CN102487817A - In vitro rapid propagation method of fraxinus rhynchophylla and propagation medium thereof - Google Patents

Abstract

The invention relates to an in vitro rapid propagation method of fraxinus rhynchophylla and a propagation medium thereof, and aims to solve the problems of complicated steps, low germination rate of dormant buds, low propagation rate of stem buds, low rooting rate, low transplanting survival rate, and high cost existing in existing rapid propagation methods of fraxinus rhynchophylla. The method comprises: conducting water culture to dormant branches of fraxinus rhynchophylla, cutting stem sections with buds in new branches for pretreatment, carrying inoculation to a propagation medium so as to obtain clustered stem buds; performing excision with single stem bud as a unit, then cutting the stem bud into stem sections with terminal buds, which are inoculated into a rooting medium; performing open seedling hardening to a regenerated plant in a culture room, cleaning the medium attached to a root system, then transplanting the plant to a culture substrate for cultivation till complete unfolding of grown new leaves so as to obtain a fraxinus rhynchophylla plantlet, thus finishing in vitro rapid propagation of fraxinus rhynchophylla. The method of the invention has the advantages of short reproduction cycle, rapid speed, high efficiency, stem bud propagation rate up to 100%; low reproduction cost; over 90% of stem bud rooting rate and transplanting survival rate.

Description

In vitro and rapid propagation method of Astragalus japonica and its proliferation medium

technical field

The invention relates to an in vitro rapid propagation method of Astragalus japonica and a propagation medium thereof.

Background technique

Fraxinus rhynchophylla is a large deciduous tree of the genus Ash in the Oleaceae family, and is an important economic hard broad tree species in the eastern mountains of Northeast my country. Astragalus has strong stress resistance, hard and dense material, beautiful pattern, and the bark is the traditional Chinese medicine "Qinpi", which can clear liver heat, stop dysentery, improve eyesight, and treat enteritis mainly; the seeds can be used to extract oil and make soap, which is an important tree species for decorative timber, medicinal tree species and landscaping tree species.

In production practice, there are problems of insufficient high-quality mother trees and no guarantee of seed quality and quantity in the propagation of Aspergillus japonica by seeds. The quantity of Aspergillus japonica seedlings obtained and multiplied only by seed propagation is far from meeting the demand for high-quality seedlings in afforestation production practice. If the mode of twig cutting or grafting is utilized for propagation, there are problems of insufficient plant material, long propagation cycle (at least 6 months), low propagation efficiency and high cost, so it is difficult to reproduce a large amount of High-quality seedlings can meet the demand for high-quality seedlings of Astragalus chinensis in afforestation production practice.

A large number of high-quality seedlings of Astragalus candidum can be multiplied in a short period of time by means of tissue culture to meet the needs of production practice. Disclosed a kind of rapid propagation method of Aspergillus fruticosa in " the in vitro cultivation and rapid propagation of white wax " (Hong Yuanfan etc., in December, 2006, Plant Physiology Communication, the 6th phase of the 42nd volume), but this method is in bud's Induction stage has used the method for tissue culture, and step is loaded down with trivial details, has increased culture cycle, and germination rate is only 84%, also exists stem bud multiplication coefficient low (multiply coefficient is 2.5), stem bud rooting rate is low (81.7%), transplantation. The problem of low plant survival rate (83.7%). Existing Tissue Culture Technology uses complex hormones in the stem and bud proliferation medium (two kinds of cytokinins and one auxin are required), which makes the cost of expanding the high-quality seedlings of T. high.

Contents of the invention

The present invention aims to solve the problems of cumbersome steps, low dormant bud germination rate, low stem bud proliferation rate, low rooting rate, low transplanting survival rate and high cost existing in the existing rapid propagation method of Astragalus japonica. body rapid propagation method.

