Micropropagation 2010-Wiley

MICROPROPAGATION OF PLANTS

BETH LOBERANT1and ARIE

ALTMAN2

1Desert Labs, Kibbutz Yotvata,Israel

2Robert H. Smith Institute ofPlant Sciences and Geneticsin Agriculture, The HebrewUniversity of Jerusalem,Rehovot, Israel

INTRODUCTION

Terminology

Micropropagation (other synonyms include: in vitropropagation) is the most common term used for clonal,true-to-type propagation of plants by a variety of tissue,and cell and organ culture methods. The terms axenicor aseptic culture and plant tissue culture refer toother applications of plant tissue culture which are notnecessarily strictly propagation.

Micropropagation implies the aseptic culture of smallsections (i.e. explants) of tissues and organs, in closedvessels with defined culture media and under controlledenvironmental conditions. Micropropagation (in additionto genetic engineering) is at present the most commer-cially efficient and practically oriented plant biotechnol-ogy, resulting in rapid generation of a large number ofclonal plants of many plant species, which are in manycases also virus- or other pathogen-free. Moreover, micro-propagation is now the technical link in the generation oftransgenic plants and otherwise somatically bred plants.Efficient production of transgenic plants relies heavily, ifnot exclusively, on the ability to regenerate whole plantsfrom those cells, tissues, or organs into which ‘‘foreign’’DNA has been inserted and expressed. Additionally, micro-propagation and other tissue-culture techniques, as wellas new modalities in molecular biology, can allow for fastertesting of new genotypes or field selections of plants.

Brief History

The science and art of plant tissue culture, and indeedof plant biotechnology, had its roots in the vision ofthe Austrian botanist Gottlieb Haberlandt (1854–1945)who visualized in 1902 the ability ‘‘to culture isolatedvegetative cells from higher plants in simple nutrientsolutions.’’ The first successful prolonged in vitrocultivation of plant tissues and organs—and theirproliferation and differentiation—included, amongothers, tomato roots (1), tobacco, carrot calluses, andcambial tissues (2a,2b) shoot tips and meristems (3).Soon, an entire new arsenal of procedures and techniquesfor controlling plant regeneration and morphogenesis in

Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. FlickingerCopyright © 2010 John Wiley & Sons, Inc.

culture became available to the scientific community.Some notable major discoveries included chemical andhormonal control of regeneration (4), basic and appliedaspects of organogenesis and somatic embryogenesis (5),practical micropropagation and production of virus-freeplants (6), haploid plants (7,8), culture and regenerationof protoplasts (9), production of secondary metabolites (10)and large-scale cell culture in bioreactors (11), to mentionjust a few milestones. This led, following many pioneeringstudies in dozens of laboratories around the world,to the first true breakthrough of plant biotechnology,namely, commercial micropropagation (12). In vitro massproduction of clonal propagules of a small number ofagriculturally important plants, primarily ornamentals,became practical in the early 1970s (13–15). Since then,the diversity of plant species that can be propagated invitro has dramatically increased, and it is now practicedon a commercial scale worldwide, resulting in over 600millions of plants annually, 60%–75 % of them beingflowers and ornamental plants (16–20). Micropropagationis now an integral part of the plant propagation industry,complementing or replacing other methods of clonalvegetative propagation (cutting, grafting, division, andseparation), or in some cases also propagation by seeds.The history, science, and practice of plant micropropaga-tion have been dealt with extensively in several books andreviews (14,19–33).

Ongoing achievements in the in vitro culture of pollen,protoplasts, and cell suspensions, and their ability toregenerate whole plants, resulted in new disciplines ofplant science: somatic cell genetics and metabolite produc-tion; somatic hybrids; production of haploid plants; selec-tion of variants and mutants; and improved generationof metabolites. All became available for the community ofplant scientists and breeders, expanding the diversity andthe agronomic and commercial value of plants.

Objectives of Micropropagation and Related Applications

1. Large-scale clonal propagation where conventionalvegetative propagation is not possible or practical;where there is a limited supply of stock plant mate-rial; or where the traditional vegetative propagationcoefficient (rate) is very low

2. Initial rapid clonal propagation of new varieties(with offspring from sexual breeding and rare speciesselected from the wild)

3. Embryo rescue where seed and embryo germinationis done in vitro to save otherwise nonviable sexualoffspring

4. Recovery of pathogen-free propagation material5. In vitro gene-banks for crop improvement6. Breeding by somatic cell genetics (with haploid pro-

duction from in vitro cultured pollen, somatic fusionof protoplasts or in vitro selection of somaclonalvariation)

1

2 MICROPROPAGATION OF PLANTS

7. Initial generation of transgenic (genetically engi-neered) plants

It should be noted that micropropagation is an espe-cially efficient system when it includes the recovery ofpathogen-free plants and/or breeding programs: for rapidproduction of sexual hybrids, recovery of transgenic plantsand/or as an intermediate stage in somatic breeding.

BIOLOGICAL PRINCIPLES OF MICROPROPAGATION

The basic aspects of plant regeneration in vitro are com-mon to all micropropagation procedures. Micropropaga-tion relies on the regeneration of new plants from smallexplants of various sources, due to the unique totipo-tency of plant cells, and includes induced and enhancedcell division, formation of a callus tissue, further prolif-eration of the callus and/or multistep differentiation andregeneration of events which leads to organogenesis (organformation, i.e. shoots and/or roots) or to somatic embryoge-nesis (formation of seed-like embryos from somatic cells).The various stages and processes are schematically repre-sented in Fig 1.

Callus induction

Dedifferentiation

Dedifferentiation inductioncytoskeleton and cell cycle genes

Organization,meristem(oid) formation

Continued mitoticactivity

Subcultures

In vitro differentiation and regeneration

Callus proliferation

Redifferentiation

Wounding effectschanges in hormone balance(auxins, cytoskeleton, ethylene etc.)

Medium and othercomponents

Metabolic activation

New environmentalconditions

A. Altman, 2001

Explants

OrganogenesisShoots–Roots

Somaticembryogenesis

Figure 1. Processes and pathways of differentiation and regen-eration of new plants in vitro. Micropropagation involves excisionof an explant from a source plant, together with its exposure toculture medium components and new environmental conditions.This activates wounding responses and a variety of physiolog-ical, hormonal, metabolic, and molecular responses (which aredepicted in the upper left-hand side box). These processes resultin the dedifferentiation of specific explant tissues and enhancedcell division (right-hand side boxes). The increased mitotic activ-ity results in turn in callus proliferation (left-hand side boxes)and/or redifferentiation. If callus is subcultured periodically ontofresh growth medium, mitotic activity proceeds and callus con-tinues to proliferate. In addition, following dedifferentiation, anew morphogenetic pattern is established under the control ofcytoskeleton and cell cycle genes, followed by redifferentiationprocesses. This finally results in organization of new shoot and/orroot meristems (organogenesis), and/or somatic embryogenesis.Micropropagation by enhanced development of axillary shootsdoes not involve dedifferentiation and redifferentiation.

Totipotency of Plant Cells

Unlike the majority of animal cells, plant cells are totipo-tent, that is, plants possess the potential to regeneratewhole plants from individual cells, or groups of cells fromwithin a tissue or an organ, expressing the full plantgenome (35,36). This potential for plant regeneration isusually realized after an explant, that is, a small pieceof tissue, an organ, or in some cases isolated callus, havebeen excised from the source (‘‘mother’’) plant and placedon a defined culture medium in vitro. Exceptions include,for example, young seedlings that can be cultured intact,in vitro, even without prior excision. Totipotency is aunique feature of plants, as opposed to animals, whereonly certain stem cells can sometimes differentiate andyield other cell types. All live explants, or even isolatedcells, are able to give rise to a new plant when given theproper culture conditions.

Cell Division and Callus Formation

The explant, which in most cases is well differentiated andorganized, has first to undergo a process of dedifferentia-tion which is followed by renewed active cell division. Thisincreased mitotic activity is induced by a set of processesbrought about by various wounding effects and exposure tothe new nutritional and hormonal components of culturemedium and new environmental conditions. The processesinclude expression of wound-induced genes, expression ofspecific wound-associated growth factors and signalingmolecules (e.g. systemin and jasmonic acid), changes inthe tissue endogenous hormone balance, metabolic acti-vation, and others. This is followed by continued callusproliferation and/or regeneration.

