阅读说明：本技术 一种高效的海马齿再生体系建立方法 (Method for establishing efficient hippocampus regeneration system ) 是由 王丹 魏翔莺 叶桂萍 何伟红 杨楠 于 2021-06-30 设计创作，主要内容包括：本发明公开了一种高效的海马齿的再生体系建立方法,属于植物快速繁殖再生技术领域,解决海马齿再生效率低的问题。所述一种高效的生海马齿的再生体系建立方法,包括以下步骤：海马齿无菌苗的准备；海马齿不定芽的诱导及成苗；再生苗的移栽与管理。采用本发明的方法能够高效诱导海马齿不定芽生成及后续成苗。(The invention discloses a method for establishing a high-efficiency regeneration system of hippocampus, belongs to the technical field of plant rapid propagation and regeneration, and solves the problem of low regeneration efficiency of hippocampus. The efficient method for establishing the regeneration system of the raw hippocampus comprises the following steps: preparing a hippocampus aseptic seedling; inducing and seedling the adventitious bud of the hippocampus; transplanting and managing the regenerated seedlings. The method can efficiently induce the generation of the adventitious bud of the hippocampus and the subsequent seedling formation.)

1. A method for establishing an efficient hippocampus regeneration system is characterized by comprising the following steps:

(1) preparing the sea horse teeth aseptic seedlings: in a superclean workbench, the collected mature seeds of the hippocastanum are sown in a sowing culture medium after surface sterilization, and are cultured in a constant-temperature illumination incubator at 24 ℃ for 1-2 months to obtain sterile seedlings of the hippocastanum for later use;

(2) inducing and seedling the adventitious bud of the hippocampus: taking the leaves of the hippocastanum aseptic seedlings in the step (1) in a superclean bench, cutting off two ends of the leaves, cutting the middle part of the leaves into small sections with the length of 0.5cm, and placing the leaf quilt facing upwards on a differentiation culture medium; culturing in 24 deg.C incubator at dark place for 10 days, and culturing in 16h light and 8 h dark incubator for 2-3 months; observing the induction of the leaf callus and the differentiation of adventitious buds during the period, and counting the differentiation rate of the callus and the adventitious buds; then, cutting the adventitious bud from the callus, transferring the cut adventitious bud to a rooting culture medium, culturing for 20-30 days, cutting a single adventitious bud, and inducing the adventitious bud to grow into a root and a seedling on the rooting culture medium;

(3) transplanting and managing regenerated seedlings: taking the regenerated seedlings rooted in the step (2) out of the culture medium, washing the culture medium on the surface of the root system with tap water, planting the washed culture medium in a plug tray, watering the plug tray thoroughly, and covering a preservative film for moisturizing and hardening the seedlings; after one week, the preservative film is gradually uncovered, normal culture is carried out in a climatic chamber with the temperature of 24 ℃ and the humidity of 75 percent, and the nutrient solution is irrigated once every other week.

2. The method for establishing a high-efficiency hippocampus regeneration system according to claim 1, wherein: the surface sterilization of the seeds in the step (1) is performed by using 75vol% ethanol for 30-90s, then using 10vol% NaClO for sterilization for 5-15 min, continuously shaking during the sterilization to fully clean the seeds, and finally using sterile water to wash for 3-5 times.

3. The method for establishing a high-efficiency hippocampus regeneration system according to claim 1, wherein: the seeding culture medium in the step (1) comprises the following components: 2.22g/L MS culture medium, 30g/L sucrose and 7g/L agar, and the pH value is between 5.8 and 6.0.

4. The method for establishing a high-efficiency hippocampus regeneration system according to claim 1, wherein the differentiation medium in step (2) comprises: 4.43g/L MS culture medium, 1.5-3.0 mg/L zeatin, 0-0.75 mg/L indoleacetic acid, 30g/L sucrose and 3g/L plant gel, and the pH value is 5.6-6.0.

5. The method for establishing a high-efficiency hippocampus regeneration system according to claim 1, wherein the rooting medium in the step (2) comprises the following components: 4.43g/L MS culture medium, 0-0.4 mg/L IAA, 30g/L sucrose and 3g/L plant gel, and the pH value is 5.6-6.0.