The in vitro rapid propagation method of Aspergillus japonica of the present invention, carries out according to the following steps:

1. Hydroponic the dormant branches of Astragalus japonica at room temperature, change the water once a day, cultivate until the dormant buds of the dormant branches germinate to form new branches, cut the budded stems of the new branches for pretreatment, and then inoculate them into the multiplication On the medium, cultivate under the conditions of temperature 20-30°C, humidity 40%-80%, and light intensity 20-80 μmol·m ^-2 ·s ^-1 , with light for 12-24 hours per day, and every 10-30 days Replace the proliferation medium, cultivate for 1 to 2 months, and obtain clustered stem buds; the proliferation medium contains 1.0 to 4.0 mg/L cytokinin, 0 to 1.0 mg/L auxin, 20 to 40 g/L sucrose and 5 ~7g/L agar;

2. Select the clustered stem buds that are normally developed, and cut them in units of individual stem buds. Cut the cut single stem buds into stem segments with terminal buds with a length of 1.5 to 5.0 cm, and then cut the stems with terminal buds The stem segments are inoculated on the rooting medium, cultivated under the conditions of a temperature of 20-30°C, a humidity of 40%-80%, and a light intensity of 20-80 μmo1·m ^-2 ·s ^-1 , with 12-24 hours of light per day, The rooting medium is replaced every 10-30 days, cultivated for 1-2 months, and regenerated plants are obtained; the rooting medium contains 0.5-2.0 mg/L auxin, 20-40 g/L sucrose and 5-7 g/L agar;

3. Put the regenerated plants with a height of 3cm, well-developed roots and expanded leaves in the cultivation room for 3-5 days, then wash the medium attached to the roots, and then transplant them into the cultivation medium in the container and water them. Cover the mouth of the container with a film, and culture it under the conditions of temperature 20-30°C, humidity 60%-80%, light intensity 40-80μmol·m ^-2 ·s ^-1 , and light 12-24h per day until the new growth grows. After the leaves are fully unfolded, remove the covering plastic film to obtain the seedlings of Astragalus japonica, that is, to complete the in vitro rapid propagation of Astragalus japonica;

Wherein the proliferation medium in step 1 is an improved B5 medium, and the pH value is 5-6;

In step 1, the cytokinin is benzyl adenine, zeatin, kinetin or thiadizuron;

The auxin in step 1 is naphthalene acetic acid or indole butyric acid;

The rooting medium in step 2 is 1/2MS medium or WPM medium, and the pH value is 5～6;

In step 2, the auxin is naphthaleneacetic acid or indolebutyric acid;

In the third step, the cultivation medium is composed of 50% peat soil, 40%-50% vermiculite and 0%-10% perlite according to volume percentage.

The proliferation medium of the above in vitro rapid propagation method of Astragalus japonica is an improved B5 medium, which consists of 3000mg/L KNO ₃ , 150mg/L CaCl ₂ 2H ₂ O, 740mg/L MgSO ₄ 7H ₂ O , 150mg/L NaH ₂ PO ₄ 2H ₂ O, 0.75mg/L KI, 3mg/L H ₃ BO ₃ , 33.5mg/L MnSO ₄ 4H ₂ O, 2mg/L ZnSO ₄ 7H ₂ O, 0.25 mg/L of NaMo ₄ .2H 2 ₀ , 0.025 mg/L of CuSO ₄ .5H ₂ O, 0.025 mg/L of CoCl ₂ .6H ₂ O, 33.8 mg/L of FeSO ₄ .7H ₂ O , 37.3mg/L Na2-EDTA, 100mg/L inositol, 10mg/L thiamine hydrochloride, 1.0mg/L niacin, 0.1mg/L pyridoxine hydrochloride and distilled water.

The in vitro rapid propagation method of Aspergillus japonica in the present invention has little damage to the female parent, short propagation cycle, fast propagation speed, high reproductive efficiency (the multiplication of every month can reach 10 times), and utilizes the original The terminal buds or axillary buds directly proliferate the buds, and the resulting offspring have good genetic stability; the germination of dormant buds does not require tissue culture, which reduces the difficulty and cost of the method, and the germination rate of dormant buds reaches 100%; using improved B5 culture As a proliferation medium, the growth state of stem buds is good, the proliferation rate of stem buds reaches 100%, the proliferation coefficient of stem buds reaches more than 3, and the multiplication factor can reach 10 every month; only one kind of cells is needed in the stem bud proliferation medium The combination of mitogen and one auxin reduces the propagation cost; both the rooting rate of stems and buds and the transplanting survival rate of regenerated plants can reach more than 90%. The goals of shortening the breeding period, improving the breeding efficiency, reducing the cost and effectively expanding the number of super seedlings of Astragalus can be realized.

