A tissue culture method of rumex patientia

Abstract

The present application relates to the technical field of plant tissue culture, and particularly relates to a tissue culture method of Rumex patientia L. The tissue culture method of Rumex patientia L. comprises taking axillary buds of Rumex patientia L. as explants, and involves establishment of a sterile system, induction of axillary bud germination, multiplication of cluster buds and induction of rooting. The tissue culture method of Rumex patientia L. can improve the induction success rate of Rumex patientia L., shorten the induction period, improve the multiplication coefficient and rooting rate of Rumex patientia L., so as to realize rapid propagation of Rumex patientia L. and promote market application of Rumex patientia L.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for tissue culture of Rumex rubrum. Background Technology

[0002] Red-veined sorrel (Rumex sanguineus) is a perennial herbaceous plant belonging to the genus Rumex in the family Polygonaceae. It is an excellent ornamental plant with beautiful leaves and red veins, making it highly valued for its color in spring and autumn. It is suitable for edging flower borders or as a potted plant. The rhizome of red-veined sorrel can be used medicinally and its extracts can be used as dye. Its young leaves are also edible, giving it significant value in Western vegetable markets.

[0003] However, as a species in the genus *Sorrel* with unique ornamental value, the tissue culture technology for *Rumex rubra* has not yet been systematically studied and reported. Existing tissue culture methods for *Sorrel* species are mostly designed for specific closely related species or hybrids. Due to genotypic differences between different species, directly applying existing methods makes it difficult to guarantee the tissue culture efficiency and regeneration quality of *Rumex rubra*. Therefore, developing an efficient and stable tissue culture method for rapid propagation of *Rumex rubra* has significant practical implications and potential for technological innovation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for tissue culture of Rumex rubrum and a culture medium for tissue culture of Rumex rubrum.

[0005] Specifically, the present invention provides the following technical solutions: This invention provides a tissue culture method for red-veined sorrel, the method comprising: using axillary buds of red-veined sorrel as explants, and sequentially performing axillary bud germination induction culture, cluster bud proliferation culture, and rooting induction culture.

[0006] In the tissue culture method of the present invention, the axillary buds of healthy and disease-free red vein sorrel mother plants are used as explants for tissue culture.

[0007] The overall process of the tissue culture method for *Rumex rubra* provided by this invention is as follows: After disinfecting the axillary buds taken from healthy, disease-free *Rumex rubra*, the axillary buds are induced to germinate using an induction medium under suitable culture conditions to obtain clustered buds. Then, the clustered buds are induced to proliferate using a proliferation medium under suitable culture conditions. The clustered buds are then separated into individual plants, and rooting is induced on a rooting induction medium to form rooted seedlings.

[0008] In the above tissue culture method, the culture medium used to induce axillary bud germination is MS medium as the basic medium and includes the following components: 6-BA 1.0-1.5 mg / L, NAA (naphthaleneacetic acid) 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L; The culture medium used for the proliferation culture of clustered shoots is MS medium as the basic medium and includes the following components: 6-BA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L.

[0009] The present invention has found that using the above-mentioned specific culture medium as the culture medium for the axillary bud germination induction stage and the cluster bud proliferation stage respectively can enable the two culture stages to be well connected and cooperate, effectively improve the differentiation of cluster buds, and at the same time improve the proliferation efficiency of cluster buds.

[0010] Among them, in the culture medium for inducing the proliferation of axillary buds, the addition of 6-BA as a plant hormone alone, compared with the combined effect of adding 6-BA and NAA, showed that the addition of 6-BA alone as a plant hormone had a better proliferation effect and did not affect the growth of axillary buds during the proliferation process. Therefore, only 6-BA was added to the culture medium for inducing the proliferation of axillary buds.

[0011] In some embodiments of the present invention, the culture medium used for inducing axillary bud germination is prepared by adding 1.0-1.5 mg / L 6-BA, 0.1-0.5 mg / L NAA, 25-30 g / L sucrose, and 5.0-5.5 g / L agar to MS medium; the culture medium used for the proliferation of clustered buds is prepared by adding 0.1-0.5 mg / L 6-BA, 25-30 g / L sucrose, and 5.0-5.5 g / L agar to MS medium.

[0012] In the above tissue culture method, the culture medium used for inducing root growth is based on 1 / 2 MS medium and contains the following components: sucrose 25-30 g / L and agar 5.0-5.5 g / L.

[0013] Preferably, no plant hormones are added to the above-mentioned root-inducing culture medium.