6. The method for establishing a high-efficiency regeneration system for the hippocampus according to claim 1, wherein the soil for cultivation in the transplanting hole tray in the step (3) is prepared by mixing a substrate and sand in a ratio of 2: 1, mixing, and spreading in a plug tray for later use.

7. The method for establishing the high-efficiency hippocampus regeneration system according to claim 1, wherein the formula of the nutrient solution for irrigation in the step (3) is as follows: macroelement of 0.10 g/L KNO₃，0.03 g/L MgSO₄，0.23 g/L Ca(NO₃)₂·4H₂O，0.02 g/L NH₄H₂PO₄，0.007 g/L Na₂FeEDTA; and trace elements: 3 μmol/L H₃BO₃，0.5 μmol/L MnCl₂·4H₂O，0.2 μmol/L CuSO₄·5H₂O，0.3 μmol/L ZnSO₄· 7H₂O, pH value is 5.4-5.8.

8. The use of a method of claim 1 for the production of a sea horse teeth biomaterial by establishing a highly efficient sea horse teeth regeneration system.

Technical Field

The invention belongs to the technical field of rapid propagation and regeneration of plants, and particularly relates to a method for establishing a high-efficiency hippocampus regeneration system.

Background

The hippocampus which is a mangrove associated plant can keep good growth in severe environments such as high salt, drought and heavy metal pollution, and has important research and application values on the restoration of ecological environment. The hippocampus japonicus is used as a pioneer plant for environmental remediation, and the excavation of excellent genes in the hippocampus and the creation of a novel hippocampus germplasm with higher environmental remediation efficiency by combining a transgenic technology are a key direction of the current plant remediation research. The establishment of the hippocampus efficient regeneration system is an important problem for gene function research and transgenic work development, and has important application value in aspects of hippocampus germplasm innovation and new variety breeding.

Disclosure of Invention

The invention aims to provide a method for establishing a high-efficiency hippocampus regeneration system.

In order to achieve the purpose, the invention adopts the following technical scheme.

An efficient hippocampus regeneration system establishing method comprises the following steps:

(1) preparing the sea horse teeth aseptic seedlings: in a superclean workbench, the collected mature seeds of the hippocastanum are sown in a sowing culture medium after surface sterilization, and are cultured in a constant-temperature illumination incubator at 24 ℃ for 1-2 months to obtain the hippocastanum aseptic seedlings for later use;

(2) inducing and seedling the adventitious bud of the hippocampus: taking the leaves of the hippocastanum aseptic seedlings in the step (1) in a superclean bench, cutting off two ends of the leaves, cutting the middle part of the leaves into small sections with the length of 0.5cm, and placing the leaf quilt facing upwards on a differentiation culture medium; culturing in 24 deg.C incubator at dark place for 10 days, and culturing in 16h light and 8 h dark incubator for 2-3 months; observing the induction of the leaf callus and the differentiation of adventitious buds during the period, and counting the differentiation rate of the callus and the adventitious buds; then, cutting the adventitious bud from the callus, transferring the cut adventitious bud to a rooting culture medium, culturing for 20-30 days, cutting a single adventitious bud, and inducing the adventitious bud to grow into a root and a seedling on the rooting culture medium;

(3) transplanting and managing regenerated seedlings: taking the regenerated seedlings rooted in the step (2) out of the culture medium, washing the culture medium on the surface of the root system with tap water, planting the washed culture medium in a plug tray, watering the plug tray thoroughly, and covering a preservative film for moisturizing and hardening the seedlings; after one week, the preservative film is gradually uncovered, normal culture is carried out in a climatic chamber with the temperature of 24 ℃ and the humidity of 75 percent, and the nutrient solution is irrigated once every other week.

The surface sterilization of the seeds in the step (1) is performed by using 75vol% ethanol for 30-90s, then using 10vol% NaClO for sterilization for 5-15 min, continuously shaking during the sterilization to fully clean the seeds, and finally washing the seeds with sterile water for 3-5 times.

The seeding culture medium in the step (1) comprises the following components: 2.22g/L MS culture medium, 30g/L sucrose and 7g/L agar, and the pH value is between 5.6 and 6.0.

