Method for inducing adventitious bud differentiation of motherwort stem with axillary bud

Abstract

The invention relates to the technical field of motherwort, in particular to a method for inducing adventitious bud differentiation of stem segments with axillary buds of motherwort, which comprises the following steps: step 1, cutting the stem segments with axillary buds of motherwort seedlings into small segments of 0.5-1cm as explants; 2, the explant is placed in an MS culture medium to be cultured, differentiation of adventitious buds is induced through cytokinin and auxin, the cytokinin is 6-BA and TDZ, the auxin is NAA, after the explant is cut into small sections, optimization experiments can be conducted under different culture media and culture conditions, and therefore the conditions most suitable for induction and regeneration of leonurus japonicus calluses are found.

Description

Technical Field

[0001] The invention relates to the technical field of motherwort, and in particular to a method for inducing adventitious bud differentiation through a stem segment of motherwort with axillary buds. Background Art

[0002] Motherwort is a plant of the genus Leonurus in the family Lamiaceae. It is included in the 2005 edition of the Pharmacopoeia and is one of the Chinese medicines that can be used as health foods published by the National Health Commission. It is clinically used for irregular menstruation, postpartum blood stasis, cardiovascular disease, blood disease, etc. It is a species with great development potential. In recent years, some research has also been conducted on the standardized cultivation technology of motherwort, especially the cultivation measures to increase the total alkaloid content of the medicinal active ingredient, thereby improving the quality of the medicinal material and realizing the standardized cultivation of fresh motherwort.

[0003] However, Motherwort is a hermaphroditic plant. Long-term self-trading in the natural state has led to problems such as low seed germination rate, difficulty in germinating and seedling formation, and a decrease in the content of effective medicinal ingredients.

[0004] Therefore, establishing an efficient and stable in vitro regeneration system is an urgent problem to be solved in the breeding and industrial development of Leonurus japonicus. Through the combination culture medium of different types and concentrations of plant growth regulators, callus tissue is induced, and the adventitious bud differentiation of its embryonic callus tissue is induced through differentiation culture medium in order to establish a complete regeneration system of Leonurus japonicus, laying the foundation for the advancement of Leonurus japonicus tissue culture breeding technology. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract of the specification and the title of the invention of this application to avoid blurring the purpose of this section, the abstract of the specification and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems existing in the prior art, the present invention is proposed.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The method for inducing adventitious bud differentiation from a stem segment of Leonurus japonicus with axillary buds is characterized by comprising the following steps:

[0009] Step 1: Cut the stem segments of Leonurus japonicus seedlings with axillary buds into small segments of 0.5 cm to 1 cm as explants;

[0010] Step 2: placing the explant in MS culture medium and culturing it, using cytokinins and auxins to induce adventitious bud differentiation, wherein the cytokinins are 6-BA and TDZ, and the auxin is NAA.

[0011] In the above design, when plant parts such as stems, roots or leaves are subjected to physical stimulation such as cutting or damage, the cells will start the formation of callus tissue. Adding auxin and cytokinin to the culture medium can effectively induce the formation of callus tissue. Callus tissue can be used to quickly propagate plants, especially those that are difficult to propagate by seeds or other means. After the stem segments of motherwort with axillary buds are cut into small segments, the contact area of ​​each segment increases, which is conducive to the absorption of nutrients and growth regulators, thereby improving the efficiency of inducing callus tissue. Cutting can help activate dormant axillary buds. The cut stems are easy to stimulate the growth of axillary buds, which is conducive to the formation of adventitious buds, thereby promoting reproduction. Moreover, after being cut into small segments, optimization experiments can be carried out under different culture media and culture conditions to find the most suitable conditions for the induction and regeneration of motherwort callus tissue. MS medium contains essential inorganic salts, vitamins and amino acids, providing basic nutrients for plant growth; 6-BA can promote cell division and bud formation, and TDZ can promote callus growth; NAA is a synthetic auxin that can promote root formation, as well as regulate growth and differentiation, especially inducing adventitious roots and promoting callus redifferentiation.

[0012] The stem segments of the motherwort with axillary buds that differentiate into adventitious buds are cultured and rooted to obtain motherwort seedlings that can be used for rapid planting.

[0013] Preferably, the concentration of 6-BA is 3 mg·L -1 The concentration of TDZ is 0.5 mg·L -1 The concentration of NAA is 0.1 mg·L -1 ; Using blank as control, three explants were inoculated in each culture medium, and each experimental group was repeated 15 times. The differentiation rate of axillary bud stem segments was statistically calculated, and the best hormone combination was obtained by variance analysis.

[0014] Preferably, sucrose is added to the MS medium, and the concentration of the sucrose is 30 g·L -1 Sucrose can provide energy for cells and promote cell growth and differentiation.