[0014] This invention reveals that, by combining the aforementioned axillary bud germination induction culture and cluster bud proliferation culture, a 100% rooting rate can be achieved during the rooting induction stage without the addition of plant hormones. Cluster buds, under the combined action of the rooting medium and various hormones synthesized by themselves, can promote rooting and seedling formation.

[0015] More preferably, the culture medium used for inducing rooting is based on 1 / 2 MS medium and contains the following components: 25-30 g / L sucrose and 5.0-5.5 g / L agar.

[0016] In some embodiments of the present invention, the culture medium used for induced rooting culture is obtained by adding 25-30 g / L sucrose and 5.0-5.5 g / L agar to 1 / 2 MS medium.

[0017] The preferred pH of the culture medium used for the above-mentioned induction of axillary bud germination culture, cluster bud proliferation culture, and induction of rooting culture is 5.8-6.0.

[0018] Among the tissue culture methods described above, the following conditions are preferred for inducing axillary bud germination culture, cluster bud proliferation culture, and inducing rooting culture: culture temperature of 23-27℃, light intensity of 1500-2300 Lux, and light duration of 9-10 h / d.

[0019] In the tissue culture methods described above, the explants are disinfected before inducing axillary bud germination. The disinfection process includes rinsing with water, followed by disinfection with 75% alcohol and treatment with 0.1% mercuric chloride solution.

[0020] The preferred treatment method is immersion and shaking.

[0021] Preferably, the alcohol disinfection time is 30-60 seconds; the mercuric chloride solution treatment time is 4-8 minutes.

[0022] Preferably, after alcohol disinfection and mercuric chloride solution treatment, the explants are washed with sterile water.

[0023] In some embodiments of the present invention, a method for tissue culture of Rumex rubrum is provided, the method comprising: obtaining and disinfecting explants, inducing axillary bud germination, proliferation of clustered buds, and inducing rooting.

[0024] On the other hand, the present invention also provides a culture medium for tissue culture of Rumex rubrum, the culture medium comprising axillary bud germination induction medium, cluster bud proliferation medium and rooting induction medium; The axillary bud germination induction medium uses MS medium as the basic medium and includes the following components: 6-BA 1.0-1.5 mg / L, NAA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L; The bud proliferation medium uses MS medium as the basic medium and includes the following components: 6-BA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L; The rooting induction medium uses 1 / 2 MS medium as the basic medium and contains the following components: sucrose 25-30 g / L and agar 5.0-5.5 g / L.

[0025] The present invention also provides the application of the culture medium described above for the culture of Rumex rubrum tissue in the culture of Rumex rubrum tissue.

[0026] Preferably, the application involves using axillary buds of *Rumex rubrum* as explants for tissue culture.

[0027] The beneficial effects of this invention are as follows: This invention selects axillary buds from the mother plant of *Rumex rubrum* as explants for tissue culture of *Rumex rubrum*. Experiments have verified that when axillary buds are used as explants, clustered buds emerge normally within 3-4 weeks and grow rapidly. However, when leaves are used as explants, although callus formation occurs, adventitious buds are not induced. This invention also develops suitable culture media and methods for inducing axillary bud germination, clustered bud proliferation, and rooting using axillary buds as explants. Experiments have verified that in the axillary bud germination induction step, the combined use of 6-BA and NAA shortens the induction time and increases the induction rate while ensuring normal growth of clustered buds. Furthermore, in the clustered bud proliferation stage, using 6-BA alone, with a lower dosage than that used for inducing axillary buds, ensures robust plant growth, eliminates the need for NAA, and improves both proliferation and rooting rates. The tissue culture method of *Rumex rubra* of this invention can effectively improve the induction success rate of *Rumex rubra* shoot clusters, increase the proliferation coefficient and rooting rate of *Rumex rubra*, thereby achieving rapid propagation of *Rumex rubra* and promoting its commercial application. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. The accompanying drawings described below are some embodiments of this invention.

[0029] Figure 1 This is a schematic diagram illustrating the induction of axillary buds to form clustered buds during the tissue culture process of *Rumex rubrum* using the tissue culture method of Example 2.

[0030] Figure 2 This is a schematic diagram of the subculture and proliferation of clustered shoots during the tissue culture process of *Rumex rubrum* using the tissue culture method of Example 2.

[0031] Figure 3 This is a schematic diagram illustrating the induction of rooting during the tissue culture process of *Rumex rubrum* using the tissue culture method described in Example 2. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] The alcohol used in the following examples is 75% Oujie disinfectant alcohol, purchased from Hangzhou Outop Biotechnology Co., Ltd.; the mercuric chloride is a standard solution of mercuric chloride purchased from Tianjin Guangfu Fine Chemical Research Institute.

