Along with the development and advancement of understanding the culture of today's network techniques have used tissue culture for various purposes, including seed industry. Micro propagation techniques (mikropropagasi) has long been used and is one interesting and classic examples of the application of tissue culture techniques. This technique is done by planting shoots eksplan of its meristemnya network known as culture techniques shoots (shoot tip culture) or the lateral buds to grow by one or more books (single node and multiple node culture). Last technique is also known by the term invitro layering.
Micro propagation can generally be interpreted as an attempt to grow the plants in aseptic medium and reproduce such parts of the plant to produce the perfect plant in large quantities. Its main objective is to produce plants in large numbers and a short time.
This technique is also known as clonning efforts to produce clones of plants from vegetative tissue. Therefore plants produced through this effort clonning is identical or similar to its parent. To know more about mikropropagasi will discuss about:
a. Technical Benefits Mikropropagasi
b. The Invitro Propagation Method
c. Factors Affecting the Success of Invitro Plant Propagation
d. Mikropropagasi kinds.
Technical Benefits Mikropropagasi
Benefits Mikropropagasi in Plant Breeding
Mikropropagasi techniques in plant breeding is generally used, among others, to:
1) Generate a disease-free plants (nematodes, Mycoplasma, viroid, viruses, and fungi)
2) Generate new cultivars or superior plants, a new hybrid, clone selection and local, and elite genotypes
3) Generate a male sterile strain of elders
4) Generate a spontaneous mutant induction
5) Making genetic variation.
In addition to these benefits, the multiplication by tissue culture techniques also have shortcomings, among others that this effort had required special skills, production support facilities in particular, need special methods to optimize the production of each crop varieties and species, and because the adult method This is a lot of manpower is generally higher production costs.
In addition it can happen that the plant variety produced from invitro propagation of plants produced so different from the parent. This variation is known as the variations due to variations appear somaklonal of clones produced from somatic organs (vegetative). This can happen because the plants continuously and in a long time planted and propagated in media containing certain growth hormones.
This variation may also arise when eksplan grown in medium that enables the formation of callus. Cell division is very fast on callus can cause genetic changes in cells due to irregularities callus genetic information received by each of these callus cells. Somaklonal variation can be reduced by:
1) Limit the number of sub-culture and perbanyakannya,
2) Planting in the medium without growth hormone for one or two sub-cultures and periods
3) If possible avoid the multiplication of the callus culture. However, in some types of plants, especially plants that can not be propagated by cuttings and difficult stimulated formation adventifnya shoots the callus culture of diregenerasikan through organogenesis and embryogenesis is an alternative that allows propagation. If somaklonal variations can not be avoided, should be testing the properties of plants before clones diregenerasikan sold commercially.
b. Micro Propagation Methods In Invitro
Vegetative propagation methods of invitro plant is a development of techniques of vegetative propagation have been done conventionally as cuttings, layering and grafting. Conventional propagation purpose, such as cuttings, is to stimulate the formation of adventif organs (roots on stem cuttings, roots and shoots in the leaf cuttings and root cuttings) on the material and the number of seeds cuttings obtained from cuttings material is generally only one. On the vegetative propagation of invitro generally used not only to stimulate the formation of plant organs (roots, stems and leaves) but also before memperbanyaknya small plants (plantlet) was transferred from culture tubes to the field.
Methods that can be done in the plant mikropropagasi done in stages starting from stock preparation and treatment plant (phase 0) to the acclimatization phase (phase IV).
Above have been described that mikropropagasi done in several stages. These stages are not only explains mikropropagasi procedures, but also explains the changes in the environment and culture, such as changes in the composition and concentration of growth regulator used in the media. There are five stages in mikropropagasi, namely:
Stage 0: the preparation phase /preparation
Phase 1: induction phase
Stage 2: stage multiplication multiplication
Stage 3: stage embeddedness
Stage 4: stage transplants (moving) to the terrestrial media.