Differentiation and Regeneration

Regeneration of new organs (organogenesis) and/orembryos (somatic embryogenesis) depends on a setof redifferentiation processes. These are followed byorganization of new shoot and/or root meristems, or aprecisely controlled bipolar cell division which givesrise to somatic embryos. Regeneration and organizationare extremely complicated and controlled biologicalprocesses, which await full elucidation. They include,among others, factors such as the determination ofcell polarity, cell cycle factors, hormonal and metabolicsignaling, proper temporal and spatial activation ofspecific genes, and controlled cytoskeleton organization,which may determine the plane of cell division. The majorpathways of new plant regeneration, that is, the basis formicropropagation, are shown in Fig 2.

Axillary Bud Proliferation

Axillary bud proliferation in vitro is usually considereda convenient route for micropropagation even if the finalnumber of newly regenerated plantlets may be consid-erably lower than in other pathways. Since axillary budproliferation typically does not include a callus stage, theprobability of somaclonal variation is lower than in trueorganogenesis. The system is therefore considered ‘‘safer’’for the preservation of clonal characteristics. In fact, this

MICROPROPAGATION OF PLANTS 3

Shoot tips Axillary bud elongation Rooting Plants

Roots Buds

Explant Buds Roots Plants

Callus

Callus Cell cultureExplant Plants

Somaticembryos

1. Enhanced development of axillary buds

2. Organogenesis (buds and/or roots)

3. Somatic embryogenesis

Figure 2. Major pathways of micropropagation (generation ofnew plants in vitro). Micropropagation, that is, generation of newplants in vitro from a variety of explants, depends on three majorpathways: (1, top) enhanced development of existent axillarybuds (resulting mainly from their activation, that is, releasefrom whole-plant control, in excised shoot tips, and from stemnode explants), and/or (2, middle) regeneration and formationof adventitious organs, that is, organogenesis (new roots and/orshoots), either directly without intermediary callus formation orvia callus, and/or (3, bottom) by regeneration and formation ofsomatic embryos (directly or via a callus stage). In some plantspecies all three pathways can occur simultaneously, in othersonly one or two pathways are available. Usually, only one pathwayoffers a propagation coefficient high and reliable enough to beemployed commercially.

pathway does not involve regeneration of new buds, sincebud meristems already exist in the axils of leaves andin the shoot tip (25,28,29,36). However, because of apicalcontrol they usually do not develop in intact plants untilthe stem elongates and grows. Thus, the short stem tipthat is used as an explant already contains many axillarybuds, at different stages of development, condensed in asmall volume. A very large number of otherwise quiescentaxillary shoot buds grow extensively when the shoot tips,or even small apical meristems, are excised and culturedin an appropriate medium, usually containing high con-centrations of cytokinins. Both the excision of the explantand the cytokinin-rich medium ‘‘activate’’ the bud, lead-ing to massive proliferation and elongation of many sideshoots. After induction, these shoots are separated forfurther culture and rooting (Fig 2).

Organogenesis: Formation of Shoots, Roots, and SpecializedStructures

Organogenesis from explants, or from callus and cell cul-tures, results in de novo formation of shoots and/or roots.These two events may take place simultaneously duringculture, but frequently either shoots or, less frequently,roots are formed first. Regeneration of the complementaryorgan occurs only later, either in the same medium or aftersubculture to another medium and under environmentalconditions that favor the formation of the specific organ(Fig 2). Organogenesis starts with a distinct organizationof a group of a few meristematic cells (meristemoids),directly within the explant or from the callus, that later

turn into a shoot or a root meristem (29,31,36,37). The typeof explant, composition of the culture medium (especiallythe balance of growth regulators), and the environmentalconditions during culture affect the formation of either ashoot or a root meristem. Once shoot or root meristemshave been organized they begin developing; forming smallshoots and roots. Further stages of organization, differ-entiation, and growth include the formation of functionalvascular connections between the developing shoots androots, finally giving rise to plantlets in vitro. This sequenceof events and operations results in new plantlets thatare removed from the culture vessels, acclimatized underex vitro conditions and subsequently, cultivated underconventional greenhouse or field conditions. In some plantspecies, organogenesis brings about the formation of modi-fied shoots and roots, usually, storage organs such as bulbsand tubers. Here, the shoot or the root meristem forms asdescribed previously, but instead of further growth andelongation, minibulbs, minitubers, or minicorms (depend-ing on the specific genotype) soon develop. These can growfurther or become dormant, depending on the cultureconditions. Such storage organs may also develop fromaxillary buds without true regeneration, especially, whenbasal plates of bulbs, or certain corm and tuber tissues,are used as primary explants.

Somatic Embryogenesis

Somatic embryogenesis is different from organogenesisin that regeneration and organization is bipolar, that is,a progenitor cell divides unequally, forming simultane-ously a shoot and a root meristem that give rise to agroup of cells known as proembryonic masses (31,35,36).As with organogenesis, differentiation and organizationof a somatic embryo take place directly in the explant orfrom the callus (Fig 2), depending on the type of explant,composition of the culture medium, and the subcultureregime. Usually, a two-stage culture is involved. First,induction of proembryonic masses, which is frequentlyfavored by an initial exposure of the tissue to 2,4-D or otherauxin-type growth regulators. Afterwards, the proembry-onic cultures are transferred to a medium with a modifiedcomposition from which the auxin has been removed orits concentration reduced, in addition to a change in othermedia components, in which the somatic embryos fullydifferentiate. Somatic embryogenesis is often favored byculture in agitated liquid medium. The patterns of dif-ferentiation are very conserved (35,38). First, ‘‘globular’’embryonic structures are formed. This is followed by the‘‘heart’’ stage in which the shoot and root meristems canbe clearly distinguished at the two poles, and continuesto the ‘‘torpedo’’ stage at which elongation of the shoot,and especially the root, takes place, and vascular connec-tions between the two are established. This coordinateddevelopment results, after some time, in the formation of awell-organized somatic embryo or plantlet. Careful controlof medium composition and subculture regimes is requiredin many cases to obtain synchronized development of thesomatic embryos.


THE PRACTICE OF MICROPROPAGATION

Technical Aspects of Micropropagation

The Explant. To undertake plant micropropagation,appropriate cells, tissues or organs, explants, must betaken from source plants and successfully cultured.Explant quality is primary to the success of any micro-propagation scheme. Choosing particular cells, tissues ororgans as explants is a function of the plant species andcultivar, micropropagation goals, tissue-culture methods,and the specific condition of the source plants or explants.Research and development of a viable micropropagationscheme will often involve extensive experimentationwith explant source, type, size, and treatment. Routinelyused explants range from barely visible true meristems,excised laboriously under a dissecting microscope to largermeristematic clusters, shoot tips, leaf, stem, or flowersegments as well as seeds, pollen, or anthers (Fig 2).The chosen plant tissue or organ, once removed from thesource, must be handled carefully to avoid unnecessaryphysical damage, external contamination, or deteriorationbecause of extended time and/or poor storage conditionsprior to reaching the laboratory. Once in the laboratory,plant tissue should be stored appropriately and treated assoon as possible, usually with external cleaning, peeling,scrubbing, and rinsing as necessary, followed by surfacesterilization.

Asepsis (Axenic Culture). Conventional plant microprop-agation requires an aseptic environment and axenic cul-tures. This is ideally achieved with surface sterilizationof contamination or disease-free explants, followed byinitiation, establishment, and subculture under asepticconditions using presterilized culture media and growthvessels. Aseptic workspaces must be created, and bufferedand isolated from other areas of the laboratory; therebyestablishing progressively cleaner spaces involving morestringent behavior and operations as the central, asepticareas of the plant transfer room are approached. The rangeof techniques for achieving asepsis in plant micropropa-gation is large. All facilities, however, will require a planthat defines and separates distinct work areas accord-ing to function and degree of asepsis. Most commercialcompanies employ the following principles:

Clean Room Technology. According to The InternationalStandards Organization (ISO), a clean room is defined as‘‘a room in which the concentration of airborne particles iscontrolled, and which is constructed and used in a mannerto minimize the introduction, generation, and retentionof particles inside the room, and in which other relevantparameters, for example, temperature, humidity, and airpressure, are controlled as necessary’’ (39). In a commer-cial micropropagation laboratory, it is necessary to createworkspaces with minimal levels of particulate matter,including fungal and bacterial spores. Designated levelsof clean air are maintained by creating an out flowinggradient under positive pressure directed through filtersand discharged into the facility at central points, prevent-ing the passive entrance of more contaminated or ‘‘fresh’’

air and particulate matter. Plant material is stored andworked on in rooms often maintained at ISO standard8/Class 100,000. Manipulation of plant material is usuallycarried out in laminar flow workspaces at ISO standard5/Class 100 (40). Outward from the transfer room, spacesbecome less and less aseptic. Filters, airflow, and levelsof asepsis are examined regularly by certified technicianswho employ internationally accepted methods and equip-ment. Design, construction, equipment, disposables, andmethods utilizing the principles of clean room technologyhave become essential to commercial micropropagation.