[0034] The *Rumex rubra* mother plants used in the following examples were obtained from Beijing Huaxiang Flower Technology Research Institute Co., Ltd.

[0035] This invention provides a method for tissue culture of *Rumex rubrum*, specifically including the following steps: 1. Acquisition and disinfection of explants (1) Obtaining explants: Select healthy mother plants free from pests and diseases, and select axillary buds at the base of the plant; (2) Disinfection of explants: Rinsing: Rinse the axillary buds obtained in step (1) under running tap water for 10 minutes and air dry; Alcohol disinfection: After drying, immerse and shake in 75% alcohol for 30-45 seconds, then rinse with sterile water for 1-2 times, each time for 1-2 minutes. Treatment with 0.1% mercuric chloride solution: Immerse the axillary buds with axillary buds in a 0.1% mercuric chloride solution and shake for 4-8 minutes, then rinse with sterile water 3-5 times, 1-2 minutes each time. Cutting: Remove the damaged part at the base of the axillary bud after disinfection, and then divide it into small segments with buds, which are 1-2 cm long, to obtain explants to be induced.

[0036] 2. Inducing axillary bud germination The explants to be induced obtained in step 1 are vertically inserted into the culture medium for inducing axillary bud germination to induce axillary bud germination and obtain clustered buds.

[0037] The culture medium used in this step has the following formulation: MS medium, 6-BA 1.0-1.5 mg / L, NAA (naphthaleneacetic acid) 0.1-0.5 mg / L, sucrose 25-30 g / L, agar 5.0-5.5 g / L, pH 5.8-6.0; the culture temperature is 23-27℃, the light intensity is 1500-1800 Lux, and the light duration is 9-10 h / d.

[0038] 3. Proliferation of clustered buds The clustered buds obtained in step 2 were divided into stem segments containing a single axillary bud, and inoculated into a subculture medium for inducing the proliferation of clustered buds to obtain the proliferated clustered buds.

[0039] The culture medium used in this step has the following formulation: MS medium, 6-BA 0.1-0.5 mg / L, sucrose 25-30 g / L, agar 5.0-5.5 g / L, pH 5.8-6.0; culture temperature 23-27℃, light intensity 1500-1800 Lux, and light duration 9-10 h / d.

[0040] 4. Inducing rooting The proliferated buds obtained in step 3 are separated into individual buds and inoculated into a rooting culture.

[0041] The culture medium used in this step is formulated as follows: 1 / 2 MS medium, sucrose 25-30 g / L, agar 5.0-5.5 g / L, pH 5.8-6.0; the culture temperature is 23-27℃, the light intensity is 1800-2300 Lux, and the light duration is 9-10 h / d.

[0042] The following examples are in conjunction with Examples 1-3 and Comparative Examples 1-4. Figure 1-3 The technical solution of the present invention will be further described in detail through testing and experiments.

[0043] Example 1 This embodiment provides a tissue culture method for Rumex rubrum, specifically including the following steps: 1. Acquisition and disinfection of explants (1) Obtaining explants: Select healthy mother plants free from pests and diseases, and select axillary buds from the mother plants; (2) Disinfection of explants: Rinsing: Rinse the axillary buds obtained in step (1) under running tap water for 10 minutes and air dry; Disinfection: Immerse the axillary bud in 75% alcohol and shake for 30 seconds, then rinse with sterile water for 1 minute each time; then immerse in 0.1% mercuric chloride solution and shake for 6 minutes, then rinse with sterile water for 1 minute each time. Cutting: Remove the damaged part at the base of the axillary bud after disinfection, and then divide it into 2cm segments with buds to obtain explants to be induced.

[0044] 2. Inducing axillary bud germination The explants obtained in step 1 were inoculated into a culture medium for inducing axillary bud germination. After 3-4 weeks, clustered buds with axillary buds were obtained.

[0045] 3. Proliferation of clustered buds The bud clusters obtained in step 2 are divided into stem segments containing a single axillary bud and inoculated into a proliferation medium for subculture. Subculture is performed every 3-4 weeks to obtain a sufficient number of proliferating bud clusters.

[0046] 4. Inducing rooting The clustered buds obtained in step 3 were divided into single buds and transferred to a bud-rooting culture medium to induce the development of adventitious roots, ultimately obtaining complete regenerated plants.

[0047] The conditions and parameters involved in each of the above steps, as well as the culture medium formulations used, are shown in Table 1.

[0048] Table 1. Parameters and culture medium formulations used in each step of Examples 1-3 and Comparative Examples 1-4. Example 2 This embodiment provides a tissue culture method for *Rumex rubrum*, which has the same steps and procedures as in Example 1, except for the condition parameters and culture medium formulations used in some steps, as shown in Table 1. Except for the condition parameters and culture medium listed in Table 1, the method steps of Example 2 are the same as those of Example 1.

