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TISSUE CULTURE | Clonal Propagation, In Vitro
1360 TISSUE CULTURE / Clonal Propagation, In Vitro European and Mediterranean Plant Protection Organization (EPPO) Guidelines. http://www.eppo.org. George EF (1993) Plant Propagation by Tissue Culture: Part 1 – The Technology. Basingstoke: Exegetics. George EF (1996) Plant Propagation by Tissue Culture: Part 2 – In Practice. Basingstoke: Exegetics. Trigiano RN and Gray DJ (eds) (2000) Plant Tissue Culture Concepts and Laboratory Exercises. 2nd edn. Roca Baton: CRC Press.
Clonal Propagation, In Vitro B S Ahloowalia, International Atomic Energy Agency, Vienna, Austria Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Clonal propagation of plants refers to the production of genetically identical plants through nonsexual methods. Many plants are propagated from vegetative parts such as bud grafts, slips, stem cuttings, bulbs, tubers, and runners. For example, date palm (Phoenix dactylifera) trees are propagated from offshoots and rooting them in soil. Yet in other plants, leaves develop adventitious buds on the margins and leaf veins, which on sprouting develop into new plants. The plants produced in this manner are exact copies or clones of the mother plant, and genetically identical unless a mutation occurs in the cells and tissues of the original plant. Vegetative propagation is used for the multiplication of many important crops, shrubs, and trees. These include crops such as cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), ginger (Zingibar officinale), sugar cane (Saccharum spp.), taro yam (Colocasia esculenta), turmeric (Curcuma domestica), and yam (Dioscorea spp.). Several ornamentals, e.g., azalea (Rhododendron spp.), carnation (Dianthus spp.), chrysanthemum (Chrysanthemum spp.), jasmine (Jasminum officinale), roses (Rosa hybrida and chinensis), and tulips (Tulipa spp.), and many fruits such as banana and plantain (Musa spp.), pineapple (Ananas comosus), passion fruit (Passiflora edulis), and kiwi fruit (Actinidia chinensis), are also propagated from vegetative parts. In many other fruits, such as apple (Malus domestica), citrus (Citrus sinensis, C. paradisi, and several other Citrus species), lychee (Litchi chinensis), mango (Mangifera indica), and pear (Pyrus communis), shoots and buds are grafted to produce clones of the original mother tree. Bud sports or spontaneous
mutations are also propagated in this manner to obtain new varieties.
In Vitro Propagation In vitro propagation is very much like multiplication of plants from vegetative parts except that plants are produced by culturing tiny pieces on a synthetic medium in a suitable container instead of soil in a pot. The in vitro propagation of plants is initiated by culturing pieces of stems, leaves, apical and axillary buds, immature seeds, embryos, and cotyledons. The basis of in vitro propagation is rapid multiplication of plants by culturing tiny pieces on media under controlled environment and aseptic (germ-free) conditions. Since strict aseptic conditions have to be maintained in the production of in vitro cultured plants, the culture and transfer of cuttings and plants is carried out under germ-free conditions in laminarflow cabinets, and the containers in which plants are cultured are kept tightly sealed. The laminar-flow cabinets are designed to produce sterile air. The instruments such as scalpels, scissors, and forceps used for cutting and transfer of plant pieces are sterilized by alcohol dip and flaming or dry heat. Likewise, the media are also sterilized. Most culture media contain basic major and minor salts, vitamins, sugar, plant growth regulators, and agar with pH adjusted between 5.6 and 5.8. The major salts provide nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium, and minor salts elements such as copper, manganese, molybdenum, sulfur, and zinc, which along with vitamins are essential for the growth and differentiation of plant cells and tissues. The sugar in the culture medium provides carbon for photosynthesis. However, the composition of media (particularly the type and amount of sugar and growth regulators) is changed according to different plant species. Several commercial companies sell ready-made basal media in powdered form. Time saving, accuracy, and standardization of composition are the main advantages for using such media. The ready-made basal media are without growth regulators and sugars. The amounts of various ingredients, particularly those of plant growth regulators, are changed to suit the proliferation and growth of different types of plant and sometimes even of different varieties. The media and glass containers are sterilized, usually with steam in a pressure cooker or an autoclave. Many laboratories use glass jars such as jam jars, which can be used repeatedly by washing and autoclaving or disposable plastic containers such as Watson ModuleTM cups which come presterilized. Tiny pieces of plants or tissues, which have been
TISSUE CULTURE / Clonal Propagation, In Vitro
surface sterilized, are then transferred to the cooled, solidified medium, and kept under controlled conditions of light, day length, humidity, and temperature to provide summer-like conditions for shoot proliferation, root formation, and plant growth. The controlled environment conditions for culture and growth allow plant propagation on a year round basis, in a small space and production of high-quality disease- and insect-free plants in large numbers. This process is called micropropagation. This technique is particularly suitable for large-scale multiplication of plants propagated from vegetative parts, especially ornamentals, such as African violet (Saintpaulia ionantha), chrysanthemums, roses, Streptocarpus, carnation, and Gerbera. Many crops such as banana and plantain, cassava, garlic (Allium sativum), ginger, pineapple, potato, sweet potato, and yams (Colocasia esculenta and Dioscorea spp.) can also be micropropagated. Conventionally these plants are propagated from cuttings, corms, bulbs, and tubers, and repeated multiplication in soil leads to infection with viruses, fungi, bacteria, and nematodes. In contrast, the micropropagated plants are free from pathogens and of high quality.
