the callus induction culture is further subjected to a disinfection process, which includes the following steps:

[0074] Soak the explants in a laundry detergent solution for 5 minutes, then rinse with clean water for 45-60 minutes. Next, soak them in 75% medical alcohol for 30 seconds, rinse with sterile water, and then soak them in a 0.1% mercuric chloride solution with 3-5 drops of Tween 80 for 5-10 minutes. Rinse to obtain the sterilized explants.

[0075] In one specific embodiment, the disinfection process includes the following steps: Wrap the appropriately cut Saxifraga stolons in gauze and soak them in a beaker containing laundry detergent for 5 minutes, then rinse with clean water for 45-60 minutes. Place the rinsed Saxifraga stolons on a clean bench and soak them in 75% medical alcohol for 30 seconds. After discarding the alcohol solution, immediately rinse once with sterile water. Then, use sterile instruments to transfer the Saxifraga stolons to a beaker containing 0.1% mercuric chloride solution, and use a dropper to add 3-5 drops of Tween 80 for soaking for 5-10 minutes. Afterward, remove the Saxifraga stolons and rinse them 3-5 times with sterile water. Finally, use sterile filter paper to absorb the moisture from the stolons, obtaining the disinfected explant.

[0076] The soaking time of the present invention is 5-10 min. More specifically, the soaking time includes, but is not limited to, a range of 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or any combination thereof.

[0077] It is understandable that the reagents used in the disinfection process may damage the cut tissue of the Saxifraga stolon, causing the cut tissue to be damaged and discolored. This damaged and discolored tissue may cause secondary contamination of the Saxifraga stolon. Therefore, in order to reduce the risk of secondary contamination and improve the survival rate of explants, the damaged and discolored parts of the explants caused by disinfection can be removed.

[0078] The present invention does not limit the specific operation of removing the damaged and discolored part. Common aseptic removal methods in the art can be used to remove the damaged and discolored part of the explant.

[0079] The above disinfection process ensures that the explants undergoing callus induction culture are in a sterile state, effectively preventing contamination by bacteria or other microorganisms during the callus induction culture process, and improving the survival rate and induction rate of explant callus induction culture.

[0080] Furthermore, in one specific embodiment of the present invention, the callus induction culture includes the following steps:

[0081] The sterilized explants were inoculated onto the induction medium. The induction medium was placed in the dark for cultivation from day 1 to day 7. From day 8 onwards, the explants were cultured under fluorescent lamps with a light intensity of 2500-3000 lx and a light duration of 12 h / d to obtain callus tissue with a length of 0.5-1.0 cm and a width of 0.5-1.0 cm.

[0082] The culture temperature for callus induction culture is 23-27℃.

[0083] In detail, the sterilized explants were inoculated onto the induction medium. For the first 7 days, the induction medium was placed in the dark for culture, which helped the explants adapt to the new environment and start growth. Then, from the 8th day, they were cultured under fluorescent lights, with the light intensity and time controlled, to induce callus tissue with a length of 0.5-1.0 cm and a width of 0.5-1.0 cm.

[0084] The light intensity of the present invention is 2500-3000 lx. More specifically, the light intensity includes, but is not limited to, a range of 2500 lx, 2600 lx, 2700 lx, 2800 lx, 2900 lx, 3000 lx, or any combination thereof.

[0085] The culture temperature of this invention is 23-27℃. More specifically, the culture temperature includes, but is not limited to, 23℃, 23.5℃, 24℃, 24.5℃, 25℃, 25.5℃, 26℃, 26.5℃, and 27℃.

[0086] The combination of dark and fluorescent culture methods for callus induction can efficiently and rapidly induce the formation of callus of appropriate length and width from explants. Dark culture helps reduce the stress and damage caused by light stimulation to explants, allowing them to initiate the callus formation process in a more stable environment and promoting cell proliferation and differentiation. Fluorescent culture, on the other hand, simulates natural light conditions, promoting normal callus growth.

[0087] Furthermore, in a specific embodiment of the present invention, the callus induction culture and the adventitious shoot induction culture are further separated by callus proliferation culture, and the adventitious shoot induction culture is further separated by adventitious shoot proliferation culture.