The components of the differentiation medium in the step (2) are as follows: 4.43g/L MS culture medium, 1.5-3.0 mg/L Zeatin (ZT), 0-0.75 mg/L indoleacetic acid (IAA), 30g/L sucrose and 3g/L plant gel, and the pH value is 5.6-6.0.

The rooting culture medium in the step (2) comprises the following components: 4.43g/L MS culture medium, 0-0.4 mg/L indoleacetic acid, 30g/L sucrose and 3g/L plant gel, and the pH value is 5.6-6.0.

The soil for cultivation transplanted into the plug in the step (3) is prepared by mixing a substrate and sand in a ratio of 2: 1, mixing, and spreading in a plug tray for later use. The formula of the nutrient solution for irrigation is as follows: macroelement 0.10 g/L KNO₃，0.03 g/L MgSO₄，0.23 g/L Ca(NO₃)₂·4H₂O，0.02 g/L NH₄H₂PO₄，0.007 g/L Na₂FeEDTA and trace elements (3. mu. mol/L H)₃BO₃，0.5 μmol/L MnCl₂·4H₂O，0.2 μmol/L CuSO₄·5H₂O，0.3 μmol/L ZnSO₄· 7H₂O), the pH value is 5.4-5.8.

The method for establishing the efficient hippocampus regeneration system is applied to preparation of the hippocampus biomaterial.

The invention has the advantages that:

an efficient hippocampus regeneration system is established, the operation steps are simple, the differentiation rate of callus and adventitious buds is high, and the creation period of excellent and new hippocampus germplasm can be greatly shortened by combining a transgenic technology.

Description of the drawings:

FIG. 1 shows the establishment of the regeneration system of the hippocampus. (A) Culturing the hippocampus aseptic seedlings; (B-C) inducing callus using leaf discs; (D-E) callus differentiation to produce adventitious buds; (F) the adventitious bud grows into a seedling on a rooting culture medium.

FIG. 2 is a diagram of the transplanting and hardening of the regeneration seedlings of the hippocampus.

Detailed Description

For a better understanding of the present invention, reference is made to the following examples and accompanying drawings which are set forth to illustrate, but are not to be construed as the limit of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Example 1

An efficient hippocampus regeneration system establishing method comprises the following steps:

(1) preparing the sea horse teeth aseptic seedlings:

firstly, preparing a seeding culture medium: 2.22g/L MS culture medium, 30g/L sucrose and 7g/L agar, the pH value is 6.0, and the mixture is poured into a sterile can bottle for standby after being autoclaved and sterilized in a super clean bench.

In a superclean workbench, disinfecting the surface of the collected mature seed of the hippocastanum, sowing the seed in a sowing culture medium, and culturing for 1 month in a constant-temperature illumination incubator at 24 ℃ to obtain an aseptic seedling of the hippocastanum for later use;

the surface disinfection of the seeds is to adopt 75vol% ethanol for disinfection for 90s, then adopt 10vol% NaClO for disinfection for 5 min, continuously shake during the disinfection to ensure that the seeds are fully cleaned, and finally wash the seeds with sterile water for 5 times.

(2) Inducing and seedling the adventitious bud of the hippocampus: firstly, preparing a differentiation medium: 4.43g/L MS basal medium +1.5 mg/L ZT +30 g/L sucrose +3g/L plant gel, and pouring into a sterile culture dish and a can bottle for later use in a super clean workbench after autoclaving.

Taking the leaves of the hippocastanum aseptic seedlings in the step (1) in a super-clean workbench, cutting off two ends of the leaves, cutting the middle part of the leaves into small sections with the length of 0.5cm, and placing the leaf quilt on a differentiation culture medium in a face-up mode. Culturing in 24 deg.C incubator at dark place for 10 days, culturing in 16h light and 8 h dark incubator for 90 days, and inducing leaf disc differentiation callus. During this period, excised callus was subcultured on differentiation medium until adventitious buds were produced.

The adventitious buds are cut from the callus and transferred to a rooting medium (4.43 g/L MS basic medium +0.2 mg/L IAA +30 g/L sucrose + 7g/L agar), the adventitious buds are cultured for 20 days, single stem segments are cut after the adventitious buds grow up, and the single stem segments are inserted into the rooting medium to induce the adventitious buds to grow roots and grow seedlings.

