A method for rapid asexual propagation of Salvia sphaerocephala

Abstract

A method for rapidly obtaining Salvia serrata plants through asexual reproduction, wherein plants can be rapidly obtained through root segment induction, are not affected by seasons, have good repeatability, high stability, rapid reproduction, and a large coefficient. The invention has a short seedling induction cycle, can be planted in the same year and can complete flowering, shortens the growth cycle, and provides technical and application guarantees for production promotion, scientific experiments, and breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant propagation and cultivation, in particular to the propagation and cultivation of sage, and more particularly to a method for rapid asexual propagation of large-leaf sage. Background Art

[0002] Salvia Salvia L.) is the largest genus in the Lamiaceae family. The plants of this genus play an important role in medicine, food, cosmetics, and ornamental fields. There are about 1,000 species distributed around the world. Salvia grandifolia ) is a species of the genus Salvia endemic to China, used as Danshen, and its root is used as medicine. Danshen has the effects of removing blood stasis, promoting new blood, promoting blood circulation, regulating menstruation and relieving pain, nourishing blood and calming the mind, cooling blood and eliminating carbuncle; it is used for uterine bleeding, irregular menstruation, blood stasis, temple pain, neurasthenia and insomnia, sores, carbuncle pain and swelling, traumatic injuries, coronary heart disease, etc.

[0003] At present, there are few research reports on Salvia salviae L. at home and abroad, especially in terms of resource investigation and metabolism. Xiao Lei et al. reported that they had isolated the first compound discovered in this genus (Xiao Lei et al. Study on chemical components of Salvia salviae L. Chinese Journal of Pharmacy, 2011, 46(13):977-979.).

[0004] Because the habitat of the bigleaf sage prairie is roadsides and sunny grass slopes, it is very easy to be destroyed (see Fig.11 ), its population reproduction method is mainly through seed reproduction, and there are many uncertainties in population maintenance. Sage is a species endemic to China with important and potential economic, ecological and scientific research value. In scientific research, ecological protection and technology promotion and utilization, it is urgent to solve other reproduction methods of this species besides seed reproduction to obtain asexual reproduction and regeneration strains. Summary of the invention

[0005] The inventor's team has conducted research on Salvia resources from the aspects of resource investigation, introduction and conservation, systematic evolution, and secondary metabolism since 2010, and found that this species has important research value as an alternative to Salvia miltiorrhiza, and has huge market prospects in terms of compounds, germplasm resource innovation, and horticultural applications.

[0006] Therefore, the present invention is a kind of endangered species of the genus Salvia native to China, Salvia officinalis ( Salvia grandifolia ) A method for rapid asexual reproduction. The purpose of the present invention is to provide a method for rapid asexual reproduction of Salvia serrata, which solves the problem that the existing Salvia serrata can only be propagated by seeds and cannot quickly obtain excellent individual plants. This method provides guarantees for mass production promotion and scientific experiments. The main method is to induce efficient germination through root segments, quickly obtain new plants and complete flowering in the same year, shorten the growth cycle, and the seedling rate can reach 99%. The operation is simple.

[0007] To achieve the above object, the present invention provides a method for rapid asexual propagation of Salvia sphaerocephala, characterized in that it comprises the following steps:

[0008] 1) Select 1-2 year old root strips of Salvia officinalis plants with excellent traits, robust growth and no pests and diseases as the source propagation materials for future use, cut the root strips into 2-3 cm root segments, and ensure that the cuts are flat when cutting horizontally;

[0009] 2) Soak the cut root segments in carbendazim;

[0010] 3) Soak the lower part of the root segment in a prepared mixture of 0.2-0.5 mg / L naphthaleneacetic acid and 0.5-2 mg / L indolebutyric acid for 3-15 minutes;

[0011] 4) Insert the root segment into the prepared seedling box matrix at its bottom, where the seedling box matrix is ​​a mixture of 0-3 mm vermiculite and 3-6 mm perlite;

[0012] 5) Cover the seedling box, place it in an environment with a temperature of 18-26℃, spray carbendazim once, and induce germination under normal light until the seedlings are induced;

[0013] 6) After inducing the seedlings to form, transplant and maintain them.