[0015] Preferably, agar is added to the MS medium, and the concentration of the agar is 7 g·L -1 Agar acts as a coagulant, allowing the culture medium to solidify and form a solid state, which is convenient for the fixation and growth of the stem segments with axillary buds of the Leonurus japonicus plant.

[0016] Preferably, the culture temperature is 23-25°C and the light intensity is 35-40 μmol·m －2 s －1 , relative humidity 40%-70%, light duration 10-12h·d －1; The pH value is set between 5.8-6; placing the explant in a suitable environment can effectively promote the absorption of nutrients and the differentiation of adventitious buds.

[0017] Preferably, plump motherwort seeds are placed in a clean bench, treated with 75% ethanol for 30 seconds, rinsed with sterile water for 3-5 times, then treated with 2% sodium hypochlorite for 20 minutes, and rinsed with sterile water for 3-5 times to obtain sterile seeds, and the sterile seeds are cultured until axillary buds appear to obtain the motherwort seedlings; this can effectively remove pollutants and bacteria on the surface of the motherwort seeds, which is crucial for subsequent tissue culture and regeneration processes, reduce microbial contamination, and improve the success rate of adventitious bud differentiation culture and the results of the experiment.

[0018] Preferably, when the stem segments of the motherwort seedlings with axillary buds are cut into small segments, a cutting knife is used for cutting, and the cutting knife includes a knife holder, and the knife holder includes a horizontal bracket located in the horizontal direction, a vertical bracket located in the vertical direction, and an inclined bracket bearing a knife surface. There are two inclined brackets, which are respectively connected to the two ends of the horizontal knife holder, and the two inclined brackets are mirror images of each other and are both located below the horizontal bracket. A knife surface is mounted on the inclined bracket, and the knife surface forms an angle of 30° with the horizontal plane; the stem segments of the motherwort seedlings with axillary buds are cut at an oblique angle instead of vertically, which can increase the contact area and help the absorption of hormones and the formation of adventitious buds; the length of the explant cut, the inclination angle of the cutting surface, the spacing between the cutting surfaces, etc. have better consistency; the oblique brackets are mirror-symmetrical, so that the forces at both ends are consistent, which can effectively ensure the service life of the cutting knife and the consistency of the two ends during cutting.

[0019] The growth point of adventitious buds is a group of cells in the plant that is specifically responsible for growth and development. It has the ability to divide and differentiate, and contains actively dividing cells that are responsible for generating new buds, leaves, roots and other structures. It may produce more buds, so these nodes should be kept intact when cutting.

[0020] Preferably, the lower edge of the blade is the blade, and the thickness of the blade gradually decreases from the upper edge to the lower edge, so as to reduce the squeezing and damage to the tissue and ensure that the cut stem segment can effectively produce adventitious buds.

[0021] Preferably, the lower edge of the blade has teeth, which are divided into two groups, the teeth in the same group have the same orientation, the teeth in different groups have an angle, and the two groups of teeth are alternately arranged, so that the surface of the cutting surface is regularly uneven.

[0022] Preferably, the preparation method of the cutting knife is: step 1, take a rectangular metal plate; step 2, punch out two cutting lines at both ends of the metal plate, the two cutting lines divide the ends of the metal plate into side segments on both sides and a middle segment in the middle, fold the two side segments inward to form an angle of 90° with the metal plate, use the side segments as vertical brackets, fold the middle segment inward to form an angle of 30° with the metal plate, and use the middle segment as a bevel cutting bracket; the structure is simple and easy to produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 It is a schematic diagram of the structure of the cutting knife before folding;

[0025] Figure 2 This is a schematic diagram of the structure of the cutting knife after folding. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more understandable, the specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings.

[0027] The method for inducing adventitious bud differentiation from a stem segment of Leonurus japonicus with axillary buds comprises the following steps:

[0028] Step 1, cut the stem segments of the motherwort seedling with axillary buds into small segments of 0.5cm to 1cm as explants. Preferably, full motherwort seeds are placed in a clean bench, treated with 75% ethanol for 30s, rinsed with sterile water for 3-5 times, then treated with 2% sodium hypochlorite for 20min, rinsed with sterile water for 3-5 times to obtain sterile seeds, and the sterile seeds are cultured to the appearance of axillary buds to obtain the motherwort seedlings. Further preferably, after the sterile seeds are cultured to the appearance of 3-5 axillary buds, the motherwort seedlings are cut in a clean bench, and the length of 0.5cm to 1cm and the axillary buds in the middle are selected as explants, and it is noted that the axillary buds closest to the root (the stem segments there are thicker and shorter) and the axillary buds closest to the terminal buds (the stem segments there are too small) are not to be used to ensure the consistency of the explants. During cutting, the motherwort seedlings are cut at an angle with a cutting knife 1, and the growth points of the adventitious buds are avoided during cutting.

