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Review Environ. Control in Biol., 36 (2), 59-75, 1998

Environmental Effects on the Growth of Plantlets in

Micropropagation

Qunyh Thi NGUYENand Toyoki KozAI*

Laboratory of Plant Cell Technology, Institute of Tropical Biology, Ho Chi Minh City, Vietnam
* Laboratory of Environmental Control Engineering , Faculty of Horticulture, Chiba University, Matsudo,
Chiba 271-8510; Japan

(Received January 13, 1998)

Research on environmental effects on the growth of plantlets in micropropagation is

reviewed to give an overview on the subject conducted in the past decade, mainly to
environmental biologists and engineers. Effects of aerial environmental factors including
light, carbon dioxide concentration, temperature, relative humidity, oxygen and ethylene on
the growth and photosynthesis of plantlets in vitro are discussed. Then, effects of culture
medium environmental factors including sugar concentration, concentrations of ion compo-
nents, osmotic potential and pH of the culture medium on the growth of plantlets in vitro
are discussed. Lastly, effects of biological factors such as explant size and explant density
on the growth of plantlets in vitro are discussed. Effects of aerial, culture medium and
biological factors on the photoautotrophic (totally photosynthesis dependent) growth of
plantlets are emphasized.

INTRODUCTION

Micropropagation is a plant tissue culture technique used for obtaining a large number of
genetically identical plantlets. Research on micropropagation has been focused mainly on
the bio-chemical effects of medium composition (sugar, mineral salts, phytohormones, vita-
mins, amino acids, etc.) on the differentiation, rooting and growth of explants under aseptic
conditions. As a result, research on the effects of physical environments on the growth of
plantlets in micropropagation is far behind that of plants in greenhouses and fields.
Recent studies, however, have revealed that physical environmental factors considerably
affect the growth and development of plantlets in micropropagation. In the past decade, a
significant number of original papers have been published on the environmental effects on the
growth of micropropagated plantlets. Those papers suggest that environmental control in
micropropagation will become an essential technique to obtain good-quality plantlets on a
desired date with low production costs. However, very few review papers on related issues
have been published recently. This article gives an overview of research on the subject
conducted in the past decade, mainly for environmental biologists and engineers who are not
so familiar with the subject.

AERIAL ENVIRONMENT

1. Light
1.1 Light flux density. Photosynthetic photon flux density (PPFD), or, more simply,

Vol.36, No.2 (1998) (1)59

 photosynthetic photon flux (PPF) at wavelengths between 400 and 700 nm, is one of the most
important factors involving the photosynthesis of chlorophyllous cultures, especially shoots
and plantlets (Fujiwara and Kozai, 1995). A high PPF has a beneficial effect on promoting
photosynthesis and growth, provided that CO2 concentration in the culture vessel is not as low
as the CO2 compensation point of the cultures (Kozai et al., 1990).
High PPF accompanied with CO2 enrichment has been shown to promote photoautotro-
phic growth of Solanum tuberosum plantlets and simultaneously induce specific adaptations
of photosynthetic response potential, while some other cultivars did not respond favorably
(Cournac et al., 1992). A higher PPF results in a greater dry weight of the plantlet associated
with a greater difference between CO2 concentrations inside and outside the culture vessel, due
to a higher net photosynthetic rate (a higher CO2 uptake rate) of plantlets (Kozai and
Sekimoto, 1988).
The PPF and percent PPF transmission in culture vessels are affected by the type and light
energy output of light sources, the material and shape of the vessel, the position of the vessel
on the culture shelf, the position of the light sources with or without reflectors, the optical
characteristics (reflectivity, etc.) of the culture shelf and the type of closures, etc. (Fujiwara et
al., 1989). The PPF in culture vessels with a closure, even with a high light transmissivity,
was significantly lower than that on the empty culture shelf (Fujiwara and Kozai, 1995). The
remaining question is how to develop an appropriate lighting system that can provide a high
and relatively even distribution of PPF at a reduced electricity cost.
1.2 Spectral distribution. The spectral distribution of light received by cultures is known
to be primarily determined by both the spectral characteristics of the light source and the
culture vessel material (Fujiwara and Kozai, 1995). The spectral distribution of light from
different light sources significantly differ from each other. Fluorescent lamps, which produce
much less far-red radiation than high-pressure sodium lamps, have been the primary light
source used in micropropagation. The spectrum generally matches the requirements of
cultures (Kozai, 1991b). Line light sources such as fluorescent lamps generally give a relative-
ly uniform horizontal distribution of PPF on the culture shelf when they are placed parallelly
on the shelf at a height of 40-50 cm above the cultures.
Photon flux ratios of red to far-red regions and of blue to red regions are considered as
important factors for photomorphogenesis of plants. Red and far-red light applied as part of
the culture regimen led to better rooting of azalea microcuttings than those cultured under
conventional cool-white fluorescent lamps (Read and Economou, 1982). Fukuda and
Kurata (1992) proved that red light had the effects of promoting shoot formation and growth,
and inhibiting root induction of carrot embryos, though these adventitious shoots were ill
shaped.
Dooley (1991) reported that the spectral light transmissivities of culture vessel materials
differed from each other. Thus, both the spectral distribution of the light source and the
spectral light transmissivities of culture vessel materials should be considered when designing
a lighting system for a culture room. More attention should be paid to the selection of light
sources and culture vessels to provide light with the required wavelengths and photon flux for
photomorphogenesis and/or photosynthesis of cultures.
Light-emitting diodes (LED), especially red LED and far-red LED, proved to have
advantages over fluorescent lamps in regulating photomorphogenesis, growth and the develop-
ment of plantlets in micropropagation (Fujiwara and Kozai, 1995). Red and far-red were
proved to have a significant effect on potato shoot length when the ratio of red photon flux to
PPF is greater than 0.5 and far-red photon flux to PPF is less than 0.02 (Miyashita et al.,
1995). Kirdmanee (1995) found that the internode length of Eucalyptus camaldulensis was
about 1.5 times greater with supplemental far-red light than without. The increased internode