Disinfection and Sterilization. Everything that comes incontact with plant material is a potential contaminant.Sterilization, disinfection, personal hygiene, and house-keeping protocols are integral factors in creating and main-taining an aseptic environment and a contamination-freeproduct. Commercial laboratories are equipped with auto-claves and/or bulk media sterilizers that are used for mostmedia, container, and tool sterilization. Heat labile chem-icals are filter sterilized and microwave ovens are oftenused for small quantities of medium. Additional large-scalesterilization may be carried out using gamma irradiationor ethylene oxide gas treatment. Glass bead sterilizers,electric incinerators, and Bunsen burners are employedto disinfect tools during plant manipulations in the hood;workers wear clean clothing, special in-house shoes orshoe coverings, and may cover their hair and beards orwear face masks and rubber gloves for work with plantmaterial. Chemical disinfectants are used throughout thefacility for cleaning all work and storage surfaces, floors,and equipment.

Surface Sterilization of Plant Material. Plant parts con-taining explants, that is, shoot tips, seeds, and stems,are first cleansed, often with combinations of detergents,disinfectants, agitation, and water rinses. The explant isthen excised followed by disinfection and additional rins-ing with sterile, distilled water. Disinfection treatmentsusually include household bleach and/or alcohol. Specificchemicals, concentrations, and duration of surface steril-ization procedures differ according to type, size, and originof plant material. If deemed necessary, and where heavyexternal, or endogenous contamination is suspected, addi-tional physical or chemical treatments including heat,extended agitation, or antibiotics may be applied.

Culture Media and Vessels. A sterile nutrient sourcein appropriate growth vessels must be provided to initi-ate and sustain in vitro plant growth. An appreciationand knowledge of the nutritional requirements of culturedcells and tissues is invaluable, not only in a decision on thetype of media to use, but also in preparation and storage(41). Since 1934, when P.R. White (42) demonstrated thecontinuous growth of tomato roots in vitro, there has beenconstant improvement in the understanding and effec-tiveness of the use of synthetic plant growth media (22).Synthetic growth medium is the primary source of nutri-tion for plants and plant tissues in vitro. Basal mediacomponents are distilled/deionized water; sugar(s) as aprimary carbon source; inorganic salts, providing macro-


and microelements; vitamins, some essential, others bene-ficial; and plant hormones or growth regulators. The mostwidely used, standard medium formulation is Murashigeand Skoog (MS) (43). The discovery, isolation, and synthe-sis of plant growth hormones (e.g. auxins, cytokinins, andgibberellins) have provided the ability to achieve hormonalcontrol of plant growth, development, and regeneration.The balance between types and concentrations of growthregulators in in vitro growth stages is a primary com-ponent of the regulation of dedifferentiation with callusproduction; differentiation leading to embryogenesis oradventitious shoot and/or root generation and multiplica-tion; elongation, and so on. Additional factors, includingcomplex, undefined materials, such as coconut milk, yeastextract, or protein hydrolysates, may be beneficial forcertain plants or growth stages.

A major challenge in large-scale commercial laborato-ries is uniformity among batches of media. Professionalexpertise in choosing and ordering media components aswell as strict inventory control are basic essentials. Pre-venting or minimizing human error in this detail orientedwork requires a quality assurance program including wellwritten and understood protocols for equipment mainte-nance and media production; checklists, and well orga-nized control procedures.

Commercially produced in vitro plants grow primarilyon semisolid media. Gelling of media solutions is usuallyachieved with agar, agar substitutes such as Gelrite, ormixtures of both (19). Liquid culture often uses bioreac-tors, which may hold from 1 to 2000 L of growth mediumand plant material. These micropropagation systems avoidgelling agents and the costs, and the uncertainties some-times associated with them (44).

Essential equipment for media preparation includean autoclave, pH meter, analytical and semianalyticalscales and measuring instruments and containers; heat-ing, stirring, and pouring equipment; refrigeration; awater distillation/deionization system; storage facilities;and a washing up area for tools and equipment. A large,commercial laboratory will employ industrial equipmentand methods to maintain and monitor inventories ofmedia components and media; produce and sterilize largebatches; and add media to, label, store, plant, and disposeof thousands of vessels daily.

The choice of glass and presterilized or autoclavableplastic growth vessels, and appropriate closures, mostdesigned or adapted for plant cell/tissue culture, is large.In vitro growth and development is influenced by lighttransference, relative humidity (RH), temperature, andgas exchange. The microenvironment, inside the culturevessel, may be influenced not only by the generationand absorption of gases by the plantlet and the culturemedium, but also by the gas exchange between the roomand the vessel (25,45). Limitations in production methodsstill require that most containers be small, usually hold-ing from 1 to 100 plantlets. Storage space for containersat various stages of use is a significant issue. Commercialmicropropagationists continue the search for and/or createvessels and systems, which will be readily available anddemonstrate low unit cost, uniformity, nonphytotoxicity,and ease in storage, sterilization, and handling.

As in other aspects of micropropagation, there is con-tinual research to fine tune and improve growth media,as well as lower costs. This is coupled with increasingrecognition of the risks and stresses of in vitro applicationof consistently high concentrations of minerals, salts, vita-mins, and hormones. This is in addition to potentially toxictrace chemicals in agar and other prepared or syntheticingredients. Consistent with work to create nonaxenic cul-tures and to reduce stress on in vitro plantlets, there areefforts to achieve satisfactory results with lower concen-trations of sugars, minerals, and hormones (46,47).

Environmental Conditions. In the commercial micro-propagation laboratory, environmental conditions andtheir control are critical elements. The expense of heating,cooling, humidifying/dehumidifying, and filtering the airand of illuminating the plant material has frequentlybeen a limiting factor in the cost basis of micropropagatedplants. Three sets of environmental conditions must bemonitored and considered. (i) The current season andenvironment, including temperature and humidity, inthe nonaseptic, ambient areas of the laboratory; (ii) Thecontrolled environment of the growing room, includingsanitation/asepsis, temperature, humidity, air flow andcirculation, light quality and intensity, and day length;and (iii) the microenvironment of the growth vesselincluding asepsis, temperature, humidity, gas exchange,light intensity and quality, nutrition, and potentialsources of stress, for example, salt concentrations,available water, and contamination. Here, we will addressstandard procedures and conditions (44,48–50).

Temperature. In vitro growth conditions for most plantsrequire ambient temperatures of 22–27◦C. Some species,growth stages or storage conditions may require differentor variable temperature regimens. Room temperature issomewhat lower than illuminated shelf temperature withthe gap depending on the level of air circulation, amountof thermal energy (heat) produced by the light sourcesabove the shelves, and the amount of insulation betweena light source and the shelf above. Growth room heatingand cooling, and maintenance of temperature stability,especially in extreme climates or weather, is a significantchallenge (25).

Relative Humidity. Microhumidity in the plant growthvessel, generally expected to reach 98%–100%, is the mostsignificant humidity factor. There is evidence that it maybe more effective to maintain RH in the vessel at 88%–94%(25,51) or less. This might be achieved by using less mediaper container, higher concentrations of gelling agents, gasexchange filters in growth vessels, and/or maintaining alower humidity in the growth room. Growth room ambienthumidity is usually 50%–80%. Room RH lower than 40%can result in growth medium desiccation, increased saltconcentration, and drying out of plant material. Room RHhigher than 85% may increase microbial contaminationand vitrification. In extreme climates or areas with signif-icant variations in weather conditions, a humidity controlsystem may be necessary.


