Plant cell suspension culture refers to the suspension of plant cells or smaller cell clusters in liquid culture medium, which can maintain good dispersibility during the culture process. Plant culture in vitro can produce callus. Loose calli are suspended in liquid culture medium, and dispersed suspension cultures can be formed after being cultured for a period of time under shaking conditions. A good county floating culture should have the following characteristics: (1) is mainly composed of single cells and small cell clusters; (2) Cells have rich ability of growth and division, and the proliferation rate is fast; (3) Most cells should have the characteristics of meristem cells in morphology. Most of them have the same diameter, large nuclear-cytoplasmic ratio, dense cytoplasm and low aneuploidy.
In the process of liquid suspension culture, we should pay attention to the subculture of cells in time, because when the culture grows to a certain period, it will enter the static stage of division. For most suspension cultures, the cell density reaches the maximum at 18 ~ 25 days, and the first subculture should be carried out at this time. In subculture, large cell clumps and inoculum residues should be removed. If a cell suspension culture system is established from plant organs or tissues, it includes callus induction, subculture, single cell separation and suspension culture. At present, this technology has been widely used in the study of cell morphology, physiology, heredity and apoptosis, especially providing an ideal material and approach for the operation of genetic engineering at the plant cell level. The transformed plant cells are induced to differentiate into plants, and individuals carrying the target genes are obtained.
Main reagents:
Add 0.2mg NAA (naphthylacetic acid) liquid medium to B5 medium (take 3.2g B5 powder medium, 30g sucrose and 200ul NAA stock solution with volume mass of 65438 0 mg/ml, dissolve it in about 800ml distilled water, put it in a magnetic stirrer, mix it evenly, and measure the pH value with a pH meter. Adjust the pH value to 5.8 with 1mol/l KOH, add distilled water to constant volume to 1L, seal with aluminum foil, sterilize with 12 1℃ for 20min, store in the refrigerator at 4℃, and take a water bath or naturally raise to room temperature before inoculation).
Main equipment:
1. purification table
2. Pressurized kettle
Step 3 rotate the vibrator
4. Water bath pot
5. Inverted microscope
6. Tweezers
spirit lamp
8. triangle bottle
9. Pipet
10.pH meter
1 1. Constant temperature culture room
12. Chimney
13. Stainless steel screen
14. Blood cell counting board, etc.
Experimental materials:
Arabidopsis thaliana callus
Experimental steps:
1. Take out the vigorous and soft calluses with tweezers, put them in a triangular flask and crush them gently. Every 100ml triangular flask is filled with 10~ 15ml sterile medium, and each flask is inoculated with 1~ 1.5g callus, so as to ensure enough cells in the initial culture.
2. Put the inoculated triangle on the rotary shaker. Shake flask culture was carried out at 65438 000 rpm and 25 ~ 28℃.
3. After 6~ 10 days of culture, if the cell proliferation is obvious, you can add 10ml fresh medium to the culture bottle, and if necessary, divide the culture into two bottles with a big mouth straw to continue the culture. (If the cells don't proliferate obviously, it may be that the starting materials are not suitable, so we should consider re-inoculating callus during the vigorous proliferation period). The first subculture can be carried out.
4. Filtration of suspension culture: After subculturing for several generations according to the "3" method, the culture solution should be mainly composed of single cells and small cell clusters (no more than 20 cells). If it still contains effective large cell clusters, it can be filtered with a metal mesh screen with an appropriate aperture, and then the filtered floating cells in the county will continue to be cultured.
5. Cell computing. Take a certain volume of cell fluid, add twice the volume of 8% chromium trioxide (CrO3), and put it in a water bath at 70℃ for 65438±05min. After cooling, the cell suspension was repeatedly blown with a pipette to fully disperse the cells. After mixing, take a drop of county liquid and put it on the blood cell counting plate for counting.
6. Making cell growth curve: In order to understand the growth dynamics of floating culture cells in the county, the following methods can be used to draw the growth curve:
1) fresh weight method At different times of subculture, a certain volume of suspension culture was taken, after centrifugal collection, the fresh weight of cells was weighed, and the growth curve of fresh weight was drawn with fresh weight as the ordinate and culture time as the abscissa.
2) The dry weight method can dry the cells after weighing the fresh weight, and then weigh the dry weight. With dry weight as the ordinate and culture time as the abscissa, the regeneration curve of cell stem cells was drawn.
The above two methods require sampling once every two days, seven times, and each sample is repeated three times. During the whole experiment, no fresh culture solution was changed into the culture bottle.
7. Examination of cell viability. For beginners, it is often necessary to detect the proportion of living cells. At different stages of culture, you can take a drop of cell fluid and put it on the glass slide, and drop a drop of 0. Stained with 1% phenol saffron solution (prepared with culture medium) and observed under a microscope. Several living cells are uncolored, while dead cells are quickly dyed red. You can also use 0. When stained with 1% fluorescent diacetate solution, all living cells will show blue fluorescence under the induction of ultraviolet light, and experienced operators can judge the life and death of cells according to cell morphology and cytoplasmic circulation.
8. Identification of cell regeneration ability: In order to know whether the cells cultured in suspension still have regeneration ability, the cultured cells can be transferred to agar-solidified medium to form callus at the base again, and then the differentiation of plants can be induced on the differentiation medium.
Precautions:
1. The above steps are all sterilized, and the culture medium, utensils and utensils can be used only after autoclaving.
2. If the culture medium is turbid or milky white, it means it has been polluted.
3. During each subculture, observe all kinds of cells and other residues in the culture under the inverted microscope, consciously leave round cells and discard long cells.