Rooting induction method for motherwort

Abstract

The invention relates to motherwort cultivation. The leonurus japonicus rooting induction method is characterized by comprising rooting induction: when adventitious buds grow to 1-3 cm, the adventitious buds are cut off from explants and transferred into a rooting culture medium to be cultured, the rooting culture medium takes an MS culture medium as a basic culture medium, and IBA with the concentration of 0.1 mg.L <-1 > is added into the rooting culture medium. The components of the culture medium are selected, and the rooting rate of the selected rooting culture medium is high.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture and forestry, and in particular to the cultivation of motherwort. Background Art

[0002] Motherwort (Leonurus japonicas Houtt.) is a plant of the genus Leonurus in the family Labiatae. It is included in the 2005 edition of the Pharmacopoeia and is one of the Chinese medicines that can be used in health foods published by the National Health Commission. There are relatively few varieties of motherwort, including white motherwort, red motherwort, purple motherwort, and fine-leaved motherwort. Motherwort tastes spicy, slightly bitter, and slightly cold in nature. It has the effects of promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, and clearing away heat and detoxifying. Modern medical research shows that motherwort has many effects such as thrombolysis, anticoagulation, lipid reduction, blood viscosity reduction, red blood cell aggregation reduction, platelet aggregation inhibition, microcirculation improvement, anti-oxidant free radicals, and reduction of intracellular calcium overload. It is clinically used for irregular menstruation, postpartum blood stasis and pain, cardiovascular disease, blood disease, etc. It is a species with great development potential.

[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. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention is proposed.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The method for inducing rooting of motherwort is characterized by comprising the steps of: cutting the adventitious buds from the explants when the adventitious buds grow to 1 to 3 cm, transferring the buds to a rooting medium for cultivation, wherein the rooting medium uses MS medium as a basic medium, and a 0.1 mg·L -1 IBA.

[0007] Preferably, the method further comprises the following steps: step 1, cutting the stem segments of the motherwort seedlings with axillary buds into small segments of 0.5 cm to 1 cm as explants; step 2, culturing the explants in a differentiation medium to obtain adventitious buds, wherein the differentiation medium uses MS medium as the basic medium, and the differentiation medium is supplemented with a concentration of 3 mg·L -1 6-BA, concentration of 0.5 mg·L -1 TDZ, concentration is 0.1mg·L -1 NAA.

[0008] Preferably, 30 g·L sucrose is added to the differentiation medium. -1 , agar 7 g·L -1The pH value of the MS 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% to 70%, light duration 12h·d -1 .

[0009] Preferably, the method further comprises adventitious bud proliferation culture: after 30 days of adventitious bud differentiation culture, the explants are transferred to MS medium without any hormones for proliferation culture for 15 days to obtain robust seedlings, and the adventitious buds of the seedlings are induced to root.

[0010] Preferably, 30 g·L sucrose is added to the MS medium without any hormones. -1 , agar 7 g·L -1 The pH value of the MS 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% to 70%, light duration 12h·d -1 .

[0011] Preferably, the method further comprises the step of training the plants: the rooting medium is placed in a triangular flask, and after 20 days of rooting induction culture, the bottle cap is opened to perform seedling hardening, and tissue culture seedlings are obtained after 3-7 days of seedling hardening.

[0012] Preferably, the method further comprises transplanting the plants: taking the tissue culture seedlings out of the triangular flask, washing the agar at the roots of the seedlings, and transplanting the seedlings into a culture cup filled with vermiculite for cultivation.

[0013] Preferably, 30 g·L sucrose is added to the rooting medium. -1 , agar 7 g·L -1 The pH value of the MS 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% to 70%, light duration 12h·d -1 .

[0014] Beneficial effects:

[0015] 1. The present invention induces callus tissue through a combination culture medium of different types and concentrations of plant growth regulators, and induces the differentiation of its embryonic callus tissue through a differentiation culture medium in order to establish a complete regeneration system of Motherwort, laying a foundation for the advancement of Motherwort tissue culture breeding technology.

[0016] 2. The present invention cultivates motherwort through tissue culture technology, which is beneficial to the breeding and rapid reproduction of motherwort compared with traditional seedling raising technology, and can also ensure its genetic stability.

