CN108782205B - Cultivation method for strong arabidopsis seedlings - Google Patents

Cultivation method for strong arabidopsis seedlings Download PDF

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Publication number
CN108782205B
CN108782205B CN201810693155.6A CN201810693155A CN108782205B CN 108782205 B CN108782205 B CN 108782205B CN 201810693155 A CN201810693155 A CN 201810693155A CN 108782205 B CN108782205 B CN 108782205B
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arabidopsis
seedlings
arabidopsis thaliana
culture
strong
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CN201810693155.6A
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CN108782205A (en
Inventor
张显
杨永超
王中元
杨小振
王永琦
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Hanzhong Agriculture Technology Promotion Center
Wenshan Zhuang And Miao Autonomous Prefecture Academy Of Agricultural Sciences
Northwest A&F University
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Hanzhong Agriculture Technology Promotion Center
Wenshan Zhuang And Miao Autonomous Prefecture Academy Of Agricultural Sciences
Northwest A&F University
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Priority to CN201810693155.6A priority Critical patent/CN108782205B/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A01G13/02—Protective coverings for plants; Coverings for the ground; Devices for laying-out or removing coverings
    • A01G13/0256—Ground coverings
    • A01G13/0281—Protective ground coverings for individual plants, e.g. for plants in pots

Abstract

The invention discloses a cultivation method of strong arabidopsis seedlings, which comprises the following steps: putting filter paper into a culture dish, soaking the filter paper with clear water, putting the arabidopsis thaliana seeds on the filter paper, and putting the filter paper into a refrigerator for vernalization for 2-4 days at 3-4 ℃; sowing the vernalized arabidopsis seeds in a nutrition pot filled with a seedling substrate soaked by clear water by using toothpicks; 5-6 seeds are sowed in each nutrition pot, covered by a preservative film, placed in a stainless steel iron plate, then placed in a culture container for culture for 15-20 days, then the preservative film is removed, the culture is continued for 5-7 days, 4 plantlets with good growth vigor are left, and the rest plantlets are pulled out. And (3) after sowing, culturing for 3-4 weeks under the conditions that the photoperiod is 10 hours and the darkness is 14 hours, and then continuously culturing under the conditions that the photoperiod is 16 hours and the darkness is 8 hours to obtain the strong seedlings of the arabidopsis thaliana. The invention adopts common materials, simplifies the cultivation steps of the arabidopsis thaliana, reduces the cost and time, and improves the strong seedling rate of the arabidopsis thaliana.