[0088] The proliferation medium used for callus proliferation culture included MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA;

[0089] The adventitious bud proliferation culture medium used includes MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

[0090] In detail, after callus induction culture, the obtained callus is inoculated onto a proliferation medium for callus proliferation culture to obtain proliferated callus.

[0091] In one specific embodiment, the explant with callus tissue is removed from the culture bottle, and the parts on both sides of the explant that have not formed callus tissue are cut off using sterile instruments. The callus tissue of appropriate size is then inoculated onto the proliferation culture medium for proliferation culture to obtain proliferated callus tissue. The planar area of ​​the proliferated callus tissue is 3 times that of the callus tissue before proliferation.

[0092] In this invention, the planar area refers to the area viewed from above when the callus tissue is placed naturally.

[0093] The proliferation medium of the present invention includes MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA. For example, the proliferation medium includes, but is not limited to, MS + 0.01 g / L ZT + 0.6 mg / L KT + 3.0 mg / L 2,4-D + 0.5 mg / L NAA, MS + 0.02 g / L ZT + 0.7 mg / L KT + 4.0 mg / L 2,4-D + 0.8 mg / L NAA, MS + 0.03 g / L ZT + 0.8 mg / L KT + 5.0 mg / L 2,4-D + 1.0 mg / L NAA, or any other proliferation medium that conforms to the above range.

[0094] This invention does not limit the culture conditions for callus proliferation culture. Appropriate culture conditions can be selected according to actual conditions. For example, the light intensity is 2500-3000 lx, the culture temperature is 23-27℃, and the light time is 12h / d.

[0095] The proliferating callus tissue was inoculated onto an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious bud clusters.

[0096] In one specific embodiment, the proliferating callus tissue is cut into cubes with a planar area of ​​1.0cm × 1.0cm using sterile instruments, and the cubes are inoculated onto adventitious bud induction medium for adventitious bud induction culture to obtain adventitious bud clusters.

[0097] The adventitious bud clusters obtained through adventitious bud induction culture were transferred to adventitious bud proliferation medium for adventitious bud proliferation culture, resulting in proliferated adventitious bud clusters.

[0098] In one specific embodiment, the adventitious bud clusters are removed from the culture flask using sterile instruments and cut into blocks with a planar area of ​​1.5cm × 1.5cm. The blocks of adventitious bud clusters are then transferred to an adventitious bud proliferation medium for adventitious bud proliferation culture, resulting in proliferated adventitious bud clusters. The planar area of ​​the proliferated adventitious bud clusters is 18.8 times that of the adventitious bud clusters before proliferation.

[0099] It is understandable that by extending the culture time, adventitious bud clusters with a greater difference in area ratio before and after proliferation can be obtained.

[0100] The adventitious bud proliferation medium in this invention is MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3. For example, the adventitious bud proliferation medium includes, but is not limited to, MS + 0.05 mg / L ZT + 0.1 mg / L TDZ + 1.5 mg / L IBA + 0.2 g / L GA3, MS + 0.08 mg / L ZT + 0.2 mg / L TDZ + 2.0 mg / L IBA + 0.3 g / L GA3, MS + 0.1 mg / L ZT + 0.4 mg / L TDZ + 3.0 mg / L IBA + 0.4 g / L GA3, or any other adventitious bud proliferation medium that meets the above range.

[0101] This invention does not limit the culture conditions for the adventitious bud proliferation culture process. Appropriate culture conditions can be selected according to the actual situation. For example, the light intensity is 2500-3000 lx, the culture temperature is 23-27℃, and the light time is 12h / d.

[0102] By selecting specific proliferation media and adventitious bud proliferation media, the proliferation culture of callus tissue and adventitious bud clusters was achieved, increasing the proliferation coefficient and contributing to the efficient propagation of Saxifraga stolonifera.

[0103] Furthermore, in one specific embodiment of the present invention, the adventitious bud proliferation culture further includes a seedling rooting culture.

[0104] The rooting medium used for strong seedlings includes MS + 0.5 mg / L IBA + 1 g / L Ac.

