★ Callus: It originally refers to a group of parenchyma cells formed on the wound surface after injury, but in tissue culture it refers to a group of parenchyma cells that grow out of order from explants on artificial culture medium.
★ Plant cell: Any plant cell with a complete nucleus has all the genetic information necessary to form a complete plant bead and has the ability to develop into a complete plant. (Haberlandt, 1902)
★ Micro (fast) propagation:
Mother plant (complete) → explant (a small part of mother plant, seeds can also be used) → inoculation on culture medium → long bud (subculture) → long root (rooting in test tube can also be used) → hardening seedlings and domestication → complete plant.
★ A Brief History of Tissue Culture Development: Cell Theory: Schleiden and Shi Wan. 1. Exploration: In the early 20th century, Haberlandt put forward "cell totipotency" (1902); 1904, Henning successfully cultivated the embryos of radish and horseradish. Laibach (1925, 1929) successfully cultivated flax interspecific hybrid embryos, which proved that embryo culture can be used for distant hybridization of plants. 1922, Robiins (USA) and Kotte (Germany) were successfully cultured in vitro. 2. Foundation: Gautheret, White and Nobecourt, founders of tissue culture. White and Gottley discovered vitamin B and auxin; Skoog( 1944), Skoog and Cui (195 1) found that the ratio of adenine and auxin controlled the formation of buds and roots, and Overbeek et al. (194 1) used coconut juice (CM) as an additive for the first time. 1952, Morel and Martin confirmed for the first time that virus-free plants can be obtained by shoot tip culture in vitro. 1953- 1954, the haploid culture of Muir was successful; 1955, Miller isolated kinetin (KT); 1957, Skoog and Miller put forward the concept that plant hormones control organ formation; 1958, Steward first confirmed Haberlandt's thought of cell totipotency; Wicksen and Thurman pointed out that CTK broke the dormancy of axillary buds; Murakami develops rapid propagation technology; In 1958- 1959, Reinert and Steward formed somatic embryos in carrot callus culture. 3. Rapid development: 197 1 year, the Ministry of Military Affairs obtained regenerated plants from tobacco protoplasts for the first time; Carlson obtained the first somatic hybrid of tobacco in 1972; 1964, Guha and Maheshwari cultured anther embryos in vitro. 1960, Morrel proposed in vitro asexual propagation of orchids. ..... (see book or courseware for details)
★ Significance of tissue culture: 1. Basic theoretical research (the accuracy and repeatability of the experimental system are widely used in metabolic physiology and other biochemical research of cells and tissues (such as differentiation)). 2. Applied research (rapid propagation of clones, commercialization of test-tube seedlings, genetic breeding, germplasm preservation, overcoming distant hybridization, germplasm resource innovation and obtaining transgenic plants).
★ Application prospect of tissue culture: 1, application in crop breeding (1, anther and pollen culture 2, embryo culture 3, cell fusion 4, genetic engineering 5, cultured cell mutant 6, germplasm preservation) 2, application in virus-free rapid propagation of crops (potato, orchid) 3, application in production of useful plant products 4, application in heredity and germplasm preservation.
★ Plant hormone regulation: auxin/CTK > 1 (promoting rooting); = 1 (calluses); & lt 1 (promoting germination)
★ Dedifferentiation: In tissue culture, the undifferentiated static cells are put on a certain culture medium, and then the cells enter the division state again. The process by which mature cells become meristems is called dedifferentiation.
★ Redifferentiation: A mature plant cell can be dedifferentiated to form a complete plant.
★ Redifferentiation pathway: 1, organogenesis mode (refers to the formation of stems, buds and roots in different parts of explants or calli, which are unipolar structures, and each has vascular bundles connected with the explants or calli, but there is no * * * same vascular bundle between adventitious buds and adventitious roots to connect them. ) 2. Embryogenesis mode (explants produce embryoids directly or through callus or suspension culture. )
★ embryoid: refers to the bipolar embryoid structure that originated from the non-zygotic cells in tissue culture and formed through embryogenesis and embryonic development. Its characteristic is: 1, which is different from zygotic embryo because it is not produced by the fusion of bisexual cells. 2. It is different from parthenogenesis/male embryo because it is not the product of apomixis. 3. It is different from the stem, bud and root formed by organogenesis, because it has experienced a development process similar to that of zygotic embryo, and the mature embryoid is bipolar.
★ organogenesis pathway: 1, axillary buds are produced by shoot tip or stem segment culture. 2. Direct adventitious bud formation: small pieces of organs and tissues are cultured on the culture medium to directly induce adventitious buds. 3. Indirect adventitious bud formation: After small pieces of organs are cultured on culture medium, they are dedifferentiated to form callus, and then adventitious buds or adventitious roots are induced by differentiation.
