CN100427581C - Solid-state culture method of hair vegetable cells - Google Patents

Abstract

The invention discloses a method for solid-state culture of Nostocella cells. It solves the problem of relatively rapid growth of Nostocs edulis under artificial control or natural conditions. Its technical scheme is: inoculate the activated Nostococci cell suspension on the surface of the solid-state culture substrate, spray a certain amount of pre-prepared culture solution, cultivate the Nostocella cells under artificial control or natural conditions, and carry out dry-wet rhythm culture, Control suitable culture conditions to realize complete artificial cultivation or field expansion cultivation. This technology breaks through the traditional culture method and accelerates the proliferation speed. The Nostocella cell culture obtained by the invention has strong drought resistance and can be preserved for a long time; it can absorb carbon dioxide in the air and fix nitrogen during growth, release oxygen into the atmosphere, and has the effect of purifying the air; no pesticide is needed, and no toxicity is produced substances; its expanded cultivation is conducive to maintaining the ecological balance in arid and semi-arid areas and the improvement of desertified soil, and has high economic and environmental value.

Description

Solid-state culture method of hair vegetable cells

technical field

The invention relates to a cell culture technology, in particular to a cell culture technology of Nostocs edulis, in particular to a solid-state culture method of cells of Nostocs edulis which can be directly inoculated on sand grains or the natural sandy soil surface of desert soil.

Background technique

Nostoc flagelliforme is a prokaryotic organism belonging to Procaryotae, Cyanophyta, Cyanophyceae, Hormogonales, Nostocaceae, and Nostoc (Nostoc). It is rich in nutrition, has certain medicinal value, has great economic development value and potential, and has been favored by people for a long time because its pronunciation is similar to "facai". Nostoc is mainly distributed in the desert and semi-desert areas of Northwest my country and some provinces in North China. Nostoc can not only perform photosynthesis, but also fix nitrogen. It is a terrestrial cyanobacteria that lives under special ecological conditions. It is resistant to drought, high temperature, It has strong adaptability to harsh environments such as ultraviolet radiation and barrenness. It is the main nitrogen-fixing resource in the desert ecological environment and plays an important role in ecological restoration. Due to the extremely slow growth of Nostocella in the wild, coupled with the large-scale unrestrained and destructive harvesting, not only the resources are sharply reduced, but also the grassland ecosystem is seriously damaged. In order to protect the resources and ecological environment of Nostocs, for a long time, people have been trying to adopt artificial cultivation methods to increase their biomass and resources to meet the growing market demand, and a large number of physiological and ecological studies have been carried out, for example, the application No.: 02138828.8 and 03119101.0 of the Chinese patent applications, carried out the liquid suspension culture work of Nostocchia cells, and made some progress. However, complete artificial cultivation has not been achieved. The current culture work is basically based on the algae collected in the field, and there is no report on the solid-state culture of Nostocci cells.

Contents of the invention

The present invention is a research aimed at the above-mentioned problems, and its purpose is to provide a method that is simple and easy to operate, which is beneficial to the protection and artificial production of the endangered species of Nostocchia, in the hope that the artificial cultivation of Nostocchi algae is progressing slowly at present. Under the circumstances, realize complete artificial cultivation, and can expand production under close to natural conditions or directly inoculate on sand grain or desert soil natural sandy soil surface layer, make it the solid-state culture method of the Nostocella cell that can grow relatively fast.

In order to achieve the above object, the technical solution of the present invention to solve the technical problem of the solid-state culture method of the Nostocella cells is to take the following technical measures:

1. Culture medium: the formula is NaNO ₃ 0.5-1.5g/L, MgSO ₄ 7H ₂ O 75mg/L, CaCl ₂ 2H ₂ O 30-40mg/L, NaSiO ₃ .9H ₂ O 55-65mg/L, K ₂ HPO ₄ 35-40 mg/L, pH 7.5-8.0. The nutrient salt of the culture medium is chemically pure and can be dissolved in tap water. When preparing, try to avoid the formation of precipitates, and slight precipitation is acceptable, and the culture medium does not need to be sterilized at high temperature.

2. Preparation of the cell liquid of Nostocchia: Centrifuge or filter the cell solution of Nostocchia in liquid culture at 4000rpm, discard the supernatant, add fresh culture solution to resuspend it, or soak the dried Nostocia cells in culture solution to make It restores activity and is used as a solid-state culture species.

3. Sand pretreatment: Sieve the sand through a 40-mesh sieve, wash it with water, dry it at 100°C, and lay it flat on a shallow tray with a thickness of about 0.5-1.5cm.

4. Cell culture of Nostocella sativa: inoculate the cell solution of Nostocchis 400ml/ ^m2 on the surface of the sand grains, and spray the culture solution or water every day according to the total dosage of 1-1.5L/ ^m2 for 3 times during the light period , Cover the sand table with a transparent plastic film to keep the surface of the sand grains moist. Remove the film when there is no light, dry the surface of the sand grains, and carry out dry-wet rhythm culture. When cultivating in the field, spray culture solution or water when the surface temperature is lower than 40°C during the day to keep the soil surface moist, and keep it dry at night or when the surface temperature is higher than 40°C.