Detailed ways

The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

Specific implementation mode one: the in vitro rapid propagation method of willow japonica in this embodiment is carried out according to the following steps:

1. Hydroponic the dormant branches of Astragalus japonica at room temperature, change the water once a day, cultivate until the dormant buds of the dormant branches germinate to form new branches, cut the budded stems of the new branches for pretreatment, and then inoculate them into the multiplication On the medium, cultivate under the conditions of temperature 20-30°C, humidity 40%-80%, and light intensity 20-80 μmol·m ^-2 ·s ^-1 , with light for 12-24 hours per day, and every 10-30 days Replace the proliferation medium, cultivate for 1 to 2 months, and obtain clustered stem buds; the proliferation medium contains 1.0 to 4.0 mg/L cytokinin, 0 to 1.0 mg/L auxin, 20 to 40 g/L sucrose and 5 ~7g/L agar;

2. Select the clustered stem buds that are normally developed, and cut them in units of individual stem buds. Cut the cut single stem buds into stem segments with terminal buds with a length of 1.5 to 5.0 cm, and then cut the stems with terminal buds The stem segments are inoculated on the rooting medium, cultivated under the conditions of a temperature of 20-30°C, a humidity of 40%-80%, and a light intensity of 20-80 μmol·m ^-2 ·s ^-1 , with 12-24h of light per day, The rooting medium is replaced every 10-30 days, cultivated for 1-2 months, and regenerated plants are obtained; the rooting medium contains 0.5-2.0 mg/L auxin, 20-40 g/L sucrose and 5-7 g/L agar;

3. Put the regenerated plants with a height of 3cm, well-developed roots and expanded leaves in the cultivation room for 3-5 days, then wash the medium attached to the roots, and then transplant them into the cultivation medium in the container and water them. Cover the mouth of the container with a film, and culture it under the conditions of temperature 20-30°C, humidity 60%-80%, light intensity 40-80μmol·m ^-2 ·s ^-1 , and light 12-24h per day until the new growth grows. After the leaves are fully unfolded, remove the covering plastic film to obtain the seedlings of Astragalus japonica, that is, to complete the in vitro rapid propagation of Astragalus japonica;

Wherein the proliferation medium in step 1 is an improved B5 medium, and the pH value is 5-6;

In step 1, the cytokinin is benzyl adenine (BA), zeatin (ZT), kinetin (KT) or thiadizuron (TDZ);

The auxin in step 1 is naphthaleneacetic acid (NAA) or indole butyric acid (IBA);

In step 2, the rooting medium is 1/2MS medium (the medium in which the content of macroelements is halved in the MS medium) or WPM medium, and the pH value is 5-6;

In step 2, the auxin is naphthaleneacetic acid (NAA) or indole butyric acid (IBA);

In the third step, the cultivation medium is composed of 50% peat soil, 40%-50% vermiculite and 0%-10% perlite according to volume percentage.

In step one of the present embodiment, the method for pretreating the budded stem section of a new branch is to soak it in an ethanol solution with a volume concentration of 70% to 80% for 20 to 60 seconds, then rinse it with sterile water for 3 to 7 times, and then Stir and soak in mercuric chloride solution with a mass concentration of 0.05% to 0.2% for 3 to 10 minutes, then rinse with sterile water for at least 5 times, and place in sterile water for later use. Before inoculation, blot the water on the surface of the stem section with sterile filter paper, and cut the stem section with a length of 1.5-2.0 cm and 1-2 buds.

In step 1 of this embodiment, the fresh medium is replaced every 10-30 days. Before each replacement of the fresh medium, the formed stem buds can be divided into 2-5 cm long stem segments with buds, and then inoculated into fresh medium respectively. Above, it can be repeated in a cycle to obtain more stem buds with multiple multiplications, until the number of stem buds that can meet the demand is produced.

