ACTIVITY 3
IMPLEMENTATION OF PLANT TISSUE CULTURE
I. INTRODUCTION
BackgroundTissue culture is one way of vegetative propagation of plants. Tissue culture propagation is a technique by isolating parts of plants such as leaves, buds, and growing these parts in an artificial medium aseptically rich nutrients and plant growth regulators in a closed opaque container so that the plants can reproduce themselves and generations to complete plants.
The
main principle of the technique of plant tissue culture is perbayakan
using vegetative parts of plants using artificial media is done in a
sterile place.BackgroundTissue culture is one way of vegetative propagation of plants. Tissue culture propagation is a technique by isolating parts of plants such as leaves, buds, and growing these parts in an artificial medium aseptically rich nutrients and plant growth regulators in a closed opaque container so that the plants can reproduce themselves and generations to complete plants.
While
the stages of tissue culture-tanhapan itself starts from the selection
and preparation of the parent plant source of explants, initiation of
cultures, propagule multifikasi and multiplication, shoot elongation and
root growth and acclimatization. At
these stages take place mainly at the stage multifikasi and elongation
media for explant must be replaced, replacement of the old media to new
media called a subculture. On this occasion, the author will report the results of lab work subculture on orchids and chrysanthemums.B. PurposeThis lab aims to:A. Students can learn the stages of tissue culture.2. Students are able to do subculture on orchids.
II. REVIEW REFERENCES
The selection and preparation of the parent plant source of explantsBefore committing to a plant tissue culture, the first activity to be done is to choose the parent material to be reproduced. Plants should be obvious kind, species, and varieties and should be healthy and free of pests and diseases. Explant source plant breeders must be conditioned and specially prepared in a greenhouse or a greenhouse in order to be cultured explants can grow healthy and well and free of contaminants at the source of the in-vitro cultured. Parent plant environment is more hygienic and cleaner could improve the quality of the explants. Routine maintenance should be done include: pruning, fertilizing, and spraying with pesticides (fungicides, bactericide, and insecticide), so the new shoots that grow and become more healthy and free from contaminants. Besides changing the physiological status of the parent plant explant source is sometimes necessary to manipulate parameters such as light, temperature, and growth regulators. Manipulation can be done with the parent plant with fotoperiodisitas conditions and a given temperature to overcome dormancy and the addition of PGR such as cytokines to stimulate the growth of new buds and to enhance the reactivity of the explants on culture initiation stage (Yusnita, 2003).2. Initiation of culturesThe main purpose of the in-vitro propagation of this stage is the creation of a free explant cultures of microorganisms and the initiation of new growth (Wetherell, 1976). Also added by Yusnita, 2004, that at this stage seeking an aseptic culture or aksenik. Aseptic means free of microorganisms, while aksenik means free from undesirable microorganisms. In this phase is also expected that the cultured explants will initiate new growth, so it will allow for the selection of the most powerful growth of plants, for propagation (multiplication) in the later stages of culture (Wetherell, 1976). For landed a culture that is free from contamination, explants should be sterilized. Sterilization is an attempt to remove contaminants on the surface of microorganisms attached to the explants. some chemicals that can be used to sterilize the surface of the explant is NaOCl, CaOCl2, ethanol, and HgCl2..3. Multiplication or reproduction propagulesThis stage aims to double its propagules or plant material propagated as bud or embryo, and maintain it in certain circumstances so that at times can be continued to the next stage (Yusnita, 2004). At this stage, the multiplication can be done by stimulating the growth of axillary buds and stems branch or stimulate the formation of adventitious shoots in plant