5. Culture conditions: temperature 24-26°C, light 60-180 μmol·m ^-2 s ^-1 , light-dark cycle 12h:12h; or cultivate under natural conditions in the field.

6. Harvesting: When the lettuce cells grow into clusters on the sand table or the field culture reaches the harvest season, go to the sand and harvest.

7. Drying: Sun-dried, air-dried or oven-dried below 45°C.

8. Preservation: dry storage at room temperature.

9. Continued cultivation: rehydrate the dried hair vegetable cells in new culture medium for continued cultivation.

The advantages and positive effects of the present invention are: due to the adoption of the cell solid culture method, the traditional algae culture method is changed, the method is simple and easy to operate, and the culture solution does not need to be sterilized at high temperature. , the multiplication rate of Nostoc chinensis is faster, which is conducive to the protection and artificial production of Nostoc chinensis, an endangered species. Owing to having carried out dry-wet rhythm culture, therefore, the Nostocillium cell culture that the present invention cultivates and obtains has stronger drought-resistant ability similar to wild Nostocaria, and can be preserved under dry conditions for a long time, and can be directly obtained through expanding cultivation. For production, it is inoculated on the natural sandy soil surface of sand grains or desert soil; applying this technology to cultivate Nostoconia cells can absorb carbon dioxide in the air and fix nitrogen, and release oxygen into the atmosphere, which has the effect of purifying the air and is different from modern agriculture Pesticides and herbicides are used in production, and no pesticides and herbicides are required in production, and no toxic substances will be produced during the cultivation process; this technology breaks through the traditional way of cultivating algae, and its expanded cultivation is conducive to maintaining dry and semi- The ecological balance in arid areas and the improvement of desertified soil have certain practical significance for the balance of grassland ecosystem, and have high economic value and environmental protection value.

Detailed ways

Embodiment 1: The solid-state culture of Nostoc sativa cells in a sand table, the specific method is:

A. Preparation of culture medium: NaNO ₃ 0.5g/L, MgSO ₄ 7H ₂ O 75mg/L, CaCl ₂ 2H ₂ O 30mg/L, NaSiO ₃ .9H _{2 O} 55mg/L, K ₂ HPO ₄ 35mg/L, pH7.5, mix well and set aside.

B. Preparation of Nostocchia cell liquid: Centrifuge or filter the Nostocchia cell culture solution, and collect Nostocyst cells. According to the ratio of 10g (wet weight) of Nostocchia cells to 1L of culture solution, resuspend them, or use dry Nostocchi cells, soak them for 24 hours at the ratio of 2.5g of cell powder to 1L of culture solution, to restore their activity, and use them as solid-state culture Use species.

C. Sand pretreatment: sieve the sand through a 40-mesh sieve, wash with water, and dry at 100°C until constant weight. Spread flat on glass, plastic, metal or wooden platters, 0.5cm thick.

D. Cell culture: Inoculate the cell liquid of Nostocchis 400ml/m ² on the surface of the sand grains, spray the culture liquid or water three times a day according to the total amount of 1L/m ² during the light period, and cover the sand table with transparent A plastic film keeps the surface of the sand grains moist. Remove the film when there is no light, dry the surface of the sand grains, and carry out dry-wet rhythm culture.

E. Culture conditions: temperature 24°C, light 60 μmol·m ^-2 s ^-1 , light-dark cycle 12h:12h.

F. Harvesting: Remove the sand and harvest when the cells of Nostocella grow into clusters or cover the surface of the sand table.

G. Drying: Sun-dried, air-dried or oven-dried below 45°C.

H. Preservation: Preserve the collected Nostocella cell culture in a dry and ventilated environment at room temperature.

I. Continue to cultivate: rehydrate the dried hair vegetable cells in a new culture medium, and continue to cultivate.

Example 2:

A. Preparation of culture medium: NaNO ₃ 1.0g/L, MgSO ₄ 7H ₂ O 75mg/L, CaCl ₂ 2H ₂ O 35mg/L, NaSiO ₃ 9H _{2 O} 60mg/L, K ₂ HPO ₄ 37.5mg/L , pH7.25 (pH7.8), mix well and set aside.

C. Sand pretreatment: sieve the sand through a 40-mesh sieve, wash with water, and dry at 100°C until constant weight. Spread flat on glass, plastic, metal or wooden platters, 1.0cm thick.

D. Cell culture: Inoculate the cell liquid of Nostocyba sativa on the surface of the sand grains at an inoculation amount of 600ml/ ^m2 , spray the culture liquid or water every day during the light period according to the total amount of 1.2L/ ^m2 , and cover the sand table A clear plastic film keeps the top layer of sand grains moist.

E. Culture conditions: temperature 25°C, light 120 μmol·m ^-2 s ^-1 , light-dark cycle 12h:12h.

All the other are with embodiment 1.

Example 3:

A. Preparation of culture medium: NaNO ₃ 1.5g/L, MgSO ₄ 7H ₂ O 75mg/L, CaCl ₂ 2H ₂ O 40mg/L, NaSiO ₃ .9H _{2 O} 65mg/L, K ₂ HPO ₄ 40mg/L, pH8.0, mix well and set aside.