Phase 0: preparation (preparation), the selection and preparation of the parent tree
This stage is before eksplan taken for propagation. Parent trees will be used as a source of eksplan should be chosen carefully. This tree is a tree of the species or that will be multiplied verietas, Vigor has a healthy and free from symptoms of pest or disease attack. Parent tree or plant part that will be taken should be treated as a special eksplan to mikropropagasi succeed.
Treatments include:
1) Planting in the green house or a pot to reduce sources of contaminants
2) Providing the appropriate environment or chemical treatments to improve
speed multiplication in invitro conditions
3) Indexing or other procedures to determine the existence of systemic disease by viruses or bacteria
4) stimulation of growth dormant buds.
At the beginning of tissue culture work on the biggest problem faced is overcoming contamination. Eksplan taking place very influential on the size of the risk of contamination by fungal infections. Eksplan taken from a secure greenhouse conditions would be far kehegienisannya can reduce the risk of contamination by fungal infection than if eksplan taken from the field. But there is more difficult to avoid contamination of bacteria, because it is often difficult to distinguish whether the contamination originated from bacteria endogin or eksogin.
Ideally the parent plant that will serve as eksplan should be planted in the greenhouse kehegienisannya awake. This not only can reduce populations of microorganisms that live on the surface of the plant, but also helps to produce a quality crop.
At stage 0 includes also some interventions that can make more appropriate eksplan or better prepared as an initial material. Factors that influence the parent plant is dimikropropagasi light, temperature and growth regulator substances.
Stage 1: initial stage or induction (initiation)
This initial stage is very important and decisive for the success mikropropagasi. The success of this first stage seen from the success of planting eksplan aseptic conditions (free of any contaminants) and should be followed with appropriate early growth eksplan planting purposes (eg: an extension of shoots, the early growth of shoots, or the growth of callus on eksplan). After 1-2 weeks of incubation, culture contaminated by bacteria or fungi (either on media or eksplannya) removed. This stage is completed and culture can be moved to the next level if that is not contaminated eksplan has grown in line with expectations (eg lateral buds or shoots growing adventif). For contamination eksplan experiencing severe or difficult to be sterilized so eksplan be planted prior to the incubation media or the establishment media that contain only sugar and so only for the purpose of insulation is not contaminated eskplan before phase 1 is initiated at mikropropagasi.
The purpose of this stage is to produce axenic cultures. For most jobs mikropropagasi, eksplan selected is aksilar or terminal buds. Factors that influence the success of this phase are:
1) Age the parent plant
2) Age of physiological eksplan
3) stage of development eksplan
4) The size of eksplan.
Hypersensitive reaction
When plant tissue is exposed to stressful situations such as mechanical injury, complex phenolic metabolism stimulated. These interventions cause hypersensitivity reactions, such as:
1) Removing the contents of cells damaged
2) The reactions in the neighboring cells, but without showing symptoms of the injury itself
3) And / or die prematurely from the cells in a specific environment where the injury or infection.
In general, complex phenolic metabolism have 3 types of reactions in response to stress or injury, namely:
1) Oxidation of phenolic complex formation (the appearance and materials quinon compounds polymerisasi)
2) The formation of monomeric derivatives
3) Formation of phenolic polymer derivatives.
Formation of phenolic monomers in the network can race to accumulate a large number of products, or the emergence of new products that play a role in the protection mechanisms of tissue injury. The role of this pruduk can form a physical barrier against invasion (like lignin), or compounds mikrobia inhibitor of growth (such as quinon or fitoalexin).
Generally, networks of complex phenolic compounds containing a high concentration, it is difficult for the network dikulturkan. Phenol compounds are unstable and the product is very easy to oxidize and phenol can be oxidized fitotoksit.
Strategies to reduce or eliminate these complex phenolic compounds are:
1) Washing or cleaning products with a complex phenolic compounds to clean it by soaking into the water on the long-term or mengabsorbsinya with activated charcoal or polyvinylpyrrolidone compounds)
2) Inhibit the enzyme work khelat agent fenolase
3) reduce the activity and the ability fenolase substrate by using low pH, with the addition of antioxidant compounds such as ascorbic acid, citric acid or menginkubasikan culture in a dark room
4) reduce the stress on eksplan, especially at the time of sterilization or planting, the parent plant hygiene can reduce stress
5) Use of specific mikroelemen can stimulate the formation of phenol compounds, such as Mn2 + (acting as cofactor peroksidasi) and Cu2 + (is part of the enzyme complex fenolase). Therefore, for networks that produce excessive phenolic complex is suggested to reduce the concentration or not to use these elements in the media.
