Plant Cell, Tissue and Organ Culture 51: 49–56, 1997. 49c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Environmental control for the large-scale production of plants through invitro techniques

Toyoki Kozai1, Chieri Kubota1 & Byoung Ryoung Jeong2

1Department of Bioproduction Science, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, JAPAN;2Department of Horticulture, College of Agriculture, Gyeongsang National University, Chinju, KOREA 660–701

Received 24 July 1996; accepted in revised form 8 March 1997

Key words: CO2 enrichment, gas exchange, in vitro environment, light, photoautotrophicmicropropagation, relativehumidity, temperature

Abstract

Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to havehigh photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather thanon sugar-containing medium, provided that the environmental factors, such as CO2 concentration, light intensityand relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets invitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitroplantlets.

Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants havebeen developed. Advantages of sugar-free over conventional (heterotrophicor photomixotrophic)micropropagationsystems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets invitro have less physiological and morphological disorders, biological contamination in vitro is less, plantletshave a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be usedbecause of less biological contamination. Hence, production costs could be reduced and plant quality could beimproved significantly with photoautotrophic micropropagation. Methods for the measurement and control of invitro environments and the beneficial effects of environmental control on photosynthetic growth, development, andmorphogenesis in large-scale production of micropropagated plantlets are presented.

Abbreviations: Cin – CO2 concentration in the culture vessel during photoperiod; DIF – difference betweenphotoperiod and dark period temperatures; E – number of air exchanges per hour; Ei – environment in vitro;Ee – environment ex vitro; LED – light emitting diode; RH – relative humidity; NPR – net photosynthetic rate;PPFD - photosynthetic photon flux density

Introduction

Micropropagation, dealing with the propagation ofplantlets in vitro, has many advantages over con-ventional vegetative propagation, and its applicationin horticulture, agriculture and forestry is currentlyexpanding worldwide. However, commercial use ofmicropropagation is still limited because of its rela-tively high production costs resulting mainly from highlabor costs, low growth rate in vitro and poor survivalrate of the plantlet during acclimatization.

The goal of micropropagation is to mass-producegenetically identical, physiologically uniform, devel-opmentally normal, and pathogen-free plantlets whichcan be acclimatized in a reduced time period and at alower cost. Development of both automated environ-mental control systems and improved in vitro culturesystems are essential for a significant reduction in pro-duction costs (Aitken-Christie et al., 1995).

Recently, extensive research and development havefocused on automation and robotization of microprop-agation processes (Aitken-Christie et al., 1995). How-

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ever, research on the effect and control of environ-mental factors in vitro has been limited, partly due tothe fact that conventional culture vessels are small andairtight, making the control and measurement of theenvironmental factors difficult. In this article, envi-ronmental factors and their effects on the growth anddevelopment of the plantlets/shoots/explants (termed‘plantlets’ hereafter) in vitro are discussed.

Reasons for environment control

A culture vessel may be thought of as a miniaturegreenhouse or growth chamber and the explant cul-tured in vitro as a miniature vegetative cutting (Read,1990). However, the physical environment in vitroin conventional tissue culture systems is quite dif-ferent from that in a greenhouse and often results inundesirable physiological and pathological problems(Debergh and Maene, 1984).

The conventional in vitro environment is character-ized by Aitken-Christie et al. (1995) as having the fol-lowings: high relative humidity (RH), constant temper-ature, low photosynthetic photon flux density (PPFD),large diurnal fluctuation in CO2 concentration, the highconcentration of sugar, salts and growth-regulatingsubstances in the medium, the accumulation of toxicsubstances, and the absence of microorganisms. Theseconditions often cause low rates of transpiration, pho-tosynthesis, water and nutrient uptake and CO2 uptakebut a high rate of dark respiration, all of which resultsin poor growth. It has been reported that a controlledmicroenvironment promotes plant growth and devel-opment, reduces morphological and physiological dis-orders and encourages more rapid and vigorous plantgrowth and development during acclimatization stage(Jeong et al., 1995). This is also expected to reduceproduction costs significantly.