During the cultivation process after transplantation in Step 3 of this embodiment, water is sprayed into the container once a day to maintain a high humidity.

The components of the improved B5 medium, WPM medium and 1/2MS medium in this embodiment are shown in Table 1.

Table 1

In this embodiment, the in vitro rapid propagation method of Astragalus japonica has little damage to the female parent, short reproductive cycle, fast reproductive speed, high reproductive efficiency (the multiplication per month can reach 10 times), and utilizes the original The terminal buds or axillary buds directly proliferate the buds, and the resulting offspring have good genetic stability; the germination of dormant buds does not require tissue culture, which reduces the difficulty and cost of the method, and the germination rate of dormant buds reaches 100%; using the improved B5 The culture medium is used as a proliferation medium, the growth state of the stem buds is good, the proliferation rate of the stem buds reaches 100%, the proliferation coefficient of the stem buds reaches more than 3, and the multiplication factor can reach 10 every month; only one kind of stem bud proliferation medium is needed The combination of cytokinin and one auxin reduces the propagation cost; the rooting rate of stems and buds and the transplanting survival rate of regenerated plants can both reach more than 90%. The goals of shortening the breeding period, improving the breeding efficiency, reducing the cost and effectively expanding the number of super seedlings of Astragalus can be realized.

Embodiment 2: This embodiment differs from Embodiment 1 in that: the stem segment with buds in step 1 is a stem segment with terminal buds or axillary buds and a length of 2.0-3.0 cm. Others are the same as in the first embodiment.

Specific embodiment 3: The difference between this embodiment and specific embodiment 1 or 2 is that in step 1, the temperature is 22-28°C, the humidity is 50%-70%, and the light intensity is 40-60 μmol·m ^-2 ·s Cultured under the condition of ^-1 . Others are the same as in the first or second embodiment.

Embodiment 4: The difference between this embodiment and Embodiment 1 or 2 is that in Step 1, the culture is carried out under the conditions of temperature 25°C, humidity 55%, and light intensity 50 μmol·m ⁻² ·s ⁻¹ . Others are the same as in the first or second embodiment.

Embodiment 5: This embodiment differs from Embodiment 1 to Embodiment 4 in that the proliferation medium in step 1 contains 3.0 mg/L cytokinin, 0.5 mg/L auxin, 30 g/L sucrose and 6 g/L agar. Others are the same as one of the specific embodiments 1 to 4.

Embodiment 6: The difference between this embodiment and one of Embodiments 1 to 5 is that in step 2, the temperature is 22-28°C, the humidity is 50%-70%, and the light intensity is 40-60 μmol·m ^-2 · s ^-1 condition. Others are the same as one of the specific embodiments 1 to 5.

Embodiment 7: The difference between this embodiment and one of Embodiments 1 to 5 is that in step 2, the temperature is 25°C, the humidity is 60%, and the light intensity is 50 μmol·m ^-2 ·s ^-1 . . Others are the same as one of the specific embodiments 1 to 5.

Embodiment 8: This embodiment differs from Embodiment 1 to Embodiment 7 in that the rooting medium in step 2 contains 1.0 mg/L auxin, 30 g/L sucrose and 6 g/L agar. Others are the same as one of the specific embodiments 1 to 7.

Embodiment 9: This embodiment differs from Embodiments 1 to 8 in that: in step 3, the temperature is 25°C, the humidity is 70%, the light intensity is 60 μmol·m ^-2 ·s ^-1 , and the light is 16 hours per day. Cultivate under conditions until the new leaves are fully expanded. Others are the same as one of the specific embodiments 1 to 8.