Process of In Vitro Cloning The production of plants through micropropagation is a four-step process: (1) explant culture, (2) shoot proliferation, (3) rooting, and (4) plant hardening and transfer to soil. Explant Initiation
Tiny pieces (explants) between 2 and 10 mm long, taken usually from various plant parts (organs) such as shoot tips, stem nodes, and leaves, are the starting material to initiate plant micropropagation. Initiation of explants is the very first step in micropropagation. A good clean explant, once established in an aseptic condition, can be multiplied several times; hence, explant initiation in an aseptic condition should be regarded as a critical step in micropropagation. Very often, explants fail to establish and grow, not due to the lack of a suitable medium but because of contamination from bacteria and fungi. The explants can also be obtained as small pieces from stems, apical leaves and axillary buds, immature whole seeds, or their parts such as embryos and cotyledons and hypocotyledons, and in some cases even roots, inner core and scales from the bulbs, floral buds, and floral parts. The tiny pieces are surface sterilized to get rid of the fungi, bacteria, and insects. To achieve surface sterilization, the explants are first washed in sterile water, rinsed in ethanol,
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and then immersed in solutions of chemicals with chlorine base. Calcium or sodium hypochlorite-based solutions, 1–3% (w/v) are usually used for soft herbaceous materials, such as leaves and shoots. A 5–7% solution of DomestosTM disinfectant (which contains 10.5% w/v sodium hypochlorite, 0.3% sodium carbonate, 10.0% sodium chloride, and 0.5% w/v sodium hydroxide, and a patented thickener) is also effective for surface sterilization of many explants. Other surface sterilants used include mercuric chloride (though its use should be avoided as far as possible, since it is highly toxic) and potassium permanganate. The explants are rewashed three to four times with sterile distilled water after sterilization with chemicals. The explants produce shoots or roots and even somatic embryos, depending upon the chemicals in the medium called plant growth regulators or phytohormones. There are two sets of growth regulators that are most critical in the process of micropropagation: the auxins such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), and cytokinins such as kinetin (6-(furfurylamino) purine), 6-benzyl aminopurine (BA or BAP), zeatin ((E)-2-methyl-4-(1H-purin-6ylamino)-2-buten-1-ol), and 2iP (6-(rr-dimethylallylamino) purine). The relative ratio of cytokinin to that of auxin in the culture medium determines the pathway the explants take. By using relatively high concentrations of auxins (particularly the synthetic auxin-like compound 2,4-D and (2,4-dichlorophenoxyacetic acid), an explant can be turned into a lump or a mass of cells – a callus. The callus is then proliferated by culturing on a series of media containing different types and varying amounts of growth regulators that make the cells differentiate into adventitious buds, shoot primordia, and even somatic embryos that resemble sexual embryos. This process of obtaining plants from cells through callus cultures is called organogenesis and that of embryos as somatic embryogenesis. The pathway of cloning plants through callus culture often leads to the production of plants, a proportion of which do not resemble the parent, and are often genetically different from the parent. Many such types of plants originate from somaclonal variation that involves changes in the structure or function of DNA. In practice, a majority of plants are micropropagated from explants taken from shoot tips and nodal cuttings with apical or axillary buds. Pieces taken from these parts are used for establishing mother cultures that are used as a source of further cuttings to multiply plants. Located in the shoot tip are a group of cells called the shoot meristem. The shoot meristem cells are capable of division and making
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more meristems or adventitious buds that give rise to multiple shoots. Shoot Proliferation
During this stage there is a rapid numerical increase and growth of structures destined to form shoots. Once explants are established, they are cultured on media that promote multiple shoot formation by forming either adventitious buds or shoot primordia (also known as stage II). To achieve high rate of shoot meristem proliferation, explants are cultured on media containing relatively high cytokinin and low auxin. So good is the shoot proliferation capacity in a small space that as many as 150–200 orchid buds can be produced on 20 ml medium in one 7-cm tall plastic cup container with 5 cm diameter base. Efficient multiplication invariably requires growth regulators in the medium.
stages of establishment in soil. Bunch planting allows better survival and root growth. The soil should be low in nutrient level and well aerated at this stage. Other growth conditions such as temperature, light, and moisture need to be adequate but not excessive. The micropropagated plants should not be exposed to direct sunlight for the first 5 to 6 days. Frequent and overwatering should be avoided during the first 14 days of soil transfer. Each plant has its own specific requirement of humidity and temperature after transfer to soil. For example, strawberry (Fragaria x ananassa) microplants prefer low temperature (12–161C) and low relative humidity, whereas those of orchids (Orchidaceae) prefer high temperature (20–221C) and high humidity. Microplants usually are slow to grow in the beginning; however, once established in soil, in vitro propagated plants grow like normal plants, and usually outperform the conventionally propagated plants in vegetative growth.
