Plant tissue culture is the principle of totipotency of plant cells, tissues or parts of the plant to take a single cell, in the appropriate medium through cultivation can grow into a complete plant. Totipotency of plant cells Totipotency of plant cells is the cells of each plant or plant cell has the complete _set_ of genes, so in certain culture conditions can develop into every cell of a plant like the parent. Utilization of plant tissue culture (A) to increase genetic variability, improved crop (B) the reproduction of plants (C) the industrial production of useful compounds (D) of germplasm storage Plant Tissue Culture http://www.jlau.edu.cn/jiaowu/jpk/jpk/id6_xxzd_33.asp In recent years, tissue culture as a research technique, has been widely used in many disciplines, it is not only important for theoretical research and has demonstrated a very broad application prospects. First, the principle After de-differentiation of plant tissue to form callus, after re-differentiation, callus differentiation but also re-structured tissues and organs, and ultimately form a complete plant. Skoog that back in 1957 found that the type of medium, plant hormones and their ratio, and then play an important role in differentiation. Second, reagents and equipment (A) of the reagent • ethanol. • iaa or 2, 4 ╟ d. • hgcl 2 (or sodium hypochlorite). • 6 - amino-benzyl adenine (6-ba) • ms medium (see appendix). (B) equipment Training room, autoclave, water bath, scalpels, Erlenmeyer flasks (100ml), beakers, measuring cylinders, petri dishes, cotton, inoculation box or clean table, analytical balance, long tweezers, scissors, volumetric flask, pipette tubes, kraft paper. Third, the experimental procedures 1. Preparation of the medium (1) for callus induction: ms medium (sucrose content was 10 g / l, 2,4 ╟ d content of 2 mg / l, agar 10 g / l). (2) Test medium: the medium in ms by adding iaa Table 33 ╟ 1 and 6 ╟ ba. First with a small amount of indole acetic acid 0.1 mol / l naoh dissolution, 6 - amino-benzyl adenine with a dollop of 0.1 mol / l hcl dissolved, and then diluted with distilled water, then add the medium. Table 33 ╟ 1 test medium content of iaa and 6 ╟ ba No. iaa (mg / l) 6-ba (mg / l) The relative ratio 1 0 2.0 2 0.2 2.0 1:10 3 0.5 2.0 1:4 4 1.0 2.0 1:2 5 2.0 2.0 1:1 6 2.0 1.0 2:1 7 2.0 0.5 4:1 8 2.0 0.2 10:1 9 2.0 0 2. Medium sterilization Will add agar medium heat with a good solution, adjusted to ph 5.8, hot-packing in 100 ml flask, the bottle about 20 ml. Cooling medium to be solidified, a layer with a layer of kraft paper and wrapping weighing bottle (of) mouth, and fastened with cotton thread, and then autoclave 121 ℃ (1 kg / cm 2) 20 min under sterile . Remove the flask on the table, the cool reserve. Vaccination of all equipment required for operation (such as long forceps, scalpels, scissors, etc.) and sterile water, to be sterilized at the same time. 3. Callus induction (1) take the number of robust tobacco stem segments, each about 5 cm long, 0.1% in the beaker using mercuric chloride (mercuric chloride) soaked 20 min, washed out with sterile 3 to 4 times, placed in sterile culture dish, in the case of immunization requirements by aseptic stripped of skin (inoculation of UV light sterilization prior case 30 min), with a scalpel cut into 5 mm thick discs (disposal and the last one to start one), with it inoculation of long tweezers in the induction medium, pay attention to the incision toward the wafer medium, inoculated 4 bottle, bottle tied after inoculation. (2) will have access to plant tissue (explants) of the triangular flask, cultured at 25 ℃ greenhouse, l ~ 2 week check times, removing the contaminated material has been flask of bacteria, generated after 3 to 4 weeks callus. (3) _Select_ the callus grew well in the triangular flask, using a scalpel to cut the callus and transferred to test medium with different hormones (also can, together with the original transfer of the explants), put a bottle ~ 2, is still training the greenhouse at 25 ℃, 1 or 2 times a week observing in the different treatment flask, callus differentiation, until the roots and shoots grow. The young plants grow is the "shoots" can be transplanted in the pot. A brief history of plant tissue culture Plant tissue culture is the early 20th century, 30 developed a bio-technology. It is prepared in artificial medium, cultured in sterile plant organs, tissues, cells, protoplast method and other materials. Scientists at the completion of plant hormones on organ, and other aspects of culture medium to improve the results achieved, greatly promoted the development of tissue culture techniques to make the technology practical for rapid propagation, strain improvement. The early 20th century, 50, French scientists succeeded in using tissue culture infected dahlia plants in addition to the off-borne viruses, thus the production of virus-free seedlings provided a feasible way. Now with the removal of plant tissue culture techniques to the virus has been widely used in production. The mid-20th century, 50, due to the discovery of cytokinin, the state of tissue culture explant bud morphogenesis as the factors that can be artificially regulated, so that under conditions in tissue culture regeneration a reality. 60 years after entering, tissue culture technology in the basic theory, practical aspects of continued progress, have been in plant somatic hybridization, haploid breeding, germplasm conservation, rapid propagation, artificial seed production, and other aspects of production of secondary metabolites With the encouraging results. Today, tissue culture technology has become a solid foundation, easy to master, the application of a wide range of technical means. Callus and the formation of callus (callus) originally referred to the local plants stimulated by trauma, the wound surface, the nascent organization. It consists of living parenchyma cells can be derived from any organ of plants within the various tissues in living cells. Trauma in the plant parts, callus can help wound healing; in the graft, the rootstock and scion may promote healing of the vascular tissues by the new rootstocks and scions to communicate; in the cutting, the callus can be differentiated from the wound the adventitious roots or buds, thus forming a complete plant. In plant organs, tissues, cells in vitro, the conditions are suitable for callus can grow. Process of its occurrence: in explants induced by living cells to restore their potential pluripotency, into meristematic cells, then its derived cells into the parenchyma and the formation of callus. From plant organs, tissues, cells produced by in vitro callus, under certain conditions, can further induce organ regeneration or the formation of embryoid plants. In haploid breeding, can also be produced pollen callus or somatic embryos differentiate into haploid plants. Even plants can be induced by protoplast culture or organ regeneration. Therefore, the concept of the callus is not limited to plant part of the new organization of trauma. In plant tissue culture, explants from a typical callus formation, generally go through three stages: initiation, mitosis and forming. Start of a cell preparing for division of the period. Exogenous plant growth hormones on the induction of cell starts to split works well. Commonly used NAA, IAA, cytokinins and so on. Typically use a 1:1 ratio of cytokinin and auxin to induce callus formation of plant material, such as ms +6- ba6-ba is a synthetic cytokinin 6 base called adenine. 0.5 mg / l + ibaiba is a synthetic auxin IBA for short. 0.5 mg / l. Mitotic cells is through the induction of the explants after dedifferentiation, keep dividing, the process of proliferation of daughter cells. Dividing callus is characterized by: cell division fast, loose and light in color and transparent. Differentiation refers to the splitting of the end, cells began to appear on a series of morphological and physiological changes, so have different callus morphology and function of cells. These cell types are thin-walled cells, meristematic cells, pigment cells, fibroblasts and so on. Cells after the explants started a series of changes such as division and differentiation, the formation of a disordered structure of the callus. If the original medium and cultured callus, malnutrition due to the medium or the accumulation of toxic metabolites, leading to callus stopped growing, and even the aging black and die. If you want to continue the growth of callus proliferation, must be regularly (2 to 4 weeks) will divide them into small pieces and inoculated into fresh medium, this callus to the long term to maintain strong growth. Morphogenetic callus way through the start, division and differentiation of callus produced, which despite its cell differentiation, but not organs of the body. Only by meeting certain conditions, the cells will occur callus redifferentiation, resulting in buds and roots, and then develop into whole plants. Induction of buds in tissue culture have generally used a higher ratio of cytokinin and low auxin, such as ms +6- ba1 mg / l + iaa (iaa IAA is a 3 - indole acetic acid for short. ) 0.1 mg / l. And can be used when rooting 1/2ms + iaa0.1 mg / l and so on. Of course, different plant species, different growth state, there will be great changes in the ratio of hormones, which in practice need to explore, to gain experience.