Specific embodiment ten: the in vitro rapid propagation method of the present embodiment of Astragalus japonica is carried out according to the following steps:

1. Hydroponic the dormant branches of Astragalus japonica at room temperature, change the water once a day, cultivate until the dormant buds of the dormant branches germinate to form new branches, cut the budded stems of the new branches for pretreatment, and then inoculate them into the multiplication cultured on the culture medium under conditions of temperature 25°C, humidity 50%, and light intensity 40 μmol·m ^-2 ·s ^-1 , with 16 hours of light per day, and replacement of the proliferation medium every 20 days, and cultured for 1 month to obtain Clustered stem buds; the proliferation medium is the improved B5 medium containing 4.0mg/L benzyl adenine, 0.1mg/L indoleacetic acid, 20g/L sucrose and 6g/L agar;

2. Select the clustered stem buds that are normally developed, and cut them off in units of a single stem bud. Cut the single stem bud after cutting into a stem section with a terminal bud with a length of 2.0 cm, and then cut the stem section with a terminal bud Inoculate on the rooting medium, cultivate under the conditions of temperature 25°C, humidity 50%, and light intensity 40 μmol m ^-2 s ^-1 , with 16 hours of light every day, and replace the rooting medium every 20 days, and cultivate one 1 month, obtain regeneration plant; Described rooting medium is the 1/2MS medium that contains 0.5mg/L naphthaleneacetic acid, 20g/L sucrose and 6g/L agar;

3. Put the regenerated plants with a height of 3cm, well-developed roots and expanded leaves in the cultivation room for 3 days, then wash the medium attached to the roots, transplant them into the cultivation medium in the container and water them, and cover them with plastic film The mouth of the container is cultivated under the condition of temperature of 25°C, humidity of 60%, light intensity of 40 μmol·m ^-2 ·s ^-1 , and light of 16 hours per day until the new leaves are fully unfolded, and the covering plastic film is removed. Obtaining the seedlings of Astragalus japonica, that is to complete the in vitro rapid propagation of Astragalus japonica;

Wherein the pH value of proliferation medium and rooting medium is 5.8;

In the third step, the cultivation medium is composed of 50% peat soil, 40% vermiculite and 10% perlite by volume percentage.

In step one of the present embodiment, the method for pretreating the budded stem section of a new branch is to soak it in an ethanol solution with a volume concentration of 75% for 30s, then rinse it with sterile water for 5 times, and then use a concentration of 0.1% ethanol The mercuric chloride solution was stirred and soaked for 6 minutes, then rinsed with sterile water at least 5 times, and placed in sterile water for later use. Before inoculation, use sterile filter paper to blot the surface moisture of the stem section, and cut the stem section with a length of 1.5 cm and 1-2 buds.

In this embodiment, the number of multiplication per month can reach 10 times, the rooting rate of stem buds can reach 95%, and the transplanting survival rate of regenerated plants can reach 93%.

The proliferation medium described in step 1 of this embodiment was replaced with B5 medium containing 4.0 mg/L benzyl adenine, 0.1 mg/L indole acetic acid, 20 g/L sucrose and 6 g/L agar, containing 4.0 mg/L WPM medium containing 4.0 mg/L benzyl adenine, 0.1 mg/L indole acetic acid, 20 g/L sucrose and 6 g/L agar containing 4.0 mg/L benzyl adenine, 0.1 mg/L indole acetic acid, 20 g/L sucrose and the QL medium of 6g/L agar, others are the same as the present embodiment. The effects of different types of media on the proliferation rate of stem buds and the growth of stem buds are shown in Table 2.

The influence of different kinds of culture medium of table 2 on stem bud proliferation rate and stem bud growth

Note: Different lowercase letters indicate significant differences (P<0.05)

The results showed that the growth state of the stem buds using the improved B5 medium was the best, and the proliferation rate of the stem buds reached 100%.

Specific implementation mode eleven: the in vitro rapid propagation method of willow japonica in this embodiment is carried out according to the following steps:

1. Hydroponic the dormant branches of Astragalus japonica at room temperature, change the water once a day, cultivate until the dormant buds of the dormant branches germinate to form new branches, cut the budded stems of the new branches for pretreatment, and then inoculate them into the multiplication cultured on the culture medium under conditions of temperature 25°C, humidity 50%, and light intensity 40 μmol·m ^-2 ·s ^-1 , with 16 hours of light per day, and replacement of the proliferation medium every 20 days, and cultured for 1 month to obtain Clustered stem buds; the proliferation medium is the improved B5 medium containing 4.0mg/L benzyl adenine, 0.1mg/L indoleacetic acid, 20g/L sucrose and 6g/L agar;