60(2) Environ. Control in Biol.

 length was correlated with the increased cell length of stem epidermis.
1.3 Lighting direction. In conventional tissue cultures, the light source is placed 30-40
cm above the vessels, giving downward light to the plantlets in the vessels below. This
downward lighting system does not always provide the best performance regarding the growth
and quality of the cultures and electricity costs for lighting and cooling the culture room
(Fujiwara and Kozai, 1995).
In a sideward lighting system, the vessels are arranged in line and the light source is placed
between the lines just beside the vessels, giving light to the plantlets mainly sideward through
vessel side walls. The sideward lighting system helps to promote the growth of plantlets in
vitro and to control plantlet height at reduced costs. Light intensity in vessels of this system
was about five times higher than that of the downward system, when the number of fluorescent
tubes used in the sideward lighting system was the same as that used in the downward lighting
system (Kozai et al., 1992b ; Hayashi et al., 1994). Leaves of in vitro plantlets received the
light energy more evenly and uniformly in the sideward than in the downward lighting system.
This results in relatively larger leaves at the bottom and smaller leaves at the top (Hayashi et
al., 1993). Plantlets in the sideward lighting system had relatively short internodes and
slightly greater numbers of leaves than in the conventional system. Moreover, the required
space can be minimized because the culture vessels can be stacked vertically (Kitaya et al.,
1995).
The required culture space can be further reduced by using a sideward lighting system
with both diffusive optical fiber belts and a point light source paired with a shape-designed
reflector in place of fluorescent lamps ( Kozai, 1991a ; Kozai et al., 1992b ; Kozai and Smith,
1995). This system helps to reduce the cooling load of the culture room and to obtain an
improved light spectral distribution without increasing air and leaf temperature when a
thermal filter is installed between the point light source and the cross-section of diffusive
optical fibers in order to remove thermal radiation. With this system, the thermal radiation
emitted from the point light source is eliminated by the thermal filter and only the photosyn-
thetically active radiation (PAR) is passed through the transparent optical fibers into the
culture room (the light source, reflector and thermal filter are all placed outside the culture
room). The plantlets can receive an even light distribution, and the cooling cost can be
reduced by about 75% because only the PAR is sent into the culture room.
Another modification of this system is a combination of diffusive optical fiber belts and
a microwave-powered lamp, which is a small (30 mm in diameter) lamp with high light output
(450 klumen, 130 lumen W-1)(Dolan et al., 1992; Kozai and Kitaya, 1993; Kozai et al.,
1995b).

2. Carbon dioxide concentration

Culture vessel closures have been traditionally designed mainly to prevent the entry of
contaminants, but consequently restrict the gas exchange between inside and outside vessels
(Desjardins, 1995). Due to the limited gas exchange, C02 concentration in the culture vessel
containing chlorophyllous tissues decreases sharply within 2 h after the beginning of the
photoperiod, then reaches a stable concentration, which is significantly lower than the stan-
dard atmospheric C02 concentration (350 j mol mol-1), but higher than the C02 compensa-
tion point (50-100 ,umol mol-1) until the end of photoperiod (Fujiwara et al., 1987; Kozai
and Sekimoto, 1988; Hayashi et al., 1993). Kozai et al. (1986) proved that the stable C02
concentration during the most of photoperiod decreased with passage of days close to the C02
compensation point, as chlorophyllous tissues in an air-tight vessel grew to plantlets.
Under such low C02 concentrations in the vessel, the net photosynthetic rate of plantlets
in the vessel is close to zero, resulting in a negative daily C02 balance of plantlets in the vessel.