Environmental factors affecting the growth anddevelopment of plantlets in vitro

The important aerial and root zone environmental fac-tors in culture vessels are summarized in Table 1. Theuse of vessels and caps (i.e., containers) in microprop-agation isolates the environment in vitro (Ei) from theenvironment ex vitro (Ee). However, the Ei is directlyand indirectly influenced by the Ee. Since the aerialenvironment in vitro is in more direct contact with theEe (i.e., through air exchange), it is more directly under

the influence of the Ee than the root zone environmentin vitro.

The root zone environment in vitro is indirectlyaffected by the Ee in most cases. The degree of influ-ence of the Ee on the Ei depends largely on the gasexchange between the two environments. By control-ling the Ee, one can directly and indirectly control theEi. However, changes in the Ee may not immediatelyhave a significant effect on the Ei depending on thedegree of the interaction because of the buffer posedby the container. The environmental factors describedbelow have an influence on the growth and develop-ment of the plantlets throughout the culture period.Therefore, the levels of those factors at the beginning aswell as during the culture period, should be controlledto maximize output. Most of the physical factors canbe maintained at a desired set point from the beginningto the end of culture, while most of the chemical fac-tors are either increasing or decreasing steadily overtime with growth of the plantlets unless a continuoussupply system (i.e., a bioreactor) is employed.

Effect of light, temperature, RH and supportingmaterial on the growth and development of theplantlet in vitro

Light

Spectral distribution of light from different lightsources varies significantly (Bickford and Dunn,1978). To control plant photomorphogenesis, differ-ent types of light emitting diodes (LED) can be used toemit either blue, red or far-red light at a low cost. Theapplication of LED for growing plantlets would be apractical alternative to conventional lighting systems(Bula et al., 1991; Miyashita et al., 1995).

White fluorescent lamps have been the primarylight source used in micropropagation, since their spec-trum generally matches the requirements of in vitrocultures and they give a relatively uniform horizon-tal distribution of PPFD over the entire culture shelf. Itshould be noted that there is a large difference in PPFDbetween the inside and outside of a culture vessel. Thedistribution of PPFD in the culture vessel is largelydependent upon the vessel and closure types (Aitken-Christie et al., 1995; Solarova et al., 1996) and thespatial arrangement of the vessels (Aitken-Christie etal., 1995). The light source is usually installed abovethe vessels and the plantlets receive downward illumi-nation. As the plantlet grows, an increasing amount

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Table 1. In vitro aerial and root zone environmental factors.

Aerial environment

(1) Temperature: Low, high, mean, DIF (difference in photoperiod

and dark period temperatures) and fluctuation over time

(2) Light: Spectral distribution, length of photoperiod and

dark period, photosynthetic photon flux density and lighting direction

(3) Thermal radiation and re-radiation

(4) Gaseous composition: CO2, O2, ethylene, humidity and other gases

(5) Air movement: Air flow pattern, air flow speed

Root zone environment

(1) Physical environment: Temperature, water (matric and osmotic) potentials,

gas and liquid diffusivity, shear stress (i.e., in a bioreactor), hardiness or compactness,

supporting materials and root zone volume

(2) Chemical environment

A. Mineral nutrition: Concentration, total availability and depletion rate,

relative ratio between ions and solubility of the ions

B. Organic matter composition and supply: Sugar, phytohormones, osmoticum, gelling agents,

vitamins and other additives

C. pH

D. Dissolved O2 and other gases

E. Ion diffusivity and depletion zone

F. Exudates: Phenolics and H+ and other ions

(3) Biological environment: Competitors and contaminants, symbiotic

microorganisms and exudates from the cultures (cell components and enzymes)

of light energy is intercepted by the upper parts of theplantlet and only a small amount of energy reaches thelower parts. Potato plantlets with reduced shoot lengthand increased leaf area were produced using sidewardrather than downward lighting (Aitken-Christie et al.,1995). To direct light from the sides, optical fibers orother thin or tiny light sources can be used. With side-ward lighting, the plant will receive a larger amount oflight energy uniformly throughout all plant parts evenwith less electrical energy for lighting.