Description

Cultivation method for strong arabidopsis seedlings
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a cultivation method of strong arabidopsis seedlings.
Background
The arabidopsis thaliana belongs to the angiosperma, dicotyledonae and biennial herbs, has the advantages of small plant size, short growth period, more seeds, small genome, easy obtaining of transgenic arabidopsis thaliana plants and the like, is a self-pollinated plant, is highly homozygous for genes, improves the mutation rate by using physical and chemical factors, and is easy to obtain various metabolic function defects. Due to the advantages, arabidopsis thaliana is a good material for genetic and molecular biological research, and is known as 'fruit fly in plant' by scientists. In the scientific research, arabidopsis is frequently used, and research shows that in the process of transforming arabidopsis by adopting a floral dip method, the transformation efficiency can be greatly improved by using the wild arabidopsis with strong growth, and in addition, a certain number of good-growing arabidopsis plants are needed in further experiments by adopting arabidopsis protoplast subcellular localization and stable genetic transgenic arabidopsis progeny.
At present, arabidopsis thaliana is usually cultivated under aseptic conditions and then transplanted into a matrix for continuous cultivation, but the cultivation under aseptic conditions requires flat plate manufacturing and seed disinfection, the operation is complicated, a large amount of time and energy are consumed, and the risk of pollution exists; in addition, root systems can be damaged in the transplanting process, and an obvious seedling revival period exists, so that the test progress is delayed; in addition, the currently used substrate is generally prepared from perlite, vermiculite and turf, and a nutrient solution is additionally applied, so that the cost is increased, and due to evaporation, the concentration of the nutrient solution in the substrate is gradually increased, salt stress to arabidopsis thaliana at different degrees is generated, the growth of arabidopsis thaliana is inhibited, and the proportion of the cultivated robust arabidopsis thaliana is small.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a cultivation method of strong arabidopsis seedlings, which not only simplifies the cultivation steps and reduces the cost, but also cultivates arabidopsis plants with large rosette, strong and thick stem, large flower quantity and many seeds.
In order to achieve the purpose, the invention adopts the following technical scheme:
a cultivation method of strong seedlings of arabidopsis thaliana comprises the following steps:
s1, placing filter paper into a culture dish, soaking the filter paper with clear water, placing the Arabidopsis seeds on the filter paper, and performing vernalization for 2-4 days at 3-4 ℃ in a refrigerator to obtain vernalized Arabidopsis seeds;
s2, soaking the seedling substrate with clear water, and placing the seedling substrate in a nutrition pot;
s3, sowing the vernalized Arabidopsis seeds obtained in S1 into a nutrition pot containing a seedling culture medium in S2 by using toothpicks, sowing 5-6 seeds in each nutrition pot, covering with a preservative film, placing into a stainless steel iron plate, and thenPutting the seedlings into a culture container for culturing for 15-20 days, then removing the preservative film, continuously culturing for 5-7 days, leaving 4 seedlings with good growth vigor, and pulling out the rest seedlings to obtain the primarily cultured arabidopsis seedlings, wherein the culture conditions of S3 are as follows: 10 hours of illumination and 14 hours of darkness, wherein the temperature under the illumination condition is 21-23 ℃, the relative humidity is 70-80%, and the light intensity is 130-150 mu E/m2The temperature under dark conditions is 19-21 ℃ and the relative humidity is 70-80%;
s4, subjecting the arabidopsis thaliana seedlings obtained in the step S3 to 16-hour light irradiation and 8-hour dark irradiation in a photoperiod, wherein the temperature is 21-23 ℃, the relative humidity is 70-80%, and the light intensity is 130-150 mu E/m2And/s, continuously culturing under the culture conditions that the temperature is 19-21 ℃ and the relative humidity is 70-80% in the dark condition, and culturing to obtain the strong arabidopsis seedlings.
Preferably, in S2, the seedling substrate is a commercially available common vegetable seedling substrate.
Preferably, in S2, the commercially available common vegetable seedling substrate comprises perlite, vermiculite, grass peat and decomposed farmyard manure.
Preferably, in S2, the depth of the seedling raising substrate in the nutrition pot is 2-3 cm below the opening of the nutrition pot.
Preferably, in S3, the culture container is one of an incubator and a culture chamber.
Preferably, in S1, the arabidopsis seed is one of a wild-type arabidopsis seed and a homozygous transgenic arabidopsis progeny seed.
Preferably, in S3 and S4, water is supplemented according to the growth condition of the arabidopsis seedlings in the culture process, and the specific method comprises the following steps: and (3) pouring clear water into the stainless steel plate, wherein the clear water soaks the seedling culture substrate through the holes at the bottom of the nutrition pot to replenish the arabidopsis seedlings.
Preferably, in S3, no water is applied during the plastic wrap period.
The invention has the following beneficial effects and advantages:
(1) the invention simplifies the cultivation steps of the arabidopsis thaliana, does not need to cultivate the arabidopsis thaliana under the aseptic condition and then transplant the arabidopsis thaliana into the substrate for cultivation, thus the flat plate and the seed disinfection are not needed to be manufactured.
(2) According to the invention, a commercially available common vegetable seedling substrate is used as the seedling substrate, additional fertilization is not needed in the whole growth process, and only water management is needed, so that salt stress of different degrees on arabidopsis due to the increase of the evaporation concentration of a nutrient solution is avoided, the growth inhibition of arabidopsis is avoided, and the cost is reduced.
(3) According to the invention, the preservative film is adopted to cover the sowed nutrition pot in the primary culture process of arabidopsis thaliana, so that the effect of moisture preservation can be achieved, and compared with the common PVC film, the preservative film has better air permeability, moisture permeability and light transmittance, so that a better growth environment is provided for the growth of arabidopsis thaliana.
(4) The invention cultures Arabidopsis thaliana in 2 light periods during the culture process. According to the invention, culture conditions of 10-hour light and 14-hour dark are adopted firstly, so that the vegetative growth of arabidopsis is faster, after 3-4 weeks of culture, the arabidopsis grows robustly, and then the culture conditions of 16-hour light and 8-hour dark are adjusted, so that the bolting and flowering of arabidopsis can be effectively promoted, and the proportion of culturing the robustly arabidopsis is increased.