[0049] Example 3 This embodiment provides a tissue culture method for *Rumex rubrum*, which has the same steps and procedures as in Example 1, except for the condition parameters and culture medium formulations used in some steps, as shown in Table 1. Except for the condition parameters and culture medium listed in Table 1, the method steps of Example 3 are the same as those of Example 1.

[0050] Comparative Examples 1-3 Comparative Examples 1-3 each provide a tissue culture method for *Rumex rubrum*, which differs from Example 1 in the condition parameters of some steps and the culture medium formulation used, as shown in Table 1. Except for the condition parameters and culture medium listed in Table 1, the method steps of Comparative Examples 1-3 are the same as those of Example 1.

[0051] Comparative Example 4 Comparative Example 4 provides a tissue culture method for *Rumex rubrum*, which differs from Example 1 in the selection of explants, as shown in Table 1. Except for the conditions and parameters listed in Table 1, the method steps of Comparative Example 4 are the same as those of Example 1.

[0052] Experimental Example Red-veined Rumex was cultured using the tissue culture methods provided in Examples 1-3 and Comparative Examples 1-4. During the culture, the contamination rate, browning mortality rate, and other parameters at each culture stage were recorded during the explant disinfection phase. The induction rate of shoot clusters, the shoot cluster proliferation coefficient, and the shoot cluster rooting rate were calculated. Specific test results are shown in Table 2. Simultaneously, the induction of shoot clusters and the growth of differentiated seedlings during the Red-veined Rumex culture process were recorded. Specific results are shown in Table 3. A schematic diagram illustrating the induction of axillary bud germination during the Red-veined Rumex tissue culture process using the tissue culture method of Example 2 is shown below. Figure 1 As shown in the diagram, during the tissue culture process of *Rumex rubrum* using the tissue culture method of Example 2, a schematic diagram of the proliferation and differentiation of clustered shoots to form clustered shoots is shown. Figure 2 As shown in the diagram, during the tissue culture process of *Rumex rubrum* using the tissue culture method of Example 2, the rooting of the clustered shoots is illustrated in the diagram. Figure 3 As shown.

[0053] Table 2 Results of tissue culture of *Rumex rubrum* (1) Table 3 Results of tissue culture of *Rumex rubrum* (2) Based on the tissue culture methods of Examples 1-3 and Comparative Examples 1-4, as well as the results in Tables 2 and 3, this invention selects axillary buds as explants and selects appropriate culture medium formulations and culture conditions at each tissue culture stage to carry out tissue culture of Rumex rubrum, which shortens the induction period, increases the proliferation rate of clustered buds, and achieves 100% rooting rate without hormones during the rooting stage, which is beneficial for later transplanting survival.

[0054] First, selecting suitable explants from the mother plant for the tissue culture of *Rumex rubrum* directly affects the final result. Examples 1-3 and Comparative Example 4 were conducted under the same tissue culture conditions and without pests or diseases. Examples 1-3 used axillary buds as explants, while Comparative Example 4 used leaves. Tables 2 and 3 show that using axillary buds as explants resulted in the normal emergence and rapid growth of clustered buds within 3-4 weeks. However, using the tissue culture method in Comparative Example 4, although callus formation occurred using leaves as explants, adventitious buds were not induced. In conclusion, the selection of explants significantly impacts the effectiveness of *Rumex rubrum* tissue culture, and choosing suitable mother plant parts determines the success of establishing the tissue culture system.

[0055] Furthermore, in the step of inducing axillary bud germination, 6-BA and NAA must be used in combination to achieve the best induction effect. Tables 1, 2, and 3 show that in Comparative Example 1, the amount of 6-BA added was 0. Compared with Example 2, the axillary bud induction rate decreased by 27%, the induction time for clustered buds increased by one week, and the proliferation coefficient in the next stage decreased significantly, from 3.3 in Example 2 to 1.8. Therefore, by adding an appropriate amount of 6-BA in this stage, the axillary bud induction rate can be improved, the culture period shortened, and the proliferation rate significantly increased. However, in Comparative Example 3, only 6-BA was added, and the amount of NAA added was 0, resulting in wrinkled leaves, stunted plants, and abnormal growth in the clustered seedlings during the proliferation culture stage. This indicates that in the step of inducing axillary bud germination, the combined use of 6-BA and NAA can shorten the induction time of bud points and improve the induction rate while ensuring the normal growth of clustered buds.