6) Menginkubasikan culture in low-temperature room
7) We recommend that before eksplan treatment planted in the media placed on the media without growth regulator substances to reduce pencoklatan occurrence eksplan or penghitaman on.
Stage 2: stage propagation (multiplication)
The purpose of this stage is to acquire and grow shoots. Axenik culture that had been produced on stage 1 and stage 2 was transferred to the medium-rich eksplan cytokines that can produce a lot of shoots at stage 3 then later shoots are transferred to the rooting medium to stimulate root growth.
At this stage, eksplan can also form a callus (callogenesis) or form a bud (caulogenesis). On callus growth and embrioid often produced embrioid each individual will be forming new plants (somatic embryogenesis), or sometimes produce meristemoid that will grow into shoots (organogenesis). Callogenesis often causes the genetic aberration known as somaklonal variations, so that the resulting plants are not identical to the parent plant.
Shoots obtained at this stage is used as an ingredient next multiplication, and therefore at this stage do a lot of sub-culture to double the amount produced plantlet. At this stage, shoots produced cut with node single-node/multiple culture techniques and by taking a bud for propagation eksplan. Materials are then planted in the new media generally contain cytokines at concentrations higher than auksin. At this stage you can use liquid media (media without order), semi-solid or solid media.
With appropriate modifications, the media, new shoots will grow from eksplan pieces. This stage is generally done as much as 8-10 times that will be produced a large number of shoots (shoots thousands) of one eksplan at initiation stage. Shoots which are then raised or mikropropagasi rooted at the next stage.
Stage 3: preparation prior to plantlet acclimatization (breeding stage)
Bud or plantlet generated from the 2nd stage is generally still very small or shoots that have not completed the roots that have not been able to support growth invivo conditions. Therefore, in this stage each of the resulting plantlet grown for enlargement, rooting and stimulation fotosintesisnya activities. Techniques to get ready for plantula moved to the media at this stage of terrestrial 4 among others, are:
1) Media for rooting and shoot extension. Rooting media are used without the addition of growth regulating substances. Cluster of shoots produced in phase 2 is stored in the media without ZPT with very high humidity
2) Individual shoots (propagule) disubkultur to reduce the concentration of media with or without the addition of increasing concentrations of cytokines and auksin and sometimes by reducing the concentration of inorganic compounds. In some types of rooting plants can be carried out by the results menempakan bud stage 2 (propagule) are placed in liquid medium aeration is better than in the solid media. Or by propagule transfer to media containing auksin for 1-2 days, then again to the media disubkultur without auksin (root induction stimulated by the auksin, but root growth can be inhibited by the presence in the media auksin). Or dipped in a solution of propagule pangakaran (auksin) for a while and then grown in medium without auksin
3) the elongation stage can be reached by putting in the medium to propagule without cytokines or with very low concentrations selamas 2-4 weeks. In some plants use the addition of GA3 in the medium. Next propagule transferred to other media such as the previous technique
4) Use praaklimatisasi media and culture environment with greater intensity of illumination light for the stimulation of photosynthetic activity such as use of media with low concentrations of sugar / no sugar, the addition of light intensity, and treatment with carbon dioxide.