(5) The method can culture the arabidopsis thaliana plant with large rosette, strong and thick stem, large flower quantity and more seeds.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the following examples, the seedling substrate is a commercial common vegetable seedling substrate, and comprises perlite, vermiculite, grass peat and decomposed farmyard manure; watering is not carried out in the plastic film covering period of S3, and water is supplemented according to the growth condition of the arabidopsis seedlings in the culture processes of S3 and S4, and the specific method comprises the following steps: and (3) pouring clear water into the stainless steel plate, wherein the clear water soaks the seedling culture substrate through the holes at the bottom of the nutrition pot to replenish the arabidopsis seedlings.
Example 1
A cultivation method of strong seedlings of arabidopsis thaliana comprises the following specific steps:
s1, placing filter paper into a culture dish, soaking the filter paper with clear water, placing wild type Arabidopsis seeds on the filter paper, and performing vernalization in a refrigerator at 4 ℃ for 2 days to obtain vernalized Arabidopsis seeds;
s2, soaking the seedling substrate with clear water (the dropping degree of the seedling substrate after being picked up by hands is equal), and then placing the seedling substrate in a nutrition pot, wherein the depth of the seedling substrate is 3cm below the pot opening;
s3, sowing the vernalized Arabidopsis seeds obtained in S1 in a nutrition pot containing a seedling culture medium in S2 by using a toothpick, sowing 6 seeds in each nutrition pot, covering the nutrition pot with a preservative film, placing the nutrition pot in a stainless steel iron plate, then placing the nutrition pot in an incubator for culturing for 20 days, then removing the preservative film, continuously culturing for 7 days, reserving 4 plantlets with good growth vigor, and pulling out the rest to obtain the preliminarily cultured Arabidopsis seedlings, wherein the culture conditions of S3 are as follows: 10 hours of illumination, 14 hours of darkness, a temperature of 22 ℃ under illumination, a relative humidity of 80 percent and a light intensity of 150 mu E/m2(ii)/s, temperature in dark 20 ℃ and relative humidity 80%;
s4, subjecting the young seedling of Arabidopsis thaliana obtained in S3 to 16-hour light and 8-hour dark light at a light period of 22 deg.C, a relative humidity of 80%, and a light intensity of 150 μ E/m2And/s, continuously culturing for 2 weeks under the culture conditions that the temperature is 20 ℃ and the relative humidity is 80% in the dark, and obtaining the strong seedlings of the arabidopsis thaliana.
Example 2
A cultivation method of strong seedlings of arabidopsis thaliana comprises the following specific steps:
s1, placing filter paper into a culture dish, soaking the filter paper with clear water, placing wild type Arabidopsis seeds on the filter paper, and performing vernalization in a refrigerator at 3 ℃ for 3 days to obtain vernalized Arabidopsis seeds;
s2, soaking the seedling substrate with clear water (the dropping degree of the seedling substrate after being picked up by hands is equal), and then placing the seedling substrate in a nutrition pot with the depth of 2cm below the pot opening;
s3, sowing the vernalized Arabidopsis seeds obtained in S1 in S2 with toothpicksIn a nutrition pot containing a seedling culture substrate, 5 seeds are sown in each nutrition pot, covered by a preservative film, placed in a stainless steel iron plate, then placed in an incubator for cultivation for 15 days, then the preservative film is removed, the cultivation is continued for 5 days, 4 seedlings with good growth vigor are left, and the rest are pulled out, so that the primarily cultivated arabidopsis thaliana seedlings are obtained, wherein the cultivation conditions of S3 are as follows: 10 hours of illumination, 14 hours of darkness, a temperature of 21 ℃ under illumination, a relative humidity of 70 percent and a light intensity of 130 mu E/m2(ii)/s, temperature in dark is 19 ℃ and relative humidity is 80%;
s4, subjecting the young seedling of Arabidopsis thaliana obtained in S3 to 16-hour light and 8-hour dark light at a light period of 21 deg.C, 70% relative humidity and 130 μ E/m light intensity2And/s, continuously culturing for 2 weeks under the culture conditions that the temperature is 19 ℃ and the relative humidity is 80% in the dark, and obtaining the strong seedlings of the arabidopsis thaliana.
Example 3
A cultivation method of strong seedlings of arabidopsis thaliana comprises the following specific steps:
s1, putting filter paper into a culture dish, soaking the filter paper with clear water, putting the seeds of homozygous transgenic arabidopsis thaliana offspring on the filter paper, and vernalizing the seeds in a refrigerator at 4 ℃ for 4 days to obtain vernalized arabidopsis thaliana seeds;
s2, soaking the seedling substrate with clear water (the dropping degree of the seedling substrate after being picked up by hands is equal), and then placing the seedling substrate in a nutrition pot, wherein the depth of the seedling substrate is 3cm below the pot opening;
s3, sowing the vernalized Arabidopsis seeds obtained in S1 in a nutrition pot containing a seedling culture medium in S2 by using a toothpick, sowing 6 seeds in each nutrition pot, covering the nutrition pot with a preservative film, placing the nutrition pot in a stainless steel iron plate, then placing the nutrition pot in an incubator for culturing for 18 days, then removing the preservative film, continuously culturing for 6 days, reserving 4 plantlets with good growth vigor, and pulling out the rest to obtain the preliminarily cultured Arabidopsis seedlings, wherein the culture conditions of S3 are as follows: 10 hours of illumination, 14 hours of darkness, 23 ℃ of temperature under illumination, 80% of relative humidity and 140 muE/m of light intensity2(ii)/s, temperature in dark is 21 ℃, relative humidity is 70%;
s4 exposing the primary cultured Arabidopsis seedlings obtained in S3 to lightThe period is 16 hours of light, 8 hours of dark, the temperature under the light condition is 23 ℃, the relative humidity is 80 percent, and the light intensity is 140 mu E/m2And/s, continuously culturing for 2 weeks under the culture conditions that the temperature is 21 ℃ and the relative humidity is 70% in the dark, and obtaining the strong seedlings of the arabidopsis thaliana.
It should be noted that when the following claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in embodiments 1 to 3, the preferred embodiments are described in the present invention for preventing redundancy, but once a person skilled in the art knows the basic inventive concept, other changes and modifications can be made to the embodiments. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (7)