[0056] Furthermore, the choice of plant hormones during the axillary shoot proliferation stage also affects the results of Rumex rubrum tissue culture. Tables 1, 2, and 3 show that Comparative Example 2, which used NAA in addition to 6-BA, exhibited a significantly lower proliferation coefficient compared to the previous example, decreasing from 3.3 in Example 2 to 2.2. Moreover, the proliferation culture resulted in significant callus formation at the plant base, thus affecting the induction of rooting in the next stage, and the rooting rate also decreased to 89%. This demonstrates that the choice and dosage of plant hormones directly affect plant growth and proliferation rate during the axillary shoot proliferation stage. Therefore, during the axillary shoot proliferation stage, using 6-BA alone at a dosage of 0.1-0.5 mg / L, lower than the dosage used in the first stage of axillary shoot induction, ensures robust plant growth, eliminates the need for NAA, and improves both proliferation and rooting rates.

[0057] In summary, the tissue culture method for *Rumex rubra* provided by this invention uses axillary buds as explants and appropriate culture media for each stage of culture. By inducing the emergence of clustered buds, the cycle is short and the proliferation coefficient is high. At the same time, *Rumex rubra* has a good rooting effect with a rooting rate of 100%, which improves the transplant survival rate of *Rumex rubra* and thus accelerates the market application of this variety.

[0058] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for tissue culture of *Rumex rubrum*, characterized in that, The method includes: using axillary buds of *Rumex rubrum* as explants, sequentially inducing axillary bud germination, cluster bud proliferation, and inducing rooting; the culture medium for inducing axillary bud germination includes both 6-BA and NAA; the culture medium for cluster bud proliferation includes only 6-BA, without the addition of NAA; the amount of 6-BA used in the culture medium for cluster bud proliferation is less than the amount used in the culture medium for inducing axillary bud germination.

2. The method for tissue culture of *Rumex rubrum* according to claim 1, characterized in that, The explant is an axillary bud at the base of the petiole.

3. The method for tissue culture of *Rumex rubrum* according to claim 1, characterized in that, The culture medium used for inducing axillary bud germination is MS medium as the basic medium and includes the following components: 6-BA 1.0-1.5 mg / L, NAA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L; The culture medium used for the proliferation culture of the clustered shoots is MS medium as the basic medium and includes the following components: 6-BA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L.

4. The method for tissue culture of *Rumex rubrum* according to claim 3, characterized in that, The culture medium used to induce axillary bud germination contained 1.2 mg / L 6-BA, 0.3 mg / L NAA, 25 g / L sucrose, and 5.2 g / L agar. The culture medium used for the proliferation culture of the clustered shoots contained 0.3 mg / L 6-BA, 25 g / L sucrose, and 5.2 g / L agar.

5. The method for tissue culture of *Rumex rubrum* according to claim 3, characterized in that, The culture medium used for the induced rooting culture is based on 1 / 2 MS medium and contains the following components: sucrose 25-30 g / L and agar 5.0-5.5 g / L.

6. The method for tissue culture of *Rumex rubrum* according to claim 5, characterized in that, The pH of the culture media used for inducing axillary bud germination, bud proliferation, and rooting was 5.8-6.

0.

7. The method for tissue culture of *Rumex rubrum* according to any one of claims 1 to 6, characterized in that, The culture temperature for inducing axillary bud germination, bud proliferation, and rooting was 23-27℃, the light intensity was 1500-2300 Lux, and the light duration was 9-10 h / d.

8. The method for tissue culture of *Rumex rubrum* according to any one of claims 1 to 6, characterized in that, Disinfect the explants before inducing axillary bud germination culture; The disinfection process includes: rinsing with water, followed by disinfection with 75% alcohol and treatment with 0.1% mercuric chloride solution; The alcohol disinfection time is 30-60 seconds; the mercuric chloride solution treatment time is 4-8 minutes.

9. A culture medium for tissue culture of *Rumex rubrum*, characterized in that, A method for tissue culture of *Rumex rubrum* as described in any one of claims 1 to 6; wherein the culture medium comprises an axillary bud germination induction medium, a cluster bud proliferation medium, and a rooting induction medium; The axillary bud germination induction medium uses MS medium as the basic medium and includes the following components: 6-BA 1.0-1.5 mg / L, NAA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L; The bud proliferation medium uses MS medium as the basic medium and includes the following components: 6-BA 0.1-0.5 mg / L, sucrose 25-30 g / L, and agar 5.0-5.5 g / L; The rooting induction medium uses 1 / 2 MS medium as the basic medium and contains the following components: sucrose 25-30 g / L and agar 5.0-5.5 g / L.

10. The application of the culture medium according to claim 9 in the culture of Rumex rubrum tissue.

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