CN112931218A

CN112931218A - Method for aseptically sowing and culturing hippocampus seeds

Info

Publication number: CN112931218A
Application number: CN202110385636.2A
Authority: CN
Inventors: 王丹; 魏翔莺; 郑秀霞; 叶桂萍; 何伟红
Original assignee: Minjiang University
Current assignee: Minjiang University
Priority date: 2021-04-10
Filing date: 2021-04-10
Publication date: 2021-06-11

Abstract

The invention relates to a method for aseptically sowing and culturing hippocampus seeds, which comprises the following steps: 1) collecting the seed of the hippocampus; 2) preparing a hippocampus germchit sterile seeding culture medium; 3) sterilizing the surface of the hippocampus seed; 4) and (5) aseptically sowing the hippocampus. The invention establishes the method for sterilizing the surface of the hippocampus dentis seeds, can reduce the pollution rate, improve the germination rate of the hippocampus dentis seeds, quickly obtain a large amount of sterile seedling seedlings with stable genetic property, and provide a high-quality experimental material for the subsequent research of a hippocampus tissue culture rapid propagation system.

Description

Method for aseptically sowing and culturing hippocampus seeds

Technical Field

The invention relates to a method for disinfection culture of hippocampus japonicus seeds, belonging to the field of plant culture.

Background

Haima tooth (A)Sesuvium portulacastrum) Also called as waterfront vegetable and sow thistle, is a perennial fleshy herbaceous halophyte with a flowering period of 4-7 months. The hippocampus is a typical coastal plant, has wide salt tolerance range, and can complete the life history in seawater, fresh water, and salinized and non-salinized environments. The hippocampus has strong stress resistance, can resist drought, salt and various heavy metal ions, has unique genetic background, is a good research material for salt and drought resistance, and the like, and can be used as an ecological restoration pioneer plant for ecological restoration of coastal wetlands and seawater environments. In China, Hainan province, Guangdong province and southeast AsiaIn China, the sea horse teeth are eaten as vegetables, also contain a plurality of effective medicinal components, and have certain economic value. Therefore, the hippocampus is a rare plant with both research value and development value.

In the research work of the hippocampus, the hippocampus which is sampled in the field is mostly used, the field environment is complex, so that the hippocampus has different genetic backgrounds, and experimental errors are caused. The tissue culture method for inducing callus by taking roots, stems and leaves as explants is complex and has long period. Therefore, a rapid and efficient breeding method for the hippocampus is lacked at present.

Disclosure of Invention

To solve the above technical problems. The invention provides a method for cultivating sea horse teeth seeds in a disinfection manner, which provides technical support for efficiently obtaining sea horse teeth sterile plants.

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

a method for disinfecting and planting sea horse teeth seeds comprises the following steps:

1) collecting the hippocampus seeds: the method comprises the following steps of collecting hippocampus seeds from hippocampus cultivated in a sea reclamation floating bed after a Pu field, collecting black seeds after mature seed pods crack, drying in a constant-temperature incubator at 37 ℃, collecting the seeds in a centrifuge tube, and storing the seeds in a refrigerator at 4 ℃ for later use;

2) preparing a culture medium: preparing a solid culture medium, sterilizing at 121 ℃ for 20-25 minutes, subpackaging in culture dishes or tissue culture bottles, and cooling for later use, wherein each dish is 25 ml;

3) seed disinfection: the seeds are separately placed in 2 ml centrifuge tubes, 25-35 grains per tube, 1ml 75% alcohol is added, the upside is reversed for 10 times, the seeds are washed, and the liquid is removed by a pipette. Then 1ml of sterile water is added, the mixture is turned upside down 10 times, the seeds are washed, the liquid is sucked off by a pipette, and the step is repeated for 2-3 times. Adding 1ml of commercial 84 disinfectant (the available chlorine content is 3.92-5.30%), disinfecting for 30 minutes, changing the disinfectant every 6 minutes, disinfecting for 1-5 times, and continuously turning the centrifugal tube upside down in the disinfection process to ensure that the surfaces of the seeds are fully contacted with the disinfectant. Then, cleaning the seeds for 3-5 times by using sterile water, and placing the sea horse teeth seeds with the sterilized surfaces into a sterile 9 cm culture dish for sowing;

4) sowing and culturing: the seeds were gently gripped with sterilized and cooled tweezers and placed on solid medium, 5 seeds per dish. Culturing the sown seeds in artificial-climate illumination incubator at 23-26 deg.C for 16 hr in 8 hr dark place with illumination intensity of 80 lmol.m^-2.s^-1Humidity is 60%; culturing for 15-30 days, transferring the germinated seeds to a tissue culture bottle, and continuously culturing for growing.