Stage 4: acclimatization
This acclimatization stage is the stage of plantet transfer to invitro conditions invivo conditions. This stage is very important and must be done carefully, because if not done properly then most produced plantet can die / disappear. Plantlet removed from the bottle and attached to the roots cleaned, soaked in a fungicide solution and planted in compost or clean porous medium to stimulate the formation of serabutnya roots. To prevent deaths due to transpiration plantlet, plantlet disungkup with plastic or placed in a room with high humidity, the temperature of the room and placed a shaded place with the light intensity of 30%. In some cases, the leaves of plants sprayed with anti transpirant (eg Abscicic acid) to prevent too great evaporation from the leaves. Slowly, humidity, light intensity is reduced and added to stimulate photosynthesis.
c. Factors that Impact on Successful Mikropropagasi
1) plant genotype
One of the factors that influence growth and morphogenesis eksplan in invitro culture is eksplan genotypes isolated from plants. The research results showed that the response of each plant eksplan varies depending on the species, even the varieties, or plant origin such eksplan. The influence of these genotypes are closely related to other factors that influence growth eksplan, such as nutritional needs, growth regulating substances, and cultural environment. Therefore, the media composition, growth regulating substances and the growth environment required by each crop varieties vary although tissue culture technique used the same.
Plant genotypic response differences can be observed in the difference eksplan each variety to grow and regenerate. Each crop varieties differ in their ability to stimulate the growth of shoots aksilar, both the number of buds and shoots aksilarnya growth rate. It is also common in the formation of callus, callus growth and regeneration of callus into complete plants either through the formation of organs adventif or somatic embryos. Organ regeneration and development of somatic embryos adventif and also very much determined by the parent plant varieties. The difference is due to genetic effects due to differences in the genetic control of each variety and sex of the parent plant.
2) Media culture
Differences of media composition, the composition of growth regulating substances and the type of media used will greatly affect the growth and eksplan the dikulturkan regeneration.
a) The composition of the media. Media composition differences, such as the type and composition of inorganic salts, organic compounds, growth regulating substances influence eksplan response time dikulturkan. Medium composition differences are usually strongly influenced the direction of growth and regeneration eksplan. Nevertheless, the media has been formulated not only apply to one species and plant eksplan. Several types of formulations used and even the media in general for different types and varieties of plants eksplan, such as MS medium. But there are also several types of media that are formulated for specific crops such as WPM, VW etc.. These media can be used for various purposes such as seed germination, shoots culture, callus culture, callus regeneration via organogenesis and embryogenesis. Media are required for seed germination, stimulation aksilar shoots are generally more modest than the media for callus regeneration through organogenesis and embryogenesis.
b) The composition of growth hormone. Composition and concentration of growth hormone was added to the medium influenced the direction of growth and eksplan the dikulturkan regeneration. Composition and concentration of growth hormone are added to the culture medium depends on the type of dikulturkan and eksplan purpose pengkulturannya. Optimal concentration of growth hormone is added to the medium depends also from the eksplan and dikulturkan endogenous growth hormone content contained in these eksplan. Appropriate composition may be estimated through experiments that have been done before with the experiment to determine the composition of growth hormone according to the needs and direction of the desired growth eksplan.
Growth hormone is used for the invitro propagation is auksin groups, cytokines, giberelin, and the growth retardant. A common Auksin is IAA (Indole Acetic Acid), IBA (Indole Butyric Acid), NAA (Naphtalena Acetic Acid), and 2,4-D (2,4-dichlorophenoxy Acetic Acid). In addition some researchers at several plants using the CPA as well (Chlorophenoxy Acetic Acid). Cytokines that are widely used Kinetin (Furfuryl Amino Purine), BAP / BA (Benzyl Amino Purine / Benzyl adenine), 2 iP (2-isopentenyl adenine). Several other cytokines are also used zeatin, thidiazuron and PBA (6 (benzylamino) -9 - (2-tetrahydropyranyl)-9H-Purine). Giberellin group growth hormone most commonly used is to GA3, but there are some researchers who use the GA4 and GA7, while the growth retardant is often used Ancymidol, Paraclobutrazol and TIBA, ABA and CCC.
c) Physical state media. Media commonly used in tissue culture is the medium of solid, semi-solid medium and liquid medium. Physical conditions will affect the growth of media culture, the pace of growth and differentiation. This medium physical conditions affecting growth partly because of its effect on the osmolarity of the solution in the media and the availability of oxygen for growth eksplan the dikulturkan.