[0014] In a preferred embodiment, in step 2), the cut induced root segments are soaked in a 800-1200-fold dilution of carbendazim for 0.5-2 minutes, preferably a 1000-fold dilution of carbendazim, and the soaking time is 1 minute.

[0015] In a preferred embodiment, in step 3), the lower part of the root segment is immersed in a prepared mixed solution of 0.4-0.6 mg / L naphthaleneacetic acid and 0.8-1.2 mg / L indolebutyric acid for 4-6 minutes. Preferably, the lower part of the root segment is immersed in a prepared mixed solution of 0.5 mg / L naphthaleneacetic acid and 1 mg / L indolebutyric acid for 5 minutes.

[0016] In a preferred embodiment, the specific operation of step 4) is to insert the root segment into the prepared seedling box matrix according to its lower part, one root per hole, and the insertion depth of the matrix is ​​preferably 2-3 cm, ensuring that the upper incision is flush with the matrix and not covered. Preferably, the seedling box matrix is ​​a mixture of 0-3 mm vermiculite and 3-6 mm perlite in a ratio of 1:0.5-2, preferably 1:1, and the perlite should be sieved and washed to remove dust and ensure uniform particles.

[0017] In a specific embodiment, the ambient temperature is observed during the germination induction process to ensure that it is within a set temperature range.

[0018] In a preferred embodiment, in step 5), carbendazim is sprayed once again after 5-10 days, followed by normal maintenance; preferably, in step 5), carbendazim is sprayed at a 1500-2500 times dilution, preferably a 2000 times dilution.

[0019] In a preferred embodiment, in step 6), when the root segment induces the upper leaves to be 1-1.5 times longer than the root segment, the plant is transplanted and maintained in the usual manner.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: the present invention is the first to target extremely small populations of the genus Salvia, which have important potential utilization value, and is even more significant for species with high difficulty in establishing an asexual reproduction system for Salvia macrophylla. The present invention has screened the induction propagation matrix ratio and the year of propagation material through relevant experiments to obtain the best germination induction method, thereby improving and solving the problem that the species cannot obtain asexual propagation and regeneration strains. The present invention has a short seedling induction cycle, can be planted in the same year and can complete flowering, shortening the growth cycle, and providing technical and application guarantees for production promotion, scientific experiments and breeding. The method of the present invention is not affected by seasons, has good repeatability and high stability, and can reproduce seedlings by taking a root segment of 2-3 cm. It has fast reproduction and a large coefficient, and has good application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1-year-old rooting cuttings of Sagebrush.

[0022] Figure 2 2-year-old rooting cuttings of Sagebrush.

[0023] Figure 3 Three-year-old rooting strip of Sagebrush (hollow rot in the middle).

[0024] Figure 4 It is a diagram of the method for cutting propagation material (root) of the present invention.

[0025] Figure 5 It is a cut view of the upper part of the propagation material (root) section of the present invention.

[0026] Figure 6 This is a diagram of the method for inducing propagation materials (roots) in a matrix according to the present invention.

[0027] Figure 7 It is a diagram of the induced growth process (callus, green shoots, seedlings) of the propagation material (root) of the present invention.

[0028] Figure 8 This is a bud point diagram for inducing germination of the propagation material (root) of the present invention.

[0029] Fig. 9 This is a diagram of inducing the propagation material (root) of the present invention into seedlings.

[0030] Fig.10 This is a picture of the propagation material (root) of the present invention after induction into seedlings and planting.

[0031] Fig.11 Bigleaf sage habitat.

[0032] Fig.12 The stems and leaves of Sagebrush are hairy. DETAILED DESCRIPTION

[0033] The following is combined with the specific examples of Salvia officinalis ( Salvia grandifolia ) is the object and the accompanying drawings, and the technical solution in the present invention is further described. The present invention is finally completed through scientific experimental exploration.