1. A cultivation method of strong seedlings of arabidopsis thaliana is characterized by comprising the following steps:
s1, placing filter paper into a culture dish, soaking the filter paper with clear water, placing the Arabidopsis seeds on the filter paper, and performing vernalization for 2-4 days at 3-4 ℃ in a refrigerator to obtain vernalized Arabidopsis seeds;
s2, soaking the seedling substrate with clear water, and placing the seedling substrate in a nutrition pot;
s3, sowing the vernalized Arabidopsis seeds obtained in S1 in a nutrition pot containing a seedling culture medium in S2 by using a toothpick, sowing 5-6 seeds in each nutrition pot, covering the seeds with a preservative film, placing the seedlings in a stainless steel iron plate, then placing the seedlings in a culture container for culture for 15-20 days, then removing the preservative film, continuing culture for 5-7 days, leaving 4 seedlings with good growth vigor, and pulling out the rest seedlings to obtain preliminarily cultured Arabidopsis seedlings, wherein the culture conditions of S3 are as follows: 10 is smallThe light is irradiated in the dark for 14 hours, the temperature under the illumination condition is 21-23 ℃, the relative humidity is 70-80%, and the light intensity is 130-150 mu E/m2The temperature under dark conditions is 19-21 ℃ and the relative humidity is 70-80%;
s4, the preliminary cultured arabidopsis thaliana plantlets obtained in the step S3 are subjected to 16-hour illumination and 8-hour darkness under the photoperiod, the temperature under the illumination condition is 21-23 ℃, the relative humidity is 70-80%, and the light intensity is 130-150 mu E/m2Continuously culturing at 19-21 ℃ under the dark condition and at the relative humidity of 70-80% to obtain strong arabidopsis seedlings through cultivation;
in the steps S3 and S4, water is supplemented according to the growth condition of the arabidopsis seedlings in the culture process, and the specific method comprises the following steps: and (3) pouring clear water into the stainless steel plate, wherein the clear water soaks the seedling culture substrate through the holes at the bottom of the nutrition pot to replenish the arabidopsis seedlings.
2. A cultivation method of strong seedlings of Arabidopsis thaliana as claimed in claim 1, wherein in S2, the seedling substrate is a commercial common vegetable seedling substrate.
3. A cultivation method of strong seedlings of Arabidopsis thaliana according to claim 2, wherein in S2, the commercial ordinary vegetable seedling substrate comprises perlite, vermiculite, grass peat and decomposed farmyard manure.
4. A method for cultivating a strong seedling of Arabidopsis thaliana as claimed in claim 1, wherein in S2, the depth of the seedling substrate in the nutrition pot is 2-3 cm below the mouth of the nutrition pot.
5. A method for cultivating a strong seedling of Arabidopsis thaliana as claimed in claim 1, wherein in S3, the culture container is one of an incubator and a culture room.
6. A strong seedling cultivation method of Arabidopsis thaliana as claimed in claim 1, wherein in S1, the Arabidopsis thaliana seed is one of a wild type Arabidopsis thaliana seed and a homozygous transgenic Arabidopsis thaliana progeny seed.
7. A cultivation method of strong seedlings of Arabidopsis thaliana as claimed in claim 1, wherein in S3, no water is added during the plastic film covering period.
CN201810693155.6A 2018-06-29 2018-06-29 Cultivation method for strong arabidopsis seedlings Expired - Fee Related CN108782205B (en)

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