Media commonly used in mikropropagasi is semi-solid medium (semi-solid) by adding to. Semi-solid media was used for several reasons, among others: small eksplan easily seen in dense media; for eksplan culture remains at the same orientation; eksplan is above the surface of the media so that no additional aeration techniques required to culture; orientation growth of shoots and roots fixed; and callus is not broken as if placed in a liquid medium. But adding that in some cases inhibits the growth because of: a compound that may contain inhibitors that can inhibit morphogenesis several cultures or slow the growth of culture; eksudasi phenolic from eksplan absorbed by the media eksplan attached so that it can affect the growth eksplan; to be washed from the roots before diaklimatisasi; and needs more time to wash glasses for example culture bottles should diautoclave to dissolve the order before washing.
3) Environmental Growing
a) Temperature. Plants generally grow in an environment with a temperature that is not the same each time, for example in the day and night conditions with the plant having a temperature difference large enough. Such circumstances could be done in invitro culture by adjusting the temperature day and night in the living culture, but during the tissue culture laboratory was set at room temperature is a constant culture both at day or night. Generally the temperature used in invitro culture is higher than the temperature conditions invivo. The goal is to accelerate the growth and morphogenesis eksplan.
In most laboratories, which use temperature is constant, ie 25 ° C (temperature range 17-32 ° C). Tropical plants generally dikulturkan at temperatures slightly higher than the plants of the four seasons, namely 27 ° C (temperature range 24-32 ° C). When day and night temperature set differently, then the difference is generally 4-8 ° C, the typical variation is 25 ° C day and 20 ° C night, or 28 ° C day and 24 ° C night. Although nearly all plants can be grown in the temperature range, but the temperature needs for each plant species are generally different. Plants can grow well at its optimum temperature. At room temperature under the optimum culture, eksplan slower growth, but at temperatures above the optimum plant growth is also hampered due to the high rate of respiration eksplan.
b) relative humidity. Relative humidity in the culture bottle with the mouth closed bottle is generally quite high, and it ranged between 80-99%. If the bottle's mouth closed a little loose then the relative humidity in the culture bottles may be lower than 80%. While the relative humidity in the culture generally is about 70%. If the relative humidity of the culture room under 70% then the media will result in a culture bottle (which is not sealed) will quickly evaporate and dry so that eksplan and plantlet will quickly run out dikulturkan media. However, the air humidity in the culture bottles that are too high cause abnormal growth of plant leaves, weak, easily broken, small plants, but too succulent. Plant condition is called vitrifikasi or hiperhidrocity. Sub-culture to another medium or small planlet put this in a bottle with a loose lid, cover with a filter, or put silica gel in the bottle culture can help overcome this problem.
c) Light. Just as plant growth invivo conditions, the quantity and quality of light, the intensity, long wavelength radiation and light affect the growth eksplan in invitro culture. Growth of organs or tissue in culture invitro plants are generally not inhibited by light, but the callus growth is generally inhibited by light.
In invitro propagation of plants, culture in general diinkubasikan storage space by radiation. Shoots generally stimulated growth by irradiation, except in the propagation technique that begins with the growth of callus. Light source on the space of this culture is generally flourescent light (TL). This is because the TL lamps produce white light, except that TL light at room temperature did not increase drastically culture (only increased slightly). The intensity of light used in the culture generally much lower (1 / 10) of the required light intensity in the normal plants. The intensity of light in the room for the growth of the shoot cultures generally range between 600-1000 lux. Germination and root initiation is generally done at a lower light intensity.
In addition to light intensity, while irradiation or photoperiodisitas also affect the eksplan growth dikulturkan. Long exposures are generally set according to the needs of plants according to their natural condition. Of light and dark periods are generally set in the range of 8-16 hours of light and dark hours depending 16-8 crop varieties and eksplan the dikulturkan. Period of day / night (light / dark) is set automatically using a timer placed on the light switch in the culture. With this technique a constant illumination can be adjusted according to crop needs.