[0034] Example 1: Preliminary experiment

[0035] 1. Determination of the ratio of propagation material (root) induction matrix:

[0036] Because the perlite purchased from the market has dust and uneven particles when the size is 3-6mm, it is found that it affects the air permeability during operation. In order to verify the effect on the germination rate, the test in Table 1 was designed. The results show that the germination rate is good when the perlite is sieved and washed, the particles are uniform, and there is no crushing or dust.

[0037] Table 1 Effect of whether the induction matrix perlite is sieved and washed on the germination rate

[0038]

[0039] In order to screen out an optimal induction matrix ratio scheme and improve the induction germination rate, the experiment in Table 2 was designed. From the results, it can be concluded that vermiculite, peat, and perlite alone or in different ratios have a certain germination effect, among which the germination rates of vermiculite and perlite in the ratios of 1:1, 1:2 and 2:1 are higher, and the germination rate of 1:1 mixed ratio is the highest.

[0040] Table 2 Effects of different ratios of induction matrix on germination rate

[0041]

[0042] Therefore, it is determined to select 0-3 mm vermiculite and 3-6 mm perlite. The perlite should be sieved and washed with water to remove dust and ensure uniform particles. Vermiculite and perlite should be mixed in a 1:1 ratio and placed in a seedling box for later use.

[0043] 2. Determination of the ratio of propagation material (root) induction reagent:

[0044] During the operation, naphthaleneacetic acid and indolebutyric acid were prepared and mixed for later use (Table 3); from the results, it can be concluded that the root growth is best when naphthaleneacetic acid 0.5 mg / L and indolebutyric acid 1 mg / L are prepared and mixed.

[0045] Table 3 Effects of different concentrations of NA and IBA on rooting

[0046]

[0047] 3. Determination of root age:

[0048] In order to screen out the best induction root material and improve the germination induction rate, the morphological observation method of the root growth cycle was compared through experiments. The experiment in Table 4 was designed, and the results showed that: through morphological observation and comparison, it was found that the root strips that grew less than 1 year were not thick enough, not mature enough, and the quality of the root strips was relatively tender, and they were easy to mold and rot when cut; the root strips that grew more than 2 years old were too lignified, and the middle part of the root strips often had empty rot; through experiments, it was found that although the thickness of 1-year-old and 2-year-old root strips was different, it had no effect on the germination rate, so 1-2-year-old root strips were the best as induction germination materials.

[0049] Table 4 Induction germination rate of root cuttings at different growth periods

[0050] Year of root growth Germination rate Root quality Morphological characteristics Special Circumstances <1 year 0% Difference Roots are thin and too tender Prone to mildew and rot during induction 1st year student 99% good Root thickness is moderate 2nd year student 99% good Thicker roots 2 years old and above 10% Difference High degree of lignification The middle part of the root often has hollow rot

[0051] 4. Determination of different lengths of root strips:

[0052] In order to screen out the best cutting length of root strips for single plant induction, which can ensure the seedling rate and not affect the reproduction coefficient, the experiment in Table 5 was designed. The results show that when the root strips are cut below 2cm, the seedling rate will be affected. When they are greater than 3cm, there is no significant difference in the seedling rate, but from the perspective of the single plant reproduction coefficient, it is lower than when the root strips are cut 2-3cm. Therefore, based on the comprehensive test data comparison, it is best to cut the root strips into 2-3cm segments during reproduction induction, which can ensure the seedling rate and improve the reproduction coefficient of the single plant.

[0053] Table 5 Effects of different lengths of root segments on the induction rate of seedlings

[0054] Length of root cutting Seedling rate Number of root segments per plant Advantages and Disadvantages Less than 2cm 30% 30-50 Affects the seedling rate 2-3cm 99% 17-30 Increased reproduction coefficient More than 3cm 99% 15-16 Reduced reproduction coefficient

[0055] 5. Determination of different hormone soaking times:

[0056] In order to screen out the effects of different hormone soaking times on root induction, the experiment in Table 6 was designed. The results show that under the four treatment conditions, the rooting effect was the worst when not soaked, and there was no significant difference in the rooting effect of the other three soaking times. Therefore, in order to simplify the operation, save costs and improve efficiency, the lower part of the root strip segment was soaked for 5 minutes during the induction treatment.