Light. Conventional in vitro growth media providecultures with a carbon source, usually sucrose. Although,there is some photosynthetic activity in vitro, plantletsdo not rely on photosynthetic carbon fixation for growth.Traditionally, fluorescent lamps have been the primarylight source used in micropropagation. Photon densityis between 20 and 200 μmol/m2/s , with a standardphotoperiod of 12–16 h (25). Plant propagators have gonefrom Foot Candles and Lux to Micro Einsteins to measurelight intensity, and on to photosynthetically activeradiation (PAR) and photosynthetically usable radiation(PUR) (52,120) to define light quality and availability.In order to improve both the intensity and quality (wavelength) of light actually reaching plants, commercialmicropropagators are known to use supplemental light,that is, incandescent bulbs, high pressure sodium ormetal halide lamps in the laboratory, and acclimatizationfacility. In recent years, research, and subsequently,commercial interest in the use of light emitting diodes(LED) have emerged in micropropagation. It is possibleto fine tune and easily alter light spectra and intensityusing combinations of LEDs. For example, cool white,warm white, red and/or blue light LEDs may be used toeffectively illuminate in vitro plantlets on a single shelfthereby making it possible to optimize PAR in a massmicropropagation facility. Another positive characteristicof LED is the high energy conversion efficiency with lowthermal energy production (53,54).

Different species, varieties, micropropagation technolo-gies, and stages of growth often demand exceptionalenvironmental conditions. Commercial laboratories maybe equipped with multiple growing rooms and options foraltering standard parameters. Much research has dealtwith environmental factors in the growth and develop-ment of in vitro plants, and there is experimental datashowing that many of the heretofore ‘‘standard’’ methodsare far from optimal (25).

Stages and Major Operations of Micropropagation

Plant micropropagation is an integrated process in whichcells, tissues, or organs of selected plants are isolated,surface-sterilized, and incubated in a growth-promoting,sterile environment to produce many clonal propagulesor plantlets. At least five critical and ordered opera-tional stages are involved, three of them in vitro: (0)selection and preparation of the explant source; (I) estab-lishment of viable explants in culture; (II) rapid regen-eration of numerous propagules; (III) establishment ofcomplete rooted plantlets or other propagation units invitro; and (IV) acclimatization and establishment ex vitro(19,23,55). None of these stages stands alone. The pro-duction of healthy, true-to-type plants is dependent onmaximizing quality and results within each stage; at theinterfaces of adjacent stages; and over the entire process.The object of the process may be virus-free flowers, massproduction of mutation-free, high value plantation crops ora metabolite with beneficial pharmaceutical or nutritionalproperties.

Stage 0: Explant Source and Mother Plants. The successof micropropagation is largely dependent on the quality

of the source plant. Despite this, one of the most commonproblems encountered in tissue culture success has beenthe failure to pay attention to the condition of the plantfrom which the explant was taken, the stock plant (motherplant, or donor plant) (19,56). Effective selection and main-tenance of source plants should provide assurance that theplant has the following characteristics:

1. It is a certified, horticultural true-to-type represen-tative of the desired species and cultivar.

2. It is free from disease and endogenous contamina-tion; or may become pathogen-free using specifictechniques, including in vitro culture.

3. It is viable and vigorous, that is, potentially able torespond to culture conditions that induce intensivecell division, regeneration, and generally, aberrantphysiological and morphological responses whencompared to standard growing conditions.

Stock plants may be maintained in air-locked, environ-mentally controlled glass houses; screen houses or tunnels;or in well marked field plots or forests. Ideally, thesesources of identified, characterized, plant material wouldbe grown in more than one physical and/or geographiclocation to protect them from catastrophic events rangingfrom human error to earthquakes, massive contaminationor acts of war. To assure enough explants of sufficient qual-ity for commercial propagation, a source of mother/stockplants must be identified or cultivated and continuallymaintained, protected, and renewed. ‘‘Preconditioning’’ bya variety of growth regimens and horticultural proceduresmay be beneficial, for example, regulation of nutritionand irrigation; optimization of day length, light qualityand intensity, and temperature; treatments with heatand/or growth regulators; pruning; and pest control. Theseapproaches, in some ways, all mimic those practices longin use by propagators of nursery materials by cuttings (25).Maintenance and conditioning of the stock plant shouldtake into consideration the demands of the in vitro sys-tem and any further downstream processes the plant orplant material might undergo. Although, much researchremains to be done, it is understood that nutrition, light,and temperature regimes as well as overall source planthealth may significantly affect the ability of the plant tomultiply and/or root in vitro and subsequently, acclimatizeex vitro (56,57).

Stage I: Explant Establishment in Culture. Following exci-sion from the source plant, and surface sterilization, theinitial explant may range in size from 0.1 mm (e.g. meris-tems used for establishing virus-free plants) to about 1 cm(e.g. bulb scales and stems). During this stage, lastingfrom 1 week to 1–3 months, or even longer, the explantis established in culture, resulting in tissue activationand multiplication. This stage is usually carried out onagar-based media, but liquid media may be employed. Thechoice of basal media and growth regulators at this stageis of critical importance, and will vary according to plantand tissue type and to the desired multiplication method.With continual visual monitoring, this stage serves as the


baseline for screening for microbial contamination andhorticultural fitness. Specific indexing and treatment ofmicrobial and viral contamination may also be under-taken. The relatively few primary explants, and their size,render this the most cost-effective and efficient stage forevaluation, treatment, and selection of plant material.Explants and growth options vary widely (i.e. seedlings;meristem growth and subsequent multiplication; stemsor leaves to callus generating shoots; or embryogenesis).Therefore, treatment must be tailored to the particulargrowth regimen.

Stage II: Rapid Multiplication. Primary explants thathave successfully passed through Stage I are transferredaseptically to Stage II for generation of numerous clonalpropagules. Masses of tissues are repeatedly manipulatedby subculturing onto new culture media that encouragepropagule proliferation. The types of regeneration andproliferation are largely dependent on growth regulatorcombinations. A high proportion of cytokinins usuallystimulates continued multiplication of axillary or adven-titious shoots, and a higher proportion of specific auxinsis required for callus proliferation with the generationof somatic embryogenesis. The combined and balancedadjustment of growth regulators, basal media composition,and environmental conditions are optimized to achievemaximal proliferation of high quality, new, plant propag-ules. Although, basic media formulations tend to remainconstant, extensive experimentation may be necessaryto reach commercially efficient multiplication with spe-cific cultivars or varieties. The duration of this stageis potentially unlimited, but usually lasts from severalmonths to 1 to 2 years. At the chosen endpoint, usu-ally after a delimited number of subcultures, the stockculture is renewed to prevent possible accumulation ofmutations and the loss of vigor and regeneration poten-tial related to endogenous contamination or the effects oflong-term treatment with plant growth regulators. Appli-cations developed with the advent of the twenty-firstcentury are allowing micropropagationists to carry outmore objective genetic and microbiological screening ofin vitro plants, and therefore, to make decisions basedon science as opposed to conjecture. Conventional micro-propagation has been carried out on semisolid agar-basedgrowth media, primarily for the production of plantlets.The wide variety of Stage II options available, that is,adventitious and axillary shoots and somatic embryogen-esis on agar-based media; liquid culture for production ofbiomass or secondary compounds and so on require pro-duction strategies and protocols suited for each plant andtechnology.

Stage III: Plantlet Establishment, Elongation, and Rooting.After repeated subcultures and screening for microbialcontamination the resulting plantlets are transferred tothe final in vitro stage. Stage III is designed to arrestrapid multiplication and to induce the establishment ofa fully developed plantlet, for example, shoot elonga-tion, root formation and when required, formation ofstorage organs that serve as independent propagationunits (bulbs, corms, and tubers) (25). This stage may

also provide the conditions for stimulation of photosyn-thesis, stomatal function, and other physiological changesthat are required to establish autotrophic growth in theex vitro acclimatization stage. This is achieved by culturemedia and environmental modifications such as reductionof cytokinin concentrations or their total elimination, theaddition of or increase in auxins and/or reduced sugarlevels, and often, increased light intensity. Undoubtedly,recalcitrance, or general lack of success in rooting in vitroplants has been because of incomplete understanding ofthe processes, or the inability to apply existing knowl-edge. Root formation is split in precise stages: induction,cellular activation, orientation, organization, and rootingexpression, with the emergence of the first root being themost direct expression of the rooting capacity of shoots orcuttings (58). To improve efficiency of this stage and toreduce production costs, systems that shorten the processor allow much of it to take place ex vitro, or in a lessaseptic, in vitro environment, have been proposed (19,25).The goal is to bring new technologies and applications tomass propagation (59–61).

