synonymtissue culture(The tissue that meets the needs is separated from the plant body) It generally refers to the plant tissue culture (new asexual reproduction technology)
Plant tissue culture is based onplant cellThe theory of omnipotence has been developed in recent decadesAsexual reproductionNew technology.The tissue culture of plants is also called in vitro culture in a broad sense, which refers to the separation of tissues that meet the needs of plantsorganOr cells,ProtoplastEtc., viaSterile operationUnder sterile conditions, it is inoculated on the medium containing various nutrients and plant hormones for culture to obtain regenerated complete plants or produce other products with economic value.In the narrow sense, it refers to the cultivation of regenerated plants from various parts of plant tissues, such as cambium, parenchyma, mesophyll tissue, endosperm, etc. It also refers to the cultivation of callus generated from various organs during the cultivation process, and then the callus is re differentiated to form regenerated plants.
In the 1830s, German botanist Schleiden and German zoologist Schwann founded the cell theory. According to this theory, if cells are provided with the same conditions as in the organism, each cell should be able to live independently;In 1902, German botanist Haberlandcell totipotencyThe theory oftissue culture The theoretical basis of.In 1958, an exciting news spread from the United States to all over the worldTivatEt al., using carrotsPhloemThe complete plant was finally obtained by culturing the cells of, and this plant was able to blossom and bear fruit, which confirmed Haberland's prediction of cell totipotency more than 50 years ago.
Simple process of plant tissue culture:Splicing plant organs or tissues - afterDedifferentiation(also called dedifferentiation) to form callusRedifferentiationFormation of tissues or organs - the development of a complete plant through culture.
The general process of plant tissue culture:Under aseptic conditions, plant organs or tissues (such as budsStem apex(root tip or anther)cellulaseAndpectinaseThe treatment is used to remove the cell wall, expose the protoplast, and then put it in the appropriate artificialculture mediumThese organs or tissues will undergo cell division and form new tissues.However, this kind of organization is not differentiated, just a massParenchyma cell, calledCallus。Under the conditions of suitable light, temperature and certain nutrients and hormones, the callus will start to differentiate, produce various organs and tissues of the plant, and then develop into a complete plant[1]。
Plant tissue culture, namely plant aseptic culture technology, also known as in vitro culture, is based onplant cellThe theory of totipotency uses the organs (such as roots, stems, leaves, stem tips, flowers, fruits, etc.), tissues (such as cambium, epidermis, cortex, pith cells, endosperm, etc.) or cells (such as megaspores, microspores, somatic cells, etc.) of plants in vitro, andProtoplastIt is a discipline that can induce callus, adventitious buds, adventitious roots, and finally form complete plants under aseptic, suitable artificial medium and temperature and other artificial conditions.[5]
Plant cellular totipotency
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cell totipotency
Tissue culture refers to the process in which any organ, tissue or cell of a plant grows and differentiates into a complete plant in a medium containing nutrients and plant growth regulators under artificially predicted control conditions.The theoretical basis of tissue culture is that plant cells have totipotency.That is, any cell of a plant carries a set of all the genetic information that develops into a complete plant, which can be expressed to produce a complete plant in vitro culture.
A cycle represents the life cycle, which includes the generation alternation of sporophyte and gametophyte.The B cycle represents the nuclear and cytoplasmic cycle (cell cycle) of a cell. Because of nuclear cytoplasmic interaction, DNA replicates, transcribes RNA and translates it into protein, enabling the formation and maintenance of totipotency.C cycle refers to the tissue culture cycle in which the tissue or cell loses contact with the donor, and under sterile conditions, heterotrophic metabolism is carried out by artificial nutrition and hormones.At this time, the meristem can achieve cell totipotency through three ways: first, the meristem directly divides into buds to achieve the purpose of rapid propagation;The second is the formation of callus from meristem and the realization of cell totipotency through differentiation;Third, free cells or protoplasts form embryoids, from which complete plants can be directly reconstructed, orArtificial seedThen rebuild the plant[1]。
It refers to the mature or immature embryo separated from the ovuleExplantIn vitro sterile culture.