Rooting
The shoots produced from stage II are rooted by further culture on rooting medium. In most plants, root formation is promoted by adding auxin to the subsequent medium. Often, shoots produce roots on media without growth regulators or with extremely low (0.01–1 mg l 1) auxins such as IBA or IAA. In case of cloning through somatic embryos, shoot and root formation occur simultaneously. Such embryos germinate on media without any growth regulators, although some require culture on media with gibberellins and/or desiccation for maturity. Plant Hardening and Transfer to Soil
The tiny in vitro produced plants (microplants) have to be acclimatized to the natural conditions of air (carbon dioxide), humidity, and temperature, a process called weaning or hardening. Usually the microplants are cultured under high humidity and their stomata function in a manner not suitable for plant survival under low humidity and variable temperature and high light intensity. A gradual adjustment to natural conditions is achieved by keeping microplants under shade, and infrequent watering during the first 1 to 3 weeks. In practice this is achieved by keeping such plants under plastic covers that allow sufficient air circulation, provide diffused light, and maintain relatively high humidity. In some cases, it helps to grow the plants in vermiculite and irrigate them with a low-concentration nutrient solution. The hardened plants are then grown in compost or soil for a few weeks and then transferred to large pots or field. It is better to transfer the microplants as bunches rather than as separated single plants in the initial
New Industry Micropropagation allows propagation of diseasefree, high-quality plants, tubers and bulbs. During the past 30 years, a whole new industry based on in vitro plant propagation has developed. Worldwide millions of plants of fruits such as banana and plantain, citrus, pineapple, strawberry, and crops such as sugar cane, potato, sweet potato, and cassava are multiplied through micropropagation. Among ornamentals, species of Anthurium, Gerbera, orchids (Cymbidium, Dendrobium, Oncidium), roses, and many more are produced through micropropagation. Plants micropropagated in Asia where labor is relatively cheap are shipped to Europe and the United States as microplants (orchids), microtubers (e.g., potato), and microbulbs (e.g., daffodils (Narcissus pseudonarcissus) and tulips). The sophistication in micropropagation ranges from low-tech in used liquor bottles to high-tech bioreactors. In large-scale plant micropropagation laboratories, it is common to make several liters of medium in bulk and dispense exact amount in thousands of containers using mechanical devices such as a peristaltic pump. A wide variety of containers from reusable milk bottles, used liquor bottles, jam jars, glass test-tubes, plastic Petri dishes, and MagentaTM vessels to disposable plastic cups such as Watson ModulesTM (Figure 1) are used for commercial-scale culture of plants. Each type of vessel has its advantages and limitations. The reusable glass vessels are cheap to buy but require high labor and energy costs for bulk cleaning and sterilization. Disposable plastic materials such as Watson Modules come presterilized, save labor costs, allow stacking of culture thus freeing
TISSUE CULTURE / Clonal Propagation, In Vitro
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suspension in the liquid medium. With the help of computers, the medium is maintained and adjusted at the desired temperature, pH, oxygen, and CO2 content, and replenished automatically. Plant cells maintained in this manner produce somatic embryos that can be matured on solid medium. Such systems are being developed for the multiplication of trees. However, many cells undergo changes in their genetic makeup after a short duration of culture that leads to the production of plants that are completely different from the parent. The procedures for plant production from bioreactor-cultured cells are as yet limited to a few experimental plants only. Many problems of forming plant shoots and embryos and their maturation and germination into microplants, and transfer to soil, still remain to be solved.
Figure 1 Potato microplants cultured on medium in a Watson ModuleTM container. Reproduced by permission of TeagascAgriculture and Food Development Authority from Ahloowalia BS (1994) Mini-tubers for seed potato production. Farm and Food 4: 4–6.
List of Technical Nomenclature Callus
A cluster of undifferentiated plant cells that have the capacity to regenerate a whole plant in some species.
Cell
Cells are the building blocks of all living organisms. A cell is the smallest structural unit of a living organism that is able to grow and reproduce independently.
Cell culture
A technique of growing cells in laboratory conditions.
Clonal propaga- Asexual multiplication of plants that are tion genetically alike and originate from a single individual or an explant. Clone
A genetic replica of an organism obtained through non-sexual (no fertilization) reproduction process.
Explant
A tiny piece of a plant or group of cells used for culture on artificial medium for growth or maintenance.