In addition to lighting configuration, the photope-riod also influences plantlet growth. Morini et al.(1990) tested the effect of different light/dark cyclesand found that the growth of peach shoots was signif-icantly greater with a 4-hour light/2-hour dark cyclecompared with the conventional 16-hour light/8-hourdark cycle when the same amount of total radiationwas supplied.

Temperature

In a culture room, the set point temperature of the airis, in most cases, unchanged throughout the day, but

the temperature distribution is somewhat uneven in theroom and over time. The temperature inside a culturevessel is approximately 1�C higher than that outsidethe vessel during the photoperiod under conventionalculture conditions.

Kozai et al. (1992) described the effect of differ-ences between photoperiod and dark period tempera-tures (DIF) and PPFD levels on the morphogenesis andgrowth of potato plantlets in vitro under CO2 enrichedconditions (1300-1500 µmol mol�1). With the samedaily average temperature of 20�C, the air temperaturesduring the photo-/dark periods were set at 25�C/15�C(+10 DIF), 20�C/20�C (0 DIF) and 15�C/25�C (-10DIF). PPFD levels were at 74 (low) and 147 µmol m�2

s�1 (high). The shoot length was greater with increas-ing DIF under both low and high PPFD. The dry andfresh weights of plantlets were similar between DIFtreatments. DIF was shown to be an efficient way ofcontrolling plantlet height in vitro with minimum heat-ing and cooling costs.

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Temperature, light intensity and photoperiodinteraction

The level of PPFD, photo-/dark periods, DIF andtheir interactions significantly influenced the growthand development of potato and mint plantlets in vitro(Kozai et al., 1995). A PPFD level of 140 µmol m�2

s�1 produced shorter plantlets than that of 70 µmolm�2 s�1 under a 16-hour photoperiod per day. Witha 8-hour photoperiod per day, the plantlets had lessheight differences between the two PPFD level treat-ments. Negative DIF produced shorter plantlets thanthe zero and positive DIF treatments.

Low temperature storage under dim light forproduction management

Growth and quality of micropropagated transplantssometimes need to be hold for a certain period oftime, especially when labor for transplanting or cul-ture/greenhouse space is not available. Low tempera-ture storage is extensively used for harvested vegeta-bles and fruits and also some vegetatively propagat-ed plant materials. During storage of plantlets, pho-tosynthetic and regrowth abilities would have to bemaintained, but growth suppressed. Kubota and Kozai(1995) reported that broccoli plantlets were success-fully stored for 6 weeks at 5 �C under 2 µmol m�2

s�1 PPFD. Plantlets stored in darkness experiencedreduced dry weight and lost their ability to regrow.They also found that the plantlets kept at the PPFDlevel of the light compensation points maintained theirinitial dry weight and retained their photosynthetic andregrowth abilities. A PPFD level above the light com-pensation points caused a deterioration in quality dueto enhanced stem elongation and increased dry weightduring storage (Kubota et al., 1995). It should be notedthat during storage a small difference in PPFD (i.e.,2 vs. 5 µmol m�2 s�1) was attributed to significantdifferences in dry weight, height and overall quality ofthe plantlets after storage.

Relative humidity in the vessel

The exchange of water in gas and liquid phasesbetween the plant, air and medium in the vessel aswell as the characteristics of the air outside the vesselplay important roles in plant growth and development.The direction and rate of water flow are determined bythe spatial distribution of water potentials inside andoutside the vessel.

The RH is normally high in the culture vessel,which can lead to abnormal leaf development, shoothyperhydricity and suppressed transpiration (Ziv et al.,1983; Schloupf et al., 1995). There is a high depen-dence of vessel RH on the number of air changes perhour between the culture vessel and the RH in the cul-ture room (Aitken-Christie et al., 1995). Kozai et al.(1993) cultured potato plantlets for 22 days in vitrounder different RH conditions and observed increasedshoot length with increased RH. The specific leaf area(m2 gDW�1) decreased with decreased RH, whilethere was no significant difference in plant dry weightamong the different RH treatments.