shoots, either directly or via callus induction first. Just as in the initiation phase of the culture, the media must be contained in minerals, sugar, vitamins, and hormones with the exact ratio required (Wetherell, 1976). The hormones used to stimulate bud formation comes from the class of cytokines such as BAP, 2-iP, kinetin, or thidiadzuron (TDZ).4. Elongation of shoots, Induction, and Development of RootsThe purpose of this stage is to form roots and shoots of plants that are strong enough to survive until then moved from in-vitro environment to the outside environment. In this stage, the culture of the plant will gain resistance to environmental influences, so ready to acclamatization (Wetherell, 1976). Shoots produced in the multiplication phase was transferred to another medium for shoot elongation. To shoot elongation medium containing cytokines is very low or no cytokinin. Shoots can be removed individually or in groups. Elongation of shoots in groups is more economical than individually. Having grown quite long, shoots can be rooted. Pengakarannya shoot elongation and can be done at once or in stages, ie after the new extended rooted.5. Acclimatization the process of plant propagation by tissue culture, plantlets acclimatization stage is one critical step is often a constraint in the mass production of seedlings. At this stage, the plantlets or shoots micro transferred to the environment outside of the bottle like a greenhouse, plastic house, or a screen house (insect-proof greenhouses). This process is called acclimatization. Acclimatization is the process of conditioning the plantlets or shoots micro (if rooting is done ex-vitro) in the new environment outside the aseptic bottle, with a soil media, or ferns that plantlets can survive and continue to be the seed that is ready to be planted in the field. Breeding procedures with new tissue culture plantlets could be said to succeed if it can be acclimatized to the external conditions with high success.6. SubcultureSubculture is one step in plant propagation through tissue culture. Basically we cut subculture, divide and replant the explants that had grown so that the plants will multiply. Basically subculture is a relatively easy stage activity compared with other activities in tissue culture.Subcultures made for the following reasons:
Plants already meet or are tall bottles
Plants have long been in the bottle so that its growth is reduced
Plants begin to nutrient deficiencies
Media in bottles already mongeringSubculture of activities carried out in accordance with the cultured species. Each plant has the characteristics and growth rate are different. So that the manner and time are also different subcultures. Plants that should be relatively quick or subcultured is the kind of banana-Pisangan, alokasia, and caladium. Relatively old plant is Aglaonema. (Training, 2009)7. ORCHIDOrchid is one of the members of the family Orchidaceae which can be found in nearly all parts of the world, especially the tropics ranging from lowland to high, even up to the snowy mountains of the border area. A wide variety of shapes, colors, smells, and sizes with a unique characteristic to the attraction of orchids known as the beautiful flowering ornamental plants. Contonya is Arundina graminifolia, Bulbophylum binnendijkii, Calanthe sp., Paphilopedilum sp., And so forth.Orchid is one of the plants that grow slowly and have the speed varies. This is very powerful if the purpose of maintenance is to produce flowers. Orchid plants have different growth patterns with other plants. Orchid growth, both vegetative (growth buds, stems, leaves, and roots) and generative growth (the growth of primordial flowers, fruits, and seeds) are not only determined by genetic factors, but also by climatic factors and maintenance factors. (Widiastoety, 2007) Basically orchid plant is a plant that is difficult to perform self-pollinating, so the breeding is quite difficult. In addition, the seeds are small, do not contain food reserves and a very tough skin and thick, making it difficult orchids are grown without human assistance, unless the orchids that grow wild in the woods. To overcome this and grow orchids en masse, then action can be done is to marry anaman orchids (may as well get a new hybrid varieties).