[0105] In detail, after adventitious bud proliferation culture, proliferated adventitious bud blocks are obtained. Using sterile instruments, individual adventitious buds are cut from the base and transferred to a seedling rooting culture medium for seedling rooting culture, resulting in robust rooted seedlings with a large number of leaves and complete root growth.

[0106] This invention does not limit the cultivation conditions for the seedling rooting process. Appropriate cultivation conditions can be selected according to the actual situation, such as light intensity of 2500-3000 lx, cultivation temperature of 23-27℃, and light duration of 12h / d.

[0107] The seedling rooting culture medium selected in this invention can simultaneously carry out seedling strengthening and rooting culture under the synergistic effect of various components, which significantly improves the culture efficiency and shortens the culture cycle. Moreover, after the above seedling rooting culture, the adventitious bud rooting rate is as high as 100%, and the resulting rooted seedlings have more roots, more leaves, and vigorous growth.

[0108] Furthermore, in one specific embodiment of the present invention, the seedling rooting culture further includes a hardening-off culture, which includes the following steps:

[0109] After the seedlings have been rooted and grown, the culture device containing seedlings with no less than 6 leaves and more than 20 roots is transferred to the hardening environment for 3 days. Then the culture device is opened and the seedlings are cultured in the hardening environment for another 2 days.

[0110] The seedling hardening environment includes a daytime temperature of 21℃~25℃, a nighttime temperature of 15℃~18℃, and an ambient humidity of 60%~80%.

[0111] Specifically, daytime temperatures include, but are not limited to, a range of 21°C, 22°C, 23°C, 24°C, 25°C, or any combination thereof.

[0112] Nighttime temperatures include, but are not limited to, a range of 15°C, 15.5°C, 16°C, 16.5°C, 17°C, 17.5°C, 18°C, or any combination thereof.

[0113] Ambient humidity includes, but is not limited to, a range of 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, or any combination thereof.

[0114] It is understandable that, in order to prevent seedlings from wilting due to lack of water, the culture device can be sprayed with a 150ml spray bottle from time to time after it is turned on.

[0115] Through the above-mentioned hardening-off cultivation, the rooted seedlings can better adapt to adverse natural environments, improve plant quality, and increase the survival rate after transplanting.

[0116] Furthermore, in one specific embodiment of the present invention, the seedling hardening-off cultivation further includes transplanting cultivation, which includes:

[0117] After hardening off, the rooted seedlings were cleaned and sterilized in sequence. Then, the rooted seedlings were transferred to seedling trays containing substrate to cultivate Saxifraga stolonifera transplant seedlings with more than 9 leaves.

[0118] The matrix is ​​prepared by mixing perlite, peat, and vermiculite in a volume ratio of 1:2:1.

[0119] This invention does not limit the specific operation of cleaning the rooted seedlings, as long as the culture medium substances adhering to the rooted seedlings can be washed away.

[0120] In one specific embodiment, the rooted seedlings after hardening are removed from the strong seedling rooting culture medium, placed in clean water, and the culture medium material adhering to the rooted seedlings is gently washed off with a brush.

[0121] This invention does not limit the specific operation of sterilization treatment, as long as it can reduce the contamination rate of rooted seedlings.

[0122] In one specific embodiment, the cleaned rooted seedlings are soaked in a 0.3% carbendazim solution for 1 hour, and then the rooted seedlings are taken out and placed on waste newspapers that have been laid on the ground for 1-2 hours to absorb the moisture from the leaves and stems.

[0123] This invention does not limit the cultivation conditions for transplanting. Appropriate transplanting cultivation conditions can be selected according to actual conditions, such as light intensity of 2500-3000 lx, cultivation temperature of 23-27℃, and light duration of 12h / d.

[0124] It is understandable that when transferring sterilized rooted seedlings to seedling pots for cultivation, it is important to maintain good soil and environmental humidity, such as daytime temperature of 21℃~25℃, nighttime temperature of 15℃~18℃, and humidity of 60%~80%. Transplanting allows the rooted Saxifraga seedlings to better adapt to the soil environment and further promotes root growth. This is beneficial for Saxifraga seedlings to establish a healthy root system, improve their stress resistance and growth rate, and ensures that the seedlings have more than 9 leaves, which can further improve the survival rate of Saxifraga seedlings in the natural environment.