Air currents in the culture vessel

Air currents in the culture vessel are slow and thus thediffusion coefficient of air in the culture vessel is con-sidered to be small. The small diffusion coefficient forCO2 and water vapor tend to limit the photosynthesisand transpiration of the plantlets.

Air currents in the vessels was visualized using fineparticles of metaldehyde (Kitaya et al., 1995). Air cur-rent speeds in the vessel ranged between 0 and 25 mms�1. The air current speeds increased by increasing theshort wave radiation flux from 0 to 30 W m�2. Theyalso increased with the presence of activated charcoalin the agar medium. The charcoal increased the shortwave radiation absorbed on the surface of agar medi-um, resulting in a higher surface temperature of themedium with activated charcoal. The air current pat-terns were also affected by the air current speed aroundthe vessel, and by the shapes and sizes of the vesselsand plantlets in vitro.

Supporting material

Gelling agents such as agar and Gelrite are commonlyused as supporting materials in plant tissue culture. Onthe other hand, fibrous materials such as polyethylenefoam, rockwool, ceramic wool and cellulose plugs,in some cases, enhanced growth compared to gellingagents (Kirdmanee et al., 1995; Roche et al., 1996).Kirdmanee et al. (1995) showed that vermiculite (arti-ficial soil consisting of a number of hydrous silicates)gave better growth of Eucalyptus plantlets in vitro thanagar, Gelrite and liquid media. It is considered that thehigher porosity (volumetric percent of air), higher gasdiffusivity, and thus higher oxygen concentrations inthe medium contributed to the enhanced growth of theplantlets in vitro.

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Photoautotrophic growth and development ofplantlets in vitro

Traditionally, explants and regenerated shoots in cul-ture have been considered to have poor photosynthet-ic ability. Assuming that plantlets require sugar inthe culture medium as an energy source, they havebeen cultured under predominantly heterotrophic con-ditions (on an artificially supplied carbon source suchas sucrose) or photomixotrophicconditions (on an arti-ficially supplied and photosynthetically produced car-bon source).

However, it has been revealed that chlorophyl-lous plantlets had remarkable photosynthetic abilityand grew better, in some cases, under photoautotroph-ic conditions (without an artificially supplied carbonsource) than hetero- or photomixo-trophic conditionswhen the physical and chemical environments in theculture were properly controlled for photosynthesis(Aitken-Christie et al., 1995). Plantlets regeneratedfrom embryos or adventitious buds at a heterotrophicor photomixotrophic phase are expected to be smooth-ly converted into a photoautotrophic phase, if theyare provided with a microenvironment for photosyn-thesis (Seko and Nishimura, 1996). The environmentfor enhanced photoautotrophic growth is also expect-ed to be beneficial for the uptake of mineral elementsby plantlets because of the increased transpirationalactivities.

Gas exchange characteristics of the culture vessel

The type of vessel closure affects the gaseous com-position as well as the light environment, and henceaffects the hyperhydricity and growth of plantlets inculture. The air exchange characteristics of the ves-sel are best expressed by the number of air exchanges(infiltration) per hour (E). The E value, defined as thehourly air exchange rate of the vessel divided by theair volume of the vessel, is a physical property of thevessel and is basically constant. The number of nat-ural air exchanges for a flat bottom glass test tube(air volume: 45 ml) closed with an aluminium foilcap, plastic formed cap or silicon foam rubber plugwere 0.18, 1.5 and 0.6 per hour, respectively (Aitken-Christie et al., 1995). The E value can be increasedby three to six times by using a gas-permeable micro-porous polypropylene film as part of the vessel clo-sure. The gas concentration in the vessel containing aplantlet and medium varies with the gas concentrationoutside the vessel, the E value, and the gas production

and absorption characteristics of the plant and medium(Aitken-Christie et al., 1995).

The loose fitting closures were reported to be bet-ter than the tight ones for reducing hyperhydricity inGypsophila paniculata (Dillen and Buysens,1989) andcarnation (Hakkaart and Versluijs, 1983). They werealso found to promote the growth of carnation andpotato plantlets (Solarova et al., 1996).