III. METHODOLOGY
Time and place of executionDay / Semester: Tuesday / VITime: pkl. 16:30 to 19:00 pmPlace: Agricultural LaboratoryB. Tools and materialsTool:- Dissecting set- Light Bunsen- Scissors- Plaster- Tissu- Petridish- Spray bottleIngredients:- Media ms0- Alcohol 96%- Alcohol 70%- Orchid plantlets ready subcultured- Media ms0 + charcoalC. Work Procedures
Prepare all the necessary tools and materials
Doing subculture of orchid plants in a way:A. Hand spraying using 70% alcohol before work.2. Took the paper and placed in sterile glass board as the base.3. Sterilize the media by bringing it close to the lights and prepare methylated.4. Orchid plantlets take one by one using tweezers, and rid it of impurities and the rest of the media that shipped with the roots.5. Enter one by one into the media orchids ms0 + charcoal + banana juice which has been prepared as much as 7-10 cigarettes per bottle and arranged so neatly arranged with a distance proportional to not scramble nutrients.6. Closing the culture bottles that had been planted and provide labels containing information about the plant's name, date of planting, and the name of the grower.7. Growth of the culture bottle store room.8. To pack all the tools that have been used and returned to the place before.9. Make the observation of the subcultures performed one week after subculture.10. Discussion, deliberation and create lab reports
IV. DISCUSSION
Initial conditions at the time of starting plant tissue culture activities is very good but a week later the cultured orchids seem damaged by fungi and microorganisms ditumbuhinya bullies, this can be caused by a lack of sterile equipment used by the researchers during the manufacturing process or during the process of culture orchids growing in media.2. As for the orchid plant at the time of the initial condition is that the quantity of explant subcultures on media previously have been very solid. It is necessary to change the media that plants can be grown with optimal because no shortage of nutrients to be absorbed. At the time of subculture, orchid plants is easier and less susceptible to light or heat. Besides this plant is quite attached to the media, not to plug too hard on the media. This is due to the roots of orchids are easier to adjust to the conditions of the media. After one week of growth recorded in the event of any change in appearance and size of these plants. The orchid plant looks more fresh, while its size is longer than when before subcultured.
V. CONCLUSION
Based on observations, it can be concluded that:Explants planting done in the LAF (Laminar Air Flow). The use of tools already in a sterile condition. Planting is done by dipping a scalpel and tweezers into a 96% alcohol and then burned in the Bunsen flame. After that new tools can be used for planting.2. In the explants Orchid suspected contamination by bacteria and fungi. Proved the presence of mucus that is thick and yellow and white in the media and around the explant.3. Subcultures should be done as considering some factors for the survival of the explants.
Subcultures do our best to avoid wounding the explants when transplanted into new media. Both plants are subcultured have not undergone significant changes in its growth. Both of these plants do not have contamination both in the media and plants. Speed and accuracy when performing subcultures influence the results obtained.
II. REVIEW REFERENCES
The selection and preparation of the parent plant source of explantsBefore committing to a plant tissue culture, the first activity to be done is to choose the parent material to be reproduced. Plants should be obvious kind, species, and varieties and should be healthy and free of pests and diseases. Explant source plant breeders must be conditioned and specially prepared in a greenhouse or a greenhouse in order to be cultured explants can grow healthy and well and free of contaminants at the source of the in-vitro cultured. Parent plant environment is more hygienic and cleaner could improve the quality of the explants. Routine maintenance should be done include: pruning, fertilizing, and spraying with pesticides (fungicides, bactericide, and insecticide), so the new shoots that grow and become more healthy and free from contaminants. Besides changing the physiological status of the parent plant explant source is sometimes necessary to manipulate parameters such as light, temperature, and growth regulators. Manipulation can be done with the parent plant with fotoperiodisitas conditions and a given temperature to overcome dormancy and the addition of PGR such as cytokines to stimulate the growth of new buds and to enhance the reactivity of the explants on culture initiation stage (Yusnita, 2003).2. Initiation of culturesThe main purpose of the in-vitro propagation of this stage is the creation of a free explant cultures of microorganisms and the initiation of new growth (Wetherell, 1976). Also added by Yusnita, 2004, that at this stage seeking an aseptic culture or aksenik. Aseptic means free of microorganisms, while aksenik means free from undesirable microorganisms. In this phase is also expected that the cultured explants will