Vol.36, No.2 (1998) (3)61

 This means that the integral of net photosynthetic rate over the photoperiod is smaller than
integral of dark respiration rate during the dark period.
The low CO2 concentration in the vessel, in turn, reveals the photosynthetic competence
of plantlets in vitro, which results in a considerable interest of photoautotrophic micro-
propagation (Kozai, 1991a, c, d). In photoautotrophic micropropagation, sugar is not added
to the medium, and the growth of plantlets depends solely on photosynthesis. Strictly
speaking, no organic substances including phytohormones, vitamins and amino acids, except
for minerals, are added to the medium in this new method.
Photoautotrophic micropropagation is generally associated with higher light intensity
and higher CO2 concentrations than in conventional micropropagation, in order to increase
photosynthesis of plantlets in vitro. The CO2 enrichment in photoautotrophic micropropaga-
tion for chlorophyllous plantlets has clearly proved effective in promoting the photosynthetic
ability and thus growth of in vitro shoots/plantlets (Kozai et al., 1987a, b ; Fujiwara et al.,
1995; Kirdmanee et al., 1995a, b, c ; Heo et al., 1996; Seko and Kozai, 1996).
The CO2 concentration inside the vessel has been increased by increasing the vessel
ventilation rate or by actively controlling the C02 concentration inside the vessel. The use of
microporous polypropylene filters with high gas permeability for increasing the vessel ventila-
tion rate is gaining popularity for passively maintaining a relatively high CO2 concentration
in the vessel (Kozai et al., 1988). Active control of CO2 concentration inside the vessel has
also been achieved by directly injecting the gas into the vessel or by using a large vessel with
a forced ventilation system for introducing CO2 enriched culture room air into the vessel
(Fujiwara et al., 1988; Kubota and Kozai, 1992).
Photoautotrophic micropropagation can have a beneficial effect only for chlorophyllous
cultures which show a positive CO2 balance during the photoperiod at a CO2 concentration of
350 jcmol mol-1 or higher (Jeong et al., 1993). A relative constant CO2 concentration in a
culture vessel can be observed only when the CO2 exchange rate between inside and outside of
the vessel is balanced with the CO2 exchange rate of the cultures in the vessel (Fujiwara and
Kozai, 1995).
Niu et al. (1996a, b), Niu and Kozai (1997) developed models for predicting the hourly
and daily net photosynthetic rate (Pa) and CO2 concentration inside the vessels for Cymbidium
and potato plantlets in vitro in order to determine optimal conditions to maximize the daily
P, of in vitro plantlets by using computer simulations.

3. Temperature and DIF

The air temperature in culture vessels is considered to be almost the same as that outside

of the vessel or that of the culture room during almost all the dark period (Fujiwara and

Kozai, 1995). In the photoperiod, however, the air temperature difference will be close to or

more than 1 when the irradiance or the net radiation flux density at the boundary of the

vessel was higher than 35 W m-2. Therefore, in order to maintain the temperature in the

vessels at a desired temperature, the air temperature in the culture room should be lowered by

about 1C (Fujiwara and Kozai, 1995).

Kozai et al. (1992c) described the effect of the difference in air temperatures (DIF=Tp-
Td) between the photoperiod (Tp) and the dark period (Td) on the stem or shoot elongation of
potato plantlets in vitro under enriched CO2 (1300-1500amol mol-1) and photoautotrophic
conditions. The shoot length was shorter with decreasing DIF at both low and high PPF (74
and 147 pmol m-2 s-1), though shorter in high PPF than in low PPF in each of three DIF
treatments (+ 10 DIF, 0 DIF, -10 DIF). However, the dry and fresh weights of plantlets
were similar among the three treatments. A similar result was obtained with Mentha rotun-
difolia plantlets in vitro (Jeong et al., 1996). The manipulation of DIF for controlling the

62(4) Environ. Control in Biol.

 shoot length and/or the plantlet height can be an important technique in micropropagation to
produce quality transplants for greenhouses or open fields.

4. Relative humidity
Relative humidity (RH) in the culture vessel is an important environmental factor that
affects the water relations of plantlets or organs in vitro. RH is generally higher than 95% in
an air-tight vessel containing plantlets (Fujiwara and Kozai, 1995).
The relation between (100-RH)% and water-vapor pressure deficit (WPD) is linear at
high RHs (e.g., RH>90%) and room temperatures (ca., 20). The WPD is defined as the
difference between the water vapor pressure of the air in question and the saturated water-
vapor pressure of the air (100%RH) at the same temperature.
The transpiration rate of shoots/plantlets is nearly proportional to WPD (Fujiwara and
Kozai, 1995) and to their total leaf area or surface area. Then, WPD and thus the transpira-
tion rate of plantlets for 98, 96 and 94%RH are, respectively, 2, 4 and 6 times greater than those
for 99%RH at room temperatures. Absolute values of WPD and transpiration rate at high
RHs are relatively small but their relative differences are significant. Moreover, the uptake
rates of water and some kinds of minerals such as Ca increase with increasing the transpiration
rate. Thus, a small difference in RH at high RHs causes a significant difference in uptake
rates of water and some kinds of ions.
Furthermore, high RH has been attributed to the poor development and disorders of the
physiological and morphological structures of plantlets resulting in the high mortality of
plantlets after being transferred into ex vitro conditions (Kozai et al., 1993). A reduced RH
(e.g., 95 to 90%) has been shown to improve plant growth, epicuticular wax deposition and
stomatal function. Again, a small difference in RH at high RHs causes a significant difference
in physiological and morphological characteristics of plantlets. By reducing RH in culture
vessels, vigorous potato plantlets with shorter shoot length and higher percentage of dry matter
have been obtained without any significant decrease in plantlet dry weight (Kozai et al.,
1993).
Tanaka et al. (1992) also showed that both RH in the vessel and transpiration rate of the
plantlets increased over time, when the in vitro cuttings grew into plantlets, due to the rapid
increase in leaf area. The rate of leaf area increase was greater than the rate of WPD increase.
It was then presumed that the abaxial leaf resistance to transpiration was greater as RH in the
vessels was lower. Thus, the plantlets growing under a condition of low RH might be able
to control excess water loss from their leaves and to withstand water stress when transplanted
even directly from in vitro conditions to greenhouses or open fields (Tanaka et al., 1992).
Therefore, a relative humidity of around 85-90% in the culture vessel at the rooting stage or the
last stage in vitro may help to produce qualified plantlets more able to withstand water stress
after being transplanted from in vitro to ex vitro conditions (Tanaka et al., 1992).
Another concern is that RH in culture vessels varies in space and time in accordance with
air temperature fluctuations, and is influenced by the gas exchange characteristics of the vessels
(Kozai et al., 1996b). Moreover, the lighting cycle affecting the air temperature inside the
vessels might have an effect on the diurnal change in RH and the daily average RH in the
vessels as well (Fujiwara and Kozai, 1995).