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No. 2
组织培养
组织培养
组织培养
Plant tissue culture is the principle of totipotency of plant cells, tissues or parts of the plant to take a single cell, in the appropriate medium through cultivation can grow into a complete plant. Totipotency of plant cells Totipotency of plant cells is the cells of each plant or plant cell has the complete _set_ of genes, so in certain culture conditions can develop into every cell of a plant like the parent. Utilization of plant tissue culture (A) to increase genetic variability, improved crop (B) the reproduction of plants (C) the industrial production of useful compounds (D) of germplasm storage Plant Tissue Culture http://www.jlau.edu.cn/jiaowu/jpk/jpk/id6_xxzd_33.asp In recent years, tissue culture as a research technique, has been widely used in many disciplines, it is not only important for theoretical research and has demonstrated a very broad application prospects. First, the principle After de-differentiation of plant tissue to form callus, after re-differentiation, callus differentiation but also re-structured tissues and organs, and ultimately form a complete plant. Skoog that back in 1957 found that the type of medium, plant hormones and their ratio, and then play an important role in differentiation. Second, reagents and equipment (A) of the reagent • ethanol. • iaa or 2, 4 ╟ d. • hgcl 2 (or sodium hypochlorite). • 6 - amino-benzyl adenine (6-ba) • ms medium (see appendix). (B) equipment Training room, autoclave, water bath, scalpels, Erlenmeyer flasks (100ml), beakers, measuring cylinders, petri dishes, cotton, inoculation box or clean table, analytical balance, long tweezers, scissors, volumetric flask, pipette tubes, kraft paper. Third, the experimental procedures 1. Preparation of the medium (1) for callus induction: ms medium (sucrose content was 10 g / l, 2,4 ╟ d content of 2 mg / l, agar 10 g / l). (2) Test medium: the medium in ms by adding iaa Table 33 ╟ 1 and 6 ╟ ba. First with a small amount of indole acetic acid 0.1 mol / l naoh dissolution, 6 - amino-benzyl adenine with a dollop of 0.1 mol / l hcl dissolved, and then diluted with distilled water, then add the medium. Table 33 ╟ 1 test medium content of iaa and 6 ╟ ba No. iaa (mg / l) 6-ba (mg / l) The relative ratio 1 0 2.0 2 0.2 2.0 1:10 3 0.5 2.0 1:4 4 1.0 2.0 1:2 5 2.0 2.0 1:1 6 2.0 1.0 2:1 7 2.0 0.5 4:1 8 2.0 0.2 10:1 9 2.0 0 2. Medium sterilization Will add agar medium heat with a good solution, adjusted to ph 5.8, hot-packing in 100 ml flask, the bottle about 20 ml. Cooling medium to be solidified, a layer with a layer of kraft paper and wrapping weighing bottle (of) mouth, and fastened with cotton thread, and then autoclave 121 ℃ (1 kg / cm 2) 20 min under sterile . Remove the flask on the table, the cool reserve. Vaccination of all equipment required for operation (such as long forceps, scalpels, scissors, etc.) and sterile water, to be sterilized at the same time. 3. Callus induction (1) take the number of robust tobacco stem segments, each about 5 cm long, 0.1% in the beaker using mercuric chloride (mercuric chloride) soaked 20 min, washed out with sterile 3 to 4 times, placed in sterile culture dish, in the case of immunization requirements by aseptic stripped of skin (inoculation of UV light sterilization prior case 30 min), with a scalpel cut into 5 mm thick discs (disposal and the last one to start one), with it inoculation of long tweezers in the induction medium, pay attention to the incision toward the wafer medium, inoculated 4 bottle, bottle tied after inoculation. (2) will have access to plant tissue (explants) of the triangular flask, cultured at 25 ℃ greenhouse, l ~ 2 week check times, removing the contaminated material has been flask of bacteria, generated after 3 to 4 weeks callus. (3) _Select_ the callus grew well in the triangular flask, using a scalpel to cut the callus and transferred to test medium with different hormones (also can, together with the original transfer of the explants), put a bottle ~ 2, is still training the greenhouse at 25 ℃, 1 or 2 times a week observing in the different treatment flask, callus differentiation, until the roots and shoots grow. The young plants grow is the "shoots" can be transplanted in the pot. A brief history of plant tissue culture Plant tissue culture is the early 20th century, 30 developed a bio-technology. It is prepared in artificial medium, cultured in sterile plant organs, tissues, cells, protoplast method and other materials. Scientists at the completion of plant hormones on organ, and other aspects of culture medium to improve the results achieved, greatly promoted the development of tissue culture techniques to make the technology practical for rapid propagation, strain improvement. The early 20th century, 50, French scientists succeeded in using tissue culture infected dahlia plants in addition to the off-borne viruses, thus the production of virus-free seedlings provided a feasible way. Now with the removal of plant tissue culture techniques to the virus has been widely used in production. The mid-20th century, 50, due to the discovery of cytokinin, the state of tissue culture explant bud morphogenesis as the factors that can be artificially regulated, so that under conditions in tissue culture regeneration a reality. 60 years after entering, tissue culture technology in the basic theory, practical aspects of continued progress, have been in plant somatic hybridization, haploid breeding, germplasm conservation, rapid propagation, artificial seed production, and other aspects of production of secondary metabolites With the encouraging results. Today, tissue culture technology has become a solid foundation, easy to master, the application of a wide range of technical means. Callus and the formation of callus (callus) originally referred to the local plants stimulated by trauma, the wound surface, the nascent organization. It consists of living parenchyma cells can be derived from any organ of plants within the various tissues in living cells. Trauma in the plant parts, callus can help wound healing; in the graft, the rootstock and scion may promote healing of the vascular tissues by the new rootstocks and scions to communicate; in the cutting, the callus can be differentiated from the wound the adventitious roots or buds, thus forming a complete plant. In plant organs, tissues, cells in vitro, the conditions are suitable for callus can grow. Process of its occurrence: in explants induced by living cells to restore their potential pluripotency, into meristematic cells, then its derived cells into the parenchyma and the formation of callus. From plant organs, tissues, cells produced by in vitro callus, under certain conditions, can further induce organ regeneration or the formation of embryoid plants. In haploid breeding, can also be produced pollen callus or somatic embryos differentiate into haploid plants. Even plants can be induced by protoplast culture or organ regeneration. Therefore, the concept of the callus is not limited to plant part of the new organization of trauma. In plant tissue culture, explants from a typical callus formation, generally go through three stages: initiation, mitosis and forming. Start of a cell preparing for division of the period. Exogenous plant growth hormones on the induction of cell starts to split works well. Commonly used NAA, IAA, cytokinins and so on. Typically use a 1:1 ratio of cytokinin and auxin to induce callus formation of plant material, such as ms +6- ba6-ba is a synthetic cytokinin 6 base called adenine. 0.5 mg / l + ibaiba is a synthetic auxin IBA for short. 0.5 mg / l. Mitotic cells is through the induction of the explants after dedifferentiation, keep dividing, the process of proliferation of daughter cells. Dividing callus is characterized by: cell division fast, loose and light in color and transparent. Differentiation refers to the splitting of the end, cells began to appear on a series of morphological and physiological changes, so have different callus morphology and function of cells. These cell types are thin-walled cells, meristematic cells, pigment cells, fibroblasts and so on. Cells after the explants started a series of changes such as division and differentiation, the formation of a disordered structure of the callus. If the original medium and cultured callus, malnutrition due to the medium or the accumulation of toxic metabolites, leading to callus stopped growing, and even the aging black and die. If you want to continue the growth of callus proliferation, must be regularly (2 to 4 weeks) will divide them into small pieces and inoculated into fresh medium, this callus to the long term to maintain strong growth. Morphogenetic callus way through the start, division and differentiation of callus produced, which despite its cell differentiation, but not organs of the body. Only by meeting certain conditions, the cells will occur callus redifferentiation, resulting in buds and roots, and then develop into whole plants. Induction of buds in tissue culture have generally used a higher ratio of cytokinin and low auxin, such as ms +6- ba1 mg / l + iaa (iaa IAA is a 3 - indole acetic acid for short. ) 0.1 mg / l. And can be used when rooting 1/2ms + iaa0.1 mg / l and so on. Of course, different plant species, different growth state, there will be great changes in the ratio of hormones, which in practice need to explore, to gain experience.