initiate new growth, so it will allow for the selection of the most powerful growth of plants, for propagation (multiplication) in the later stages of culture (Wetherell, 1976). For landed a culture that is free from contamination, explants should be sterilized. Sterilization is an attempt to remove contaminants on the surface of microorganisms attached to the explants. some chemicals that can be used to sterilize the surface of the explant is NaOCl, CaOCl2, ethanol, and HgCl2..3. Multiplication or reproduction propagulesThis stage aims to double its propagules or plant material propagated as bud or embryo, and maintain it in certain circumstances so that at times can be continued to the next stage (Yusnita, 2004). At this stage, the multiplication can be done by stimulating the growth of axillary buds and stems branch or stimulate the formation of adventitious shoots in plant shoots, either directly or via callus induction first. Just as in the initiation phase of the culture, the media must be contained in minerals, sugar, vitamins, and hormones with the exact ratio required (Wetherell, 1976). The hormones used to stimulate bud formation comes from the class of cytokines such as BAP, 2-iP, kinetin, or thidiadzuron (TDZ).4. Elongation of shoots, Induction, and Development of RootsThe purpose of this stage is to form roots and shoots of plants that are strong enough to survive until then moved from in-vitro environment to the outside environment. In this stage, the culture of the plant will gain resistance to environmental influences, so ready to acclamatization (Wetherell, 1976). Shoots produced in the multiplication phase was transferred to another medium for shoot elongation. To shoot elongation medium containing cytokines is very low or no cytokinin. Shoots can be removed individually or in groups. Elongation of shoots in groups is more economical than individually. Having grown quite long, shoots can be rooted. Pengakarannya shoot elongation and can be done at once or in stages, ie after the new extended rooted.5. Acclimatization the process of plant propagation by tissue culture, plantlets acclimatization stage is one critical step is often a constraint in the mass production of seedlings. At this stage, the plantlets or shoots micro transferred to the environment outside of the bottle like a greenhouse, plastic house, or a screen house (insect-proof greenhouses). This process is called acclimatization. Acclimatization is the process of conditioning the plantlets or shoots micro (if rooting is done ex-vitro) in the new environment outside the aseptic bottle, with a soil media, or ferns that plantlets can survive and continue to be the seed that is ready to be planted in the field. Breeding procedures with new tissue culture plantlets could be said to succeed if it can be acclimatized to the external conditions with high success.6. SubcultureSubculture is one step in plant propagation through tissue culture. Basically we cut subculture, divide and replant the explants that had grown so that the plants will multiply. Basically subculture is a relatively easy stage activity compared with other activities in tissue culture.Subcultures made for the following reasons:
Plants already meet or are tall bottles
Plants have long been in the bottle so that its growth is reduced
Plants begin to nutrient deficiencies
Media in bottles already mongeringSubculture of activities carried out in accordance with the cultured species. Each plant has the characteristics and growth rate are different. So that the manner and time are also different subcultures. Plants that should be relatively quick or subcultured is the kind of banana-Pisangan, alokasia, and caladium. Relatively old plant is Aglaonema. (Training, 2009)7. ORCHIDOrchid is one of the members of the family Orchidaceae which can be found in nearly all parts of the world, especially the tropics ranging from lowland to high, even up to the snowy mountains of the border area. A wide variety of shapes, colors, smells, and sizes with a unique characteristic to the attraction of orchids known as the beautiful flowering ornamental plants. Contonya is Arundina graminifolia, Bulbophylum binnendijkii, Calanthe sp., Paphilopedilum sp., And so forth.Orchid is one of the plants that grow slowly and have the speed varies. This is very powerful if the purpose of maintenance is to produce flowers. Orchid plants have different growth patterns with other plants. Orchid growth, both vegetative (growth buds, stems, leaves, and roots) and generative growth (the growth of primordial flowers, fruits, and seeds) are not only determined by genetic factors, but also by climatic factors and maintenance factors. (Widiastoety, 2007) Basically orchid plant is a plant that is difficult to perform self-pollinating, so the breeding is quite difficult. In addition, the seeds are small, do not contain food reserves and a very tough skin and thick, making it difficult orchids are grown without human assistance, unless the orchids that grow wild in the woods. To overcome this and grow orchids en masse, then action can be done is to marry anaman orchids (may as well get a new hybrid varieties).