[0017] 3. The present invention selects the components of the culture medium, and the selected rooting culture medium has a high rooting rate, and the selected differentiation culture medium has a high differentiation rate. More importantly, the main components of the rooting culture medium and the differentiation culture medium of the present invention are the same, which can reduce the production cost or procurement cost. It also provides a premise for the flexible use of the culture medium.

[0018] 4. The present invention optimizes the culture environment of the culture medium. After the optimization, the culture environments of the root induction culture and the adventitious bud proliferation culture are consistent, which facilitates the regulation of the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a photo of day 0 of differentiation induction;

[0020] Figure 2 This is a photo taken on the 10th day of induction of differentiation;

[0021] Figure 3 This is a photo taken on the 20th day of induction of differentiation;

[0022] Figure 4 This is a photo taken on the 30th day of induction of differentiation;

[0023] Figure 5 These are photos taken after 15 days of proliferation;

[0024] Figure 6 For photos of the 30th day of rooting;

[0025] Figure 7 For photos of the 30th day of rooting;

[0026] Figure 8 This is a photo taken 20 days after transplanting. DETAILED DESCRIPTION

[0027] 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.

[0028] The method for inducing rooting of motherwort comprises the following steps: when the adventitious buds grow to 1-3 cm, cutting the adventitious buds from the explants, transferring the buds to a rooting medium for cultivation, wherein the rooting medium uses an MS medium as a basic medium and auxin is added to the rooting medium.

[0029] The rooting medium used MS medium as the basic medium, and the auxin was IBA, with a concentration of 0.05 mg·L-1 and 0.1 mg·L -1 and 0.15 mg·L -1 Three adventitious buds were planted in each culture medium, and each experimental group was repeated 20 times. Sucrose 30 g·L was added to the culture medium. -1 , agar 7 g·L-1 , the pH value was set between 5.8 and 6, the culture temperature was 25°C, and the light intensity was 35 to 40 μmol·m -2 s -1 , relative humidity 40% to 70%, light duration 12h·d -1 . For photos of the cultivation process, see Figure 6 , Figure 7 As shown. After 30 days of cultivation, the number of roots at the bottom of the buds was observed and the rooting rate was calculated. SPSS17.0 statistical software and Excel were used for data statistical analysis. Rooting rate = number of roots / number of inoculated buds × 100%, as follows:

[0030] IBA mg / L Rooting rate (%) 1 0.05 26.7 2 0.1 80 3 0.15 45

[0031] From the above table, we can see that different IBA concentrations have different effects on the rooting rate. -1 When the concentration was 0.05 mg·L -1 When the concentration of IBA increased, the rooting rate was the lowest, which was 26.7%. The rooting rate increased first and then decreased with the increase of IBA concentration.

[0032] The method of inducing rooting of Motherwort also includes adventitious bud differentiation:

[0033] Step 1: Cut the stem segments of the motherwort seedlings with axillary buds into small segments of 0.5 cm to 1 cm as explants. When cutting, use a cutting knife to cut the motherwort seedlings at an angle, avoiding the growth points of the adventitious buds.

[0034] The cutting knife includes a knife frame, which includes a horizontal support located in the horizontal direction, a vertical support located in the vertical direction, and an inclined support that carries a knife surface. There are two inclined supports, which are respectively connected to the two ends of the horizontal knife frame. The two inclined supports are mirror images of each other and are both located below the horizontal support. A knife surface is mounted on the inclined support, 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 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, 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, 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. The cutting knife 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 and the consistency of the two ends during cutting. 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.

[0035] Step 2: placing the explant in a differentiation medium to obtain adventitious buds, wherein the differentiation medium uses MS medium as a basic medium, and cytokinins and auxins are added to the differentiation medium.

[0036] The differentiation medium was MS medium with 30 g·L sucrose added. -1 , agar 7 g·L -1 The pH value of the MS 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% to 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 15 replicates for each experimental group. For photos of the culture process, see Figure 1 , Figure 2 , Figure 3 , Figure 4 The following table shows the differentiation rate of axillary bud stem segments after 30 days of culture, where differentiation rate = number of differentiated adventitious buds / number of inoculated buds × 100%.