In vitro
In a test-tube or other laboratory apparatus. (Opposite of in vivo – in the living organisms.)
In vitro culture
Storage and growing of living material in tissue culture.
Microplants
Plants produced through in vitro culture or micropropagation.
Future Developments
Micropropagation
Many innovations are taking place in the technology of cloning plants through in vitro culture. These include the automated containers, ‘‘bioreactors,’’ in which cells rather than plants are cultured as
Multiplication of plants from tiny pieces on synthetic medium under controlled environment and aseptic conditions.
Organogenesis
Production of organs, such as adventitious buds, primordial shoots, and roots from the cells and callus cultures.
Figure 2 Micropropagated plants of potato after 10 (left) and 21 days (right) of transfer to soil in Watson ModuleTM containers. Reproduced by permission of Teagasc-Agriculture and Food Development Authority from Ahloowalia BS (1994) Mini-tubers for seed potato production. Farm and Food 4: 4–6.
space in culture rooms, and can be turned into pots for soil growing of microplants (Figure 2); however, they cannot be reused for plant culture.
1364 TISSUE CULTURE / Organogenesis Plant propagation
Production of plants from vegetative parts such as cuttings and slips without sexual generation.
Shoot meristem
A group of special cells in the apical region of the shoots, which are capable of dividing and give rise to axillary buds, lateral shoots, and stem.
Somaclonal variation
Variation that originates in tissue cultures. It may be physiological, genetic, or epigenetic.
Somatic embryo Embryos of nonsexual origin in plants obtained from cultures of cells and calluses. Tissue culture
A technique in which portions of a plant (or animal) are grown on a synthetic culture medium.
Vegetative prop- Reproduction of plants using a nonagation sexual process involving the culture of plant parts such as stem and leaf cuttings.
See also: Production Systems and Agronomy: Nursery Stock and Houseplant Production. Root Development: Genetics of Primary Root Development; Root Growth and Development. Tissue Culture and Plant Breeding: Regeneration of Fruit and Ornamental Trees via Cell and Tissue Culture.
Further Reading Ahloowalia BS (1995) Watson Module: A new tool for plant micropropagation. BioLink 2: 17. Ahloowalia BS (2000) High-tech propagation of horticultural plants. In: Chadha KL et al. (eds) Biotechnology in Horticulture and Plantation Crops, pp. 26–37. New Delhi: Malhotra Publishing House. Debergh PC and Zimmerman RH (1991) Micropropagation: Technology and Application. Dordrecht: Kluwer Academic Publishers. Dixon RA (1985) Plant Cell Culture: A Practical Approach. Oxford: IRL Press. Evans DA, Sharp WE, Ammirato PV, and Yamada Y (1983) Handbook of Plant Cell Culture, vol. 1, Techniques for Propagation and Breeding. New York: MacMillan. George EF, Puttock DJM, and George HJ (1987) Plant Culture Media, vol. 1, Formulations and Uses. Edington: Exgenetics Ltd. Kyte L (1987) Plants from Test Tubes: An Introduction to Micropropagation. Portland: Timber Press. Murashige T (1974) Plant propagation through tissue cultures. Annual Review of Plant Physiology 255: 135–166. Thorpe TA and Harry IS (1997) Application of tissue culture to horticulture. In: Altman A and Ziv M (eds) Horticultural Biotechnology: In Vitro Culture and Breeding, pp. 39–49. Leuven: Acta Horticulturae.
Torres K (1988) Tissue Culture Techniques for Horticultural Crops. New York: Van Nostrand Reinhold. Wetter LR and Constabel F (1982) Plant Tissue Culture Methods. Saskatoon: National Research Council of Canada.
Organogenesis G-J de Klerk, Center for Plant Tissue Culture Research, Lisse, The Netherlands Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction In plants, differentiated somatic cells may reinitiate the ontogenetic program: when given the proper stimuli, they develop into adventitious meristems. These new meristems generate adventitious roots, shoots, or embryos. The formation of adventitious roots and shoots is referred to as (adventitious) ‘‘organogenesis,’’ whereas ‘‘somatic embryogenesis’’ denotes the formation of adventitious embryos. In vitro, adventitious organogenesis may occur at very high frequencies, and is one of the corner stones of biotechnological breeding and propagation methods. Biotechnological breeding techniques such as genetic engineering and haploid production involve adventitious regeneration of a complete plant from somatic cells. Many micropropagation protocols involve the formation of adventitious shoots from, for example, leaf or scale fragments. Before planting ex vitro, microcuttings are treated with auxin to obtain adventitious roots. Regeneration of somatic embryos from cell suspensions will – if broadly applicable – revolutionize plant propagation. This article deals with adventitious organogenesis in vitro from the scientific and practical points of view.