2. Select the clustered stem buds that are normally developed, and cut them off in units of a single stem bud. Cut the single stem bud after cutting into a stem section with a terminal bud with a length of 2.0 cm, and then cut the stem section with a terminal bud Inoculate on the rooting medium, cultivate under the conditions of temperature 25°C, humidity 50%, and light intensity 40 μmol m ^-2 s ^-1 , with 16 hours of light every day, and replace the rooting medium every 20 days, and cultivate one 1 month, obtain regeneration plant; Described rooting medium is the 1/2MS medium that contains 0.5mg/L naphthaleneacetic acid, 20g/L sucrose and 6g/L agar;

3. Put the regenerated plants with a height of 3cm, well-developed roots and expanded leaves in the cultivation room for 3 days, then wash the medium attached to the roots, transplant them into the cultivation medium in the container and water them, and cover them with plastic film The mouth of the container is cultivated under the condition of temperature of 25°C, humidity of 60%, light intensity of 40 μmol·m ^-2 ·s ^-1 , and light of 16 hours per day until the new leaves are fully unfolded, and the covering plastic film is removed. Obtaining the seedlings of Astragalus japonica, that is to complete the in vitro rapid propagation of Astragalus japonica;

Wherein the pH value of proliferation medium and rooting medium is 5.8;

In the third step, the cultivation medium is composed of 50% peat soil and 50% vermiculite by volume percentage.

In step one of the present embodiment, the method for pretreating the budded stem section of a new branch is to soak it in an ethanol solution with a volume concentration of 75% for 30s, then rinse it with sterile water for 5 times, and then use a concentration of 0.1% ethanol The mercuric chloride solution was stirred and soaked for 6 minutes, then rinsed with sterile water at least 5 times, and placed in sterile water for later use. Before inoculation, use sterile filter paper to blot the surface moisture of the stem section, and cut the stem section with a length of 1.5 cm and 1-2 buds.

In this embodiment, the germination rate of dormant buds can reach 100%, the stem bud proliferation rate can reach 100%, the stem bud proliferation coefficient can reach 3, the number of multiplication per month can reach 10 times, and the stem bud rooting rate can reach 95%. The survival rate of transplanted regenerated plants can reach 90%.

Specific embodiment 12: the in vitro rapid propagation method of the present embodiment willow, follow the steps below:

1. Hydroponic the dormant branches of Astragalus japonica at room temperature, change the water once a day, cultivate until the dormant buds of the dormant branches germinate to form new branches, cut the budded stems of the new branches for pretreatment, and then inoculate them into the multiplication cultured on the culture medium under conditions of temperature 25°C, humidity 50%, and light intensity 40 μmol·m ^-2 ·s ^-1 , with 16 hours of light per day, and replacement of the proliferation medium every 20 days, and cultured for 1 month to obtain Clustered stem buds; the proliferation medium is the improved B5 medium containing 4.0mg/L benzyl adenine, 20g/L sucrose and 6g/L agar;

2. Select the clustered stem buds that are normally developed, and cut them off in units of a single stem bud. Cut the single stem bud after cutting into a stem segment with a terminal bud with a length of 2.0 cm, and then cut the stem segment with a terminal bud Inoculate on the rooting medium, cultivate under the conditions of temperature 25°C, humidity 50%, and light intensity 40 μmol m ^-2 s ^-1 , with 16 hours of light every day, and replace the rooting medium every 20 days, and cultivate one 1 month, obtain regeneration plant; Described rooting medium is the 1/2MS medium that contains 0.5mg/L naphthaleneacetic acid, 20g/L sucrose and 6g/L agar;

3. Put the regenerated plants with a height of 3cm, well-developed roots and expanded leaves in the cultivation room for 3 days, then wash the medium attached to the roots, transplant them into the cultivation medium in the container and water them, and cover them with plastic film The mouth of the container is cultivated under the condition of temperature of 25°C, humidity of 60%, light intensity of 40 μmol·m ^-2 ·s ^-1 , and light of 16 hours per day until the new leaves are fully unfolded, and the covering plastic film is removed. Obtaining the seedlings of Astragalus japonica, that is to complete the in vitro rapid propagation of Astragalus japonica;

Wherein the pH value of proliferation medium and rooting medium is 5.8;

In the third step, the cultivation medium is composed of 50% peat soil, 40% vermiculite and 10% perlite by volume percentage.