[0125] The following detailed description of the method for efficiently propagating Saxifraga stolonifera using stolons, provided by the present invention, is illustrated through specific embodiments.

[0126] The *Saxifraga stolonifera* of this invention was obtained from the planting resource nursery of the Yangtze Rare Plant Research Institute of China Three Gorges Corporation, located in Yichang City, Hubei Province, near the Three Gorges Dam. The mother plant of *Saxifraga stolonifera* was in the following growth state: Figure 1 As shown.

[0127] Example 1

[0128] 1. Pretreatment and disinfection of Saxifraga stolonifera stolons

[0129] Tender stolons of 1-month-old Saxifraga stolonifera were selected from the plant resource nursery. 2cm segments with one, two, and no buds were cut from the base of the stolons and used as explants. Each explant was wrapped in gauze and soaked in a beaker containing laundry detergent for 5 minutes, followed by rinsing with running water for 50 minutes. The explants were then placed on a clean bench and soaked in 75% medical alcohol for 30 seconds. After discarding the alcohol solution, they were immediately rinsed once with sterile water. The explants were then transferred to a 0.1% mercuric chloride solution with 5 drops of Tween 80 and soaked for 6 minutes, followed by rinsing with sterile water 5 times. Finally, the water in the stolons was absorbed with sterile filter paper, and damaged or discolored parts were removed using sterile scissors.

[0130] 2. Callus induction culture and callus proliferation culture

[0131] 1) The effect of the number of buds on induction culture

[0132] Using the pretreatment and disinfection methods described above, 30 explants with different numbers of buds were treated in each group, repeated 3 times, resulting in 90 samples per group. The explants with different numbers of buds were inoculated into sterilized induction medium, which included MS medium, 0.02 g / L ZT, 0.7 mg / L KT, 4.0 mg / L 2,4-D, 0.8 mg / L NAA, 30 g sucrose, and 7 g agar, with a pH of 6.0. The culture was carried out in the dark for the first 7 days, followed by daylight culture at a light intensity of 2500-3000 lx, a temperature of 25℃, and a photoperiod of 12 h / d for 30 days, yielding explants with callus tissue. (Details are as follows...) Figure 2 As shown.

[0133] The callus induction rate of explants with different numbers of buds was statistically analyzed. The number of contaminations of explants with different numbers of buds was detected by intuitive statistical method, and the contamination rate was calculated. See Table 1 for details.

[0134] Table 1

[0135]

[0136] It can be seen that, under the same induction medium, the germination rate of explants with a certain number of buds is no less than 92.2%, while the germination rate of explants with one bud can reach 97.8%.

[0137] 2) Effects of different induction media on induction culture

[0138] Using the above pretreatment and disinfection methods, 450 explants with one bud were treated and used as samples. The explants were then inoculated onto 15 sterilized induction media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3, with 30 samples treated per induction medium.

[0139] The culture conditions for each sample were the same: dark culture for the first 7 days, and then daylight culture for the next 8 days, with a light intensity of 2500-3000 lx, a culture temperature of 23-25℃, and a light exposure time of 12 h / d.

[0140] The specific composition of the 15 induction media is shown in Table 2. All 15 induction media included MS medium, 30g sucrose, and 7g agar. The pH of the 15 induction media was 5.5-6.0.

[0141] After 25 days of culture, explants with callus tissue were obtained. The number and induction rate of explants induced by different induction media were statistically analyzed. The number of explants induced was the number of explants with callus tissue, and the induction rate was the ratio of the number of explants with callus tissue to the initial number of explants. The specific statistical results are shown in Table 2.