Stage IV: Acclimatization. in vitro culture submitsplants to aberrant and stressful environmental andnutritional conditions. Acclimation to these conditionsleads to formation of plantlets with morphology, anatomy,and physiology different from naturally grown plants,including reduced leaf wax deposits, nonfunctionalstomata, aberrant photosynthetic activity, reduced roothair development, and so on. After transfer from in vitroto ex vitro culture stage IV, acclimatization is needed tocorrect abnormalities and to ensure proper growth andsurvival of the plants. Effective control of transpiration,and adequate photosynthetic capacity have not yet beenestablished (52,62); therefore, a several week regimen in acontrolled environment is provided. Initially, plantlets areplaced under low light and temperatures and humidityclosely resembling in vitro conditions; an incubator for exvitro plants (55). As cuticular waxes, stomatal function,and new, functional roots develop, photosynthetic activityis increased and plants become autotrophic. Over thefollowing weeks, light intensity is gradually raised andambient temperature and humidity are regulated tothe greenhouse environment. Rooting hormones maybe used to additionally stimulate root developmentand antimicrobials may be applied to control diseaseemergence. Most laboratories and nurseries transplantinto a uniform medium that adequately supports theplant, has a suitable pH, is well buffered, is reproducible,and is sufficiently porous to allow adequate drainage andaeration (19). in vitro plants that are unable to survivethis stage must be considered unsuccessful, therebylosing the entire investment in the micropropagationprocess (25).

ADVANTAGES AND DISADVANTAGESOF MICROPROPAGATION

For many species, micropropagation offers significantadvantages in quality, quantity, and economics over


conventional vegetative propagation. However, inherentin micropropagation are disadvantages and significantlacunae in the knowledge of how plant tissue culturedoes and does not work, and why. These cannot beignored if the potential of plant biotechnologies is tobe achieved. The search for better methods and morefavorable outcomes in commercial micropropagationcontinues.

Advantages

Production of a Very Large Number of Clonal Propaguleswithin a Relatively Short Time Span Relative to the Same Plantusing Conventional Techniques. Depending on multiplica-tion rates, thousands and millions of in vitro plants can beproduced from relatively few selected source plants.

Production of Disease-Free Plant Material with thePossibility of Eliminating Viral, Bacterial, and FungalContamination. Primary reasons for employing microprop-agation and in vitro techniques, and their biotechnologicalbases, are that they provide important tools for creatingand working with pathogen-free explants and multi-plication material. Where elimination of contaminantsis not possible, methods for detecting the presence ofendogenous contamination in vitro allow for treating orminimizing the damage, or disposing of unfit cultures in atimely manner (63–66).

Production of a Large Stock of True-to-Type Clonal Prop-agation Material. A basic premise in commercial micro-propagation is the ability to control and guarantee aconsistently high degree of likeness between source plantcharacteristics and the final plant product, whether thisis size, shape, flower color, the presence and concentrationof metabolites, or other characteristics.

The Ability to Safely Ship Large Quantities of Plant Mate-rial Quickly, Efficiently, and Relatively Inexpensively. Tensof thousands of in vitro or several thousand acclima-tized plants can be packed into a cubic meter of airshipping space. This effectively eliminates or minimizesthe distance between research and development centers,micropropagation laboratories, weaning facilities, and/orfields where large-scale agriculture and horticulture cantake advantage of good weather and available and lessexpensive land, water, and labor.

The Possibility of Bringing New Technologies or NewlyBred Plants and Selections to Market, Rapidly and in LargeQuantities. Plant micropropagation techniques, integratedinto emerging technologies, frequently provide the vehiclefor bringing quicker and often more qualitative resolutionto questions in horticultural, agricultural, chemical, med-ical, and pharmaceutical sciences. Breeding and selectionof commercial plant products have been lengthy, oftenlifetime processes. Micropropagation and new modalitiesin molecular biology and the tracking and testing of newgenotypes or field selections may be achieved in months ora few years.

Disadvantages

In 1988, Pierik listed ‘‘Handicaps for Large-Scale Commer-cial Application of Micropropagation’’ (67). They includedthe following in vitro issues: frequent mutations; lackof basic knowledge of organogenesis or embryogenesis;exceptional difficulty with woody species; internal infec-tions; vitrification, and toxic exudates; increased ethyleneand CO2 levels; neglect of the role of physical growth fac-tors (light, temperature, humidity, and the gas phase);losses on transfer from in vitro to acclimatization; laborcosts and lack of realistic mechanization; many newlydeveloped techniques are not economically viable; com-mercial production is often insufficiently controlled.’’ Inthe twenty-first century, nearly 20 years later, commer-cial micropropagationists continue to deal with the sameor similar issues, although from a position of greaterunderstanding and respect for the complexities of com-mercialized plant tissue culture.

Contamination. Some of the greatest economic losses,direct and indirect, in commercial micropropagation arecaused by both endogenous and environmentally induced,contamination of plant cultures (66).

Higher Than Acceptable Levels of Somaclonal Variation.Plants that have been clonally propagated in vitro mayexhibit a wide array of commercially unacceptable geneticand epigenetic variation. This is termed somaclonal vari-ation(18). Very often, somaclonal variation is identifiedonly when the resulting mature plant is expected to floweror fruit. When masses of micropropagated plants do notperform as expected, both the specific project and com-mercial micropropagation fail. The industry has sufferedsignificantly as a result of undesirable plant variants andmany business ventures and projects have failed as aresult.

Losses Incurred During Transfer of Plant Material fromStage III to Stage IV, the Acclimatization Stage. The successand viability of a tissue-culture based commercial micro-propagation program is measured not just by multiplica-tion coefficient, somaclonal variation rates and in vitro‘‘vigor,’’ but by the ability of a plantlet to make the tran-sition from a heterotrophic/mixotrophic in vitro systemto a photoautotrophic state in the greenhouse and field.When in vitro plants in suboptimal condition leave the lab-oratory, or inadequate acclimatization methods are used,significant waste and economic failure often result. Ineffect, the whole system has failed. As discussed earlier,there are many ways in which plant tissue-culture meth-ods affect the quality of the in vitro plant; however, aconsensus is slowly growing that the poor recovery inacclimatization is a consequence of low photosyntheticactivity in vitro (52). This altered morphology and physiol-ogy is also responsible for the plantlet’s poor recovery aftertransfer to ex vitro conditions and poor establishment touncontrolled field conditions.


Plant Stress. In so far as tissue culture is an unnaturalprocess, the plant tissues are exposed following excisionand during culture to stresses and stress combinationsthat they may not have encountered in nature in theirlong evolution (50). Stress may cause some of the problemsdiscussed earlier (especially, somaclonal variations andlosses during the transfer from in vitro conditions to thefield). However, as discussed earlier, initial stress, includ-ing wounding, can trigger dedifferentiation, cell division,and regeneration.

High Production Costs. Expensive technology, facilitiesand overheads, and the labor-intensive nature ofmicropropagation, often render it economically unfeasibleand engender costs that are untenable for certain plants,varieties, products or markets.

ECONOMIC AND FINANCIAL ASPECTS OF COMMERCIALMICROPROPAGATION

The economic potential for in vitro propagation of plantswas recognized in the 1970s, when it became clear thatconventional propagation methods and equipment wereoften inadequate because of the increasing incidence ofplant disease and abiotic stress and the decreasing qualityof soil and water. It was soon realized that benefits couldbe gained from crop improvement and mass productionprograms utilizing in vitro plant technologies. However,the potential of rapid in vitro clonal propagation of a givenplant genotype does not necessarily mean that this tech-nique is also practical or economically feasible. Moreover,the number of plant species that can be regenerated intissue cultures in the laboratory far exceeds the numberof plants that are actually being micropropagated on acommercial scale. The practical application of microprop-agation relative to alternative methods of propagation isdependent on the following:

1. A very high propagation rate of true-to-type plants2. The current market value of the plant and its poten-

tial in horticulture or agriculture3. The cost-effectiveness of micropropagation relative

to alternative, conventional methods of multiplica-tion for the same plant

4. The level of plant quality that can be consistentlyoffered to the market

Before embarking on the commercial production ofa particular cultivar, the following question must beaddressed: Is there a practical micropropagation tech-nology for this species or cultivar, and is micropropagationexpected to provide solutions to the problems that havearisen in conventional propagation? The latter two issuesare manifested in the application of micropropagation tothe specific groups of plants (19,25,64). Generally, thepotential of micropropagation has been realized in plantsthat are normally vegetatively propagated, and where themarket price of the individual plant is relatively high.The defining characteristics of industrialized plant micro-propagation are size, self-sufficiency in a broad range

of activities and disciplines (e.g. production, plant nurs-ery maintenance, research and development, and salesand marketing), and engagement in vertically integratedagritechnology projects. A successful industrialized planttechnology company will have achieved expertise in mostaspects of developing and growing a particular product.When this is coupled with a business-like approach tomarketing, management, and customer service, there is agreater likelihood of economic success.