III. METHODOLOGY
Time and place of executionDay / Semester: Tuesday / VITime: pkl. 16:30 to 19:00 pmPlace: Agricultural LaboratoryB. Tools and materialsTool:- Dissecting set- Light Bunsen- Scissors- Plaster- Tissu- Petridish- Spray bottleIngredients:- Media ms0- Alcohol 96%- Alcohol 70%- Orchid plantlets ready subcultured- Media ms0 + charcoalC. Work Procedures
Prepare all the necessary tools and materials
Doing subculture of orchid plants in a way:A. Hand spraying using 70% alcohol before work.2. Took the paper and placed in sterile glass board as the base.3. Sterilize the media by bringing it close to the lights and prepare methylated.4. Orchid plantlets take one by one using tweezers, and rid it of impurities and the rest of the media that shipped with the roots.5. Enter one by one into the media orchids ms0 + charcoal + banana juice which has been prepared as much as 7-10 cigarettes per bottle and arranged so neatly arranged with a distance proportional to not scramble nutrients.6. Closing the culture bottles that had been planted and provide labels containing information about the plant's name, date of planting, and the name of the grower.7. Growth of the culture bottle store room.8. To pack all the tools that have been used and returned to the place before.9. Make the observation of the subcultures performed one week after subculture.10. Discussion, deliberation and create lab reports
IV. DISCUSSION
Initial conditions at the time of starting plant tissue culture activities is very good but a week later the cultured orchids seem damaged by fungi and microorganisms ditumbuhinya bullies, this can be caused by a lack of sterile equipment used by the researchers during the manufacturing process or during the process of culture orchids growing in media.2. As for the orchid plant at the time of the initial condition is that the quantity of explant subcultures on media previously have been very solid. It is necessary to change the media that plants can be grown with optimal because no shortage of nutrients to be absorbed. At the time of subculture, orchid plants is easier and less susceptible to light or heat. Besides this plant is quite attached to the media, not to plug too hard on the media. This is due to the roots of orchids are easier to adjust to the conditions of the media. After one week of growth recorded in the event of any change in appearance and size of these plants. The orchid plant looks more fresh, while its size is longer than when before subcultured.
V. CONCLUSION
Based on observations, it can be concluded that:Explants planting done in the LAF (Laminar Air Flow). The use of tools already in a sterile condition. Planting is done by dipping a scalpel and tweezers into a 96% alcohol and then burned in the Bunsen flame. After that new tools can be used for planting.2. In the explants Orchid suspected contamination by bacteria and fungi. Proved the presence of mucus that is thick and yellow and white in the media and around the explant.3. Subcultures should be done as considering some factors for the survival of the explants.
Subcultures do our best to avoid wounding the explants when transplanted into new media. Both plants are subcultured have not undergone significant changes in its growth. Both of these plants do not have contamination both in the media and plants. Speed and accuracy when performing subcultures influence the results obtained.
REFERENCES
Gunawan, L.W. Of 1988. Tissue Culture Techniques. New York: Tissue Culture Laboratory, Biotechnology PAU, IPB.Rahardja, P. C. Of 1995. Tissue Culture: The Modern Technique of Plant Propagation. Self publishers, Jakarta.Sriyanti, Daisy P. and Ari Wijayani. Of 2002. Tissue Culture Techniques: Introduction and Plant Propagation Tips In vegetative-Modern. Canisius, Yogyakarta.Susilowati, Ari. Shanti Listyawati. Of 2001. Diversity of Microorganisms Type Culture In vitro Contamination Sources in Sub-Lab. Biological Laboratory of Mathematics and Science Center UNS. Biodiversity Journal Volume 2 No. 1 pp. 110-114.
Gunawan, L.W. Of 1988. Tissue Culture Techniques. New York: Tissue Culture Laboratory, Biotechnology PAU, IPB.Rahardja, P. C. Of 1995. Tissue Culture: The Modern Technique of Plant Propagation. Self publishers, Jakarta.Sriyanti, Daisy P. and Ari Wijayani. Of 2002. Tissue Culture Techniques: Introduction and Plant Propagation Tips In vegetative-Modern. Canisius, Yogyakarta.Susilowati, Ari. Shanti Listyawati. Of 2001. Diversity of Microorganisms Type Culture In vitro Contamination Sources in Sub-Lab. Biological Laboratory of Mathematics and Science Center UNS. Biodiversity Journal Volume 2 No. 1 pp. 110-114.
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