[0142] Explants of callus tissue with a length and width of 0.5-1.0 cm and 0.5-1.0 cm were removed from the induction medium. The portions of the explants that had not formed callus tissue on both sides of the internodes were cut off. Callus tissue larger than 0.5 cm × 0.5 cm was divided in half and transferred to proliferation medium for proliferation culture. During the proliferation culture, the light intensity was 2500-3000 lx, the culture temperature was 25℃, the photoperiod was 12 h / d, and the culture time was 25 days to obtain proliferated callus tissue. The proliferation rate and proliferation coefficient of the callus tissue were measured and statistically analyzed. The proliferation rate is the ratio of the number of proliferating callus tissue to the number of initial callus tissue, and the proliferation coefficient is the ratio of the planar area of ​​the proliferating callus tissue to the initial planar area. The specific results are shown in Table 2.

[0143] Among them, the 15 groups of proliferation media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3 have the same formula as the 15 groups of induction media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3. For example, the composition of proliferation media 1-1 is the same as that of induction media 1-1.

[0144] Table 2

[0145]

[0146]

[0147] Table 2 shows that the No. 4-2 induction medium has significant advantages in terms of callus induction quantity, induction rate, proliferation rate, and proliferation coefficient. Specifically, the optimal combination of No. 4 formulation and No. 2 induction medium resulted in 4 more callus inductions than the optimal combinations of No. 1, No. 2, No. 3, and No. 5 formulations, respectively; the induction rate increased by 13.4%, 20.0%, 40.0%, and 70.0%; the proliferation rate increased by 9.2%, 20.8%, 23.3%, and 29.9%; and the proliferation coefficient increased by 1.7, 3.1, 3.4, and 4.0.

[0148] 3. Adventitious bud induction culture and adventitious bud proliferation culture

[0149] The proliferating callus tissue was cut into blocks with a planar area of ​​1.0 cm × 1.0 cm, and a total of 450 block samples were collected. The block samples were inoculated into 15 groups of adventitious bud induction culture media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3 for adventitious bud induction culture. Each group of adventitious bud induction culture media treated 30 samples. During the adventitious bud induction culture, the light intensity was 2700 lx, the culture temperature was 25℃, and the photoperiod was 12 h / d.

[0150] The specific composition of the 15 adventitious bud induction media is shown in Table 3. Each of the 15 adventitious bud induction media contains MS medium, 30g sucrose, and 7g agar. The pH of the 15 adventitious bud induction media is 5.5.

[0151] After culturing for 25 days, adventitious bud clusters with numerous adventitious buds were obtained, such as... Figure 3 As shown in Table 3, the induction of adventitious shoots was statistically analyzed. The number of adventitious shoots induced refers to the number of adventitious shoots, and the induction rate refers to the ratio of the number of adventitious shoots to the number of explants.

[0152] Adventitious bud clusters were removed from the adventitious bud induction medium, and block-shaped adventitious bud clusters with a planar area of ​​1.5cm × 1.5cm were cut. The block-shaped adventitious bud clusters were inoculated into 15 groups of adventitious bud proliferation medium for adventitious bud proliferation culture. Each group of adventitious bud proliferation medium was used to treat 30 samples. During the adventitious bud proliferation culture, the light intensity was 2500-3000 lx, the culture temperature was 25℃, and the photoperiod was 12h / d.

[0153] The 15 groups of adventitious bud proliferation media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3 have the same composition as the 15 groups of adventitious bud induction media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3. For example, the composition of adventitious bud proliferation media numbered 1-1 is the same as that of adventitious bud induction media numbered 1-1.

[0154] After 25 days of cultivation, adventitious bud clusters were obtained, such as... Figure 4 As shown, the proliferation of adventitious bud clusters was statistically analyzed. The proliferation rate is the ratio of the number of adventitious bud clusters that have proliferated to the number of adventitious bud clusters that participated in the adventitious bud proliferation culture. The proliferation coefficient is the ratio of the total number of adventitious buds that have proliferated to the total number of callus tissues. See Table 3 for details.

[0155] Table 3

[0156]

[0157]

[0158] Table 3 shows that the adventitious shoot induction medium formula No. 4-2 has significant advantages in terms of the number of adventitious shoots induced, induction rate, proliferation rate, and proliferation coefficient. Specifically, the optimal formula No. 4-2 increases the number of callus induced by 13, 27, 40, and 75 compared to the optimal formulas No. 1-2, No. 2-2, No. 3-3, and No. 5-1, respectively; the induction rate increases by 43.3%, 90.0%, 133.3%, and 250.0%; the proliferation rate increases by 11.8%, 19.5%, 32.9%, and 76.1%; and the proliferation coefficient increases by 5.3, 9.5, 11.2, and 17.2, respectively.