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The concept of plant tissue culture
植物组织培养的概念
植物组织培养的概念
植物组织培养的概念
Plant tissue culture concepts (general) concept of plant tissue culture (general) also known as in vitro, that isolated from the plant meet the needs of the organization. Organs or cells, protoplasts, etc., through the sterile operation, carried out under controlled conditions in artificial Training for a complete regeneration of the economic value of production of plants or other products of technology. Plant Tissue Culture Concepts (narrow) refers to the various parts of plant tissues, such as the cambium. Parenchyma. Mesophyll. Endosperm cultured in regeneration, etc., but also refers to the training process to generate various organs from the callus culture Differentiation of callus formation and then recycled through the plant.
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Brief History of Plant Tissue Culture
组织培养 植物组培发展简史
Plant tissue culture and cell culture began in the latter half of the 19th century, when the concept of totipotency of plant cells has not been fully determined, but based on the natural state of some plants to produce offspring through asexual reproduction observed, people had such a kinds of ideas that could be part of plants cultured under appropriate conditions into a complete plant, for many plant scientists began trying to plant tissue culture. The initial problem is still concentrated in the plant cells have pluripotency and how to make this round of the show. Schwann cells in 1839 made a living cell of every organism in the appropriate conditions are independent of the external environment and development potential. 1853 trecul use of in vitro stem and root segments were cultured in the callus, callus is a no organ differentiation but can be active in dividing cells group, but that does not prove totipotent cells, because Callus did not give birth again by the full plant. 1901 Morgan first time, an all-round development of a cell should have a full plant capacity. The so-called totipotent cell means a complete membrane system and nucleus of living cells under suitable conditions by cell division and differentiation, and regeneration of a complete plant. White pointed out: If a given organism, all cells have roughly the same, and has pluripotency, then observed in the organic body of the cell differentiation of these cells must be organic and the surrounding microenvironment in vivo response. That is why every cell of the body did not show all-round, it is because the different location of the cells, resulting in some of its function is inhibited (suppressed), a full description of the body of the micro-environmental factors played in the cell differentiation a very important role. According to modern developmental biology and cell biology theory, cell differentiation is affected by genes both in time and space tone space, the space is a cell in the body position. Different positions of the cells, which express different genes, cells demonstrated different morphology and behavior. If a living cell isolated from plants, so separated from the original environment, inhibiting the function of cells are expected to be restored and re-exhibit pluripotency. Based on this understanding, scientists have sprouted out of the idea of plant tissue culture. 1934 White with tomato roots in vitro established the first clone of an active growth, so that the root of the in vitro experiment the first time a real success, and made the first discovery and B vitamins B1, vitamin B6 and nicotinic acid importance. At the same time, Cautheret Mao in the mountains form a layer of black poplar and willow cultivation of tissue were also found in the role of B vitamins, and make training a success. Nobecourt also used to establish a similar carrot continuous growth of tissue culture. Therefore, Haberlandt, White and Nobecourt together known as the founder of plant tissue culture. It is now used by a number of training methods and media, in principle, are they in the years 1939 to establish the methods and results of media evolution, almost all media are added in different types and different amounts of B vitamins. From plant tissue culture phase of rapid development. In 1941, Overbeek, Conklin and Blakeslee, etc. with additional coconut milk into medium obtained Datura in vitro embryo culture of success. Coconut milk composition complex, containing many different organic compounds, the later study found that in tissue culture which play a major role in the adenine hormone or analogues. In 1944, Skoog reported that DNA degradation products of adenine and adenosine can promote callus growth, the lifting of the inhibitory effect of auxin on bud formation and induce bud formation. 1948, Caplin and Steward proved coconut milk and with 2,4-D co-cultured carrot and potato on the proliferation of organizations play a significant role in promoting. Myeloid cells in the use of tobacco callus induction experiments, Skoog, Miller identified other isolated 6 - furan aminopurine can promote cell division, and named "KT" (Kinetin). After A related homologue 6 - benzylaminopurine was synthesized, it also stimulated the cultured cell division. Thus, a "cytokinin" the collective name used to refer specifically to stimulate cell division and culture of a group of 6 - amino group of a purine compounds. Later, zeatin, isopentenyl adenine and cytokinins and other plant hormones have been found, but also increased the types of cytokinin. The discovery of auxin and cytokinin complement each other to regulate cell division and differentiation, control of organ differentiation, growth hormone can induce high root formation, high cytokinin can promote the differentiation of bud, so that the work of plant tissue culture rapid breakthrough. 1958 United States and Germany, Reinert Steward cultured carrot cells were induced by the formation of embryoid bodies, Vasil and Hildebrandt in 1965 by a single isolated cells cultured in the regeneration of the whole plant, so that the theory of plant cell totipotency truly confirmed by science. Since then, batch after batch of tissues or organs of plants obtained by culture of plant regeneration. Of the 20th century, 60 years, in terms of plant tissue culture is divided into two other achievements microspore culture and protoplast culture of success. Guha and Maheshwari (1966,1967), Rourgin and Nitsch (1967) has the use of tobacco and carrot plants haploid microspore culture, and successfully achieved the chromosome doubling, so that homologous diploid plants in 5 months of harvest to the seed. Cocking, etc. The purified cellulase and pectinase treatment of tobacco cells, protoplasts obtained by adjusting the osmotic pressure of the methods to control protoplast expansion, so that training to be successful, have been regenerated. 60 years since the beginning of the 20th century, plant tissue and cell culture of the factory gradually and the commercialization stage. Now can not be exact statistics on the number of plants obtained through tissue culture regenerated plants, as may occur almost daily use of new plant species to obtain training success stories. Plant tissue culture has become a routine experimental technique widely used in plant detoxification, propagation, genetic engineering, cell engineering, genetic research, the production of secondary metabolites, and other aspects of industrialized breeding; from advanced research institutions, colleges and universities to the general biotechnology company, and even farmers specialized households are in varying degrees of use or to carry out tissue culture work.