Financial Aspects of Commercial Micropropagation

Costs. Establishing an average size commercial micro-propagation facility, and the accompanying agricultural,research, marketing, and management systems necessaryto support it, may cost more than US $1 million. Size, loca-tion, and technologies will determine initial investment,but even the most primitive home or village-based labora-tory must be backed up by a source of quality stock plantmaterial, technical expertise, and appropriate researchand development. In 40 years of commercial microprop-agation, lessons have been learned and improvementshave been made in determining feasibility and control-ling costs. However, the limited financial success of manycompanies of all sizes has led to a constant search forless expensive technology and labor. In the more devel-oped world, labor remains the major economic obstacle,and may represent up to 85% of costs (68). In developingcountries, electricity is often the greatest limiting eco-nomic factor (47). The solution most frequently seen is themove of mass production facilities to developing nationswith relatively inexpensive and available labor, land, andinfrastructure. This often necessitates relocation of techni-cal and managerial staff and usually means that researchand development facilities, and source plant material andinitiation will be carried out at other sites, often thousandsof miles away. Alternatively, more automated systems(see below) may be introduced. The qualitative advan-tages of plant biotechnologies have been well defined, buttissue-cultured products must also demonstrate a quantifi-able advantage over traditionally produced plant material.The agricultural community is slow to accept change, anddemands significant justification for the cost, time, andeffort involved. Reducing production costs and thereforeprices, has become a critical challenge for commercialmicropropagation companies (69).

Quality. Micropropagation has become an establishedtechnology and a complex business venture commandinghigh prices. In recent years, the quality issues that arecommon in traditional manufacturing have also arisenin commercial plant technology. Farmers and nurserymanagers are unwilling to accept the inconsistencies,which plagued micropropagation in its earlier develop-ment stages. Agricultural companies and more sophis-ticated growers demand products, education, and ser-vices in-line with the quality standards that are beingemployed worldwide (25,29,40,70). Economic survival willbe awarded only to those micropropagation companiesand laboratories that are able and willing to comply. Asingle flaw in the system seldom results in economic fail-ure; it is usually an accumulation of some or all of the


above advantages and disadvantages that determine theultimate success or failure of a project or a company.

World Production and Distribution

Plant tissue culture is carried out in most countries of theworld. Commercial foci are found in the United States,Europe, India, and in the Asian Pacific Rim (Japan,Taiwan, Thailand, and Australia/New Zealand). Addition-ally, commercial tissue culture is an important elementin plant biotechnology activities in South Africa, EasternEurope (where cheaper labor and overhead has createddirect competition against companies in central and West-ern Europe), and in Israel. China has significant researchin and production of micropropagated plant material,but it appears that most plant tissue culture is subsi-dized or state-run commercial production. Although, mosttissue-cultured plant material remains in its country orregion of production, 10%–20% of in vitro or acclimatizedplantlets are exported. The current trend is for in vitroresearch and development and initiation of plant mate-rial in more developed countries and facilities, followedby shipping to less developed, agriculturally based regionsof the world for downstream mass production, or moreoften for acclimatization and field production. Much ofthe European production is shipped in vitro to centersof cheaper labor and warmer climate for acclimatizationand planting out, and a large quantity of the finishedplants is subsequently shipped back to Europe for distri-bution and sale (71–74). The high turnover in the businessmakes it difficult to amass figures that are reliable in realtime, but it has been estimated that in the first decadeof the twenty-first century, from 500 million to 1 billionmicroplants will be annually produced with an expectedannual growth rate of 15% (67) Much of the ‘‘low tech’’development in commercial micropropagation is directedtoward the establishment and survival of the industry indeveloping areas of the world. How to construct and man-age commercial laboratories and acclimatization facilities;acquire equipment, disposables, and chemicals in a timelymanner; adaptation of available materials and continuingcompliance with increasing economic and quality demandsis a significant challenge to that endeavor and has beenthe subject of several papers (47,75,76).

Major Crops in Commercial Micropropagation

Within the last four decades, plant micropropagation hasdeveloped from a laboratory curiosity to a real industry(62) and commercial production of in vitro plantlets andplant products continues to progress and expand world-wide. The annual volume of plants micropropagated fromtissue culture is estimated at hundreds of millions, repre-senting tens of thousands of varieties (77).

Highlighted here are some crops that have been amongthe primary production models for commercial microprop-agation methods and systems; and problem definition andsolving techniques. Research and practical experience withthese species contributed to international standards forthe detection of plant disease and the establishment ofgermplasm banks for maintenance, breeding and protec-tion of selected, disease-free varieties. Improved, increased

and more economically viable production methods haveseen disease reduced or eliminated, yields increased anduniformity of weight, color, variegation, ripening or otherdesirable characteristics, enhanced. Producing large quan-tities of disease-free, uniform planting material on sched-ule and year round, has been a key factor in large-scaleinternational marketing of both planting material andthe final product be it flowers, food, or pharmaceuticals.The first decade of the twenty-first century is seeing alarge-scale migration of both micropropagation technologyand production capacity to Asia. The next goal is to bringtissue-cultured plant material to the small farmer and thedomestic market (74,75).

Flowers and Ornamental Species. With great horticul-tural and commercial value, standard cut flower species,ornamental house plants, and foliage crops have beenat the forefront of commercial micropropagation. Tissueculture production of ornamental plants in general andorchids in particular, forms the basis for an entire horti-cultural industry (78).

Orchids always appreciated and in demand, were oncevery rare in the world flower market. With the advent oftechnologies that allowed for in vitro seed germination andmicropropagation, the industry grew first in the UnitedStates and Europe and then Southeast Asia. Orchidsrepresent the first floricultural crop successfully masspropagated through tissue culture. Currently, orchids aremicropropagated around the world in vast numbers, asflowers and potted plants with a market value of over US$2 billion. Orchids are currently the second most valu-able potted crop in the United States, which is the largestimporter, with a 2005 value of US $144 million (79).Thailand is the world’s largest micropropagated orchidproducer with production in 2005 of 44.6 million tonsof flowers and potted plants, an increase of 33% over1999 figures. Taiwan, India, and China are all increasingproduction and technology and investing heavily in theproduction and export industry. Orchids continue to be thesubject of research, and interest in their quantity, quality,and novelty shows no signs of abating (23–25,80–82).

Plantation Crops. These crops, for example, pineapple,banana, and sugarcane remain a source of food and energythe world over. To maintain health and yield and to com-ply with international regulatory requirements for export,they require large quantities of clean, true-to-type plantingmaterial on a regular basis. These factors have driven pro-ducers to seek biotechnological solutions and worldwide,growers have succeeded in dealing with and/or solvingproblems and increasing quality, yield, and efficiency withmicropropagated planting material (25).

Bananas. Banana and plantain (a type of banana) arethe most important agricultural products in the tropics.They are the main fruit in international trade and interms of volume they are the first in exported fruit. Over100 million metric tons of fruits are produced annuallyand according to the Food and Agriculture Organizationof the United Nations (FAO) Statistics estimations, world


total exports of banana accounted for 16.8 million tons in2006, with a value of $5 billion per year (83,84).