The solid culture medium in the step (2) comprises the following components: 1/2 MS culture medium +15 g/L sucrose +8 g/L agar, pH adjusted to 5.6-6.0.

The formula of the 1/2 MS culture medium is as follows: 825 mg/L of ammonium nitrate, 950 mg/L of potassium nitrate, 220 mg/L of calcium chloride dihydrate, 185 mg/L of magnesium sulfate heptahydrate, 350 mg/L of monopotassium phosphate, 0.83 mg/L of potassium iodide, 6.2 mg/L of boric acid, 22.3 mg/L of manganese sulfate tetrahydrate, 8.6 mg/L of zinc sulfate heptahydrate, 0.25 mg/L of sodium manganate dihydrate, 0.025 mg/L of copper sulfate pentahydrate, 0.025 mg/L of cobalt chloride hexahydrate, 27.8 mg/L of ferric sulfate heptahydrate, 37.3 mg/L of disodium ethylenediamine tetraacetate, 100 mg/L of inositol, 0.5 mg/L of hydrochloric acid, 60.5 mg/L of vitamin B and 10.5 mg/L of vitamin B.

The invention has the beneficial effects that:

1. the invention improves the germination rate of the hippocastanum seeds, shortens the germination time of the seeds, controls the seed pollution rate to be below 4 percent, and the germination rate to be more than 90 percent, and the cultured hippocastanum has developed root systems and good growth vigor, and can quickly obtain a large amount of robust hippocastanum aseptic seedlings.

2. An effective hippocampus seed surface disinfection method is established, material guarantee is provided for the establishment of a subsequent hippocampus regeneration system and a transformation system, and a foundation is laid for improving the resistance and the ecological restoration function of the hippocampus from a molecular level.

Drawings

FIG. 1 shows the growth of the Oncorhynchus serratus of the present invention after 80 days of sowing. Sodium hypochlorite treatment 5 times for 30 minutes of treatment group growth.

Detailed Description

Example 1 a method for the sterile sowing and cultivation of hippocampus seeds, comprising the following steps:

1) collecting the hippocampus seeds: directly collecting black seeds after the seed of the hippocastanum becomes black and maturely, storing the seeds in dry and cool places, and using the seeds as soon as possible.

2) Preparing a culture medium: the culture medium is prepared, sterilized at 121 ℃ for 20 minutes, split-packed into culture dishes and cooled for standby, 25ml of each dish, and 30 dishes are prepared.

3) Seed disinfection: 150 seeds were prepared and divided into 5 tubes of 30 seeds each, each of which was placed in a 2 ml centrifuge tube. 1ml of 75% alcohol was added to each of the seed bottles, the mixture was inverted 10 times, and after washing the seeds, the liquid was removed by pipetting. Then, about 1ml of sterile water was added thereto, the mixture was inverted 10 times, the seeds were washed, and the liquid was aspirated off by a pipette, and this step was repeated 3 times. Adding 1ml of 84 disinfectant (the effective rate content is 4.5%) to sterilize for 6 minutes each time, turning the centrifugal tube upside down for 10 times, absorbing the liquid by using a liquid transfer gun, and repeating the sterilization for 5 times; and after cleaning with sterile water again, cutting off the tip of the gun head, adding the sterile water, sucking out the hippocampus seeds, placing the hippocampus seeds in a sterile 9 cm culture dish, and keeping part of water in the dish, wherein the seeds are conveniently clamped at the back by using the tension of the water.

4) Sowing: gently clamping seeds with sterilized and cooled tweezers, placing on the surface of culture medium, each bottle contains 5 seeds, the middle part of 4 seeds at the periphery contains 1 seed, each bottle contains 6 containers, and sealing with gas-permeable sealing film.

5) Culturing: culturing in an artificial climate closed incubator at 24 deg.C under 16 hr of light and 8 hr of dark at illumination intensity of 80 lmol.m^-2.s^-1Culturing at humidity of 60% for 20 days, transferring to tissue culture bottle of 1/2 MS culture medium after seed germination and seedling growth, and culturing until seedling grows up. And (5) counting the pollution rate and the germination rate of the hippocastanum seeds.

The culture medium in the step 2) comprises the following components: 1/2 MS medium +15 g/L sucrose +8 g/L agar, pH 5.6. After the culture medium is prepared, the culture medium is sterilized for 20 minutes under high pressure at 121 ℃, and is arranged in a culture dish and a tissue culture bottle in a super clean workbench for standby.