In step one of the present embodiment, the method for pretreating the budded stem section of a new branch is to soak it in an ethanol solution with a volume concentration of 75% for 30s, then rinse it with sterile water for 5 times, and then use a concentration of 0.1% ethanol The mercuric chloride solution was stirred and soaked for 6 minutes, then rinsed with sterile water at least 5 times, and placed in sterile water for later use. Before inoculation, use sterile filter paper to blot the surface moisture of the stem section, and cut the stem section with a length of 1.5 cm and 1-2 buds.

In this embodiment, the germination rate of dormant buds can reach 100%, the stem bud proliferation rate can reach 100%, the stem bud proliferation coefficient can reach 3, the number of multiplication per month can reach 10 times, and the stem bud rooting rate can reach 90%. The survival rate of transplanted regenerated plants can reach 93%.

Specific embodiment thirteen: In this embodiment, the proliferation medium of the in vitro rapid propagation method of Astragalus japonica is an improved B5 medium, which consists of 3000 mg/L KNO ₃ , 150 mg/L CaCl ₂ ·2H ₂ O, 740 mg/L L of MgSO ₄ ·7H ₂ O, 150mg/L of NaH ₂ PO ₄ ·2H ₂ O, 0.75mg/L of KI, 3mg/L of H ₃ BO ₃ , 33.5mg/L of MnSO ₄ ·4H ₂ O, 2mg/L of ZnSO ₄ ·7H ₂ O, 0.25mg/L of NaMo ₄ ·2H ₂ O, 0.025mg/L of CuSO ₄ ·5H ₂ O, 0.025mg/L of CoCl ₂ ·6H ₂ O, 33.8mg/L L of FeSO ₄ ·7H ₂ O, 37.3mg/L of Na ₂ -EDTA, 100mg/L of inositol, 10mg/L of thiamine hydrochloride, 1.0mg/L of niacin, 0.1mg/L of pyrimidine hydrochloride Duoxin and distilled water.

The pH value of the modified B5 medium is 5-6.

Using the improved B5 medium of this embodiment as the proliferation medium, the growth state of the stem buds is better, the plants are taller, and the stem segments are thick, the leaves are dark green and well developed; the proliferation rate of the stem buds reaches 100%.

The preparation method of the improved B5 medium in this embodiment is as follows: each component is dissolved in distilled water respectively, mixed evenly, and sterilized under high temperature and high pressure at 121° C. and 0.105 MPa for 20 minutes.

Claims (10)

1. the rapid propagation in vitro method of Fraxinus rhynchophylla is characterized in that the rapid propagation in vitro method of Fraxinus rhynchophylla, carries out according to the following steps:

One, the resting shoot with Fraxinus rhynchophylla carries out water planting in room temperature; Change primary water every day; The sleeping bud that is cultured to resting shoot is sprouted the new branch of formation; The stem with bud that cuts new branch carries out preliminary treatment, is inoculated into then on the proliferated culture medium, and be that 20～30 ℃, humidity are 40%～80%, intensity of illumination is 20～80 μ molm in temperature ^-2S ^-1Condition under cultivate, illumination every day 12～24h changed proliferated culture medium in per 10～30 days, cultivated the acquisition stem eye that grows thickly 1～2 month; Said proliferated culture medium contains 1.0～4.0mg/L basic element of cell division, 0～1.0mg/L growth hormone, 20～40g/L sucrose and 5～7g/L agar;

Two, select the stem eye that grows thickly of normal development; With single stem eye is that unit cuts off; Single stem eye after cutting off is cut into the stem section that length is the band terminal bud of 1.5～5.0cm; To be inoculated on the root media with the stem section of terminal bud then, is that 20～30 ℃, humidity are 40%～80%, intensity of illumination is 20～80 μ molm in temperature ^-2S ^-1Condition under cultivate, illumination every day 12～24h, per 10～30 days change root media, cultivate the acquisition regeneration plant 1～2 month; Said root media contains 0.5～2.0mg/L growth hormone, 20～40g/L sucrose and 5～7g/L agar;