Occurrence of Organogenesis Formation of Plant Meristems
An organism begins its existence as a fertilized egg cell, the zygote. From this single, morphologically simple cell, the embryo proper and the suspensor are formed following a series of cell divisions. In the embryo proper, the shoot and root apical meristem arise in a polar manner. In postembryonic development, the shoot apical meristem produces the stem, leaves, axillary meristems, and flowers, while the root apical meristem generates the primary root, but no lateral organs. During the plant’s life, differentiated cells may produce adventitious meristems; a
Clonal Propagation, In Vitro B S Ahloowalia, International Atomic Energy Agency, Vienna, Austria Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Clonal propagation of plants refers to the production of genetically identical plants through nonsexual methods. Many plants are propagated from vegetative parts such as bud grafts, slips, stem cuttings, bulbs, tubers, and runners. For example, date palm (Phoenix dactylifera) trees are propagated from offshoots and rooting them in soil. Yet in other plants, leaves develop adventitious buds on the margins and leaf veins, which on sprouting develop into new plants. The plants produced in this manner are exact copies or clones of the mother plant, and genetically identical unless a mutation occurs in the cells and tissues of the original plant. Vegetative propagation is used for the multiplication of many important crops, shrubs, and trees. These include crops such as cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), ginger (Zingibar officinale), sugar cane (Saccharum spp.), taro yam (Colocasia esculenta), turmeric (Curcuma domestica), and yam (Dioscorea spp.). Several ornamentals, e.g., azalea (Rhododendron spp.), carnation (Dianthus spp.), chrysanthemum (Chrysanthemum spp.), jasmine (Jasminum officinale), roses (Rosa hybrida and chinensis), and tulips (Tulipa spp.), and many fruits such as banana and plantain (Musa spp.), pineapple (Ananas comosus), passion fruit (Passiflora edulis), and kiwi fruit (Actinidia chinensis), are also propagated from vegetative parts. In many other fruits, such as apple (Malus domestica), citrus (Citrus sinensis, C. paradisi, and several other Citrus species), lychee (Litchi chinensis), mango (Mangifera indica), and pear (Pyrus communis), shoots and buds are grafted to produce clones of the original mother tree. Bud sports or spontaneous
mutations are also propagated in this manner to obtain new varieties.
In Vitro Propagation In vitro propagation is very much like multiplication of plants from vegetative parts except that plants are produced by culturing tiny pieces on a synthetic medium in a suitable container instead of soil in a pot. The in vitro propagation of plants is initiated by culturing pieces of stems, leaves, apical and axillary buds, immature seeds, embryos, and cotyledons. The basis of in vitro propagation is rapid multiplication of plants by culturing tiny pieces on media under controlled environment and aseptic (germ-free) conditions. Since strict aseptic conditions have to be maintained in the production of in vitro cultured plants, the culture and transfer of cuttings and plants is carried out under germ-free conditions in laminarflow cabinets, and the containers in which plants are cultured are kept tightly sealed. The laminar-flow cabinets are designed to produce sterile air. The instruments such as scalpels, scissors, and forceps used for cutting and transfer of plant pieces are sterilized by alcohol dip and flaming or dry heat. Likewise, the media are also sterilized. Most culture media contain basic major and minor salts, vitamins, sugar, plant growth regulators, and agar with pH adjusted between 5.6 and 5.8. The major salts provide nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium, and minor salts elements such as copper, manganese, molybdenum, sulfur, and zinc, which along with vitamins are essential for the growth and differentiation of plant cells and tissues. The sugar in the culture medium provides carbon for photosynthesis. However, the composition of media (particularly the type and amount of sugar and growth regulators) is changed according to different plant species. Several commercial companies sell ready-made basal media in powdered form. Time saving, accuracy, and standardization of composition are the main advantages for using such media. The ready-made basal media are without growth regulators and sugars. The amounts of various ingredients, particularly those of plant growth regulators, are changed to suit the proliferation and growth of different types of plant and sometimes even of different varieties. The media and glass containers are sterilized, usually with steam in a pressure cooker or an autoclave. Many laboratories use glass jars such as jam jars, which can be used repeatedly by washing and autoclaving or disposable plastic containers such as Watson ModuleTM cups which come presterilized. Tiny pieces of plants or tissues, which have been
TISSUE CULTURE / Clonal Propagation, In Vitro
surface sterilized, are then transferred to the cooled, solidified medium, and kept under controlled conditions of light, day length, humidity, and temperature to provide summer-like conditions for shoot proliferation, root formation, and plant growth. The controlled environment conditions for culture and growth allow plant propagation on a year round basis, in a small space and production of high-quality disease- and insect-free plants in large numbers. This process is called micropropagation. This technique is particularly suitable for large-scale multiplication of plants propagated from vegetative parts, especially ornamentals, such as African violet (Saintpaulia ionantha), chrysanthemums, roses, Streptocarpus, carnation, and Gerbera. Many crops such as banana and plantain, cassava, garlic (Allium sativum), ginger, pineapple, potato, sweet potato, and yams (Colocasia esculenta and Dioscorea spp.) can also be micropropagated. Conventionally these plants are propagated from cuttings, corms, bulbs, and tubers, and repeated multiplication in soil leads to infection with viruses, fungi, bacteria, and nematodes. In contrast, the micropropagated plants are free from pathogens and of high quality.