The effect of different disinfection times on the contamination rate and germination rate is shown in table 1.

The pollution rate of the embodiment is as low as 3.3 percent, and the germination rate is as high as 93.3 percent.

Example 2

An acanthus seed sterile seeding and culturing method comprises the following steps:

1) collecting the hippocampus seeds: directly collecting black seeds after the hippocampus seeds are mature and turn black, drying in a constant temperature incubator at 37 ℃, collecting in a centrifuge tube, and placing in a refrigerator at 4 ℃ for later use.

2) Preparing a culture medium: preparing a solid culture medium for sowing, carrying out autoclaving at 121 ℃ for 25 minutes, and then subpackaging in a culture dish and a tissue culture bottle for later use.

3) Seed disinfection: 150 seeds were prepared and divided into 5 tubes of 30 seeds each, each of which was placed in a 2 ml centrifuge tube. About 1ml of 75% alcohol was added to each of the seed bottles, and the mixture was inverted 10 times, and after washing the seeds, the liquid was removed by pipetting. Then 1ml of sterile water was added, the mixture was inverted 10 times, the seeds were washed, and the liquid was aspirated off with a pipette, and this step was repeated 3 times. Adding 1ml of 84 disinfectant (the effective rate content is 3.92 percent), disinfecting for 6 minutes each time, turning the centrifugal tube upside down for about 10 times, absorbing the liquid by using a liquid transfer gun, and repeatedly disinfecting for 4 times; and after cleaning with sterile water again, cutting off the tip of the gun head, adding the sterile water, sucking out the hippocampus seeds, placing the hippocampus seeds in a sterile 9 cm culture dish, and keeping part of water in the dish, wherein the seeds are conveniently clamped at the back by using the tension of the water.

4) Sowing: gently clamping seeds with sterilized and cooled tweezers, placing on the surface of culture medium, each bottle contains about 5 seeds, the middle part of 4 seeds at the periphery contains 1 seed, treating 6 dishes, and sealing with gas-permeable sealing film.

5) Culturing: culturing in an artificial climate closed incubator at 24 deg.C under 16 hr of light and 8 hr of dark at illumination intensity of 80 lmol.m^-2.s^-1And culturing at humidity of 60% for 30 days, transferring to a tissue culture bottle of 1/2 MS culture medium after the seeds germinate and grow to grow seedlings.

The seeding culture medium in the step 2) comprises the following components: 1/2 MS medium +15 g/L sucrose +8 g/L agar, pH 5.8.

The germination rate of the hippocastanum of this example was 90.0%.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A method for aseptically sowing and culturing sea horse toothseeds is characterized in that: the method comprises the following steps:

1) taking black seeds in mature cracked fruit pods, drying the seeds in a constant-temperature incubator at 37 ℃, collecting the seeds in a centrifuge tube, and placing the centrifuge tube in a refrigerator at 4 ℃ for storage;

2) preparing a solid culture medium for aseptically sowing Hippocampus seeds, sterilizing for 20-25 minutes at 121 ℃ in an autoclave, and subpackaging in a culture dish and a tissue culture bottle in a superclean bench for later use;

3) the seeds are respectively arranged in 2 ml centrifuge tubes, 25-35 grains are added into each tube, 1ml of 75% alcohol is added, the upside is reversed for 10 times, the seeds are cleaned, and liquid is removed by a pipette; adding 1ml of sterilized water, reversing the upside down for 10 times, cleaning seeds, and removing liquid by using a liquid transfer gun, wherein the step is repeated for 2-3 times; adding 1ml of 84 disinfectant, wherein the effective chlorine content of the disinfectant is 3.92-5.30%, disinfecting for 30 minutes, replacing the disinfectant every 6 minutes, disinfecting for 1-5 times, continuously turning the centrifugal tube upside down in the disinfection process to enable the surfaces of the seeds to fully contact the disinfectant, and then cleaning for 3-4 times by using sterile water;

4) sowing and culturing: gently clamping seeds with sterilized and cooled tweezers, and placing on a solid culture medium, wherein 5 seeds are placed in each dish; culturing the sown seeds in artificial-climate illumination incubator at 23-26 deg.C for 16 hr in 8 hr dark place with illumination intensity of 80 lmol.m^-2.s^-1Humidity is 60%; culturing for 15-30 days, transferring the germinated seeds to a tissue culture bottle, and continuously culturing for growing.

2. The method of claim 1, wherein said method comprises the steps of: the solid culture medium is 1/2 MS solid culture medium, and 15g/L of sucrose and 8 g/L of agar are added into the culture medium.