[0029] In the above design, when plant parts such as stems, roots or leaves are subjected to physical stimulation such as cutting or damage, the cells will start the formation of callus tissue. Adding auxin and cytokinin to the culture medium can effectively induce the formation of callus tissue. Callus tissue can be used to quickly propagate plants, especially those that are difficult to propagate by seeds or other means. After the stem segments of motherwort with axillary buds are cut into small segments, the contact area of ​​each segment increases, which is conducive to the absorption of nutrients and growth regulators, thereby improving the efficiency of inducing callus tissue. Cutting can help activate dormant axillary buds. The cut stems are easy to stimulate the growth of axillary buds, which is conducive to the formation of adventitious buds, thereby promoting reproduction. Moreover, after being cut into small segments, optimization experiments can be carried out under different culture media and culture conditions to find the most suitable conditions for the induction and regeneration of motherwort callus tissue. MS medium contains essential inorganic salts, vitamins and amino acids, providing basic nutrients for plant growth; 6-BA can promote cell division and bud formation, and TDZ can promote callus growth; NAA is a synthetic auxin that can promote root formation, as well as regulate growth and differentiation, especially inducing adventitious roots and promoting callus redifferentiation.

[0030] Reference Figure 1 , Figure 2The cutting knife 1 includes a knife holder, which includes a horizontal bracket 11 located in the horizontal direction, a vertical bracket 12 located in the vertical direction, and an inclined bracket 13 that carries the knife surface. There are two inclined brackets 13, which are respectively connected to the two ends of the horizontal knife holder. The two inclined brackets 13 are mirror images of each other and are both located below the horizontal bracket 11. A knife surface is mounted on the inclined bracket 13, and the knife surface forms an angle of 30° with the horizontal plane. The lower edge of the knife surface is the blade, and the thickness of the knife surface gradually decreases from the upper edge to the lower edge. The lower edge of the knife surface is serrated, and the serrated structure is used to more conveniently insert it into the motherwort seedling and make the surface of the cutting surface uneven, thereby increasing the area of ​​the cutting surface. Preferably, the preparation method of the cutting knife 1 is as follows: Step 1, take a rectangular metal plate; 2. Punch out two cutting lines at both ends of the metal plate, the two cutting lines divide the ends of the metal plate into side segments located on both sides and a middle segment located in the middle, fold the two side segments inward to form a 90° angle with the metal plate, use the side segments as vertical brackets 12, fold the middle segment inward to form a 30° angle with the metal plate, and use the middle segment as a bevel bracket. The blade surface can be fixed on the inclined bracket 13, or the middle segment can be directly polished to gradually change its thickness, and the middle segment can be directly used as the blade surface. While punching the cutting line, the outer edge of the middle segment of the metal plate can be punched to make the outer edge serrated 14. The cutting knife 1 of the present invention has the following beneficial effects: 1. The structure is simple and easy to produce; 2. The length of the cut explant, the inclination angle of the cutting surface, the spacing of the cutting surface, etc. have better consistency; 3. The bevel bracket is mirror-symmetrical, so that the forces at both ends are consistent, which can effectively ensure the service life of the cutting knife 1 and the consistency of the two ends during cutting. Preferably, the lower edge of the blade has teeth 14, which are divided into two groups, the teeth 14 in the same group are oriented in the same direction, the teeth 14 in different groups are oriented at an angle, and the two groups of teeth 14 are arranged alternately, so that the surface of the cutting surface is regularly uneven.

[0031] The growth point of adventitious buds is a group of cells in the plant that is specifically responsible for growth and development. It has the ability to divide and differentiate, and contains actively dividing cells that are responsible for generating new buds, leaves, roots and other structures. It may produce more buds, so these nodes should be kept intact when cutting.

[0032] Step 2, the explant is placed in MS culture medium and cultured, and adventitious bud differentiation is induced with cytokinins and auxins, wherein the cytokinins are 6-BA and TDZ, and the auxin is NAA. The whole explant can be immersed in an induction solution mixed with cytokinins and auxins, or the cytokinins and auxins can be mixed and then applied to the cut surface. In order to better stimulate axillary buds, part of the outer epidermis of the explant can be removed. Cytokinins and auxins are applied to the place where the outer epidermis is removed. Cytokinins and auxins can also be directly and evenly added to the MS culture medium.