★ Embryogenesis mode: 1, direct embryogenesis (direct differentiation from organs, tissues, cells or protoplasts in the culture into embryos without callus in the middle), and indirect embryogenesis (explants are callus first, and then mature from callus cells).
Global embryo) → Heart embryo) → Torpedo embryo) → Single cell embryo.
★ Artificial seed: refers to the use of cell totipotency to wrap somatic cells or meristems (embryoids, stems and stem segments) produced by in vitro culture in a protective outer membrane containing nutrients to form small particles that can develop into complete plants under suitable conditions.
The structure includes artificial seed coat, embryoid (meristem) and artificial endosperm.
★ Application steps for plant tissue culture: 1. Aseptic explants were obtained and sterile culture system was established. 2. Proliferation, constantly producing adventitious buds or embryoids. 3. Rooting culture. 4. Transplanting test-tube seedlings.
★ Selection principle of explants: 1, which must contain living cells. 2. Young organizations contain a high proportion of active splinter cell. 3. The mother pearl must be healthy without signs of decay or disease. 4. The mother pearl must grow actively and will not go into dormancy immediately.
★ Selection of explants: 1, stem tip (most commonly used for tissue culture of horticultural plants, with high reproduction rate and difficult genetic variation, but limited materials); 2. Stem segment (it is easy to obtain by using tender stem segment to promote axillary bud germination); 3. Leaves (young leaf tissue is differentiated from callus or adventitious buds to produce plants, which are easy to obtain and operate, but easy to mutate); 4. Flower balls and buds; 5. Seeds, roots, tubers, petals, etc.
★ Disinfection principle: Disinfectant should be in contact with explants for a long time to remove microorganisms on the surface of explants, but the damage to explants cells should be minimized.
★ Disinfection method: wash the plant materials to remove particles with the same size as the soil → Soak in 70-75% ethanol, which is beneficial to the wetting of the plant surface → Disinfect the surface with 5-20% NaClO solution (adding 1 drop of surfactant) for 5- 10min→ Rinse with sterile water for at least 3 times → Cut off the section contacted with disinfectant. Because disinfectants will kill exposed cells, thus affecting nutrient absorption → cutting explants, usually 10mm stem segments and 10mm leaves (too much hormone will weaken the effect, and too little hormone will not survive).
★ Precautions for disinfection: 1. Surface disinfectants are harmful to plant tissues, so the concentration and treatment time of disinfectants should be correctly selected to reduce tissue death. 2. After the surface of the material is disinfected, it must be washed with sterile water for more than 3 times to remove the residual bactericide, but if it is disinfected with alcohol, it is not necessary to wash it. 3. Before transferring to a sterile substrate, the cut surface that has been in contact with the disinfectant should be removed, because the disinfectant will hinder the absorption of nutrients in the substrate by plant cells. 4. When the explants are seriously polluted, the explants should be washed with running water 1 hour or above, or the seeds should be cultured first to obtain aseptic seedlings, and then tissue culture should be established with various parts of them. 5.HgCl2 has the best effect, but it is the most harmful to people. Rinse with water at least 5 times after use.
★ Shoot tip culture: cut shoot tip or shoot meristem for sterile culture.
Steps: establishment of sterile culture → bud induction → rooting culture → transplanting of test-tube seedlings (genetic variation).
Precautions: In the process of transplanting test-tube seedlings, from heterotrophic to autotrophic, from constant temperature to temperature difference, from aseptic to bacteria, from low light, from high humidity to low humidity, it is necessary to keep the water balance of the seedlings (add plastic film and use a sprayer), select appropriate substrates, and pay attention to light and temperature conditions.
Anthurium andraeanum: leaves → callus induction → bud induction → root induction.
↓↑
Proliferation → root cutting → bud proliferation → re-culture → strong seedlings → before rooting.
Induction of adventitious embryos: tissue section → medium containing 2,4-D → generation of adventitious embryos → medium with 2,4-D removed → spherical embryos → heart-shaped embryos → torpedo-shaped embryos → plants.
Induction of adventitious buds: BA is used for induction, and BA should be removed during ball, heart and torpedo.
★ Significance of embryo culture: 1. In vitro embryo culture of materials with poor endosperm development or incompatibility between embryo and endosperm is helpful to the success of distant hybridization. 2. This provides a good opportunity to study the nutritional needs of embryos at different developmental stages. 3. The regenerative potential of the whole embryo and its parts can be studied.