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The application prospects of plant tissue culture
1, the rapid propagation of some rare plant or plants of economic value greater reliance on natural conditions in a short time propagation of rare plants and plants with high economic value, subject to geographic and seasonal restrictions, it is difficult to achieve rapid and efficient purpose; particular need for a certain amount in a short time in order to create the plant should be valued, time is effective only through tissue culture methods to meet this requirement. Plant propagation by tissue culture, which is tissue culture used in the production of the main and most effective examples. The first is the successful application of the orchids. Morel in 1960 from orchid tissue culture by postemergence, and soon applied to production, the formation of a tissue-culture propagation orchid industry. The changing environment of many species of plants facing extinction, and many species of plants have become extinct, leaving only a human regret. How to save these plants, there are many animals, has become a worldwide concern. Proved by the tissue culture method can be part of the endangered plant species to be renewed and preserved; if the combination of cryopreservation techniques to make these plants are more permanent preservation. In fact, for most common plants, the use of tissue culture methods preserve their germplasm materials, is also of great significance. Because people now can not predict which plants will face extinction, lush plants may seem today, tomorrow could be the desert, flood, fire or war engulfed. 4, through the haploid pollen and anther culture plants, shorten the breeding period by anther and pollen haploid plant tissue culture can greatly shorten the breeding time. Development of new varieties to greatly simplify the process 5, the application of embryo culture in the far source hybrid, the hybrid after the formation of ovules often immature state, to stop growing, can not form a seed viability, and thus hybrid sterility, which hybridization to cause great difficulties. The late nineteenth century, twentieth, Laibach culture with embryo culture technique interspecific hybrid embryos of flax, the first hybrid plants obtained, this time successfully overcome in the distant hybrid incompatibility barriers provided a useful technique. The technology development so far, is already quite mature, we can say the majority of mature or immature plant embryos are available through the cultivation of success. Immature embryo culture allows five cell size has been a very young embryo-like structures develop into plants. Ovule culture, but few studies, a single ovule culture still exist in many of the problems, the need for deepening the study. Hybridization, because of physiological and genetic disorders and can not cross the success of in vitro fertilization can be used to overcome the upcoming female ovule in vitro culture, the mutant pollen germinated in the ovule fertilization, the resulting hybrid embryos in vitro develop into complete plants. Endosperm triploid plants obtained by culture, in order to induce the formation of triploid plants opened up a new way. Hexaploid doubled triploid obtained can be bred polyploid species. 6, cell fusion by protoplast fusion, can be partially overcome the incompatibility of sexual hybridization, somatic cell hybrids obtained in order to create new kinds of varieties bred stock. 7, the application of mutant cells cultured in a constant state of meristematic state of cells, culture conditions and it vulnerable to external pressures (such as radiation, chemicals) and mutagenic effects, from which you can filter out the useful mutants, which breeding new varieties. Currently this method has been _select_ed to the resistance, salt, high protein, high-yield and other mutants, some have been used in the production. 8, for genetics, molecular biology, cell biology, histology, embryology, genetic engineering, biological engineering activities to reveal the secret of life, need more science, more technology complement each other, in which plant tissue culture technology is indispensable, it is genetics, molecular biology, cell biology and biological engineering provides an effective and fast way. As to reveal the secret of life, we must first study the role of individual genes to study how it is assembled in cells, how to contact with other genes, such as how to express and control. Separation of a single gene, sequenced its DNA, and then base the implementation of some of these mutations, and then also need to receptor cells which genes to see the expression, to determine its function. Accept the gene to produce regenerated receptor cells to tissue culture methods need to be achieved. 9, the use of the materials as plant tissue culture bioreactor Chinese herbal medicine is a valuable as_set_ of mankind, but a variety of herbs lack of resources, insufficient production, and even on the brink of extinction. If you can use the method of tissue and cell culture production in the laboratory, no longer dependent on the natural environment, not only can solve the existing difficulties, but also the active ingredient by screening high-yielding cell lines, to improve their medicinal value. Such as the use of ginseng cell suspension culture to produce ginsenosides, has been in Japan and other countries scale. Use of plant cell and tissue culture cells as a bioreactor, can also produce certain proteins, amino acids, antibiotics, vaccines, such as raw vegetables production by hepatitis B vaccine is experimental. 10, for other unknown scientific study of modern scientific development is very fast, a lot of unexpected things are now possible, new inventions, new discoveries, new creation after another, something considered impossible today may become a reality tomorrow. Plant tissue culture is also out with a lot of untapped potential, perhaps one day people will grow in the flask in a large pumpkin. In short, the current plant tissue culture is still in development stage, far from its peak, many people have not figure out the mechanism, its potential is far from played out. I believe in the next few decades, tissue culture in our country will have greater development in the agriculture, pharmaceutical industry, processing industry, etc. will play a greater role in creating greater economic benefits.