4) The condition Eksplan
Growth and morphogenesis in mikropropagasi highly influenced by plant tissue is used as eksplan. Eksplan genetic factors other than those mentioned above, the conditions that affect the success eksplan technique mikropropagasi is eksplan type, size, age and physiological phase of the network used as eksplan.
Although each plant cell has the ability totipotensi, but each network has a capacity different to grow and regenerate in tissue culture. Therefore, the type used for eksplan each culture vary depending on the destination pengkulturannya.
Eksplan age is very influential on the eksplan ability to grow and regenerate. Eksplan generally derived from plant tissue is still young (juvenile) more easily grow and regenerate compared with differentiated network has continued. Young tissue generally have cells that actively divide with the cell wall complex that not more easy to modify in culture than the old network. Therefore, the initiation of the culture is usually done by using the young shoots, young buds, hipokotil, immature inflorescence, etc.. If eksplan taken from adult plants, the parent plant rejuvenilisasi through pruning or fertilizing can help to get young people to culture eksplan more successful.
Eksplan size also affects the culture of success. Eksplan with small sizes more easily sterilized and does not need space and lots of media, but its ability to regenerate is also smaller, so required a more complex media for growth and regeneration. Conversely the greater eksplan, the more likely to carry diseases and even more difficult to be sterilized, and the media needs more culture. Eskplan the appropriate size depends on the type of plants that dikulturkan, techniques and pengkulturannya goals.
d. Various Mikropropagasi
1) Production of plants from the shoots aksilar
Crop production by stimulating the formation of shoots mikropropagasi technique aksilar is the most common. There are 2 (two) aksilar bud production methods made the culture of shoots (shoot culture or shoot-tip culture) and culture bud eye (one eye buds: a single-node culture, more than one eye shoot: a multiple-node culture). Both culture techniques are based on the principle of stimulating the formation or emergence of side shoots by breaking the apical dominance of the apical meristem.
2) Culture shoots (shoot culture or shoot-tip culture)
Culture shoots (shoot culture) is a technique that mikropropagasi done in a way that contains mengkulturkan eksplan top meristem (apical and lateral) with excitation and propagation purposes tunas-tunas/cabang-cabang aksilar. Aksilar shoots which are then propagated through the same procedure as eksplan initially and then rooted and grown in invivo conditions.
The term used for this technique depends on the culture of shoots eksplan used. If eksplan used is the tip apical buds (± 20 mm long) just so the technique is known as shoot-tip culture, but when used is eksplan top end of the shoot apical and other parts below it called a shoot culture. Eksplan size used affects the success of culture shoots.
The smaller eksplan, the less likely to contamination by microorganisms but also less ability to regenerate and reproduce themselves. Conversely, the greater eksplan used, the greater its ability to adapt in invitro conditions, but also the greater the chances for contamination, the more need for the media and the larger container / bottle culture needed. Therefore, please note eksplan appropriate size for each variety and species of plants.
Growth of shoots, bud initiation and propagation resulting aksilar generally stimulated by adding growth hormone (mostly cytokines) into the growth media. This treatment can stimulate the growth of side shoots and break the apical dominance of shoots that dikulturkan. In addition, apical dominance can also be relieved by other treatments such as trimming the leaves contained in the books or put eskpan shoots in a horizontal position. Shoots produced aksilar then used as a miniature cuttings for subsequent propagation process. With this technique, and accompanied by a sub-culture can be obtained from plantet lot eksplan one. By limiting the number of sub-cultures to a maximum of 8-10 times the plants can be obtained clones of true-to-type.
3) Culture eyes shoots / Single-node or multiple-node culture (Invitro layering)
These shoots eye culture is one of the techniques used for invitro propagation of plants by stimulating the emergence of aksilar shoots sprout from the eyes dikulturkan. Just as culture pieces, which are used in eksplan culture can shoot the eye from lateral buds, side shoots or part of the stem containing one or more eye shoots (containing one or more books). Known two culture techniques bud eye is the eye eksplan containing more than one bud horizontally planted on solid medium (invitro technique of layering) or any book containing one bud eye is cut and planted separately in each culture bottle.