[0033] The following uses MS medium as the culture medium, and sucrose 30g·L is added to the culture medium -1， Agar 7g·L -1 The pH value of MS medium was 5.8-6, the culture temperature was 24℃-26℃, and the light intensity was 35-40μmol·m -2 s -1 , relative humidity 40%-70%, light duration 12h·d -1 A three-factor three-level orthogonal experiment was conducted under the following conditions, with blank as control, three explants inoculated in each culture medium, and each experimental group repeated 15 times. The table below shows the differentiation rate of axillary bud stem segments after 30 days of culture, where differentiation rate = number of adventitious buds differentiated / number of inoculated × 100%. Sucrose can provide energy for cells and promote cell growth and differentiation. Agar, as a coagulant, enables the culture medium to solidify and form a solid state, which is convenient for the fixation and growth of the axillary bud stem segments of the Leonurus japonicus plant. Placing the explants in a suitable environment can effectively promote the absorption of nutrients and the differentiation of adventitious buds.

[0034] concentration 6-BA mg / L TDZ mg / L NAA mg / L Differentiation rate (%) 1 1 0.5 0.1 66.7 2 1 1 0.2 60 3 1 1.5 0.3 51.9 4 2 0.5 0.2 67.3 5 2 1 0.3 64.6 6 2 1.5 0.1 73.3 7 3 0.5 0.3 75 8 3 1 0.1 64.7 9 3 1.5 0.2 72.9 ck 0 0 0 0

[0035] As can be seen from the table above, the differentiation rate of stem segments with axillary buds in several combinations is above 50%, and the overall differentiation rate is relatively high. The orthogonal test shows that the differentiation rate of combination 7 is the highest, which is 75%, and the differentiation rate of combination 3 is the lowest, which is 51.9%. The variance analysis found that the best hormone combination is: the concentration of 6-BA is 3mg·L-1, the concentration of TDZ is 0.5mg·L-1, and the concentration of NAA is 0.1mg·L-1.

[0036] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds, characterized in that: The steps include: Step 1: Cut the stem segments of Leonurus japonicus seedlings with axillary buds into small segments of 0.5 cm to 1 cm as explants; Step 2: placing the explant in MS culture medium and culturing it, using cytokinins and auxins to induce adventitious bud differentiation, wherein the cytokinins are 6-BA and TDZ, and the auxin is NAA.

2. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 1, characterized in that: The concentration of 6-BA is 3 mg·L -1 The concentration of TDZ is 0.5 mg·L -1 The concentration of NAA is 0.1 mg·L -1 .

3. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 1, characterized in that: Sucrose is added to the MS medium, and the concentration of the sucrose is 30 g·L -1 .

4. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 1, characterized in that: The MS medium is supplemented with agar, the concentration of which is 7 g·L -1 .

5. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 1, characterized in that: The pH value of the MS culture medium is 5.8-6, the culture temperature is 24°C-26°C, and the light intensity is 35-40 μmol·m -2 s -1 , relative humidity 40%-70%, light duration 12h·d -1 .

6. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 1, characterized in that: The full motherwort seeds are placed in a clean bench, treated with 75% ethanol for 30 seconds, rinsed with sterile water for 3-5 times, and then treated with 2% sodium hypochlorite for 20 minutes, and rinsed with sterile water for 3-5 times to obtain sterile seeds. The sterile seeds are cultured until axillary buds appear to obtain the motherwort seedlings.

7. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 1, characterized in that: When the stem segments of the motherwort seedlings with axillary buds are cut into small segments, a cutting knife is used for cutting. The cutting knife includes a knife holder, and the knife holder includes a horizontal bracket located in the horizontal direction, a vertical bracket located in the vertical direction, and an inclined bracket bearing a knife surface. There are two inclined brackets, which are respectively connected to the two ends of the horizontal knife holder. The two inclined brackets are mirror images of each other and are both located below the horizontal bracket. A knife surface is mounted on the inclined bracket, and the knife surface forms an angle of 30° with the horizontal plane.

8. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 7, characterized in that: The lower edge of the blade surface is the blade edge, and the thickness of the blade surface gradually decreases from the upper edge to the lower edge.

9. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 8, characterized in that: The lower edge of the blade surface is provided with teeth, which are divided into two groups. The teeth in the same group have the same orientation, and the teeth in different groups have an angle in orientation. The two groups of teeth are alternately arranged.

10. The method for inducing adventitious bud differentiation from stem segments of Leonurus japonicus with axillary buds according to claim 9, characterized in that: The preparation method of the cutting knife is: Step 1, take a rectangular metal plate; Step 2: Punch out two cutting lines at both ends of the metal plate. The two cutting lines divide the ends of the metal plate into side sections on both sides and a middle section in the middle. Fold the two side sections inward to form a 90° angle with the metal plate, and use the side sections as vertical brackets. Fold the middle section inward to form a 30° angle with the metal plate, and use the middle section as an oblique cutting bracket.

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