S. Oxygen
O
2 concentration has an important effect on the photorespiration of photosynthetic C3
plants. At normal atmospheric (21%) 02 concentration, C3 plants grown ex vitro photo-
autotrophically lose up to 50% of photosynthetically fixed carbon due to photorespiration.
However, this release of CO2 due to photorespiration is prohibited by low 02 concentrations.

Vol.36, No.2 (1998) (5)63

 The net photosynthetic rate (Ps) of Primula malacoides (C3 plant) plantlets in vitro at 1% O
2 (10 mmol mol-1) is about 3 times greater than 21% O2 and 1.5 times greater than 10% O2
when CO2 concentration was approximately 200 ,Lcmolmol-1 (Shimada et al., 1988). This
suggested that the Pn of in vitro C3 plantlets increased with decreasing O2 concentration.
The effect of long-term exposure to low O2 concentrations on the growth of Chrysanth-
enum morifolium (C3 plant), studied by Tanaka et al. (1991), demonstrated the fact that the
increase of the plantlet dry weight for 30 d was significantly greater in 10% O2than in 5,15 and
21% O2. It will become a practical application to control O2 concentration at about 10%, if
one can find a simple way to keep O2 concentration in a small and airtight vessel at low levels
for the entire culture period (Kozai et al., 1992a).
O2concentration in a culture vessel decreases in accordance with increasing CO2 concen-
tration in the vessels (Doi et al.,1989). In Caladium bicolor (C3 plant) plantlets, the decrease
in O2 concentration in the vessels was equal to the increase in CO2 concentration ; whereas, in
Dendrobium phalaenopsis (CAM plant) plantlets in vitro, the decrease in O2 concentration in
the vessels was remarkably larger than the increase in CO2 concentration. These results
clearly showed that the change in O2 concentration in the vessels depends on the type of carbon
fixation system (Fujiwara and Kozai, 1995).
It should be noted, however, that in conventional micropropagation the CO2 concentra-
tion in the vessel is generally in the range from 0.01 to 1-3% and thus the O2 concentration is
in the range between about 18 and 22%. That is, O2 concentration is never lowered to 10%
unless it is controlled artificially using O2 absorbants, for instance. On the other hand, the
change in O2 concentration from 21 to 18% does not affect the Pn of C3 plants.

6.C2H4 and gas diffusion coefficient

The gas C2H4 is known to cause numerous hormonal influences on plants. C2H4 concen-
tration in relatively air-tight culture vessels was reported to increase gradually with time up to
more than 2 pmol mol-1 from 21 to 60 d after the start of a new culture. C2H4 has been
implicated in the reduced growth of cultures of both Dahlia and Petunia (Gavinlertvanata et
al., 1979). The concentrations of C2H4 and toxic compounds (oxidation products of phenols)
tend to be high in some cases in air-tight vessels (De Proft et al., 1985). In contrast to CO2
and O2, C2H4 is only released, not absorbed, by cultures and, therefore, it should be released
to the outside of culture vessels once accumulated. A simple way to solve the problem is to
increase the number of air exchanges of culture vessels by using a gas permeable filter over a
hole on the cap or lid of the vessels, or by forced ventilation systems (Fujiwara and Kozai,
1995).
The diffusion of gas in culture vessels is also a considerably important process that helps
to facilitate : (1) the exchange of gases (including water vapor) between cultures and the air in
the headspace of the vessels, (2) the exchange of solutes (including dissolved gases) between
cultures and medium, and (3) the uniformization of the spatial distribution of gases in the
headspace and the solutes in the medium (Fujiwara and Kozai, 1995).
Under natural ventilation conditions, molecular diffusion might be dominant in the
headspace of culture vessels during most of the dark period. On the other hand, natural
convection is dominant during the photoperiod. Eddy diffusion becomes a dominant mode
of mass transfer in the headspace under forced ventilation conditions.
Kubota and Kozai (1992) suggested that forced ventilation increased the eddy diffusion
coefficient of gases and the air flow speed and, thus, enhanced the CO2 uptake of cultures
during the photoperiod to a greater level than when only CO2 concentration was increased.

64(6) Environ. Control in Biol.