Process of In Vitro Cloning The production of plants through micropropagation is a four-step process: (1) explant culture, (2) shoot proliferation, (3) rooting, and (4) plant hardening and transfer to soil. Explant Initiation
Tiny pieces (explants) between 2 and 10 mm long, taken usually from various plant parts (organs) such as shoot tips, stem nodes, and leaves, are the starting material to initiate plant micropropagation. Initiation of explants is the very first step in micropropagation. A good clean explant, once established in an aseptic condition, can be multiplied several times; hence, explant initiation in an aseptic condition should be regarded as a critical step in micropropagation. Very often, explants fail to establish and grow, not due to the lack of a suitable medium but because of contamination from bacteria and fungi. The explants can also be obtained as small pieces from stems, apical leaves and axillary buds, immature whole seeds, or their parts such as embryos and cotyledons and hypocotyledons, and in some cases even roots, inner core and scales from the bulbs, floral buds, and floral parts. The tiny pieces are surface sterilized to get rid of the fungi, bacteria, and insects. To achieve surface sterilization, the explants are first washed in sterile water, rinsed in ethanol,
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and then immersed in solutions of chemicals with chlorine base. Calcium or sodium hypochlorite-based solutions, 1–3% (w/v) are usually used for soft herbaceous materials, such as leaves and shoots. A 5–7% solution of DomestosTM disinfectant (which contains 10.5% w/v sodium hypochlorite, 0.3% sodium carbonate, 10.0% sodium chloride, and 0.5% w/v sodium hydroxide, and a patented thickener) is also effective for surface sterilization of many explants. Other surface sterilants used include mercuric chloride (though its use should be avoided as far as possible, since it is highly toxic) and potassium permanganate. The explants are rewashed three to four times with sterile distilled water after sterilization with chemicals. The explants produce shoots or roots and even somatic embryos, depending upon the chemicals in the medium called plant growth regulators or phytohormones. There are two sets of growth regulators that are most critical in the process of micropropagation: the auxins such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), and cytokinins such as kinetin (6-(furfurylamino) purine), 6-benzyl aminopurine (BA or BAP), zeatin ((E)-2-methyl-4-(1H-purin-6ylamino)-2-buten-1-ol), and 2iP (6-(rr-dimethylallylamino) purine). The relative ratio of cytokinin to that of auxin in the culture medium determines the pathway the explants take. By using relatively high concentrations of auxins (particularly the synthetic auxin-like compound 2,4-D and (2,4-dichlorophenoxyacetic acid), an explant can be turned into a lump or a mass of cells – a callus. The callus is then proliferated by culturing on a series of media containing different types and varying amounts of growth regulators that make the cells differentiate into adventitious buds, shoot primordia, and even somatic embryos that resemble sexual embryos. This process of obtaining plants from cells through callus cultures is called organogenesis and that of embryos as somatic embryogenesis. The pathway of cloning plants through callus culture often leads to the production of plants, a proportion of which do not resemble the parent, and are often genetically different from the parent. Many such types of plants originate from somaclonal variation that involves changes in the structure or function of DNA. In practice, a majority of plants are micropropagated from explants taken from shoot tips and nodal cuttings with apical or axillary buds. Pieces taken from these parts are used for establishing mother cultures that are used as a source of further cuttings to multiply plants. Located in the shoot tip are a group of cells called the shoot meristem. The shoot meristem cells are capable of division and making
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more meristems or adventitious buds that give rise to multiple shoots. Shoot Proliferation
During this stage there is a rapid numerical increase and growth of structures destined to form shoots. Once explants are established, they are cultured on media that promote multiple shoot formation by forming either adventitious buds or shoot primordia (also known as stage II). To achieve high rate of shoot meristem proliferation, explants are cultured on media containing relatively high cytokinin and low auxin. So good is the shoot proliferation capacity in a small space that as many as 150–200 orchid buds can be produced on 20 ml medium in one 7-cm tall plastic cup container with 5 cm diameter base. Efficient multiplication invariably requires growth regulators in the medium.
stages of establishment in soil. Bunch planting allows better survival and root growth. The soil should be low in nutrient level and well aerated at this stage. Other growth conditions such as temperature, light, and moisture need to be adequate but not excessive. The micropropagated plants should not be exposed to direct sunlight for the first 5 to 6 days. Frequent and overwatering should be avoided during the first 14 days of soil transfer. Each plant has its own specific requirement of humidity and temperature after transfer to soil. For example, strawberry (Fragaria x ananassa) microplants prefer low temperature (12–161C) and low relative humidity, whereas those of orchids (Orchidaceae) prefer high temperature (20–221C) and high humidity. Microplants usually are slow to grow in the beginning; however, once established in soil, in vitro propagated plants grow like normal plants, and usually outperform the conventionally propagated plants in vegetative growth.