(3) Transplanting and managing regenerated seedlings: and (3) taking the regenerated seedlings rooted in the step (2) out of the culture medium, washing the culture medium on the surface of the root system with tap water, planting the washed culture medium in a plug tray, watering thoroughly, and covering a preservative film for moisturizing and hardening the seedlings. After one week, the preservative film is gradually uncovered, and the cultivation is normally carried out in a climatic chamber with sufficient illumination, 24 ℃ and 75% humidity, and the cultivation is watered once every other week by using nutrient solution.

The soil for cultivation in the transplanting hole tray is prepared by mixing a substrate and sand in a ratio of 2: 1, mixing, and spreading in a plug tray for later use.

The formula of the nutrient solution for irrigation is as follows: macroelements: 0.10 g/L KNO₃，0.03 g/L MgSO₄，0.23 g/L Ca(NO₃)₂·4H₂O，0.02 g/L NH₄H₂PO₄，0.007 g/L Na₂FeEDTA; and trace elements: 3 μmol/L H₃BO₃，0.5 μmol/L MnCl₂·4H₂O，0.2 μmol/L CuSO₄·5H₂O，0.3 μmol/L ZnSO₄· 7H₂O, pH 5.8.

In the embodiment, the callus induction rate of the hippocampus reaches 100 percent, and the adventitious bud differentiation rate reaches 73.66 percent.

Example 2

An efficient hippocampus regeneration system establishing method comprises the following steps:

(1) preparing the sea horse teeth aseptic seedlings:

firstly, preparing a seeding culture medium: 2.22g/L MS culture medium, 30g/L sucrose and 7g/L agar, and pouring the mixture into a sterile can bottle for later use in a super clean bench after autoclaving.

In a superclean workbench, disinfecting the surface of the collected mature seed of the hippocastanum, sowing the seed in a sowing culture medium, and culturing for 1 month in a constant-temperature illumination incubator at 24 ℃ to obtain sterile hippocastanum seedlings for later use;

the surface disinfection of the seeds is to adopt 75vol% ethanol for disinfection for 30s, then adopt 10vol% NaClO for disinfection for 15 min, continuously shake during the disinfection to ensure that the seeds are fully cleaned, and finally wash the seeds with sterile water for 3 times.

(2) Inducing and seedling the adventitious bud of the hippocampus: firstly, preparing a differentiation medium: 4.43g/L MS basal medium, 2 mg/L ZT, 0.5 mg/L IAA, 30g/L cane sugar and 3g/L plant gel are poured into a sterile culture dish and a can bottle for standby after being autoclaved. Taking the leaf of the aseptic seedling of the hippocampus in a superclean bench, cutting off two ends of the leaf, cutting the middle part of the leaf into small sections with the length of 0.5cm, and placing the leaf quilt on a differentiation culture medium with the leaf quilt facing upwards. Culturing in 24 deg.C incubator at dark place for 10 days, culturing in 16h light and 8 h dark incubator for 60 days, and inducing leaf disc differentiation callus. Subsequently, excised callus was subcultured on a differentiation medium until adventitious buds were produced.

The adventitious buds are cut from the callus and transferred to a rooting culture medium (4.43 g/L MS basic culture medium +0.4mg/L IAA +30 g/L sucrose + 7g/L agar) for culturing for 25 days, and single stem segments are cut after the adventitious buds grow up and inserted into the rooting culture medium to induce the adventitious buds to grow roots and grow seedlings.

(3) Transplanting and managing regenerated seedlings: and (3) taking the regenerated seedlings rooted in the step (2) out of the culture medium, washing the culture medium on the surface of the root system with tap water, planting the washed culture medium in a plug tray, watering the plug tray thoroughly, and covering a preservative film on the plug tray for moisturizing and hardening the seedlings. After one week, the preservative film is gradually uncovered, and the cultivation is normally carried out in a climatic chamber with sufficient illumination, 24 ℃ and 75% humidity, and the cultivation is watered once every other week by using nutrient solution.

The soil for cultivation in the transplanting hole tray is prepared by mixing a substrate and sand in a ratio of 2: 1, mixing, and spreading in a plug tray for later use.