Like buds culture techniques, growth of shoots aksilar also based on the principle of breaking the apical dominance. Therefore, the growth of shoots aksilar occurs when eksplan (eye shoots) grown in medium containing cytokines in high enough concentrations of these cytokines can stop the dominance of apical buds and lead to the development aksilar shoots. Aksilar buds formed later split and can be directly planted in the rooting medium from which new plants are perfect or re-used as planting material for further multiplication. These shoots then rooted, diaklimatisasi and then planted in the field.
4) induced the formation of flower buds from the meristem
Meristem flowers can also be stimulated to form vegetative shoots in invitro conditions. Is used Eksplan flower inflorescence immature (Immature inflorescences) that are not yet formed sexual organs of males and females. The use of adult infloresence will produce the formation of flower organs rather than vegetative buds.
5) Initiation of shoot directly adventif
Adventif buds are formed from buds at the eksplan that is not a place of origin of the formation (not from the eyes of the shoot or books). Adventif shoots can be formed directly from eksplan without going through the process of callus formation first. This technique is a technique which is also a lot mikropropagasi done and can result in a number of plantlet far more than previous techniques (aksilar bud formation). Adventif bud formation process directly from eksplan networks such as roots, shoots and flowers called organogenesis.
Organogenesis occurrence spurred by the presence of components such as medium, the endogenous components during eksplan started dikulturkan, and compounds eskplan carried during initiation. In addition organogenesis also spurred by the presence of exogenous growth regulating substances in the medium. For example, the ratio between the high auksin: eksplan induces cytokines towards callus formation on roots of tobacco plants but on the contrary, if the low ratings auksin: cytokines will accelerate towards the bud. Shoots and roots formed in several thin layers of cells in several species eksplan by the concentration difference between auksin and cytokines. Root initiation can be stimulated by the addition of NAA and zeatin and bud formation stimulated by the addition of cytokines such as benzylaminopurine or zeatin without adding auksin.
According to Torrey (1966 in Dodds and Roberts, 1983) hypothesized that organogenesis from callus initiated with the formation of clusters meristem cells (meristemoid) able to respond to factors in the network to produce a primordium. Initiation of the formation of roots, shoots and embrioid also influenced by internal factors naturally.
Several factors affect rhizogenesis including auksin, carbohydrates, lighting, and fotoperiode. In some tissue culture auksin spur the formation of roots, whereas the exogenous auksin be prevented and rhizogenesis can be stimulated by anti-auksin.
The success of the formation of shoots directly adventif is highly dependent on plant parts used as well as eksplan strongly influenced by the species or varieties of plants such eskplan origin. In plants that are responsive, almost all parts of the plant (leaf, root, stem, meristem, etc..) Can be stimulated to form adventif organs, but at other plants adventif shoots can only be formed on parts of certain plants such as bulbs layer, embryo or sprouts.
Like other mikropropagasi techniques, adventif buds are formed directly through a series of stages from initiation (Stage 1). After being on eksplan aseptic conditions and the shoots begin to grow, eksplan directly to the media disubkulturkan propagation (or the same media with initiation: depending on variety) to reproduce adventif shoots sprout from the eyes of adventif been created in the previous stage. Shoots which are then separated, rooted and diaklimatisasi to produce a complete and intact plants that can grow in a natural state.
6) Direct Somatic embryogenesis
Asexual embryos or somatic embryos (somatic embryos) is not an embryo formed from the union of cells of male and female gametes or embryos in other words, the network formed from vegetative / somatic. These embryos can be formed from plant tissue dikulturkan without due process known as somatic embryogenesis. If this process is formed directly on eksplan without going through the process of callus formation first, then the process is called somatic embryogenesis directly (direct somatic embryogenesis).
7) The formation of food reserves storage organ micro -
Some types of plants can be vegetatively dikembangbiakan using storage organs such as tuber, rhizome, bulbus, etc.. Storage organs can also be produced in plants that are naturally produce these storage organs. Techniques to obtain storage organs vary widely depending on the type dikulturkan network. This micro storage organs can be used as seed for planting directly in the field or grown for the production of seed bulbs.
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