Rooting
The shoots produced from stage II are rooted by further culture on rooting medium. In most plants, root formation is promoted by adding auxin to the subsequent medium. Often, shoots produce roots on media without growth regulators or with extremely low (0.01–1 mg l 1) auxins such as IBA or IAA. In case of cloning through somatic embryos, shoot and root formation occur simultaneously. Such embryos germinate on media without any growth regulators, although some require culture on media with gibberellins and/or desiccation for maturity. Plant Hardening and Transfer to Soil
The tiny in vitro produced plants (microplants) have to be acclimatized to the natural conditions of air (carbon dioxide), humidity, and temperature, a process called weaning or hardening. Usually the microplants are cultured under high humidity and their stomata function in a manner not suitable for plant survival under low humidity and variable temperature and high light intensity. A gradual adjustment to natural conditions is achieved by keeping microplants under shade, and infrequent watering during the first 1 to 3 weeks. In practice this is achieved by keeping such plants under plastic covers that allow sufficient air circulation, provide diffused light, and maintain relatively high humidity. In some cases, it helps to grow the plants in vermiculite and irrigate them with a low-concentration nutrient solution. The hardened plants are then grown in compost or soil for a few weeks and then transferred to large pots or field. It is better to transfer the microplants as bunches rather than as separated single plants in the initial
New Industry Micropropagation allows propagation of diseasefree, high-quality plants, tubers and bulbs. During the past 30 years, a whole new industry based on in vitro plant propagation has developed. Worldwide millions of plants of fruits such as banana and plantain, citrus, pineapple, strawberry, and crops such as sugar cane, potato, sweet potato, and cassava are multiplied through micropropagation. Among ornamentals, species of Anthurium, Gerbera, orchids (Cymbidium, Dendrobium, Oncidium), roses, and many more are produced through micropropagation. Plants micropropagated in Asia where labor is relatively cheap are shipped to Europe and the United States as microplants (orchids), microtubers (e.g., potato), and microbulbs (e.g., daffodils (Narcissus pseudonarcissus) and tulips). The sophistication in micropropagation ranges from low-tech in used liquor bottles to high-tech bioreactors. In large-scale plant micropropagation laboratories, it is common to make several liters of medium in bulk and dispense exact amount in thousands of containers using mechanical devices such as a peristaltic pump. A wide variety of containers from reusable milk bottles, used liquor bottles, jam jars, glass test-tubes, plastic Petri dishes, and MagentaTM vessels to disposable plastic cups such as Watson ModulesTM (Figure 1) are used for commercial-scale culture of plants. Each type of vessel has its advantages and limitations. The reusable glass vessels are cheap to buy but require high labor and energy costs for bulk cleaning and sterilization. Disposable plastic materials such as Watson Modules come presterilized, save labor costs, allow stacking of culture thus freeing
TISSUE CULTURE / Clonal Propagation, In Vitro
1363
suspension in the liquid medium. With the help of computers, the medium is maintained and adjusted at the desired temperature, pH, oxygen, and CO2 content, and replenished automatically. Plant cells maintained in this manner produce somatic embryos that can be matured on solid medium. Such systems are being developed for the multiplication of trees. However, many cells undergo changes in their genetic makeup after a short duration of culture that leads to the production of plants that are completely different from the parent. The procedures for plant production from bioreactor-cultured cells are as yet limited to a few experimental plants only. Many problems of forming plant shoots and embryos and their maturation and germination into microplants, and transfer to soil, still remain to be solved.
Figure 1 Potato microplants cultured on medium in a Watson ModuleTM container. Reproduced by permission of TeagascAgriculture and Food Development Authority from Ahloowalia BS (1994) Mini-tubers for seed potato production. Farm and Food 4: 4–6.
List of Technical Nomenclature Callus
A cluster of undifferentiated plant cells that have the capacity to regenerate a whole plant in some species.
Cell
Cells are the building blocks of all living organisms. A cell is the smallest structural unit of a living organism that is able to grow and reproduce independently.
Cell culture
A technique of growing cells in laboratory conditions.
Clonal propaga- Asexual multiplication of plants that are tion genetically alike and originate from a single individual or an explant. Clone
A genetic replica of an organism obtained through non-sexual (no fertilization) reproduction process.
Explant
A tiny piece of a plant or group of cells used for culture on artificial medium for growth or maintenance.
In vitro
In a test-tube or other laboratory apparatus. (Opposite of in vivo – in the living organisms.)
In vitro culture
Storage and growing of living material in tissue culture.
Microplants
Plants produced through in vitro culture or micropropagation.