[0159] 4. Seedling rooting culture

[0160] The proliferated adventitious buds were removed from the adventitious bud proliferation medium, and 2cm long adventitious buds containing at least 4 leaves were vertically cut from the base. A total of 360 adventitious buds with similar growth conditions were cut and inoculated into four groups of seedling rooting media numbered A, B, C, and D for seedling rooting culture. During the seedling rooting culture, the light intensity was 2500-3000 lx, the culture temperature was 25℃, and the photoperiod was 12h / d. Figure 5 A scene depicting the cultivation of strong seedlings to take root.

[0161] Among them, the rooting medium for seedlings in group A included MS medium, 5 ml / L IBA, and 1 g / L Ac; the rooting medium for seedlings in group B included MS medium and 1 g / L Ac; the rooting medium for seedlings in group C included MS medium and 0.5 ml / L IBA; and the rooting medium for seedlings in group D was MS medium. All four groups of rooting mediums for seedlings included 30 g sucrose, 7 g agar, and pH 6.

[0162] After 30 days of cultivation, rooted seedlings were obtained. The specific number and growth status of the rooted seedlings are recorded in Table 4. Figure 6 The images show rooted seedlings obtained after 15 days of vigorous seedling rooting culture. The left side shows an overall view of the rooted seedling, and the right side shows a breakdown of the rooted seedling. Figure 7 The image shows a rooted seedling obtained after 30 days of strong seedling rooting culture. The left side shows the overall view of the rooted seedling, and the right side shows a breakdown of the rooted seedling.

[0163] Table 4

[0164]

[0165]

[0166] It can be seen that the rooting rate of the AC group seedling rooting medium is not less than 95.6%, and the rooted seedlings are growing well. The rooting rate of the A group seedling rooting medium even reaches 100%, and the rooted seedlings are growing vigorously.

[0167] 5. Seedling hardening and transplanting cultivation

[0168] Explants with a single bud were cultured according to the above-described series of culture steps to obtain rooted seedlings. 360 rooted seedlings with at least 6 leaves and 20 roots were selected and transferred to a hardening-off environment for 3 days. The bottles were then left uncapped for another 2 days of cultivation. During this period, 150mL sprayers were used to mist the culture bottles to prevent wilting due to dehydration. The hardening-off environment was characterized by a daytime temperature of 21℃–25℃, a nighttime temperature of 15℃–18℃, and an ambient humidity of 60%–80%. Figure 8 This is a scene depicting the seedling hardening-off process. Day 6... Figure 9The rooted seedlings were removed from the culture medium and placed in clean water. The culture medium residue adhering to the seedlings was gently washed off with a brush. After washing, the seedlings were first soaked in a 0.3% carbendazim solution for 1 hour, then removed and placed on newspaper laid out on the ground for 2 hours to allow the leaves and stems to dry. Based on their growth, the seedlings were classified into three grades: Grade 1 seedlings had 10 or more leaves and 20 or more roots; Grade 2 seedlings had 6-9 leaves and 10-20 or more roots; and Grade 3 seedlings had 6 leaves and fewer than 10 roots. Figure 10 The diagram shows the grading of rooted seedlings. The rooted seedlings were transplanted into four groups of substrates numbered A, B, C, and D for cultivation. Each group of substrates contained 30 seedlings of each of the three different grades. During cultivation, the light intensity was 2500-3000 lx, the cultivation temperature was 25℃, and the light duration was 12h / d.

[0169] After 30 days of cultivation, the survival and growth of rooted seedlings in different substrates were recorded (see Table 5). The rooted seedlings were then further cultivated to obtain Saxifraga stolonifera transplant seedlings with more than 9 leaves. Figure 11 , Figure 12 as well as Figure 13 These are growth charts of rooted seedlings after different cultivation days.