[0057] Table 6 Effects of different hormone immersion times on root induction

[0058]

[0059] 6. Determination of induction and culture temperature:

[0060] The best growth temperature of Salvia officinalis is 18-26℃. Through experiments, it is found that temperatures below 18℃ or above 26℃ will affect the growth of this species. The purpose of induction through root cutting is to quickly obtain asexual reproduction and regeneration strains, so providing the best growth temperature for this species can improve the relevant efficiency. Therefore, during the operation, on the basis of the guarantee of facilities with an ambient temperature of 18-26℃, it will not be affected by the season.

[0061] Example 2: Induction and germination experiment

[0062] Prepare vermiculite, perlite, seedling boxes, naphthaleneacetic acid, indolebutyric acid, carbendazim, regular razor blades, and a suitable breeding site (free of pests and diseases, ambient temperature 18-26°C) for use.

[0063] 1. Select 1-2 year old root strips of Sagebrush plants with excellent traits, strong growth and no pests and diseases as seed propagation materials, and cut the root strips into 2-3 cm root segments ( Figure 4 ), ensure smooth cut when cutting crosswise ( Figure 5 ).

[0064] 2. Soak the cut induced root segments in 1000 times diluted carbendazim solution for 1 minute.

[0065] 3. Soak the lower part of the induced root segment (the part inserted into the matrix) in the prepared mixed solution (naphthaleneacetic acid 0.5 mg / L, indolebutyric acid 1 mg / L) for 5 minutes.

[0066] 4. Insert it into the prepared seedling box matrix ( Figure 6 ), 1 hole 1 root, the depth of insertion into the matrix is ​​preferably 2-3cm (make sure the upper incision is flush with the matrix and not covered). During the operation, the order of inserting the induced root segments into the matrix should ensure that the upper part is facing up and cannot be inverted. Among them, the matrix of the seedling box is a mixture of 0-3 mm vermiculite and 3-6 mm perlite in a ratio of 1:1. The perlite should be sieved and washed with water to remove dust and ensure uniform particles.

[0067] 5. After the operation is completed, cover the seedling box, place it in an environment with a temperature of 18-26°C, then spray 2000 times of carbendazim solution once, and induce germination under normal light.

[0068] During the germination induction process of the propagation material, the ambient temperature should be frequently observed to ensure that it is within the set temperature range; spray 2000 times of carbendazim solution once a week, and then maintain it normally. The experimental results show that callus begins to appear in about 15 days, and green buds can appear in about 20 days ( Figure 7 ), germination point (see Figure 8 ), the germination rate can reach 99% (Table 7); the root strips of a single plant can be cut into 17-30 root segments, that is, the reproduction coefficient is between 17 and 30 (Table 8).

[0069] Table 7 Root segment induction germination rate test

[0070] (The number of root segment germination > 1 or = 1 is counted as the germination rate sample)

[0071] category Test root number Number of sprouting root segments Germination rate 360 359 99％

[0072] Table 8 Test on number of root sections cut from single root strips

[0073] category Number of test plants Minimum number of root segments Maximum number of root segments Average number of root segments 15 17 30 24

[0074] 6. Transplanting and maintenance after induction: When the leaf length of the upper part of the induction material (root) is 1-1.5 times longer than the root segment (2-3cm), transplanting and routine maintenance should be performed in time ( Fig. 9 Timely transplanting can promote the growth of the root system of the induced seedlings, ensure sufficient nutrient supply and prevent the seedlings from becoming stiff. Routine maintenance and management should be carried out after transplanting and planting ( Fig.10 ), to ensure that flowering can be completed that year.