 CULTURE MEDIUM ENVIRONMENT

1. Sugar concentration
Sugar, such as sucrose, fructose, glucose and sorbitol, is considered merely as a source of
carbohydrate in the culture medium for the heterotrophic growth of cultures. The actual
requirement for carbohydrate depends on the environmental conditions, particularly light
intensity and CO2 concentrations in the vessel (Williams, 1995). Sugar supply to the culture
medium has a negative effect on Pn which results in a feedback inhibition of rubisco, i.e., the
low activity of rubisco explains the low rates of Pn in vitro plantlets (Hdider and Desjardins,
1994).
Kozai (1991d) suggested sugar be reduced or eliminated from the culture medium by
increasing CO2 concentration in the vessels and maintaining cultures under a high light
intensity. The reduction or elimination of sugar will help to lower high production costs in
conventional micropropagation. This is due to the loss of plantlets in vitro by biological
contamination in the medium, the abnormal development of plantlets, the use of plant
growth-regulators, the difficult acclimatization of plantlets in vitro and the labor costs.
This conclusion arose out from experiments on Cymbidium (Kozai et al., 1987b), and
carnation (Kozai and Iwanami, 1988) which demonstrated that the plantlet dry weight
increase with time was greater in CO2-enriched than in CO2-nonenriched treatments regardless
of the sucrose concentrations. Nakayama et al. (1991) also proved in the potato culture that,
3 d after the start of culture, the Pn was 8-10 times greater in the sugar-free medium treatment
than that in the sugar-containing medium (30 g L-1 sucrose) treatment when CO2 concentra-
tion in the vessel was in the range of between 500 and 1 000 ,umol mol-1.
Moreover, in carnation cultures, the amount of sugar absorbed by the plantlets was only
about 2-8% in weight of the initial sucrose content (Kozai and Iwanami, 1988). The total
osmotic pressure caused by sugar should have increased with time as the hydrolysis of sucrose,
a disaccharide, to monosaccharides (glucose and fructose) proceeded in the medium, though
the total weight of sugar in the medium decreased by 10% (Kozai, 1991a). Fujiwara et al.
(1995) also demonstrated that the ratio of gross production by photosynthesis to dry weight
increase of potato plantlets in vitro was estimated to be four times greater than that of
saccharides uptake at both inflow-air CO2 concentrations for the culture box, 350 and 1 000
1Lcmolmol-1.

2. Ion components
Plant tissue cultures respond differently to the overall mineral supply. Some species
grow better on media with lower ionic strength as compared to MS medium (Murashige and
Skoog, 1962), whereas others grow better on MS medium (Williams, 1995). The optimum
mineral requirements can also vary between explant types and stages of culture growth. The
rate of uptake of an ion may exceed the rate of utilization creating a pool of available ions
within a plant.
Furthermore, the environmental conditions are proved to have different effects on the ion
uptake of cultures in vitro. The ion uptake pathway involves a number of steps and processes
beginning with the transport of ions. At each step of the pathway, the transport may be due
to the diffusion along a concentration gradient, a mass flow driven by the transpiration stream
or an active uptake selecting and depending on energy (Williams, 1992, 1995).
In vitro conditions may modify the relative activity of the normal transport mechanisms,
particularly where the mass flow is involved. It is likely that the mass flow would be limited
by a high relative humidity within a closed culture vessel (Williams, 1992). Kozai et al.

Vol.36, No.2 (1998) (7)65

 (1991) suggested that a reduction in relative humidity in the vessel enhances transpiration and
thus mineral uptake, and that the growth under photoautotrophic conditions is enhanced
under a low relative humidity (85-90%).
For the maximization of the culture growth, medium inorganic ion composition and
medium inorganic ion strength are critically important (Yang et al., 1995a) . Carnation
plantlets cultured in vitro grew better on a sugar-free medium with medium ionic composition
widely used for hydroponic culture than on a sugar-free medium with half strength MS or on
medium containing sugar with either half strength MS or the hydropronic medium ionic
composition (Kozai et al., 1988).
The MS medium is, in fact, optimized for heterotrophic growth and/or differentiation of
callus ; thus may not be optimal for photoautotrophic or photomixotrophic cultures . Kozai
et al. (1991) examined the importance of mineral nutrition of strawberry plantlets in vitro
under photoautotrophic and photomixotrophic conditions. A half strength of MS medium
was used as basal nutrient ion composition. The experimental results showed that PO43- was
absorbed rapidly (the percent residual of PO43- reached 3% on day 21) by the plantlets under
the photoautotrophic conditions with high CO2 concentration . It was suggested that the Pn
and, thus, photoautotrophic growth was further promoted if PO43- was available to the
plantlets at the later stage of growth. It was presumed that the initial amount of PO43- in the
medium should be higher for photoautotrophic or photomixotrophic than for heterotrophic
micropropagation. The percentage for all ions was the lowest under the photoautotrophic
condition on day 14 and 21, except SO42- on day 14, as compared with those under the
photomixotrophic or heterotrophic conditions (Kozai et al., 1991).
Nitrogen is in essential component for all plant growth. The absorption of NH4+ and
N03- is different according to species, and will influence the morphogenesis of plantlets in
vitro (Williams, 1995). Potassium is abundant in plant tissues and reflects that supply exceeds
demand by the plant in many cases. Therefore, it is not a problem in plant tissue culture ,
though some species (e.g., Manihot) are sensitive to high potassium concentrations (Williams ,
1995).
Sulfur exists in agar at a significant concentration, even though in MS medium sulfur may
be the second limiting mineral following phosphorus (Williams, 1991). Calcium plays an
important role in the control of cell wall synthesis and maintenance of membrane integrity .
An adequate supply of Ca is essential for the continued plant growth, while high Ca concen-
trations may inhibit cell extension. Cytoplasmic Ca is also involved in the regulation of
hormone responses to environmental factors such as light and temperature . Cultures may
continue to grow for some time without an external supply of Ca until endogenous supplies
are depleted. A typical symptom of Ca deficiency is shoot-tip necrosis due to poor distribu-
tion of Ca at the shoot tip rather than absolute deficiency in the plantlets (Williams , 1995).
Magnesium is a component of chlorophyll and a cofactor for many enzyme reactions .
Mg may substitute for Ca in some specific roles. Therefore, the relative supply of Mg may be
important where Ca uptake is limited. On the other hand , Na and Cl are not usually essential
to normal plant growth and are mainly included in media to balance the ionic composition
when providing other elements (Williams, 1995).
Yang et al. (1995a, b) proved that with the increaseof about 1/3 (or to 3 in case of Mgt,
H2P04- and SO42-)of the ionic strength of Ca2+, Mg2+, H2P04- and SO42-supplied in full
strength MS medium, dry weight, fresh weight, and leaf area of the strawberry plantlet in full
strength of modified Hoagland and Arnon (1950) were significantlygreater than those in 1/2
or full strength MS medium.
Micronutrients essentially exist in the culture media as catalysts for many biochemical
processes. Though required at low concentrations, sufficient quantities may be obtained to