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The classification of tissue culture
Explants divided by plant tissue culture can be divided into the following categories: 1, embryo culture, embryo culture of plants, including embryo culture, endosperm culture, ovule and ovary culture and embryo culture in vitro fertilization technology. 2, organ and tissue culture plant organ culture is all or part of an organ or organ primordia of the culture, including the stem, shoot tip, tubers, bulbs, leaves, inflorescence, petal, ovary, anther, receptacle, fruit and seeds. Tissue culture of the broad and narrow sense. General: included the cultivation of various types of explants. Narrow sense: including the cambium tissue, meristem, epidermis, parenchyma and various organs and tissues, and its resulting callus culture. 3, including the use of cell culture cell culture bioreactor, and to promote cell growth and biosynthesis of a large number of single cell culture system and the use of cloning technology for cell growth, differentiation until the formation of complete plants, single cell culture. 4, protoplast culture of plant protoplasts the cell wall was removed from the plasma membrane coated with a life force of the bare cell.
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Tissue culture step
组织培养的步骤
First, media preparation There are two ways to the preparation of media options, one to buy all the chemicals in the medium, in accordance with the needs of their preparation; the second is a good medium to purchase goods in the basic components of mixed powder, such as MS, B5 and so on. Their preparation can save costs, but a waste of time, manpower, and sometimes the quality of medicines, to the experimental trouble. To the current domestic situation, or most of their preparation. For convenience, an example is configured to MS medium the primary process medium. 1, the preparation of several liquor (1) preparation of a large number of elements in the mother liquor MS General preparation of the large number of elements are 100 times the mother liquor, and then were used diluted 100 times. Weigh NH4NO3 165g KH2PO4 17g KNO3 190g CaCl2 · 2H2O 44g MgSO4 · 7H2O 37g 1L dubbed their mother liquor. Into the reagent bottle 1L, store in refrigerator. (2) preparation of MS trace the mother liquor Preparation of the trace elements generally 100 times the mother liquor. Weigh in turn KI 0.083g Na2MoO4 · 2H2O 0.025g H3BO3 0.62g CuSO4 · 5H2O 0.0025g MnSO4 · H2O 1.69g CoCl2 · 6H2O 0.0025g ZnSO4 · 7H2O 0.86g 1L dubbed the mother liquor, into the reagent bottle 1L, store in refrigerator. CuSO4 · 5H2O and CoCl2 · 6H2O as weighed very small, if the balance did not reach millionth accuracy can be dubbed to adjust fluid. Weigh CuSO4 · 5H2O 0.05g CoCl2 · 6H2O 0.05g The adjustment of their respective dubbed 100ml liquid, and then take the amount of 5ml to also 0.0025g. (3) preparation of organic liquor MS MS 100 times the general preparation of organic liquor. Weigh in turn Inositol 10g thiamine hydrochloride (VB1) 0.01g Niacin Glycine 0.2g 0.05g Pyridoxine hydrochloride (VB6) 0.05g 1L dubbed the mother liquor, into the reagent bottle 1L, store in refrigerator. (4) preparation of ferric salt mother liquor MS MS 100 times the general preparation of ferric liquor. Weigh in turn Disodium EDTA 3.73g FeSO4 · 7H2O 2.78g 1L dubbed the mother liquor, into the reagent bottle 1L, store in refrigerator. So MS liquor liquor There are 5 major elements, trace elements together with the mother liquor MS, MS MS iron salts of organic liquor and liquor, a total of eight kinds of liquor. Mother liquor of the preparation of hormones Various auxins and cytokinins to the preparation of a separate, not mixed together, usually first with a small amount of auxin class of 95% alcohol or 1 equivalent of NaOH solution, cytokinin generally first dissolved in hydrochloric acid with 1 equivalent, then add distilled water to volume. Generally take 100mg dubbed 100ml liquor. 2, preparation of medium To configure 1L MS medium, for example, order the following: (1) first put some distilled water in the beaker. (2) above were taken into eight 10ml liquor. (3) Weigh 30g of sucrose into the general, stirring to dissolve. (4) be dissolved in distilled water with a graduated cylinder to 1L. (5) designed the program by adding a variety of hormones, a small amount of the hormone, and hormones on the growth of tissue culture plants is essential. So best to use conditional micro adjustable pipette, then drawn to reduce the error. (6) with a precision test strips or pH meter to adjust PH to 5.7-5.8. (Able to do so using the pH meter, more precise) can be equipped with an equivalent of HCL and 1 equivalent of NaOH solution to adjust PH value. 1 equivalent HCL preparation: paired with cylinder capacity to take 8.3ml 100ml solution. 1 equivalent NaOH preparation: Weigh NaOH 4g dubbed in 100ml solution. (7) weigh about 5g agar powder (good quality agar), poured into the top with a good solution on the electric furnace heated to boiling until the agar melted. (8), slightly cooled, divide into culture container. The cultivation of non-cover sealing the container to use film or kraft paper and sealed with a rubber band or string tightly enclosed. (9) into the pot disinfection and sterilization sterilization, sterilization about 20 minutes. (10) after sterilization removed from the sterile medium pot, flat on the experimental stage so that it Solidification. Second, sterilization Sterilization is one of important tissue culture. Beginners have to clear bacteria and sterile areas. With bacteria areas are: all objects exposed to the air, contact the natural water body, at least on the surface it is a bacteria. So point of view, sterile room where raw, clean table surface, simple boiled medium, we use the knife, cut the raw before the appearance of our body and connected to the table with the outside world, such as the entire digestive tract, respiratory tract, the gas that we exhale, regardless of culture vessels washed clean, and so much more are all bacteria. This refers to bacteria, including bacteria, fungi, actinomycetes, algae and other microorganisms. Bacteria is characterized by: small, unseen. Everywhere and at all times have, all-pervasive. Under natural conditions, patient and strong and living conditions require simple, high reproduction rate, conditions could be appropriate, a large number of breeding. Sterile areas are: high temperature burning, or cooking a certain time after the object, the other physical or chemical method of sterilization after the object (of course, these methods have proved to be effective), the upper atmosphere, the rock house, health animals and plants are not in contact with the outside organization, strong acid and alkali, chemical elements and the internal surface sterilization agent is sterile. It can be seen from the above: the earth's surface than a sterile world, the world much smaller bacteria. Sterilization refers to the physical or chemical means, and the pore surface to kill all the microorganisms or organisms, that is to kill all the material of all life. Related to this is the concept of disinfection, it means to kill, remove or fully inhibit the part of the microorganisms, so that no harmful effects occur, it is clear sterilized, many bacterial spores, mold spores and other thick wall will not completely kill that, because After disinfection of the environment in and items there are live micro-organisms, so the operating space through strict sterilization (vaccinations, Clean Bench, etc.) and use of utensils, and the operator's clothing and hands do not take any living micro-organisms . Under such conditions the operation, called aseptic operation. Plant tissue culture requirements for sterile conditions are very strict, even more than the cultivation of microorganisms required, because the medium is rich in nutrients, a little careless to cause bacterial contamination. To achieve complete sterilization purposes, must be taken according to the different objects of different methods of effective sterilization to ensure the bacteria cultured from the impact of the plantlets to grow normally. Commonly used method of sterilization can be divided into physical and chemical categories, namely: physical methods such as dry heat (burning and burning oven), heat (atmospheric or high pressure cooking), irradiation (UV, ultrasonic, microwave), filtering, cleaning and washing a large number of measures such as sterile water; chemical methods is the use of mercuric chloride, formaldehyde, hydrogen peroxide, potassium permanganate, to the Soviet Union water, bleach, sodium hypochlorite, antibiotics, alcohol, chemical processing. These methods and agents to work in accordance with the appropriate choice of different materials for different purposes. 