2. Organ culture
It refers to the in vitro sterile culture of plant roots, stems, leaves, flowers, fruits and other organs as explants, such as root tips and segments of roots, stem tips, stem segments and segments of stems, leaf primordium, leaves, petioles, leaf sheaths and cotyledons of leaves, petals, stamens (anthers, filaments), ovules, ovaries, fruits, etc. of flower organs.
3. Tissue culture
It refers to the tissues (such as meristem, cambium, xylem, phloem, epidermis, cortex, endosperm tissueParenchyma, pith, etc.), or induced callus is in vitro sterile culture of explants.This is the narrow sense of plant tissue culture.
It refers to in vitro sterile culture in which a single free cell (such as somatic cell separated from the tissue by using fructase, or pollen cell, egg cell) is used as the inoculum.
Non test tube micro tissue rapid propagation technology is to place explants (usually with one leaf and one bud) on indoor and outdoor ordinary sand medium for culture, and use the natural multiplication of plant axillary buds to achieve the purpose of rapid propagation.Generally, plants can grow roots in 7-15 days.This technology has low investment and few operation links.
2. In vitro tissue culture
In vitro tissue culture, explants (i.e., tissue, organ or cell in vitro) are placed inTissue culture bottleTissue culture was carried out in aseptic conditions to obtain tissue culture bottle seedlings.
The first step is to remove the unused parts of the collected plant materials and carefully clean the needed parts, such as brushing with an appropriate brush.Cut the material to an appropriate size, that is, the sterilization container can be put into it.Flush with running water under the tap for several minutes to several hours. The flushing time depends on the cleanliness of the materials.Easy floating or fine materials can be put into yarn bags for washing.Flushing with running water is especially useful in case of serious pollution.Washing powder can be added to wash, and then tap water can be used to rinse the detergent water.Washing powder can remove the dirt slightly attached to the surface of plants, remove the lipid substances, and facilitate the direct contact of sterilization liquid.Of course, the most ideal cleaning material is surfactant——tween 。
The second step is to soak and sterilize the surface of the material.It should be completed on the ultra clean table or in the inoculation box, and sterilized beakers, glass rods, 70%alcohol, disinfectantSterile water, watches, etc.Immerse in 70% alcohol for 10~30 seconds.Because alcohol can wet the surface of plant materials, and 70% alcohol has strong penetration, it is also easy to kill plant cells, so the immersion time cannot be too long.Some special materials, such as seeds, flower buds, booths with bracts, bracts, etc., dormant buds with multiple scales, etc., and mainly internal materials, can be treated with 70% alcohol for a slightly longer time.The outer layer of the processed material is peeled off after the alcohol evaporates under sterile conditions, and the internal material is taken.
The third step is to treat with sterilization agent.There are many kinds of surface sterilization agents, and 1~2 kinds can be selected according to the situation, as shown in the table.The fourth step is to useSterile waterRinse for about 3min each time. Rinse for about 3 to 10 times depending on the type of disinfectant used.Washing with sterile water is exempteddisinfectantSide effects of killing plant cells Note: ①alcoholStrong permeability, young materials are easy to lose their green color in alcohol, so the soaking time should be short to prevent alcohol from killing plant cells. ②Mature materials, especially seeds, can be soaked in alcohol for a longer time. For example, seeds can be soaked for 5 minutes. ③The penetration of mercuric chloride is weak, and it is generally soaked for about 10 minutes, which is not harmful to plant materials. ④Bleaching powder is easy to cause green loss of plant materials, so young materials should be used with caution. ⑤Add 0.08-0.12%Twain 20Or 80 (a wetting agent), which can reduce the surface tension of plant materials and achieve better disinfection effect[2]。
Training characteristics
Constant temperature culture
1. Cultivation conditions can be controlled artificially
Tissue cultureThe plant materials used are grown entirely in the artificially provided culture medium and microclimate environment, free from the adverse effects of four seasons, day and night changes in nature and disastrous climate, and the conditions are uniform, which is extremely beneficial to plant growth and convenient for stable cultivation and production every week.