66(8) Environ. Control in Biol.

 enable normal growth. It is difficult to see clearly the precise effect of micronutrients on the
culture growth because of the potential for interaction between them. However, general
symptoms of micronutrient deficiencies include shoot tip necrosis (B), leaf chlorosis (Fe, Zn,
Mn), reduced lignification (Cu, Fe) and rosetting (Zn, Mn). The supply of Fe is usually more
critical than other micronutrients because of the large levels required and the solubility
problems. Excess Mn can lead to Fe deficiency, while excess Fe or EDTA can reduce Zn
uptake (Williams, 1995).
The concentration of inorganic ions in the culture medium decreases with time as they are
absorbed by shoots/plantlets in vitro. This decrease is affected not only by the initial
concentration and uptake rate of each ion, but also by the volume of the medium per plantlet
(Kozai et al., 1991). The rate of decrease is greater when smaller amounts of medium are
used, because it is determined by the amount of absorbed ions divided by the medium volume.
In other words, the ion uptake rate, or amount of absorbed ion per unit time, is equal to the
rate of decrease multiplied by the volume. However, the ion uptake rate depends on the
medium concentration. On the contrary, the ion concentration increases with time when the
percent medium volume decrease per day due to evaporation from the medium and transpira-
tion from the plantlets is greater than the percent ion concentration decrease due to the ion
uptake per day. Some inorganic and organic substances are released from the explant and
plantlet to the medium in some cases.
Thus, optimum nutrient composition should be dependent on the initial volume of the
medium and the rates of evaporation and transpiration when the medium is not renewed
during a long-period culture (Kozai et al., 1992a). In plant tissue culture, the medium
volume is usually relative small (4-10 mL per plantlet), so that the changes in ionic concentra-
tions tend to be significant. Therefore, both initial medium volume and initial nutrient
concentration are important to determine the initial nutrient composition in the medium
(Kozai et al., 1995a).

3. Osmotic potential
Osmotic potential depends on the total molar (ions and molecules) concentration in the
culture medium. Low medium osmotic potentials are associated with a high total molar
concentration of medium solutes. Sugar and mineral salts are main contributors. The
increase of mineral and sucrose uptake, when plants grow, results in increasing osmotic
potential. However, the counteraction of the concurrent removal of water by transpiration
and evaporation may decrease osmotic potential (Williams, 1995).
The osmotic potential of the medium influences many aspects of growth and development
of cultures. Osmotic potential of the culture medium involves osmotic potentials mainly
caused by basic components, saccharide (sucrose, glucose and fructose) as a carbon source and
osmoregulatory substance (manitol and sorbitol) (Fujiwara and Kozai, 1995).
In general, the osmotic potentials of widely used tissue culture media are lower as
compared to those of hydroponic culture solutions. Thus, culture medium components are not
only the nutrient substances, but also the causative substances of osmotic potentials affecting
the water relations of plantlets in vitro (Fujiwara and Kozai, 1995).
Osmotic potentials caused by basic components and carbon source in the medium can be
changed during the culture. The osmotic potential caused by saccharides decreased when
sucrose was inverted into glucose and fructose by invertase located in the root surface ; whereas
the osmotic potential caused by basic components increased as their solutes were absorbed by
shoots. The sum of the two osmotic potentials was equal to the total osmotic potential of the
MS liquid medium, which decreased gradually with time by approximately 90 kPa over 35 d
(Kinoshita et al., 1988). Hydrolysis of sucrose into glucose and fructose can bring about a

Vol.36, No.2 (1998) (9)67

 relatively small change in osmotic potential in the medium ; whereas the incorporation of
monosaccharides into culture media may cause a relative large change in osmotic potential
(Fujiwara and Kozai, 1995).