The international economic importance of this cropmade it a prime candidate for the new technologies andmethods of the 1970s. By the 1990s, tens of millionsof micropropagated banana plants had been provided tocommercial plantations in America and Africa. Initially,most of these came from Europe and Israel. At that time,Asia became a client and then a producer of microprop-agated plants. See examples of sophisticated commercialoperations: (i) in India established with Israeli exper-tise www.JAINS..com/PDF/crop/high-tech%20banana%20book.pdf (85) and (ii) in the Philippines http://hvcc.da.gov.ph/pdf/Bananatech.pdf (86). In the 1970s America pro-duced 50% of the world’s bananas while Asia produced34%. The market share for Asian banana production beganto increase in the course of the 1990s and the 2000s tofinally reach 58% in 2007. The share of the African con-tinent in the world production has remained relativelyunchanged from the 1970s (13%) to the 2000s (11%) (87).

Bananas are also a very important staple commod-ity for many developing countries, hence the relevanceof bananas for food security. Major banana producingcountries such as India or Brazil are hardly involvedin international trade. Locally consumed fruit are majorstaple foods in sub-Saharan Africa and Latin America.

Diseases are major constraints in the production of thisimportant crop. They affect every organ of the plant andare caused by fungi, bacteria, viruses, and nematodes.They reduce yield; affect the appearance, shelf life, mar-ketability of harvested fruit and debilitate, and sometimeskill the host plant. The new and ongoing outbreak ofPanama disease or Fusarium wilt, caused by the fungusFusarium oxysporum f. sp. cubense race 4, and attack-ing the major commercial varieties of dessert bananas(84,88), has highlighted the importance of providing largequantities of disease-free banana plantlets to be grownon newly cultivated plantations in conjunction with inte-grated disease management and prevention and researchprograms.

Sugarcane. Of primary interest is sugarcane, a tradi-tional crop that provides more than 60% of the world’ssugar as well as a number of by-products. Sugarcaneplays a major role in the economy of sugarcane grow-ing areas and, hence, improving sugarcane productionwill greatly help in economic prosperity of the farmersand other stakeholders associated with sugarcane cultiva-tion. Micropropagated sugarcane plants have been used inBrazil (89) and Indonesia for almost three decades (per-sonal observation, Indonesian Sugar Research Institute,and Pasuruan Indonesia). Rising demand for both sugarand ethanol are creating competition for available landand are hampered by disease and below average yieldsin much of the world. Currently, about half of Brazil’ssugarcane crop, 514,079,729 tons in 2007, has gone intobioethanol production with the rest being refined intosugar (90).

Vegetable Crops. Traditional field and vegetable cropshave been successfully grown and improved for genera-tions using conventional seed technology. Biotechnology

has made significant contributions to the improvementof the three major food crops rice, wheat, and maize.However, protocols for commercial mass propagation suit-able for most relevant cultivars have not been perfected.In the face of rising population and poor nutrition inunderdeveloped nations, research continues to find solu-tions for these and other important food crops. A crop forwhich micropropagation has provided solutions is givenbelow.

Potato. (Solanum tuberosum L.). Potato serves as aneffective model for a variety of micropropagation meth-ods and technologies. It ranks fourth in terms of totalworld food production (63) with more than 325 milliontons produced in 2007. In 2005, for the first time, pro-duction of potatoes in the developing world exceeded thatin the developed world. Potatoes are cultivated as a val-ued crop in over 100 countries and about a third of allpotatoes are harvested in China and India with Chinathe largest producer worldwide (91). Potato accounts fora majority of micropropagation of vegetable crops in theUnited States, the fourth largest producer in 2007, withmore than 10 million propagation units produced annu-ally (16,91). When traditionally propagated many tubersbecome infected while still in the field and when replanted,pass the infection to the following generations (64). A majorinnovation for the potato industry in developed coun-tries was the widespread adoption in the 1970s of tissueculture—or micropropagation—as a means of multiply-ing disease-free plants that can then be used to producehealthy seed tubers for farmers. Micropropagation or prop-agation in vitro offers a low-cost solution to the problem ofpathogens in seed potato. Plantlets can be multiplied bycutting them into single-node pieces and cultivating thecuttings. The plantlets can either be induced to producesmall tubers directly within containers or transplanted tothe field, where they grow and yield low-cost, disease-freetuber ‘‘seed.’’ This technique is routinely used commer-cially in a number of developing and transition countries.In Vietnam, introducing improved, high yielding potatocultivars able to resist late-blight disease has seen yieldsdouble, from 10 to 20 tons per hectare. The farmers arethemselves multiplying their plantlets through microprop-agation, making the seed more affordable. For example,in Vietnam micropropagation directly managed by farm-ers contributed to the doubling of potato yields in a fewyears. Sufficient quantities of certified seed potatoes area limiting factor in increasing production and yields ina number of developing nations including Pakistan andIraq (25,77,92–96).

Micropropagation is now a ‘‘mature’’ plant biotechnol-ogy and is among the most widely used plant biotech-nologies, reportedly being applied in 21 less developedcountries in Africa, 10 in Asia, 9 in Eastern Europe, 9 inLatin America, and 8 in the Near East (97) (Table 1).

Micropropagation has become an irreplaceable toolfor many clonally propagated species for the productionof pathogen-free plantlets (among such clonally prop-agated crops are 10 of the 30 most cultivated cropsworldwide). Emphasis on the development of cost-effective


Table 1. Some Examples of Developing Countries usingPlant Propagation Techniques for Different Crops at theCommercial Level

Crop or Species Countries

MicropropagationPotato Bangladesh, Islamic

Republic of Iran, Jordan,Nepal, Syrian ArabRepublic, and Uganda

Sweet potato UgandaPlantain, cassava GabonBanana Bangladesh, Cameroon,

Gabon, Islamic Republic ofIran, Kenya, Myanmar,Uganda

Date palm Islamic Republic of Iran,Kuwait, Morocco, Tunisia,and United Arab Emirates

Jackfruit BangladeshPineapple Bangladesh, MyanmarCitrus, almond, Prunus

rootstocks, grape, olive,and pistachio

Tunisia

Anther cultureBread wheat MoroccoDurum wheat TunisiaFruits, cereals, and

ornamentalsBrazil

Embryo rescueBanana, citrus, papaya,

fruits, cereals, andornamentals

Brazil

Germplasm conservationCassava, violet BrazilOther propagation

techniquesKakrol Bangladesh

micropropagation techniques is expected to increase evenfurther (97).

ADDITIONAL ISSUES AND TRENDS IN IMPROVINGTHE ECONOMIC EFFICIENCY OF MICROPROPAGATION

Additional Issues

Micropropagation, whether research or commercial,encounters several critical issues which must be dealtwith constantly. While each is a separate issue andoften a specific problem, the issues are often integrated,for example, recalcitrance or trueness-to-type may becontrolled by undetected contamination.

Recalcitrance. Recalcitrance in plant tissue culturerefers to those plant types of specific varieties or genotypeswhich, when produced in vitro, continually fail to growor develop into healthy acclimated plants. Contraryto laboratory experience, routine and commerciallyviable micropropagation methods have traditionallybeen less successful with many different types of plants.Recalcitrant plants remain the subjects of research

and development, or are abandoned for more amenablecultivars or species and do not rise to the level ofcommercial production. The causes for recalcitranceappear to be many and varied, and endogenous con-tamination, often undetected, seems to be a majorone (65). Additionally, the commercial value of manyornamental plants is due to visual characteristics suchas variegation and coloration, which often result fromchimeras. Micropropagation may cause disorganization ofchimeras, especially sectorial and mericlinal (98), and itis often impossible to produce a sufficient quantity of hor-ticulturally attractive chimeric plants. In both researchand marketing terms, recalcitrance is the worst case andthe opportunity to reach a solution or a product remainselusive.

Somatic Variation and Trueness-to-Type. Trueness-to-type refers to the degree of genetic and/or epigeneticlikeness that micropropagated plants bear to the sourceplant from which the initial explants were taken.However, variations in micropropagated plants canoccur, under certain conditions, because of genetic and/orepigenetic changes, thus interfering with the reliability ofmicropropagation. When these changes occur in explantsthat are composed of somatic cells they are termedsomaclonal variation, but under specific conditionschanges can occur also in gametes (pollen and ovules)that serve as explants—termed gametoclonal variation(29,99). Somaclonal variation has been classified intothat which arises from preexisting variation, in theexplants from the donor plant; and variation whichmay be induced by the tissue-culture environment(50,99–103). Therefore, variation may be seen as theresult of poor or uninformed source plant selection orincomplete understanding or monitoring of productionmethods and plant material during micropropagation, orboth. Some plants exhibit relatively frequent variation(e.g. mutations, and chromosomal aberrations) in vivoand these plants also tend to exhibit higher levels ofsomaclonal variation in vitro. This characteristic isoften amplified by in vitro exposure to quantities andproportions of certain plant growth regulators and othermedium components, and/or length of time in culture,and may contribute to an overall increase in the rate ofsomaclonal variation (24,99,101).