Three, plant height is reached regeneration plant uncovered refining seedling 3～5d in culturing room of 3cm, well developed root system and mounted blade; Clean the medium that root system adheres to then; Be transplanted in the cultivation matrix in the container again and water; With plastic film covering container mouth, be that 20～30 ℃, humidity are 60%～80%, intensity of illumination is 40～80 μ molm in temperature ^-2S ^-1, illumination every day 12～24h condition under be cultured to the young leaves that grows and launch fully after, remove the plastic film of covering, obtain the Fraxinus rhynchophylla nursery stock, promptly accomplish the rapid propagation in vitro of Fraxinus rhynchophylla;

Wherein proliferated culture medium is the B5 medium of improvement in the step 1, and the pH value is 5～6;

The basic element of cell division is benzyladenine, zeatin, kinetin or Thidiazuron in the step 1;

Growth hormone in the step 1 is methyl or indolebutyric acid;

Root media is 1/2MS medium or WPM medium in the step 2, and the pH value is 5～6;

Growth hormone is methyl or indolebutyric acid in the step 2;

In the step 3 cultivation matrix by volume percentage composition form by 50% turfy soil, 40%～50% vermiculite and 0%～10% perlite.

2. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 1 is characterized in that the stem with bud in the step 1 is to have the stem section that terminal bud or axillalry bud and length are 2.0～3.0cm.

3. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 1 and 2 is characterized in that in the step 1 in temperature being that 22～28 ℃, humidity are 50%～70%, intensity of illumination is 40～60 μ molm ^-2S ^-1Condition under cultivate.

4. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 1 and 2 is characterized in that in the step 1 in temperature being that 25 ℃, humidity are 55%, intensity of illumination is 50 μ molm ^-2S ^-1Condition under cultivate.

5. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 3 is characterized in that proliferated culture medium contains the 3.0mg/L basic element of cell division, 0.5mg/L growth hormone, 30g/L sucrose and 6g/L agar in the step 1.

6. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 5 is characterized in that in the step 2 in temperature being that 22～28 ℃, humidity are 50%～70%, intensity of illumination is 40～60 μ molm ^-2S ^-1Condition under cultivate.

7. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 5 is characterized in that in the step 2 in temperature being that 25 ℃, humidity are 60%, intensity of illumination is 50 μ molm ^-2S ^-1Condition under cultivate.

8. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 6 is characterized in that root media contains 1.0mg/L growth hormone, 30g/L sucrose and 6g/L agar in the step 2.

9. the rapid propagation in vitro method of Fraxinus rhynchophylla according to claim 8 is characterized in that in the step 3 in temperature being that 25 ℃, humidity are 70%, intensity of illumination is 60 μ molm ^-2S ^-1, illumination every day 16h condition under be cultured to the young leaves that grows and launch fully.

10. the proliferated culture medium of the rapid propagation in vitro method of the described Fraxinus rhynchophylla of claim 1 is characterized in that the proliferated culture medium of the rapid propagation in vitro method of Fraxinus rhynchophylla is the B5 medium of improvement, by the KNO of 3000mg/L ₃, 150mg/L CaCl ₂2H ₂The MgSO of O, 740mg/L ₄7H ₂The NaH of O, 150mg/L ₂PO ₄2H ₂The KI of O, 0.75mg/L, the H of 3mg/L ₃BO ₃, 33.5mg/L MnSO ₄4H ₂The ZnSO of O, 2mg/L ₄7H ₂The NaMo of O, 0.25mg/L ₄2H ₂The CuSO of O, 0.025mg/L ₄5H ₂The CoCl of O, 0.025mg/L ₂6H ₂The FeSO of O, 33.8mg/L ₄7H ₂The Na of O, 37.3mg/L ₂The nicotinic acid of the inositol of-EDTA, 100mg/L, the thiamine hydrochloride of 10mg/L, 1.0mg/L, pyridoxine hydrochloride and the distilled water of 0.1mg/L are processed.