2. Short growth cycle and high reproduction rate
Tissue cultureDue to the artificial control of culture conditions, different culture conditions are provided according to different requirements of different parts of different plants, so the growth is fast.In addition, the plant is also relatively small, usually 20-30 days a cycle.Therefore, although tissue culture requires certain equipment and energy consumption, plant materials canGeometric seriesFor reproduction and production, the cost is generally low, and high-quality seedlings or virus-free seedlings with consistent specifications can be provided in time.
3. Convenient management, conducive to factory production andAutomatic control
Plant tissue culture is to provide a certain temperature, light, humidity, nutrition, hormone and other conditions artificially in a certain place and environment, which is very conducive to highly intensive and high-density factory production, as well as automatic control production.It is the development direction of future agricultural factory seedling raising.Compared with pot planting and field cultivation, it saves a series of complicated labor such as weeding, watering, fertilization, disease and insect control, and can greatly save manpower, material resources and land needed for field planting[3]。
Cultivation advantages
1. Small space, not limited by region and season;
2. Cultivating virus-free crops;
3. Short culture cycle;
4. Special biochemical products can be made from callus in tissue culture;
5. It can breed in large numbers in a short time for rescueEndangered plant；
6. It can be induced to differentiate into required organs, such as roots and buds;
7. Solve the problem that some plants produce little or no seeds;
8. There is no variation, and all the genetic characteristics of the original mother parent can be maintained;
9. Low investment and high economic benefits;
10. There are many breeding methods and trial varieties.
Culture material collection
Materials shall be selected appropriately according to the purpose of culture. The selection principle is easy to induce and less bacteria.To selectplant tissue Internal sterile materials.In this respect, materials should be taken from robust plants, not materials with wounds or pests.On the other hand, materials should be taken in sunny days, preferably at noon or afternoon, and never in rainy days, cloudy days or when the dew is still wet.Because robust plants and tissues with strong photosynthesis and respiration in sunny days have their own disinfection effect, such tissues are generally sterile.
Sterilization of culture materials:
The plant materials selected from the outside or inside all contain various microorganisms to varying degrees.Once these pollution sources are brought into the culture medium, they will cause the pollution of the culture medium.Therefore, plant materials must undergo strict surface sterilization treatment, and then be transferred to the culture medium through aseptic operation procedures.
reagent
Ethanol.
Indoleacetic acid (IAA) or 2,4-D (auxin analogue).
Mercury chloride (mercuric chloride) or sodium hypochlorite.
6-benzylaminoadenine (6-BA)
MS medium
0.1 mol/L NaOH and 0.1 mol/L HCl
Preparation of culture medium
(1) Callus induction medium: MS medium (sucrose content 10 g/L, 2,4-D content 2 mg/L,agar10 g/L）。
(2) Test medium: add IAA and 6-BA to MS medium.
Indole acetic acid(IAA) was first dissolved with a small amount of 0.1 mol/L NaOH, and 6-benzylaminoadenine was first dissolved with a small amount of 0.1 mol/L HCl, then diluted with distilled water, and added to the culture medium.
Add the prepared culture medium into agar, heat and dissolve it, adjust it to pH 5.6~5.8, and then sub pack it into 100 mL tissue culture bottles while hot, each bottle is about 30 mL.After the culture medium is cooled and solidified, cover the group cap, or cover the sealing film and fasten it with cotton thread, and then sterilize it in a autoclave at 121 ℃ (1 kg/cm2) for 20 min.Take out the tissue culture bottle and put it on the table for cooling.All equipment (such as long tweezers, scalpels, scissors, etc.) and sterilized water required for inoculation operation shall be sterilized at the same time.
Business applications
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The application in the commercial field is mainly in major flower production bases orDendrobium candidum, Clematis clematis and other medicinal materials production.