[0075] Comparative Examples and Comparison of Different Methods

[0076] The inventors also tried stem cutting method and tissue culture method. Among them, the effect of using semi-lignified stems of the current year in stem cutting method was not particularly good (Table 9). In tissue culture method, excellent regeneration strains were obtained; specific methods: ① using raw mercury and ② sodium hypochlorite with concentrations of 5%, 10% and 15% combined with 75% alcohol, two methods to disinfect the surface of leaf and stem explants, because the surface of leaves and stems are densely covered with hairs (see Fig.12 ), all of which cannot avoid the contamination of explants by foreign bacteria; root materials contain a large number of secondary metabolites and cannot be cultured as explants. Therefore, it is difficult to obtain asexually propagated regeneration strains through tissue culture (there are currently no reports of successful tissue culture).

[0077] Table 9 Comparison of different asexual reproduction methods of Salvia serrata

[0078] Asexual reproduction Propagation material Germination rate Seedling rate Single plant reproduction number Induction Season Example 2 (Root Segment Induction) 1-2 years old root 99% 99% 17-30 Not affected Stem cuttings Semi-lignified stem of the current year 60% 50% 9-15 July-August Tissue culture Leaf and stem explants unsuccessful unsuccessful unsuccessful Not affected

[0079] It can be concluded from Table 9 that the root segment induction method in the embodiment of the present invention has a high germination rate and is not affected by seasons. It is a rapid method for obtaining asexual reproduction regeneration strains.

Claims

1. A method for rapid asexual propagation of Salvia sphaerocephala, characterized in that: The steps include: 1) Select 1-2 year old root strips of Salvia officinalis plants with excellent traits, robust growth and no pests and diseases as the source propagation materials for future use, cut the root strips into 2-3 cm root segments, and ensure that the cuts are flat when cutting horizontally; 2) Soak the cut root segments in 800-1200 times diluted carbendazim for 0.5-2 minutes; 3) Soak the lower part of the root segment in a mixture of 0.2-0.5 mg / L naphthaleneacetic acid and 0.5-2 mg / L indolebutyric acid for 5-15 minutes; 4) Insert the root segment into the prepared seedling box matrix at a depth of 2-3 cm, with the upper cut flush with the seedling box matrix and not covered. The seedling box matrix is ​​a mixture of 0-3 mm vermiculite and 3-6 mm perlite. 5) Cover the seedling box, place it in an environment with a temperature of 18-26℃, spray carbendazim once, and culture it under normal light to induce germination until seedlings are induced; the carbendazim should be diluted 1500-2500 times; 6) Transplant and maintain after induction of seedlings: Transplant when the length of the upper leaves induced by the root segment is 1-1.5 times that of the root segment, and carry out routine maintenance.

2. The method according to claim 1, characterized in that In step 2), soak the cut induced root segments in a 1000-fold diluted solution of carbendazim for 1 minute.

3. The method according to claim 1, characterized in that In step 3), soak the lower part of the root segment in a prepared mixture of 0.5 mg / L naphthaleneacetic acid and 1 mg / L indolebutyric acid for 5 minutes.

4. The method according to claim 1, characterized in that The specific operation of step 4 is to insert the root segment into the prepared seedling box matrix at its lower part, one root per hole.

5. The method according to claim 1, characterized in that In step 4), the matrix of the seedling box is a mixture of 0-3 mm vermiculite and 3-6 mm perlite in a ratio of 1:0.5-2.

6. The method according to claim 5, characterized in that The substrate of the seedling box is mixed with vermiculite and perlite in a ratio of 1:1, and the perlite should be sieved and washed with water to remove dust and ensure uniform particles.

7. The method according to claim 1, characterized in that During the germination induction process, observe the ambient temperature to ensure that it is within the set temperature range.

8. The method according to claim 1, characterized in that In step 5), spray carbendazim once again after 5-10 days and then maintain normally.

9. The method according to claim 1, characterized in that In step 5), spray carbendazim at a dilution of 2000 times.

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