★ Two important issues in embryo culture: 1. Method of embryo peeling: The best time for embryo peeling is 13- 15 days after pollination. 2. Composition of culture medium: Find a suitable culture medium and add sucrose (energy to maintain appropriate osmotic pressure) in embryo culture.
★ Anther culture method:
Venue: Dachuan Greenhouse.
Materials: Most of them are mononuclear pollen cultures, and the frequency of inducing callus or embryoid is high.
Determination of pollen stage: magenta acetate-potassium iodide staining is often used, and then tabletting is performed for microscopic examination. In practice, it is often determined according to the correlation between bud length and size and pollen age.
Pretreatment: low temperature, high temperature or cross treatment
Medium: MS, Nitsch, Miller, B5, N6. Low concentration of auxin is combined with cytokinin, and high concentration of sucrose has certain effect on pollen induced growth. Adding activated carbon to the culture medium can also improve the induction frequency.
Disinfection, inoculation and culture: anther → beaker grinding (with solvent) → filtration → concentration gradient centrifugation → middle layer collection → centrifugation.
Haploid identification and doubling treatment: When the haploid was treated with 2% colchicine for 24 hours, the callus cells naturally doubled.
★ Significance of pollen anther culture: 1. Haploid cells have only one genome, and the phenotype and genotype are consistent. Once mutation occurs, whether dominant or recessive, it can be manifested in contemporary times, so haploid is an ideal material for somatic genetics research and mutation breeding. 2. In the process of interspecific hybridization breeding, F 1 generation anther culture obtained haploids, and the chromosomes became homozygous diploids immediately after doubling. It takes only two generations from hybridization to obtaining hybrid offspring without separation of individual plants, which significantly shortens the breeding cycle compared with conventional breeding.
★ Anther culture step (using improved NLN medium):
F 1 generation anther → microspore formation → microspore separation → callus formation → embryo formation → haploid plant → homozygous diploid.
↓
Embryogenesis → haploid plants → chromosome doubling to form homozygous diploids.
★ Protoplast isolation: enzymes (cellulase, isolation enzyme)
Steps: disinfection of leaf surface → removal of epidermis → leaf fragments floating in solution containing enzyme and osmotic pressure stabilizer → culture → protoplast sinking to the bottom of Petri dish → removal of enzyme solution → moving protoplast into CPW for cleaning → centrifugation → washing substrate twice → re-suspending in culture medium → removing small individuals, counting with blood cell counter → adjusting to appropriate density and re-suspending in culture medium.
★ Protoplast culture:
Medium: MS+NAA 2.0 ppm+BA 0.5 ppm+3% sucrose +9% mannitol.
Note: 1. After protoplast separation, it is very fragile and needs osmotic pressure protectant protection until the cell wall is formed.
2. Adjust the levels of auxin and cytokinin according to different research objects.
Factors affecting protoplast culture: nutrient requirement (NH4 ++ should not be too much), osmotic agent and culture density (105/mL),
Storage conditions (usually in the dark).
Culture methods: liquid substrate culture, semi-liquid substrate culture, solid substrate culture and nursing culture.
Steps of solid culture: transfer protoplast to culture medium → 1 volume of culture medium containing protoplast and 1 volume of culture medium containing agarose (40℃) → invert the culture dish and culture at 25℃ → protoplast cell wall is regenerated and divided into thin pieces.
Cell mass → cell mass is subcultured in agarose matrix, and penetrant should be reduced in the medium to facilitate the formation of callus and induce differentiation into roots and stems of plants.
Significance of protoplast isolation and culture: 1. The removal of cell wall paves the way for the fusion between plant cells, and leaves open the way for making new hybrids. 2. Protoplasts can absorb exogenous DNA, organelles, bacteria or virus particles, and the combination of these characteristics and plant totipotency lays the foundation for genetic transformation of higher plants. 3. Obtaining cell clones and excellent starting materials for breeding mutants.
★ Protoplast fusion concept: protoplasts separated from the same species or different species are fused under appropriate conditions to obtain a mixture of nuclear material and cytoplasmic material.
★ Somatic hybridization: The method of making hybrid by somatic cell fusion without sexual process at all is called somatic hybridization.
★ Protoplast fusion methods: spontaneous fusion, induced fusion (NaNO3 treatment, high PH- high concentration calcium ion treatment, PEG treatment, electrofusion).
PEG method:
Protoplasts were isolated from green leaves (1). 2. Colorless protoplasts isolated from cell suspension cultures of the same or different species), so that heterokaryon and mother can be distinguished.
