In Vitro Aerial Environments and Their Effects on Growth and

Chapter 4

T. KOZAI & C. KUBOTA

IN VITRO AERIAL ENVIRONMENTS AND THEIR EFFECTS ON GROWTH AND DEVELOPMENT OF PLANTS

Contents 1. Introduction 2. General Characteristics 2.1. CO2, C2H4 and O2 concentration 2.1.1. CO2 concentration in the culture vessel with natural ventilation 2.1.2. CO2 concentration profile in a test tube type vessel 2.1.3. CO2 concentration in the culture vessel with forced ventilation 2.1.4. C2H4 concentration in the vessel with natural and forced ventilation 2.1.5. O2 concentration in the vessel 2.2. Relative humidity 2.3. Light 2.3.1. Increase in PPF by reflective e sheet placed above the lamps 2.3.2. Reduction in PPF due to vessels with closures 2.4. Air movement 2.4.1. Air movement above the culture shelf 2.4.2. Air movement in the vessel with natural ventilation 2.4.3. Air movement in large vessels with forced ventilation 2.5. Temperature 3. References

Key words: Aerial environment, air movement, CO2 concentration, ethylene, forced ventilation, natural ventilation, relative humidity. 1. INTRODUCTION In this chapter, general characteristics of in vitro aerial environment and fundamental features of major environmental factors in photoautotrophic and conventional (photomixotrophic or heterotrophic) micropropagation are summarized and compared with the general characteristics of the greenhouse environment for plant propagation and transplant production. Effects of the number of air exchanges of the vessel on CO2 concentration, C2H4 concentration and relative humidity in the T. Kozai et al. (eds.), Photoautotrophic (sugar-free medium) micropropagation as a new propagation and transplant production system, 31-52. © 2005 Springer. Printed in the Netherlands.

T. KOZAI & C. KUBOTA vessel are also discussed. It is pointed out that the in vitro environment is considerably different from the greenhouse environment. Basic physical relationships governing the in vitro environment are introduced to facilitate understanding of the mechanism of in vitro environment and to indicate opportunities to improve the in vitro environment for photoautotrophic micropropagation. Basic ideas came from our observation that, in many cases, germinated seeds with cotyledons and stem cuttings with a leaf grow and develop faster in the controlled environment greenhouse than in the culture vessel in conventional micropropagation. Effects of environmental factors on the growth and development of plantlets in vitro are also discussed. 2. GENERAL CHARACTERISTICS The environmental factors affecting the growth and development of cultures or plantlets in vitro are divided into aerial and root zone environmental factors (Figure 1). Most of the in vitro environmental factors are mutually influenced by, and also interact with, the plantlets in vitro. Moreover, the culture room environment and the physical characteristics of culture vessels affect the in vitro environment considerably (Figure 2, Kozai et al., 1995a). Unique features of the in vitro environment, in contrast to those of the greenhouse environment, are the presence of sugar in the medium in conventional micropropagation and the absence or low density of microorganisms in the culture vessel. Asepsis of culture vessels containing cultures and medium is required in conventional micropropagation for two reasons. One is to obtain pathogen free plantlets, and the other is to prevent the rapid growth of microorganisms, including non-pathogenic ones, in sugar-containing medium, which can damage or kill the cultures. Thus, culture vessels are kept airtight to prevent microorganisms from entering. This use of airtight vessels with small air volume typically characterizes the in vitro environment in conventional micropropagation. General features of the in vitro environment in conventional micropropagation are shown in Table 1, compared with those of the greenhouse environment. Notable characteristics of the in vitro environment are low flow rates of mass or materials (sugar, minerals, CO2, etc.) and energy (heat and radiation including light) mainly due to the airtight culture vessels and a relatively stable culture room environment. Table 2 shows the typical values of major environmental factors in the vessel for conventional micropropagation in comparison with those in a controlled environment greenhouse for plant propagation and transplant production. Due to the small volumes of air and culture medium per plantlet in vitro, low exchange rates of gases and medium components in the airtight culture vessel result in considerable changes in concentrations of the gases and medium components. This relationship is generally expressed as follows:

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4. IN VITRO AERIAL ENVIRONMENTS AND THEIR EFFECTS

∆C = ∆A/V

(1)

P=∆C /C C

(2)

where ∆C is the change in concentration per unit time of a component in the air or medium in a vessel, ∆A is the change in amount of the component per unit time, and V is the volume of air or medium in the vessel. Thus, when ∆A is large relative to V, ∆C is considerable. C is the concentration of the component before it changes. P is the change in concentration of the component, ∆C, relative to C. V is generally small (50-500 ml) in conventional micropropagation compared with that in the greenhouse (600-6000 m3 or 10 x 20 x 3 m3 - 40 x 50 x 3 m3), so that P has a high value when C is relatively low compared to ∆C. Actual change in ∆C of the gaseous component is affected also by the exchange rate of the component across the vessel, as described below. 2.1. CO2, C2H4 and O2 concentration 2.1.1. CO2 concentration in the culture vessel with natural ventilation It has been found that the CO2 concentration in a relatively airtight vessel containing chlorophyllous cultures (green-colored or chlorophyllous somatic embryos of cotyledonary stage, shoots, leafy stem cuttings, plantlets, etc.) often decreases sharply to lower than 100 µmol mol-1 within a few hours after the onset of the photoperiod (Figure 3). This CO2 concentration during the photoperiod is 270-330 µmol mol-1 lower than atmospheric CO2 concentration of 370-380 µmol mol-1, and is as low as just above the CO2 compensation point of plantlets in vitro (50-100 µmol mol-1). It remains the same until the dark period begins, and increases over time during the dark period up to 5 to 10 mmol mol-1. It was also found that CO2 concentration in a petri dish type vessel containing coffee (Coffea arabusta) somatic embryos of cotyledonary stage significantly decreased with time after the start of photoperiod, and a rate of change of CO2 concentration is affected by PPF (Afreen et al., 2002).

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Figure 1. Classification of the in vitro environmental factors affecting growth and development of the cultures.

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4. IN VITRO AERIAL ENVIRONMENTS AND THEIR EFFECTS

Figure 2. Schematic diagram showing the environmental factors inside and outside the culture vessel and their relationships. Lines indicate flows of energy and materials. Rectangle symbols denote state variables and valve symbols denote rate variables. Ellipse symbols denote coefficients and parameters. The letter N denotes the number of air exchanges of the vessel (Kozai et al., 1995b).

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Table 1. General features of the in vitro environment in conventional micropropagation with respect to state and rate variables, compared with those of the ex vitro orr greenhouse environment. State variables

Rate variables

Aerial Environment

Flow of Material and Energy

1) High relative humidity (high water potential) 2) Constant air temperature 3) Low CO2 concentration in light

1) Low transpiration rate

4) High CO2 concentration in dark 5) High C2H4 concentration

4) Low net photosynthetic rate 5) Low photosynthetic photon flux

6) Small air volume per plant

6) High dark respiration rate 7) Low exchange rate of mass between inside and outside the vessel

2) Low air flow rate 3) Low net thermal radiation flux

Root zone (Medium) Environment 1) High sugar concentration

1) Low sugar uptake rate

2) High mineral ion concentration 2) Low osmotic potential

2) Low mineral ion uptake rate 3) Low water uptake rate

4) Low dissolved oxygen concentration 5) High concentration of phenolic or other toxic substances 6) Low density of microorganisms 7) High concentrations of plant growth regulators 8) Small medium volume per plant

4) Low transport rates of medium components

Definitions of state and rate variables are given in the text.

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4. IN VITRO AERIAL ENVIRONMENTS AND THEIR EFFECTS Table 2. Typical values of major environmental factors in the vessel for conventional micropropagation in comparison with those in controlled environment greenhouse for plant propagation and transplant production. Environmental factors

In the vessel

In the greenhouse

10-100

100-1000

Air current speed during photoperiod (mm s )

1-20

10-1000

Temperature (C)

20-30

10-35

Relative humidity (%)

80-100

30-100

100-10000

250-400

0.0-1.0