1, the culture medium with heat sterilization Medium in the preparation completed within 24 hours after the sterilization process. Autoclave principle is: in closed steamer, in which the steam can not leak out, increasing the pressure, so increasing the boiling point of water to the pot temperature increased. Under the 0.1MPa pressure, the pot temperature reached 121 ℃. In the steam temperature, can quickly kill all bacteria and its high heat-resistant spores. Note that the air completely ruled out the pot, so that all the pot of water vapor, can be completely sterilized. Autoclave deflated several different approaches, but the purpose is to drain the air, so even the pot temperature to ensure complete sterilization. Common method is: put off valves, power and after the pressure up to 0.05MPa, the play open valve and release air until the pressure gauge pointer is zero, then close the release valve. After the valve off and then power, pressure increased to 0.1MPa, the start time, to maintain the pressure 0.1-0.15MPa, 20 minutes. By vessels of different sizes, dwell time is different, see Table. The figures in the table is a complete sterilization is the number of insured, if the larger the container, but a small number of placement can also reduce the time. Third, inoculation Inoculation as a process of exposure, it is easy to cause pollution of the period, this period mainly by airborne bacteria and staff are the cause, inoculation room should be strictly for space disinfection. Indoor maintain regular inoculation of 1% -3% with potassium permanganate solution on equipment, walls, floors, wash the paint. In addition to using ultraviolet light and formaldehyde sterilization before use, but also in the use of alcohol during the period with 70% or 3% of the children spray to the Soviet Union, so that dust particles in the air _set_tling down. Aseptic technique according to the following steps: (1) 4 hours before inoculation, vaccination with formaldehyde fumigation room, and open the UV lamp for sterilization therein; (2) 20 minutes before inoculation, open the clean table fan and UV lamp table; (3) Wash hands before vaccinators, in the buffer for a good special lab coat, and change into slippers; (4) on the table after swabbing with alcohol cotton ball with both hands, especially nails place. And then swabbing the work surface; (5) cotton swabbing with alcohol inoculation tools, forceps and scissors from start to finish and then fire again, and then repeated at the tip too far, too far to the dish to be dried; (6) inoculation, the vaccinators can not leave the table with both hands, unable to speak, walk and cough; (7) after inoculation to clean tables, used ultraviolet light sterilization for 30 minutes, if the continuous inoculation, every 5 days to a major strength of sterilization. Vaccination is a good sterile roots, stems, leaves and other organs from the body through cuts or cut into small pieces or small pieces, put into the process medium. Before and after the vaccination program will now be described coherently. Sterile inoculation steps: (1) the initial wash and cut the ingredients into a beaker, into the clean table, sterilized with disinfectant, and then sterile water rinse, and finally drain to the water, out placed in sterilized gauze or filter paper on. (2) dry materials, the one hand and forceps in one hand and scissors or scalpel, cut the material properly. Such as leaves cut into 0.5cm square pieces; stems cut into small pieces with a section. Micro-tip to peel into pieces containing only 1-2 young leaves of the shoot tip size. In the course of vaccination vaccination should always burning equipment to prevent cross-contamination. Fourth, culture Training refers to training materials on the culture room (with light, temperature), the so growth, division and differentiation of callus formation or further differentiation into the process of regeneration. 1, the training methods (1) Solid culture That is medium solidified with agar to develop methods of plant material. Is now the most commonly used method. Although the method of simple equipment, easy, but the uneven distribution of nutrients, growth rate is not balanced, and often occurs Browning poisoning. (2) liquid culture That is not reinforcing agent with the liquid culture medium method of plant material. As the oxygen content of less liquid, so stir or shake usually need to approach the culture medium to ensure the supply of oxygen, the use of reciprocating or rotary shaker for shaking culture, and its speed is generally 50-100r/min, this periodic immersion method, both the medium homogenization, but also ensures the supply of oxygen. 2 cultivation steps (1) primary culture Primary culture of sterile materials and designed to be clonal. That some of the explants after inoculation, the first generations of training. Primary culture, the commonly used induction or differentiation medium, the medium containing cytokinin and a little more growth hormone. Primary culture clonal established, including: stem tip, bud burst, embryoids and PLB and so on. When first developed under the direction of culture can be divided into: 1) The development of terminal buds and axillary buds This regeneration of plants suitable for propagation in the sample, you can only use the terminal bud, lateral bud or stem segments with the other branches such as seed germination can take after. Shoot tip culture in this area can be seen as a rather peculiar way. It uses an extremely young terminal bud of the apical meristem as explants for inoculation. In actual operation, including the use of apical meristems and some other organizations to develop, this will ensure easy operation and easy to survive. Determined by culture with the cultures of buds are generally longer internode, with erect stems shoot upward, Propagation is mainly used when the stem cutting method, such as carnation, petunia, chrysanthemum, and so on. However, under special circumstances will also give birth to bud, bud burst. 2) the development of adventitious buds In explants cultured by the adventitious buds, usually the first to go through the process of dedifferentiation, callus cells. Then, after further differentiation, that is, from the formation of organ primordia meristem, which constitute the organs of one-way on the vertical axis shows the polarity (which embryoid different). In most cases it is the formation of buds to form roots. Culture in the bud, they often induce or differentiation medium. Obtained by culture with cultures of adventitious buds, the general bud burst for breeding, such as gerbera, strawberry and so on. 3) The embryoid body cells in the occurrence and development Somatic embryogenesis is similar to zygotic embryos but different, it is also through the ball, heart-shaped, torpedo-shaped and cotyledonary embryo development stage, and ultimately develop into seedlings. But it is the body's cells. Embryoids from callus surface, or from the surface of the explants produce differentiated cells, or cells from suspension culture generated. 4) primary culture explants browning Explant browning is after inoculation, the surface began to browning, and sometimes even make the medium browning. Its appearance is due to plant tissue polyphenol oxidase is activated, leaving due to changes in cell metabolism. In the browning process, will produce quinone, which mostly tan, when spread to the medium, the it will inhibit the activity of other enzymes, thus affecting the exposure of cultured explants. Browning main reasons are: a, studies have shown that plant species in different varieties of browning is different. Polyphenol oxidase activity as the difference, so some flower varieties after inoculation explants browning easier, and some varieties of flowers after inoculation of explants is not easy browning. Therefore, in the training process should have a choice of different varieties were processed. b, the physiological state of the physiological state of explants different, after inoculation at different levels of browning. Generally speaking, in the young plant material browning of shallow, but adult plants have been harvested from the explant, as containing quinone more, so the browning is more serious. In general, the young organization in the degree of browning is not obvious after inoculation, and mature organizations in more serious degree of browning after inoculation. c, high concentrations of inorganic salts medium composition of certain ornamental plants would increase the degree of browning, in addition, high levels of cytokinin explants also stimulated some of the polyphenol oxidase activity, so that browning deepened. d, culture conditions inappropriate if the light is too strong, high temperature, incubation time too long, etc., can make to improve the activity of polyphenol oxidase, thus speeding up the explants were cultured in the browning. To improve the rate of tissue culture seedling must be the browning of explants to control. The following preventive measures can be used to reduce the browning phenomenon. 