Future Developments
Micropropagation
Many innovations are taking place in the technology of cloning plants through in vitro culture. These include the automated containers, ‘‘bioreactors,’’ in which cells rather than plants are cultured as
Multiplication of plants from tiny pieces on synthetic medium under controlled environment and aseptic conditions.
Organogenesis
Production of organs, such as adventitious buds, primordial shoots, and roots from the cells and callus cultures.
Figure 2 Micropropagated plants of potato after 10 (left) and 21 days (right) of transfer to soil in Watson ModuleTM containers. Reproduced by permission of Teagasc-Agriculture and Food Development Authority from Ahloowalia BS (1994) Mini-tubers for seed potato production. Farm and Food 4: 4–6.
space in culture rooms, and can be turned into pots for soil growing of microplants (Figure 2); however, they cannot be reused for plant culture.
1364 TISSUE CULTURE / Organogenesis Plant propagation
Production of plants from vegetative parts such as cuttings and slips without sexual generation.
Shoot meristem
A group of special cells in the apical region of the shoots, which are capable of dividing and give rise to axillary buds, lateral shoots, and stem.
Somaclonal variation
Variation that originates in tissue cultures. It may be physiological, genetic, or epigenetic.
Somatic embryo Embryos of nonsexual origin in plants obtained from cultures of cells and calluses. Tissue culture
A technique in which portions of a plant (or animal) are grown on a synthetic culture medium.
Vegetative prop- Reproduction of plants using a nonagation sexual process involving the culture of plant parts such as stem and leaf cuttings.
See also: Production Systems and Agronomy: Nursery Stock and Houseplant Production. Root Development: Genetics of Primary Root Development; Root Growth and Development. Tissue Culture and Plant Breeding: Regeneration of Fruit and Ornamental Trees via Cell and Tissue Culture.
Further Reading Ahloowalia BS (1995) Watson Module: A new tool for plant micropropagation. BioLink 2: 17. Ahloowalia BS (2000) High-tech propagation of horticultural plants. In: Chadha KL et al. (eds) Biotechnology in Horticulture and Plantation Crops, pp. 26–37. New Delhi: Malhotra Publishing House. Debergh PC and Zimmerman RH (1991) Micropropagation: Technology and Application. Dordrecht: Kluwer Academic Publishers. Dixon RA (1985) Plant Cell Culture: A Practical Approach. Oxford: IRL Press. Evans DA, Sharp WE, Ammirato PV, and Yamada Y (1983) Handbook of Plant Cell Culture, vol. 1, Techniques for Propagation and Breeding. New York: MacMillan. George EF, Puttock DJM, and George HJ (1987) Plant Culture Media, vol. 1, Formulations and Uses. Edington: Exgenetics Ltd. Kyte L (1987) Plants from Test Tubes: An Introduction to Micropropagation. Portland: Timber Press. Murashige T (1974) Plant propagation through tissue cultures. Annual Review of Plant Physiology 255: 135–166. Thorpe TA and Harry IS (1997) Application of tissue culture to horticulture. In: Altman A and Ziv M (eds) Horticultural Biotechnology: In Vitro Culture and Breeding, pp. 39–49. Leuven: Acta Horticulturae.
Torres K (1988) Tissue Culture Techniques for Horticultural Crops. New York: Van Nostrand Reinhold. Wetter LR and Constabel F (1982) Plant Tissue Culture Methods. Saskatoon: National Research Council of Canada.
Organogenesis G-J de Klerk, Center for Plant Tissue Culture Research, Lisse, The Netherlands Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction In plants, differentiated somatic cells may reinitiate the ontogenetic program: when given the proper stimuli, they develop into adventitious meristems. These new meristems generate adventitious roots, shoots, or embryos. The formation of adventitious roots and shoots is referred to as (adventitious) ‘‘organogenesis,’’ whereas ‘‘somatic embryogenesis’’ denotes the formation of adventitious embryos. In vitro, adventitious organogenesis may occur at very high frequencies, and is one of the corner stones of biotechnological breeding and propagation methods. Biotechnological breeding techniques such as genetic engineering and haploid production involve adventitious regeneration of a complete plant from somatic cells. Many micropropagation protocols involve the formation of adventitious shoots from, for example, leaf or scale fragments. Before planting ex vitro, microcuttings are treated with auxin to obtain adventitious roots. Regeneration of somatic embryos from cell suspensions will – if broadly applicable – revolutionize plant propagation. This article deals with adventitious organogenesis in vitro from the scientific and practical points of view.
Occurrence of Organogenesis Formation of Plant Meristems
An organism begins its existence as a fertilized egg cell, the zygote. From this single, morphologically simple cell, the embryo proper and the suspensor are formed following a series of cell divisions. In the embryo proper, the shoot and root apical meristem arise in a polar manner. In postembryonic development, the shoot apical meristem produces the stem, leaves, axillary meristems, and flowers, while the root apical meristem generates the primary root, but no lateral organs. During the plant’s life, differentiated cells may produce adventitious meristems; a