Photosynthetic response of plantlets in vitro

The CO2 concentration in the culture vessel duringthe photoperiod (Cin) has been shown to be low (Fuji-wara et al., 1987; Infante et al., 1989; Solarova, 1989;Desjardins et al., 1990; Debergh et al., 1992). TheCin in airtight vessels containing ornamental plantletsdecreased to 70 to 80 µmol mol�1 (ppm) two to threehours after the onset of the photoperiod (Fujiwara et al.,1987; Infante et al., 1989). It was also found that Cin

was as low as the CO2 compensation point of C3 plantsand was about 250 µmol mol�1 lower than normalatmospheric CO2 concentration (ca. 340 µmol mol�1).

The experimental results obtained by Fujiwara et al.(1987) and Infante et al. (1989) suggest several things.A chlorophyllous plantlet has photosynthetic ability,since Cin decreases sharply after the onset of the pho-toperiod in conventional air tight vessels. InsufficientCO2 supply into the vessel limits photosynthesis duringmost of the photoperiod. An elevated PPFD level willnot increase the net photosynthetic rate (NPR) undersuch low CO2 conditions. Plantlets are forced to devel-op heterotrophy or photomixotrophy. Plantlets devel-op photoautotrophy and can grow well under photoau-totrophic conditions with high levels of CO2 and PPFDrather than under hetero- or photomixo-trophic condi-tions. The initial growth rate is greater for an explantwith a large area of chlorophyllous tissue (Miyashitaet al., 1996).

Photosynthetic responses of in vitro Cymbidiumplantlets in situ were similar to those of plants grown inthe shade in the greenhouse (Kozai et al., 1990). Whenthe CO2 concentration was fixed at 200 µmol mol�1,the NPR in Primula malacoides plantlets in vitro in 1and 10% O2 was approximately 3 and 1.5 times higher,respectively, than that in 21% O2. This resulted from areduced rate of photorespiration (Shimada et al., 1988).

The NPR of in vitro rose plantlets (Capellades,1989) and potato (Nakayama et al., 1991) plantletsincreased when cultured on medium with a lowersucrose concentration. The leaf starch concentrationincreased when the plantlet was cultured on medium

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with an elevated sucrose concentration. The increasedleaf starch concentration was associated with a low-er NPR (Capellades, 1989). Sugar in the medium hasbeen reported to reduce the rubisco activity, and thusthe NPR of in vitro plantlets (Desjardins, 1995; Aitken-Christie et al., 1995).

The NPR was also affected by the RH (Capel-lades, 1989). The NPR in strawberry was higher whenplantlets were cultured in a vessel with forced venti-lation than with natural ventilation (Nakayama et al.,1991). Under natural ventilation, the boundary layerscaused by stagnant air may restrict CO2 diffusion intothe stomata. The NPR of the plantlets and seedlings invitro under saturated PPFD, 340 µmol mol�1 CO2 anda leaf temperature of 20 �C were similar, regardless ofthe fact that NPR affected by CO2 concentration wasslightly different (Pospisilova et al., 1987). A simu-lation predicting the effects of in vitro environmentalfactors on the CO2 concentration inside the vessel,NPR, and plantlet growth was developed by Niu et al.(1996).

CO2 enrichment under high PPFD

Carbon dioxide enrichment under high PPFD (100-200µmol m�2 s�1) was effective in promoting growth ofpotato (Kozai et al., 1988), tobacco (Mousseau, 1986)and Eucalyptus (Kirdmanee et al., 1995) plantlets cul-tured on medium with and/or without sugar. Kozai andIwanami (1988) observed enhanced growth of carna-tion plantlets under a CO2 concentration level of 1000-1500 µmol mol�1 and a PPFD level of 150 µmol m�2

s�1 regardless of the addition of sugar in the medium.Based upon these findings, it can be expected that

NPR will increase and hence growth and developmentof plantlets in vitro, if the CO2 concentration in thevessel is raised during the photoperiod. There are a fewpractical ways of elevating vessel CO2 concentration.