→ Take 4 ml of protoplasts (density 2× 105) cultured in suspension on CPW containing 13% mannitol, mix, centrifuge at 100g 105min, put the protoplasts into 0.5% substrate → Add 2 ml of 30% (w105 min). After each dilution, the protoplast weight was resuspended by slight shaking → the mixture was centrifuged at 65438 000 g for 65438 00 minutes, washed in PEG-free medium after centrifugation → centrifuged and resuspended in the same medium.
Disadvantages of PEG method: toxic, low fusion rate: less than 1%.
Symmetrical fusion: parents have not dealt with it, and their contribution to future generations is the same.
Asymmetric fusion: parents make different contributions to their offspring, radiotherapy and chemotherapy.
IOA (does not affect nuclear division, but affects cytoplasmic division)
★ Cytoplasmic hybrid: Using protoplast fusion technology, two extranuclear genetic components (organelles) from different sources are combined with a specific nuclear genome to form a cytoplasmic hybrid.
Identification methods of somatic hybrids and cytoplasmic hybrids: morphology, cytology and molecular genetics.
★ Virus-free horticultural plants:
Heat treatment for detoxification:
Principle: At the temperature higher than normal temperature, many viruses in plant tissues can be partially or completely inactivated, but they are very
Little or no damage to the host tissue.
Methods: Hot water treatment (beneficial to dormant buds) and hot air treatment (beneficial to actively growing shoot tips).
Hot air therapy: the air temperature is 35-40℃, and the duration varies from a few minutes to several weeks depending on the patient.
Note: you can't put it into high temperature at once, you should gradually heat it to adapt. And keep humidity and light.
Limit: 1. Not all viruses are sensitive to heat treatment.
2. It is effective for diseases caused by viruses and bacteroides with equal diameters and threads.
3. Only a few plants can survive after heat treatment.
★ Shoot apical meristem: refers to the part above the base of Yuan Ye, the youngest stem, with a maximum diameter of about 100μm and a maximum length of.
250 microns.
★ Shoot tip: It consists of apical meristem and its 1-3 Ye Yuan base.
★ Shoot tip virus-free culture:
Principle: The distribution of virus in plants is uneven. In infected plants, the apical meristem usually contains no or only a low concentration of virus, and the farther away other plant tissues are from the shoot tip, the higher the virus content.
Factors affecting the virus-free effect of shoot tip culture: medium, explant size (virus-free effect is negatively correlated with explant size, and shoot tip survival rate is positively correlated with shoot tip size), storage conditions (light culture is better than dark culture), and explant physiological state (active growth bud, terminal bud better than axillary bud and bud cutting time).
★ Virus-free callus culture:
Principle: In the callus formed by infected tissue, not all cells carry pathogens evenly.
Reason: 1, the replication speed of virus can't keep up with the proliferation speed of cells.
2. Some cells have acquired antiviral properties through mutation.
★ Identification of virus-free plants: appearance judgment method, indicator plant method (inoculation identification method), antiserum identification method, electron microscope examination method,
Spectrophotometry and enzyme-linked immunosorbent assay.
Indicator plant method: the dead spots produced by the virus on other plants are used as the standard for identifying the virus.
Indicator plants: Hosts specially selected for local diseases are called indicator plants.
★ Non-toxic original seed preservation: plant the seeds in the sterile soil in the greenhouse or insect-proof cover, isolate the area, and propagate through tissue culture.
Common English-Chinese Comparison of Tissue Culture
Abortion (abortion) adenine (adenine) agar (agar) anther (pollen) top (aseptic) auxin (auxin) axillary (axillary bud) callus, callus (callus) cellulartotipotency cellulase (cellulase) cellulose (cellulose) centrifugation (centrifugation) chloroplast (chloroplast) Cytoplasmic hybrid (cytochrome hybrid, cytoplasmic hybrid) Cytokinin (cytokinin) Cytoplasmic degeneration (abortion) Dedifferentiation (dedifferentiation) Redifferentiation (redifferentiation) Within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species within species. Intra-species intra-species intra-species intra-species intra-species intra-species intra-species intra-species intra-species intra-species kinetin (shoot tip culture) microspore monocotyledons (stem segment culture) organ cells (organelles) organogenesis (infiltration) pith (pith) plantlet (plantlet, seedling) pollen culture pollinated protocorm (PLB) protoplast fusion rapid propagation and regeneration.
Common abbreviations
ABA (abscisic acid) CM (coconut milk) CPW (cell-protoplast cleaning solution)
Dimethyl sulfoxide indoleacetic acid indolebutyric acid
Kinetin NAA naphthalene acetic acid polyethylene glycol
LH (liquid nitrogen) CH (hydrolyzed casein) GA3 (gibberellin)