[0170] Table 5

[0171]

[0172]

[0173] It is evident that the survival rate of rooted seedlings transplanted from the AC substrate was high, not less than 92.2%, and the survival rate of rooted seedlings transplanted from the A substrate even reached 98.9%, with tender green stolons and vigorous growth.

[0174] The transplanted saxifrage seedlings obtained through transplanting were transferred to a room-temperature greenhouse environment for ground planting, such as... Figure 14 As shown, the saxifrage is growing well.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for efficiently propagating Saxifraga stolonifera using stolons, characterized in that, At least the following steps are included: Saxifraga stolons with buds were used as explants, and the explants were subjected to culture treatments including callus induction culture and adventitious bud induction culture in sequence to obtain Saxifraga stolon transplant seedlings. The induction medium used for the callus induction culture is one of the following: MS + 0.2-0.6 mg / L ZT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA, MS + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA, MS + 1.1-1.5 mg / L ZT + 0.5-1.0 mg / L NAA, or MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA. The adventitious bud induction culture medium used is one of the following: MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA, MS + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA, MS + 0.05-0.1 mg / L ZT + 1.5-3.0 mg / L IBA, MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

2. The method according to claim 1, characterized in that, The induction medium consisted of MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA.

3. The method according to claim 1 or 2, characterized in that, The adventitious bud induction medium is MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L TDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

4. The method according to any one of claims 1-3, characterized in that, The explant has one bud point.

5. The method according to any one of claims 1-4, characterized in that, The callus induction culture process also includes a disinfection treatment, which includes the following steps: The explants were soaked in a laundry detergent solution for 5 minutes, then rinsed with clean water for 45-60 minutes. After that, they were soaked in 75% medical alcohol for 30 seconds, rinsed with sterile water, and then soaked in 0.1% mercuric chloride solution with 3-5 drops of Tween 80 for 5-10 minutes. The resulting explants were then rinsed to obtain sterilized explants.

6. The method according to claim 5, characterized in that, The callus induction culture includes the following steps: The sterilized explants were inoculated onto the induction medium. The induction medium was placed in the dark for cultivation from day 1 to day 7. From day 8 onwards, the explants were cultured under fluorescent lamps with a light intensity of 2500-3000 lx and a light duration of 12 h / d to obtain callus tissue with a length of 0.5-1.0 cm and a width of 0.5-1.0 cm. The culture temperature for inducing callus tissue is 23-27℃.

7. The method according to claim 6, characterized in that, The callus induction culture and the adventitious shoot induction culture are further separated by callus proliferation culture, and the adventitious shoot induction culture is further followed by adventitious shoot proliferation culture; The proliferation medium used for the callus proliferation culture is MS + 0.01-0.03 g / L ZT + 0.6-0.8 mg / L KT + 3.0-5.0 mg / L 2,4-D + 0.5-1.0 mg / L NAA; The adventitious bud proliferation culture medium used was MS + 0.05-0.1 mg / L ZT + 0.1-0.4 mg / L LTDZ + 1.5-3.0 mg / L IBA + 0.2-0.4 g / L GA3.

8. The method according to claim 7, characterized in that, The adventitious bud proliferation culture also includes seedling rooting culture. The seedling rooting culture medium used was MS + 0.5 mg / L IBA + 1 g / L Ac.

9. The method according to claim 8, characterized in that, The seedling rooting culture also includes a hardening-off culture, which includes the following steps: After the seedling rooting culture, the seedlings with no less than 6 leaves and more than 20 roots obtained are transferred to the hardening environment for 3 days. Then the culture device is opened and the seedlings are cultured in the hardening environment for another 2 days. The seedling hardening environment is characterized by a daytime temperature of 21℃~25℃, a nighttime temperature of 15℃~18℃, and an ambient humidity of 60%~80%.

10. The method according to claim 9, characterized in that, The seedling hardening process also includes transplanting culture, which includes: After hardening off, the rooted seedlings were washed and sterilized in sequence, and then the rooted seedlings were transferred to seedling pots containing substrate to cultivate the Saxifraga stolonifera transplant seedlings with more than 9 leaves. The matrix is ​​prepared by mixing perlite, peat, and vermiculite in a volume ratio of 1:2:1.

Images