4. Supports
Gelling agents provide several advantages for plant tissue culture production and research
as compared to liquid media. The use of gels provides the growing culture a beneficial
physical contact with a nutrient medium ; thus helps to prevent it from submersion in a liquid
medium with relative low-oxygen concentration (Smith and Spomer , 1995). In other words,
the use of a gelling agent will help to prevent hyperhydricity, and provides a good analogue
of soil for root zone investigations. Gelled media, however , create some drawbacks, such as
little adaptability to automation (gelled medium cannot be recirculated easily) , inactivation of
some chemical components, and difficulty for cleaning vessels and removing the gel from
plantlet roots after the culture period. Moreover, gels, principally agar or gelrite, have also
been cited as in vitro shoot or embryogenic cell growth inhibitors (Smith and Spomer , 1995),
because they cannot be considered as inert components of the system , and both their chemical
and physical properties can influence culture performance (Williams , 1992). Some experi-
ments proved that an agar-gelled medium inhibited the plantlet growth in vitro as compared
to that on liquid-based media (Debergh, 1983). On the other hand, shoots , which were
generated in the more humid, less aerated, restricted in vitro environment with gelling agents,
produced roots having underdeveloped vascular systems and proved to be less adaptable to
acclimatization. Other supporting materials such as cellulose plugs , rockwool cubes, and
vermiculite, which have a high air porosity, are proved to enhance the plantlet growth and ,
thus, survival percentage of plantlets ex vitro.
Takazawa and Kozai (1992) used Sorbarod, a fibrous cellulose plug , as supporting
material in culture plastic vessels and cited that the greatest average fresh weight of plantlets ,
the largest percent dry matter, the smallest top/root fresh weight ratio and the shortest shoot
were observed in Sorbarod treatment as compared with other treatments using agar as support-
ing material.
Kirdmanee et al. (1995a) demonstrated that, under both CO2 enriched and CO2 non-
enriched conditions, the palisade layer index, the Pn in vitro and the survival percentage ex
vitro of Eucalyptus camaldulensis cultured in vitro photoautotrophically were the highest in
the vermiculite followed by the plastic net, gelrite matrix and agar matrix (in descending
order). Kozai et al. (1996a) also proved that both rockwool and vermiculite supports enhan-
ced the root growth and chlorophyll concentrations of sweetpotato plantlets cultured
photoautotrophically in vitro as compared with the conventional agar gel. Nagae et al.
(1995) showed that Gerbera plantlets developed vigorously and were much bigger under
photoautotrophic conditions with CO2 enrichment and Rockwool Multiblock as a supporting
material in the Culture Pack than those on sugar-contained agar medium in bottle .
Fujiwara et al. (1993) showed that the O2 diffusion coefficient decreased about 50% when
agar in media increased from 0 to 8 g L-1, and about 25% more if adding 30 g L-1 sucrose into
8 g L-1 agar-gelled MS medium. Therefore, the poor rooting of shoots would partially result
from low dissolved O2 concentration round the shoot base in the medium (Fujiwara and
Kozai, 1995).
On the contrary, Kurata and Fukuda (1995) have indicated that the low dissolved oxygen
(DO) concentrations in liquid media in shake culture of carrot somatic embryos were prefer-
able for regenerating plantlets from embryos.

68(10) Environ. Control in Biol.

 S. pH
pH of medium is also one important factor affecting the growth, development and
morphogenesis of in vitro plantlets. However, in many plant tissue cultures, the pH of the
medium is largely taken for granted. A standard practice is to adjust the pH during prepara-
tion of the medium and then to ignore it (Williams, 1992). It is well known that the pH of
media does not usually remain constant (George, 1993), but changes both during media
preparation, particularly autoclaving, and in the presence of living plant material (Gamborg
and Shyluk, 1981; Williams et al., 1990).
The change in pH when plant material is added seems to be due to an ion imbalance
between the medium and the plant (Williams et al., 1990). This possibly results in a
substantial efflux of minerals out of the plant into the medium. The equilibrium point
depends on the species but is similar irrespective of the initial set pH (Williams, 1992). A
major factor of this change is the balance between NO3- and NH4+ supply and uptake
(Congard et al., 1986). As this balance changes over the culture period, there may be an
initial decrease in pH followed by an increase (Gamborg and Shyluk, 1981).
Congard et al. (1986) showed that the pH of plantlets depends on the developmental
stage, beginning by a decrease in the primary root formation then an increase with the
secondary roots. Kobayashi et al. (1995) also proved that pH slightly dropped within the
first 2 weeks corresponding to the root formation of Torenia and, then, rose to 7.0 as compared
to the initial pH of 5.6. The duration of rising pH corresponded to the time of formation and
development of vegetative buds. After rising, pH rapidly dropped to 5.0 corresponding to the
period of formation and development of floral buds.
Yoshihara and Hanyu (1992) demonstrated that in strawberry tissue culture using liquid
medium, pH dropped to about 4 from the initial 6 within the first 7 d, corresponding to the lag
phase of the callus growth. It also dropped to the same level in the growing stage of the intact
plant after 20 to 30 d of culture. After the droppings, the pH rose to 6 or higher in both the
cultures. However, little pH change and almost constant pH were observed in the multiple
shoot culture. The results led to the conclusion that the preferential uptake of nitrogen
sources affected the pH changes.