1, _select_ the appropriate explants in general, the best choice for strong growth in the explants, which can significantly reduce the browning. 2, the right culture conditions inorganic components, the level of plant growth substances, suitable temperature, in a timely manner can reduce the subculture of the material are browning. 3, the use of antioxidants in the medium, the use of cysteine, ascorbic acid and other antioxidants can be more effectively avoid or mitigate many of the browning of explants. In addition, the use of 0.1% -0.5% of the activated carbon to prevent browning also have a more significant results. 4, continuous transfer of material can be easily Browning interval 2-24 hours of incubation, then transferred to the new medium, so that after 7-10 days of continuous treatment, browning will be controlled or greatly reduced . (2) subculture In the initial culture obtained on the basis of buds, seedlings, somatic embryos and the original bulbs, etc., quantities are not enough, they need to further proliferation, the more and more, to play fast breeding advantage. Is the second subculture after primary culture for several generations of extended propagation training process. To breed a considerable number of non-rooted, the last side to achieve the purpose of breeding side of root. Subculture generations is based on a geometric increase in the process. If the basis of two seedlings, then after 10 generations to produce 210 seedlings. Subculture propagation methods include: cutting stems, buds cluster separation, separation embryoid, separation of the original bulbs and so on. Cut stems are commonly used in the shoot elongation of the stem, stem node culture more visible. This method is simple, to maintain the characteristics of the mother species. MS medium is often a basic medium; separation of bud burst for the buds from the callus plexus birth. Medium is often a differentiation medium. If the small bud bud cluster. Buds can be cut into small bundles and put it into MS medium, until the larger, and then separated and cultured. Proliferation medium used for each kind of plant is almost identical, as cultures in close with the best environmental conditions, under the control of nutrient supply and hormones, eliminating competition from other organisms, so that by geometric progression proliferation. In the rapid propagation of the initial culture is a necessary process, and subculture was carried out regular non-stop process. However, the amount reached after the transfer should be considered part of the rooting phase. In a sense, but reserves the proliferation of the mother plant, while the root is the proliferation of materials diverted to produce finished products. (3) subculture of the glass when the material Practice shows that when the plant material for in vitro propagation constant, some cultures of the stems, leaves often showed a translucent water-like track that imagination is often referred to as vitrification. Its appearance makes the slow growth of the plantlets, the propagation coefficient decreased. Glass tube into the physiology of seedlings disorders. Because of the emergence of the spears of glass should not be rooting, Thus, the propagation coefficient is substantially reduced. In different species, cultivars, the degree of the glass tube seedlings are also different. When the medium is high cytokinin levels, are also prone to vitrification. Add a small amount of alcohol in the culture medium, ABA and other substances, to a certain extent, reduce the vitrification phenomenon. Vitrified plantlets show, its stems, leaves the surface without wax, higher levels of polar compounds in vivo, cells poor water holding capacity, plant transpiration intensity, not normal transplanting. This was mainly due to high air humidity in the culture vessel, caused by poor ventilation, the specific solution is: a, increase the level of solutes in the medium to reduce the water potential of the medium; b, reducing the amount of nitrogen compounds in the medium; c, increased light d, increase the container ventilation, it is best to CO2 fertilization, which is to reduce the phenomenon of Vitrification significant role; e, reduced culture temperature, the temperature training, help to reduce the phenomenon of Vitrification; f, lower cytokinin content of the medium, consider adding an appropriate amount of ABA. 3, rooting When the material to a certain quantity of proliferation, it is necessary to divert part of the culture to the rooting phase. If not in time to rooting culture medium up, it will transfer the seedling to eventually becoming an aging yellow, or leaving because of overcrowding caused by discarded waste invalid seedlings increased. Root culture is to take root without rooting process that aims to give birth to the adventitious roots thick and sturdy. Rooting can be 1 / 2 or 1/4MS medium, remove all the cytokinins, and add non-food auxin (NAA, IBA, etc.). The following methods can be used rooting a, dip the base of the shoot 50 or 100 * 10-6IBA solution treatment 4-8 hours; b, in the presence of auxin medium for 4-6 days c, directly into the rooting medium containing auxin. Shoot the three methods can induce root, but the first two methods of growth and development of new roots is more favorable. And the third inhibit the growth of young roots. The reason is that when the root after the original formation of the continued existence of a higher concentration of auxin is not conducive to the growth of young roots. However, this method more feasible. In addition the following methods can be used also can take root. 1 in the proliferation medium to extend the culture time; 2, intends to reduce the number of proliferation rate and reduce the amount of cytokinin (ie proliferation and rooting into step); 3, cutting twigs stout roots directly in the nutritional bowl, This method is not rooting phase. Eliminates the need for a media production, cutting cuttings available under solution dip auxin treatment, but this method is only suitable for some easy to root crops. Also rooting more difficult when a small number, you need to place the filter paper bridge in the medium, making it slightly higher than the surface, relying on absorbent filter paper supply of water and nutrients, and induce root. From somatic embryos developed into seedlings, often the root of the original differentiated, this root can not grow by rooting stages. But by the development of embryoid particularly large number of seedlings and young individuals, it often requires a low concentration of plant hormones or not the stage of culture medium for seedling root. Rooting in vitro stage, in order to successfully migrate to the environment outside the tube, in order to adapt to the environment vitro conditions. Usually suitable for domestication of different plants at different temperatures. Such as chrysanthemum, to 18-20 ℃ appropriate. Practice shows that the growth of plants will not only involve high temperature transpiration strengthened. But also involves the problem likely to grow fungi. Growth temperature is too low to slow, or difficult to survive. Seedbed in spring when the temperature _set_ting on the hotline can be added, so that substrate temperature is slightly higher than the air temperature 2-3 ℃, which is conducive not only to promote rooting and root system, but also to advance survived. Transplant seedlings of plants outside the test tube light intensity should be higher