[0037]

[0038]

[0039] It can be seen from the table above that the differentiation rate of the stem segments with axillary buds of Leonurus japonicus 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 0.5 mg·L -1 The concentration of NAA is 0.1 mg·L -1 .

[0040] In order to better stimulate axillary buds, part of the outer epidermis of the explant can be removed and the area where the outer epidermis is removed can be buried in differentiation medium.

[0041] The method for inducing rooting of motherwort also includes adventitious bud proliferation culture: after 30 days of adventitious bud differentiation culture, the explants are transferred to MS medium without any hormones for proliferation culture for 15 days to obtain robust seedlings. Figure 5 As shown, the adventitious buds of the seedlings were induced to root. 30 g·L sucrose was added to the MS medium without any hormones. -1 , agar 7 g·L -1 The pH value of the MS 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% to 70%, light duration 12h·d -1 .

[0042] The method for inducing rooting of motherwort also includes the training of plants: the rooting medium is placed in a triangular flask, and after 20 days of rooting induction culture, Figure 7 , Figure 8 As shown, the bottle cap is opened to perform seedling hardening, and tissue culture seedlings are obtained after 3-7 days of hardening. The method also includes transplanting plants: taking the tissue culture seedlings out of the triangular bottle, washing the agar at the root of the seedlings, and transplanting them into a culture cup filled with vermiculite for cultivation. A turntable is provided under the triangular bottle, so that the triangular bottle can be rotated by the turntable to adjust the direction of the motherwort, so that the motherwort can take root more evenly.

[0043] 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 rooting of Leonurus japonicus, characterized in that: The process includes root induction: when the adventitious buds grow to 1-3 cm, the adventitious buds are cut off from the explants and transferred to a rooting medium for cultivation. The rooting medium uses MS medium as the basic medium and contains 0.1 mg·L -1 IBA.

2. The method for inducing rooting of Leonurus japonicus according to claim 1, characterized in that: The method also includes adventitious bud differentiation: step 1, cutting the stem segments of motherwort seedlings with axillary buds into small segments of 0.5 cm to 1 cm as explants; step 2, culturing the explants in a differentiation medium to obtain adventitious buds, wherein the differentiation medium uses MS medium as the basic medium, and the differentiation medium is supplemented with a concentration of 3 mg·L -1 6-BA, concentration of 0.5 mg·L -1 TDZ, concentration is 0.1mg·L -1 NAA.

3. The method for inducing rooting of Leonurus japonicus according to claim 2, characterized in that: Add 30 g·L sucrose to the differentiation medium -1 , agar 7 g·L -1 The pH value of the MS 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% to 70%, light duration 12h·d -1 .

4. The method for inducing rooting of Leonurus japonicus according to claim 2, characterized in that: The method also includes adventitious bud proliferation culture: after 30 days of adventitious bud differentiation culture, the explants are transferred to MS medium without any hormones for proliferation culture for 15 days to obtain robust seedlings, and the adventitious buds of the seedlings are induced to root.

5. The method for inducing rooting of Leonurus japonicus according to claim 4, characterized in that: Add 30 g·L sucrose to MS medium without any hormones -1 , agar 7 g·L -1 The pH value of the MS 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% to 70%, light duration 12h·d -1 .

6. The method for inducing rooting of Leonurus japonicus according to claim 1, characterized in that: It also includes plant training: the rooting medium is placed in a triangular flask, and after 20 days of rooting induction culture, the bottle cap is opened to harden the seedlings, and tissue culture seedlings are obtained after 3-7 days of hardening the seedlings.

7. The method for inducing rooting of Leonurus japonicus according to claim 6, characterized in that: The method also includes the transplanting of plants: taking out the tissue culture seedlings from the triangular flask, washing the agar at the roots of the seedlings, and transplanting them into a culture cup filled with vermiculite for cultivation.

8. The method for inducing rooting of Leonurus japonicus according to any one of claims 1 to 7, characterized in that: Add 30 g·L sucrose to the rooting medium -1 , agar 7 g·L -1 The pH value of the MS 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% to 70%, light duration 12h·d -1 .

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