Abnormal performance and improvement measures
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Performance, possible causes and improvement measures of culture in test tube or bottle
Training stage
Culture performance
Possible causes of symptoms
Alternative improvement measures
Initiation and dedifferentiation of primary culture stage
The culture is soaked in water, discolored, necrotic, and dry near the stem surface
The concentration of surface sterilizing agent is too high and the time is too long;Improper selection of explants;Inappropriate period
Try a mild sterilizing agent to reduce the concentration and time;Try other parts instead of sampling at the beginning and middle of growth
Long term culture of culture has little response
Inappropriate auxin types;Insufficient consumption;Improper temperature;Unsuitable culture medium
Increase the amount of auxin and try 2,4-D;Adjust the culture temperature
Callus grew too fast, loose, and soaked in water at later stage
Too much auxin and cytokinin;The culture temperature is too high;Low osmotic potential of culture medium
Reduce the amount of auxin and cytokinin, and properly reduce the culture temperature
Callus grows too tight, smooth or protuberant, thick and slow
Excessive use of cytokinin;Too high sugar concentration;Excessive auxin can also cause
Properly reduce the amount of cytokinin and sugar
The lateral buds do not germinate, the cortex is too enlarged, and the lenticels grow callus
The sampled branches are too tender;Too much auxin and cytokinin
Reduce the amount of auxin and cytokinin, and use older branches
Redifferentiation in subculture stage and proliferation of clustered shoots
The number of seedlings differentiation is small, the speed is slow, the branches are few, and individual seedlings grow thin and high
Increase the amount of cytokinin and lower the temperature appropriately
Seedling differentiation is more, growth is slow, some seedlings are deformed, internode is extremely shortened, seedling clusters are dense, and over miniaturization
Excessive use of cytokinin;Improper temperature
Reduce cytokinin or stop using for a period of time, and adjust the appropriate temperature
Less differentiated seedlings, abnormal seedlings, and seedlings cultured for a long time may be callus again
High auxin consumption and high temperature
Reduce the amount of auxin and lower the temperature appropriately
The leaves are thick and brittle
High amount of auxin or cytokinin
Properly reduce the amount of hormone and avoid the leaves contacting the medium
Occasionally adventitious buds differentiate from the leaf edge, leaf surface, etc. of the regenerated seedlings
Excessive use of cytokinin, or the plant is suitable for this regeneration method
Reduce the amount of cytokinin appropriately, or use this regeneration method in stages
The secondary seedling is too weak for rooting and transplanting
Too much cytokinin, too high temperature, short light, insufficient light intensity, long time no transfer, narrow growth space
Reduce the amount of cytokinin, extend the light, increase the light intensity, timely transfer subculture, reduce the inoculation density, and improve the sealing conditions
Yellow leaves and dead seedlings are often sandwiched in clumps of seedlings. Some seedlings gradually weaken and stop growing. Herbs are sometimes soaked or scalded
The gas condition in the bottle deteriorates;Excessive change of pH value;The sugar has been exhausted after a long time of not transferring, and the photosynthesis is insufficient to maintain itself;The ethylene content in the bottle increases;The culture may be contaminated;Temperature discomfort
Some measures are the same as above.Remove pollution and control temperature
The seedlings are weak in growth, yellow and deciduous in succession, and the tissues are immersed in water and cooked
Part of the reasons are the same as above.The proportion of plant hormone is not suitable, and the concentration of inorganic salt is not suitable
Some measures are the same as above.Timely subculture and properly adjust hormone ratio
The seedlings are light green, and part of them lose green
Forgot to add iron salt or the amount is insufficient;PH value is not suitable, iron, manganese and magnesium are out of proportion, light is too strong, and temperature is not suitable
Carefully prepare the culture medium, pay attention to the ingredients of the formula, adjust the pH value, and control the conditions
Induced rooting stage
The culture has not taken root for a long time, and there is no suitable callus growth in the basal incision
The type of auxin is not suitable;Insufficient consumption;Poor ventilation at rooting site;Genotype influence, improper rooting procedure;PH value is not suitable;Inappropriate concentration and coordination of inorganic salts
Improve the culture procedure, select or increase the amount of auxin, and use filter paper bridge solution for root culture
Callus grew too large and too fast, root swelled or deformed, several roots were connected in parallel or healed;Yellow seedling is inhibited or dead
Unsuitable auxin species;The dosage is too high;Or the dosage of cytokinin is too high;Inappropriate procedure, etc
Reduce the amount of auxin or cytokinin, improve the culture procedure, etc[4]