1) Use of CO2 permeable film in the closure

Several reports have indicated positive effects of usinga gas-permeable film as the closure under high PPFDon the NPR and the growth rate of plantlets in vitro(Aitken-Christie et al., 1995; Solarova et al., 1996).Plantlets of some species derived from leafy single-node cuttings grew faster when cultured photoau-totrophically in a vessel closed with a gas-permeablefilm than when cultured heterotrophically in a relative-ly airtight vessel (Aitken-Christie et al., 1995; Cournacet al., 1991). Under high PPFD, even this type of pas-

sive CO2 enrichment will significantly enhance plant-let growth in vitro. The percentage of hyperhydric-ity decreases with the use of a gas-permeable film,which probably resulted from a decreased RH and anincreased gas exchange and dehydration of the medi-um.

2) CO2 enrichment in the culture room

CO2 enrichment under high PPFD (100–200 µmolm�2 s�1) was effective in promoting the growth ofchlorophyllous plantlets regardless of the medium sug-ar concentration. A practical and formulated methodof enriching culture rooms with CO2 is described byAitken-Christie et al. (1995).

3) A large culture vessel with a CO2 supply system

Dry weight and the NPR of strawberry plantlets cul-tured on sugar-free liquid medium increased when cul-tured in a large vessel with a forced ventilation sys-tem under a PPFD of 96 µmol m�2 s�1, compared tothose of plants cultured using a conventional method(Fujiwara et al., 1988). However, forced ventilationwith atmospheric air or a N2-O2-CO2 mixture reducedpropagule weight and shoot number in stage 2 Rhodo-dendron cultured in a 400 ml vessel under a PPFD of39 µmol m�2 s�1 (Walker et al., 1988). This contradic-tion may be attributed to the lower light level used inRhododendron than in strawberry and/or growth stage,which can be an internal factor affecting photosynthe-sis and growth of plantlets, as described by Desjardins(1995).

In these systems, not only CO2 concentration butalso RH, ethylene concentration, and gas diffusionin the vessel are modified. Therefore, the observedchanges in growth of plantlets in vitro resulting fromthe use of these systems cannot be attributed solely toCO2 enrichment. However, in most cases, the changeswere probably caused primarily by the CO2 enrich-ment.

Advantages of photoautotrophic micropropagation

Some disadvantages and problems associated withheterotrophic and photomixotrophicmicropropagationare summarized as follow: (1) Addition of sugar as acarbon source in the medium increases the incidenceof biological contamination, and airtight, small ves-sels are commonly used to reduce this contamina-

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Table 2. Some advantages of photoautotrophic micropropagation.

1. Promoted growth and development of plantlets resulting from improved environmental conditions for normal growth and development.

2. Minimal use of growth regulators and other organic matter.

3. A larger vessel with environmental control and monitoring systems can be used with decreased incidence of biological contaminations.

4. Reduced loss of plantlets from biological contaminations and simplified procedures for rooting and acclimatization.

5. Reduced physiological, morphological and genetic disorders and improved plantlet quality.

6. Easier manipulation of plantlet growth and development by means of environmental control.

7. Automation, robotization and computerization practical and achievable.

8. Stable production cycles and lowered production costs.

tion. Therefore, automation, robotization and com-puterization of a micropropagation system would beimpractical and difficult; (2) The air inside the vesselis nearly saturated with water vapor and vessel CO2

and ethylene concentrations become abnormal. Thus,a high PPFD becomes ineffective in promoting plant-let growth; (3) Growth regulators are often necessaryfor plant regeneration; (4) Undesirable environmen-tal conditions induce physiological and morphologicaldisorders, growth retardation, and mutation; (5) Ulti-mately, an unstable production cycles, non-uniformplantlet growth and a high plantlet death rate duringthe acclimatization stage increase production costs.

Photoautotrophic micropropagation has advan-tages over the conventional micropropagation method(Table 2) and its potential benefits, whether automatedor not, seem to be great.

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