The formula of the nutrient solution for irrigation is as follows: macroelements: 0.10 g/L KNO₃，0.03 g/L MgSO₄，0.23 g/L Ca(NO₃)₂·4H₂O，0.02 g/L NH₄H₂PO₄，0.007 g/L Na₂FeEDTA; and trace elements: 3 μmol/L H₃BO₃，0.5 μmol/L MnCl₂·4H₂O，0.2 μmol/L CuSO₄·5H₂O，0.3 μmol/L ZnSO₄· 7H₂O, pH 5.4.

In the embodiment, the callus induction rate of the hippocampus reaches 96.3 percent, and the adventitious bud differentiation rate reaches 56.7 percent.

Example 3

An efficient hippocampus regeneration system establishing method comprises the following steps:

(1) preparing the sea horse teeth aseptic seedlings:

firstly, preparing a seeding culture medium: 2.22g/L MS culture medium, 30g/L sucrose and 7g/L agar, the pH value is 6.0, and the mixture is poured into a sterile can bottle for standby after being autoclaved and sterilized in a super clean bench.

In a superclean workbench, disinfecting the surface of the collected mature seed of the hippocastanum, sowing the seed in a sowing culture medium, and culturing for 1 month in a constant-temperature illumination incubator at 24 ℃ to obtain an aseptic seedling of the hippocastanum for later use;

the surface sterilization of the seeds is to sterilize the seeds for 60s by using 75vol% ethanol, then sterilize the seeds for 10 min by using 10vol% NaClO, shake the seeds continuously during the sterilization to ensure that the seeds are cleaned fully, and finally wash the seeds for 4 times by using sterile water.

Inducing and seedling the adventitious bud of the hippocampus: firstly, preparing a differentiation medium: 4.43g/L MS basal medium, 2.5 mg/L ZT, 0.75 mg/L IAA, 30g/L cane sugar and 3g/L plant gel are poured into a sterile culture dish and a can bottle for standby after being autoclaved.

Taking the leaves of the hippocastanum aseptic seedlings in the step (1) in a super-clean workbench, cutting off two ends of the leaves, cutting the middle part of the leaves into small sections with the length of 0.5cm, and placing the leaf quilt on a differentiation culture medium in a face-up mode. Culturing in 24 deg.C incubator in dark for 10 days, culturing in 16h light and 8 h dark incubator for 90 days, and inducing leaf disc differentiation callus. During this period, excised callus was subcultured on differentiation medium until adventitious buds were produced.

The adventitious buds are cut from the callus and transferred to a rooting culture medium (4.43 g/L MS basic culture medium +30 g/L sucrose + 7g/L agar), the adventitious buds are cultured for 30 days, single plant stem segments are cut after the adventitious buds grow up, and the single plant stem segments are inserted into the rooting culture medium to induce the adventitious buds to grow roots and grow seedlings.

(3) Transplanting and managing regenerated seedlings: and (3) taking the regenerated seedlings rooted in the step (2) out of the culture medium, washing the culture medium on the surface of the root system with tap water, planting the washed culture medium in a plug tray, watering thoroughly, and covering a preservative film for moisturizing and hardening the seedlings. After one week, the preservative film is gradually uncovered, normal culture is carried out in a climatic chamber with sufficient illumination, 24 ℃ and 75% humidity, and the plastic film is irrigated with nutrient solution once every other week.

The soil for cultivation in the transplanting hole tray is prepared by mixing a substrate and sand in a ratio of 2: 1, mixing, and spreading in a plug tray for later use.

The formula of the nutrient solution for irrigation is as follows: macroelements: 0.10 g/L KNO₃，0.03 g/L MgSO₄，0.23 g/L Ca(NO₃)₂·4H₂O，0.02 g/L NH₄H₂PO₄，0.007 g/L Na₂FeEDTA; and trace elements: 3 μmol/L H₃BO₃，0.5 μmol/L MnCl₂·4H₂O，0.2 μmol/L CuSO₄·5H₂O，0.3 μmol/L ZnSO₄· 7H₂O, pH 5.6.

In the embodiment, the callus induction rate of the hippocampus reaches 96 percent, and the adventitious bud differentiation rate reaches 30 percent.