C. Sand pretreatment: sieve the sand through a 40-mesh sieve, wash with water, and dry at 100°C until constant weight. Spread flat on glass, plastic, metal or wooden platters, 1.5cm thick.

D. Cell culture: Inoculate the cell liquid of Nostocchis 800ml/m ² on the surface of sand grains, spray the culture liquid or water three times a day according to the total amount of 1.5L/m ² during the light period, and cover the sand table A clear plastic film keeps the top layer of sand grains moist.

E. Culture conditions: temperature 26°C, light 180μmol·m ^-2 s ^-1 , light-dark cycle 12h:12h;

All the other are with embodiment 1.

Embodiment 4: the field culture of hair vegetable cell, its concrete method is:

The cell liquid of Nostocys sativa is sprayed on the sandy soil surface of desert-semi-desert steppe or hilly bare land according to the inoculum amount of 500ml/m ² , and cultivated under natural conditions. Sow seeds at the end of April or early May and harvest at the end of November. In the high temperature season of summer, the ground is covered with sunshade nets to shade the too strong light and reduce the surface temperature. Spray culture solution or water when the surface temperature is lower than 40°C during the day to keep the soil surface moist, and keep it dry at night or when the surface temperature is higher than 40°C. All the other are the same as in Example 1 (excluding artificially controlled cultivation conditions, sand grain pretreatment, etc.).

Embodiment 5: the field culture of hair vegetable cell, its concrete method is:

Lettuce cell liquid is sprayed on the sandy soil surface layer of desert-semi-desert steppe or hill bare land by 750ml/m inoculum ^amount , and all the other are with embodiment 4.

Embodiment 6: the field culture of hair vegetable cell, its concrete method is:

Lettuce cell liquid is sprayed on the sandy soil surface layer of desert-semi-desert steppe or hill bare land by 1000ml/m ² inoculum amount, and all the other are with embodiment 4.

Claims (2)

1. the process for solid culture of a hair weeds cells is characterized in that the hair weeds cells culture that fluid suspension culture obtains is inoculated in sandy soil matrix top layer, may further comprise the steps:

A. nutrient solution preparation: NaNO ₃0.5-1.5g/L, MgSO ₄7H ₂O75mg/L, CaCl ₂2H ₂O30-40mg/L, NaSiO ₃.9H ₂O 55-65mg/L, K ₂HPO ₄35-40mg/L, pH7.5-8.0, mixing is standby;

B. hair weeds cells liquid prepares: hair weeds cells medium centrifugal or filtration, and the collection hair weeds cells adds fresh medium, makes it to suspend again, perhaps dry hair weeds cells is soaked with nutrient solution, makes it recover active, uses as solid-state cultivation and plants;

C. grains of sand pre-treatment: 40 eye mesh screens sieve the grains of sand, wash, and dry to constant weight for 100 ℃, are tiled on glass, plastics, metal or the wooden tray thickness 0.5-1.5cm;

D. hair weeds cells is cultivated: hair weeds cells liquid is inoculated in sandy soil matrix top layer; Press 1-1.5L/m photostage every day ²Total consumption divides and sprays nutrient solution or water 3 times, and sand table top layer coating film makes the matrix top layer keep moistening, unglazed according to the time then remove film, make the matrix superficial drying, do wet rhythm and pace of moving things cultivation; Culture temperature 24-26 ℃, illumination 60-180 μ molm ^-2s ^-1, light dark period 12h: 12h;

E. results: grow when hair weeds cells and to go sand to gather when agglomerating;

F. dry: in dry below 45 ℃, air-dry or oven dry;

G. preservation: the hair weeds cells drying at room temperature ventilated environment of collecting is preserved down.

2. the process for solid culture of a hair weeds cells is characterized in that the hair weeds cells culture that fluid suspension culture obtains is inoculated in sandy soil matrix top layer, may further comprise the steps:

A. nutrient solution preparation: NaNO ₃0.5-1.5g/L, MgSO ₄7H ₂O75mg/L, CaCl ₂2H ₂O30-40mg/L, NaSiO ₃.9H ₂O 55-65mg/L, K ₂HPO ₄35-40mg/L, pH7.5-8.0, mixing is standby;

B. hair weeds cells liquid prepares: hair weeds cells medium centrifugal or filtration, and the collection hair weeds cells adds fresh medium, makes it to suspend again, perhaps dry hair weeds cells is soaked with nutrient solution, makes it recover active, uses as solid-state cultivation and plants;

C. hair weeds cells is cultivated: the sandy soil top layer that hair weeds cells liquid is sprayed on desert-semi-desert grassland or hills bare area, when being lower than 40 ℃, surface temperature sprays nutrient solution or water daytime, keep the soil layer top layer moistening, then keep dry state when night or surface temperature are higher than 40 ℃;

D. gather in the crops: sow every year by the end of April or at the beginning of 5 months, gathers by the end of November;

E. dry: in dry below 45 ℃, air-dry or oven dry;

F. preservation: the hair weeds cells drying at room temperature ventilated environment of collecting is preserved down.