However, these changes should, and can, to a largeextent be avoided. There are several accepted guidelineswhich can potentially reduce the hazards of somaclonalvariation, including a reduced propagation efficiency (rateof propagule production), shorter culture periods, avoidingmedia components that at high concentration are knownto cause variations (e.g. 2,4-D), and others. Institutingquality assurance protocols and monitoring proceduresare necessary components to a well integrated productionsystem.

There are many lines of research attempting toidentify sources of genetic instability as well as tocreate predictive and diagnostic tools which will aid inidentification and prevention. Identification of normallystable traits in a species or cultivar becomes a basictool in identifying mutation or genetic drift resulting


from micropropagation. An important area of currentresearch is DNA methylation, thought to be a basis formuch of the epigenetic (reproducible, reversible, andheritable) variation seen in micropropagation. There isparticular interest in variation in transgenic plants. Astransgenic technologies become more common in bothresearch-based and large-scale commercial micropropa-gation, the fine-tuning of existing diagnostic tools, thatis, polymerase chain reaction (PCR)–based, restrictionfragment length polymorphism (RFLP), and randomlyamplified polymorphic DNA (RAPD) will be required.The emerging field, ‘Micropropagation-Omics’—includesgenomics, metabolomics, and proteomics which are new,powerful molecular tools that may revolutionize plantbiology (104). Applying these emerging technologies maybring new understanding of the phenomenon and allowfor early detection of variability (64), which is highlydesirable (102,103,105).

Trueness-to-type is the most important issue for boththe micropropagation laboratory and the client. What-ever their source, commercially nonviable plant variantsremain a major technical and economic limitation in con-ventional mass micropropagation. A plant which is nottrue-to-type is not a salable product.

Contamination. Contamination-free culture schemesand disease-free planting material are primary goals ofmicropropagation. Endogenous and exogenous contam-ination of cultures from virus, bacterial, bacteria-like,fungal, and insect pests cause the most damaging andconsistent economic losses and logistical problems in themicropropagation process. When present, contaminationcan destroy the culture or significantly weaken orchange the micropropagated plants. Contaminatedcultures must usually be destroyed and the source ofthe contamination is often impossible to discern oreliminate. In 20 years, since the first meetings dedicatedto issues of contamination and quality in plant tissuecultures (63,64,66) there have been tremendous leapsin the research and application of novel technologiesfor detecting, characterizing, diagnosing, treating,minimizing, and preventing a wide range of disease andcontamination in vitro. The plant biotechnology industryis going to great efforts to understand, minimize, andtreat the sources and consequences of contaminationin and associated with micropropagated plant material(65). Improvements may be characterized by a betterunderstanding of the need for prevention at everystep in the production process. Better awareness andeducation has led to the widespread implementation ofquality standards and measurements throughout theprocess. Research continues to optimize new methodswith regard to sensitivity, specificity, and time-to-result.One solution to this problem, that may be applicable incertain cases, is in vitro production of semiautotrophicplants under not fully axenic conditions (106). Despiteprogress, endogenous contamination remains a signif-icant challenge to the economic vitality of commercialmicropropagation.

Trends in Improving the Efficiency of Micropropagation

As discussed earlier, there are some major inherent limita-tions to commercial efficiency of micropropagation. Severalconcepts that have been suggested for reducing productioncosts and the market price of individual microplants arementioned below. While not all of them have been real-ized commercially as yet, they may prove important in thefuture.

Large-Scale Production and Automation. As discussedearlier, widespread use of micropropagation for majorcrops in agriculture and forestry is still restricted becauseof relatively high production costs. Therefore, much efforthas been devoted to developing automated, robotized, andmore efficient transplant production methods. The dynam-ics of scaling-up must be harnessed to stringently engi-neered inventory control and quality assurance programs.Expanded inventories with short shelf-life are especially,difficult to manage in export-based companies. Addition-ally, because most crops are not planted or harvested on ayear-round basis, there are peaks in the production sched-ule. Thus, commercially viable, year-round employmentof staff and facilities and maintenance of large stocks ofstart-up cultures are necessary. Developing low cost, auto-mated mass-propagation systems for producing in vitroplantlets will become more and more important in thetwenty-first century (107,108). The following issues seemmost relevant:

Liquid Culture: Despite its many advantages,(47,68,109) the use of liquid culture systems is stilla subject of considerable research and for manyspecies, liquid culture is neither economically feasi-ble nor technically possible. Therefore, commercialapplications have been rather limited (47,68). Thereare several physiological limitations to propagationof many plant species in immersed culture. Amongthese are hyperhydration (vitrification), (33,110),deformation, and somaclonal variation becauseof sometimes uncontrolled multiplication (111).Although, these conditions may appear also inagar-based, semisolid, micropropagation schemes,the relatively small number of plants usually min-imizes the damage. In the event of contamination,the entire liquid culture system may be destroyed ordamaged and the technical problems and economiclosses are potentially severe.

Plantlet Complexity: Systems using semiautomated,automated, robotized, or computer-aided excisiontools and procedures for subculturing are hamperedby the complex and unique nature of the plantmaterial. Tools or systems are often appropriateonly for one plant type or culture protocol and theytend to be less successful for others; and for manycommercial laboratories the combination of thesetwo issues makes the systems undesirable. Efficientrobotization is limited to situations where return oninvestment can be assured (25).

Costs: In addition to the basic costs of establishingand operating a plant micropropagation laboratory,


the high costs associated with the research,development, and purchase of automated systemslimit their use. Established companies usuallyconsider it as an option for very high volumeproducts.

Practical Applications: Many pitfalls and unsolvedproblems remain in the scale-up and full automationof plant micropropagation. As a result, many com-mercial laboratories have successfully implementedpartial or semiautomation, for example, develop-ment of a prototype for the automated manipulationof growth containers with in vitro plants. Thisrobotic system carries out the sterile exchangeof liquid medium at an accelerated rate withminimal worker participation. There are processingsystems where media components are measured,mixed, processed, and poured into containers, usingcomputer programming, minimal human support,and environmental control systems. On the basis ofneed and experience, each laboratory creates manyoriginal, in-house solutions to the problems of time,space, and costs (112–115) .

Production of ‘‘Artificial (Synthetic) Seeds’’: Coatedor Encapsulated Somatic Embryos. The possibility forlarge-scale formation of somatic embryos has set the stagefor developing the concept and practice of ‘‘artificial seeds,’’also referred to as synthetic seeds (116). Commercialmicropropagation has proved itself useful when true seedseither are unavailable or inadequate, and productionof clonal artificial seeds may become an alternative totraditional seeds, offering a unique combination of traitsto the market (26,116). Since somatic embryos are func-tionally similar to regular embryos that are contained,partially or fully dormant, within the seed coats, aprocedure has been developed to handle them as seeds.This procedure involves large-scale production of somaticembryos, synchronization of somatic embryo developmentand encapsulation in a seed coat-like ‘‘envelope’’ with orwithout simultaneous partial dehydration of the embryos(117,118). This is followed by storage under appropriateconditions that permit further development of the plantletat a later stage. Synthetic seed production was aimedinitially to provide sufficient survival and germinationrates, but for commercial mass production these goalshave yet to be reached.

Additional Developments and Trends. Several otherdevelopments, the commercial efficiency of which is stillpremature to judge, include the following:

1. Use of disposable bioreactors

2. Explant culture on membrane rafts with novel liquidculture systems

3. Use of only partially aseptic cultures

4. Use of semiautotrophic cultures

ADDITIONAL IMPLICATIONS OF MICROPROPAGATION

In addition to the accepted use of in vitro culture and plantregeneration for commercial rapid clonal propagation, thistechnique is very important for several other purposes, asdiscussed earlier in this article (119) and in other articlesof this encyclopedia.

1. Production and maintenance of pathogen-free stockplants

2. Production of therapeutic and cosmetic metabolites3. Long-term in vitro conservation of germplasm4. Selection and generation of transgenic plants5. Tagging and patenting transgenic plants and pro-

cesses

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