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Testing the Microboxes

These experiments were set up in order to evaluate the added value of the Microbox as an in-vitro culture container. All measurements were taken at the beginning of the light phase.

Current problems in micropropagation are:

  • When using hermetically closed containers without filters: problems caused by poor gas exchange of CO2, O2 and especially ethylene concentrations can be far from optimal. Another common complication of poor gas exchange is hyperhydricity.
  • When using loose covers: occurrence of secondary infections, transported by air or by mites and trips.

Materials and methods

Containers: the incubations were performed in three types of standard polypropylene boxes with different types of closing systems.

  • Type 1: cover without filter. The cover is snapped on, but not hermetically closed
  • Type 2: plain cover, hermetically closing the box
  • Type 3: Microbox cover, equipped with a series of filters and hermetically closing the box
    (figures below).
Investigated species: Anthurium andreanum cultivar Mylene, which is known to be sensitive to ethylene gas. We prepared shoot cultures with heavy cutting, in order to produce as much ethylene as possible.

Medium: a modified medium according to Murashige and Skoog (1962), solidified with agar.

Environmental conditions:
  • Incubation temperature: 25°C
  • Light intensity: approx. 60 µmol.m-2.s-1. in a rhythm of 12h dark and 12h light. Adequate ventilation prevented the CO2 concentration from rising above 0.2%
  • Contamination pressure: the boxes were placed in an environment, which was heavily contaminated with mites and spores of Aspergillus sp.
Analytical methods:
  • CO2 concentration was measured with an infra-red gas sensor (Engicom Systems)
  • O2 concentration was determined with a paramagnetic sensor (Engicom Systems)
  • Ethylene was detected by means of gas chromatography according to Smalle and Van der Straeten (1997).

Graph 01: carbon dioxide concentration in the gas phase in the head space of the container. Fluctuations are caused by night and day rhythm.

Graph 02:oxygen concentration in the gas phase in the head space of the container

Graph 03: ethylene concentration in the gas phase in the head space of the container

Observations

As could be expected, the carbon dioxide concentration never rose above 0.6 % in all ventilated containers, which is considered beneficial for plant production.

The oxygen concentration was almost at the same level in all containers, allowing night regime reactions.

In all ventilated systems, ethylene concentration was lower than the detection limit of 0.0625 ng/ml of gas phase, which is too low to have a visible effect on the plantlets. In our comparative series with completely closed containers, ethylene concentrations as high as 1.5 ng/ml were measured. On photographs 1 and 2 the effect of high ethylene concentrations are clearly visible, such as long internodia, pale green leaves, adventitious air roots and shoot formation in the medium.

The Microbox shows an even better gas exchange than the non-closed cover (about 4 gas exchanges per day).







The Microbox, hermetically closed cover with filter




Healthy plantlets in Microbox






Hermetically closed cover without filter





Effect of high ethylene concentration and hyperhydricity
   
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