BIOLOGICAL EFFECTS

1. Plant size and physiological differences of explants

The method of plant tissue culture applying the regeneration of plantlets from callus,
single cells and protoplasts has currently aroused the question of genetic stability of these
plantlets and the loss of the organ/embryo-forming capacity (Pierik, 1988). The use of
somatic embryos or adventitious buds/shoots derived from the leaf, petiole, stem, stalk, corm,
etc., in micropropagation is also restricted due to the fact that chances of obtaining mutated
plants are higher (Pierik, 1988).
In commercial micropropagation, nodal sections, especially single-node stem cuttings,
and axillary buds are most commonly used as explants at the multiplication stage. Different
leaf area and fresh weight of explants may cause the growth variation of regenerated plantlets.
Pierik (1987) suggested that larger explants sometimes regenerated easier than smaller ones.
In photoautotrophic micropropagation, the leafy part of explants, instead of the stem part,
plays the most important role on the subsequent growth of regenerants because it helps to
produce carbohydrates by photosynthesis (Miyashita et al., 1996). This means that under
photoautotrophic conditions, the growth of a leafless explant is delayed, and the growth of
leafy explants is promoted when their leaf area or mass of photosynthetic (chlorophyllous)
organ is larger. Under photoautotrophic conditions, the leaf area of the explant had a greater

Vol.36, No.2 (1998) (11)69

 effect than the nodal position in the mother plant or fresh weight of the explant on the
subsequent growth into plantlets, provided that the environment is favorable for photosynthe-
sis (Miyashita et al., 1996).
In conventional micropropagation using sugar-containing medium , the increase of inter-
node length of explants helps to improve the initial growth rate of explants due to its greater
mass of carbohydrates. Furthermore, when the internode is longer , manipulation for multi-
plication is easier, especially in automation (Kozai et al., 1991). Marcelis-van Acker and
Leutscher (1993) observed that two-node cuttings (longer piece of stem) with large leaf area
produced larger plantlets in cutting propagation ex vitro of rose and Schefflera. They also
indicated that two-node cuttings with the upper leaf removed produced even larger plantlet
growth than single node cuttings did. Veierskov (1978) suggested that the effect of stem or
internode length was related to carbohydrates accumulated at the basis cuttings in ex vitro
propagation of pea. The rate of propagation is also strongly dependent on the number of
nodes (leaves) formed in vitro within a certain time period (Pierik , 1988). A larger explant
produced a greater number of shoots in vitro than a small explant.
It was suggested that a reduction of light intensity associated with an increase in distance
from top to base of nodal cutting position through a plant canopy and an increase of the stem
length below the node affected the subsequent shoot growth , axillary bud break and root
proliferation. In photoautotrophic micropropagation, the sideward lighting system seems to
be a such desirable technique to enhance the subsequent growth and development of plantlets
in vitro, as the system provides an even distribution of energy to the upper , middle and lower
leaves of shoots/plantlets. This, thus, results in the relatively large leaves at the bottom and
relatively small leaves at the top of shoots/plantlets (Kozai et al ., 1992b, 1995b).
The morphology of micropropagated plantlets , which derived from either axillary,
adventitious or meristematic origin is equally as important as their physical characteristics
when these plantlets are transferred as transplants to the greenhouse or the open field (Kozai
et al., 1991). The generally required morphological characteristics of transplants are: (1)
shortness and thickness of the main stem, (2) low shoot/root weight ratio , and (3) large leaf
area with normal stomata and thick cuticular layers . The most important characteristic
among them is the shortness and thickness of the main stem . However, in reality, the
conventionally micropropagated transplants tend to be tall and thin due to factors of the in
vitro environment (Kozai et al., 1991). With a proper control of physical and chemical
environments in photoautotrophic tissue culture, the growth and development (fresh and dry
weights, number of nodes, total leaf area, rooting and branching) of chlorophyllous explants/
shoots will be enhanced, while morphological and physiological disorders such as hyperhy-
dricity (or glassiness), thin cuticular wax formation of leaves will be reduced . Thus, the
uniformity of the plantlet growth increases , whereas the excess application of exogenous plant
growth regulators is reduced (Kozai, 1991b).

2. Plant density
Kozai et al. (1989) proved that , under photoautotrophic conditions, the growth (fresh
weight, dry weight, leaf area and number of unfolded leaves) of potato plantlets was substan-
tially affected by the shoot/plantlet density inside the vessel . CO2 concentration, net
photosynthetic rate and mineral component concentration decreased with increasing the plant
density. With the same conditions of number of air exchanges and high/low PPF , the growth
of potato plantlets in vitro was also proved to decrease with the increase of the plantlet density
inside the vessel after 2 weeks of culture (Niu and Kozai , 1997).

70(12) Environ. Control in Biol.

 3. Others
The chronological age of the potential explant and the season that the explant is obtained

are important only when they influence the extent of differentiation of the cells and their

physiological age. However, the age geffecth on the regeneration ability of explants is a

controversial issue in plant tissue culture (Read, 1992).

In vitro derived plantlets have recently been reported to be more regenerative than
explants taken from the field- or greenhouse-grown stock plants (Economou and Read, 1986;
Liu and Sandford, 1988; Stamp et al., 1990). It can be generally accepted that these explants
have undergone apparent physiological changes contributing to a better in vitro performance
(Read, 1992).
It is obvious that disease-free stock plants are preferred for explants in vitro. The use of
meristem culture to produce virus-free plants may be helpful in some cases. The use of
explants from virus-free stock plants in micropropagation will help to produce a large number
of healthy plants in a limited time (Read, 1992).

CONCLUSION

The effects of physical and chemical environments on the growth and development of
plantlets in micropropagation have been significantly proved through several recent studies
(Jeong et al., 1995). It is hoped that this article will contribute to the further understanding
of the essential role of environmental control in micropropagation systems in order to optimize
in vitro plantlet production at lower costs.

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Vol.36, No.2 (1998) (17)75