than before transplantation cultivation has increased, and can adapt to high intensity diffuse light, (about 4000lx or so) to maintain photosynthesis light intensity. But the light is too strong to stimulate transpiration enhanced water balance of the conflict will be more acute. V. transplanting acclimation Tissue culture plantlets were transplanted is an important part of the process, part of the job done well, it will cause to naught. In order to do the transplanting plantlets should choose a suitable substrate, and in line with the appropriate management measures to ensure the successful completion of the work of tissue culture. In vitro because it is sterile, nutrient supply, suitable for light and temperature close to 100% relative humidity, the growth of environmental conditions, therefore, in physiology, morphology and other aspects of the growth of seedlings and the natural conditions were very different. It must be hardened, for example through water control, weight loss, credit to, cooling and other measures to enable them to gradually adapt to the environment, so that the physiology, morphology, corresponding changes in the organization to make it more suitable for the natural environment, the only way transplanting plantlets to ensure the smooth success. Shangkan from leaves, shoots developed cuticle, leaves usually without trichomes, or only a few tables 皮毛, and even leaves a lot of water holes there, and that the number of stomata, size, often more than an ordinary seedlings. It can be seen, more suitable for plantlet growth in high humidity environments, transplanting them to the test tube when the external environment, the plantlets will be very high water loss rate is very likely to die. Therefore, in order to improve the above-mentioned adverse physiological vitro, morphological characteristics, it must be compatible with the outside world of acclimation, the measures usually are: to increase the humidity of the outside world, less light; on the in vitro permeability to gas, enriching carbon dioxide, fertilizer, and gradually reduce the air humidity. In addition, cultivation of domesticated substrate to be sterilized because of plantlets grown in a sterile environment, on the outside of bacteria, fungi poor resilience. In order to improve the survival rate in the culture medium can be mixed with chlorothalonil 75% WP 200-500 times to be sterilized. 1, transplanting with the matrix and the container Plantlets for planting the matrix to have the air permeability, moisture and certain fertility, easy sterilization, is not conducive to the breeding characteristics of bacteria, and can be used perlite, vermiculite, sand and so on. In order to increase adhesion and some with peat soil fertility or humus can be. Timing rata basis with the general use of perlite, vermiculite, peat or humus soil ratio of 1:1:0.5. Can also be sand: peat soil or humus is 1:1. These media should be autoclaved before use. Or at least hours of baking to eliminate one of the microorganisms. According to the cultivation of habits of different plants to the preparation, cultivation in order to obtain satisfactory results. Here are some common vitro culture medium. (1) river sand Divided into coarse river sand, fine sand of two types. Coarse river sand that is usually referred to, the particle diameter of 1-2mm. Surface of sand known as sand, the particle diameter of 0.1-0.2nm. Drainage characteristics of river sand is strong, but poor water storage capacity of fertilizer, planting is generally not used alone directly vitro. (2) peat soil Peat soil is deposited on the marsh in the decay of plant debris formed after a long period, and its good water retention, fat storage ability, were neutral or slightly acidic reaction, but usually can not alone be used to plant shoots, and the river should other types of sand mixed with each other dubbed Pentu and to use them. (3) humus Humus from plant leaves formed by the decay. One is the natural form, a man-made, artificial fall leaves may be collected and then buried in pits, irrigation to weathering under the conditions of moisture, and then screening can be obtained. Leaf mold that contains a large number of mineral nutrients, organic matter, it usually can not be used. The culture medium mixed with humus will help the hair root plants. (4) container Cultivation containers used 6 * 6cm-10 * 10cm soft plastic bowl can also be used nursery tray. The former covers an area of large, use a large amount of matrix, but the seedlings do not transplant, which requires the second transplant seedlings, but save space, the provincial matrix. 2, preparation before transplanting Culture before transplanting can be moved without opening exercise 2-3 days under natural light, so that the irradiation of light shoots to accept, it looks sturdy up, and then open practice 1-2 days seedlings, subjected to low humidity processing, to meet future natural moisture conditions. 3, transplanting and seedling management Remove the hair from the root of test-tube seedlings, the roots washed with tap water medium adhesion, the whole is removed to prevent the residual medium breed bacteria. But gently removed, should avoid root injury. Transplantation with a thick bamboo chopsticks _insert_ed a small hole in the matrix, and then _insert_ the seedlings, pay attention to more tender seedlings to prevent the hurt, the surrounding matrix compaction after transplanting the seedlings, planted before the substrate to be irrigated. Light thin the water after transplanting. Then the seedlings into an environment of high humidity. Ensure that more than 90% humidity. (2) to prevent the breeding of fungus. Vitro environment as the original is a sterile, difficult to maintain after the move out completely sterile, therefore, should try not to breed a large number of fungi to facilitate survival. So should the Substrate sterilization autoclave or hung roast. For the proper use of a certain concentration of fungicide for the effective protection of seedlings, such as carbendazim, Tuobu Jin, 800-1000 times the concentration, spraying time should be 7-10 days. Injury in the transplant seedlings when the seedlings as little as possible, wound too much, too much root damage are caused by Simiao reasons. When the water can be added 0.1% urea, or a large number of elements in aqueous solution as 1/2MS dressing, can accelerate the growth and survival of seedlings. (3) a certain temperature and light conditions. Plantlets after transplanting to maintain a certain temperature and light conditions, temperature is suitable rooting 18-20 ℃, winter and spring, low ground temperature can be electricity to heat hot. Growth will slow the temperature is too low, or not easy to survive. Evaporation temperature is too high, so that the destruction of water balance and will promote the breeding of fungus. Also available in the early management of weak light illumination, such as stamped in the Small Shed shade net or newspaper to prevent sunburn seedlings and increased water evaporation. When the small plants with new growth, the gradual strengthening of light, latter can directly use natural light. Promote the accumulation of photosynthetic products, and enhance resistance, and promote their survival. (4) maintaining the substrate proper ventilation. To _select_ the appropriate granular matrix, ensure good ventilation effect. Do not water in the management process too much, too much water should quickly drain in addition, to facilitate the root respiration. In summary, the process of transplanting plantlets, as long as the water balance, appropriate media, control bacteria and appropriate light and temperature conditions, good control, plantlets is very easy transplanting.
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Wikipedia Daquan
Tissue culture tissue culture Method of artificial culture of living tissue. To be in a sterile tissue culture conditions, the living tissue transplantation in the presence of nutrient medium (usually made of serum and embryo extract), the temperature and the temperature in the appropriate box. Through the cultivation of cells and tissues to observe the growth, reproduction and differentiation. In the training process, such as giving a variety of conditions can cause tissue changes, you can trace the process of organizational change and why. Various tissue and cell culture can also be used as a tool for culture and identification of the virus.
