Evolution of the photosynthetic capacity of Guzmania 'Hilda' (Bromeliaceae) from in vitro to in vivo growth conditions.

 
Tissue culture is a widely applied technology for the production of ornamental plants. During the in vitro phase, plants are exposed to lower light intensities and limited gas exchange, both associated with poor photosynthesis. For that reason, another carbon source is needed for adequate growth and development. Sucrose is generally supplied to culture media, stimulating heterotrophic behavior of the in vitro plants. As a consequence, transfer of the plants to an ex vitro environment implies acclimation to an autotrophic way of energy providing. To achieve excellent ex vitro performance, delivery of qualitative in vitro plants with improved carbon utilization is required. In this work, the interaction between heterotrophic and autotrophic growth is investigated by determining the influence of sucrose in the medium on carbohydrate dynamics ina culture vessel and their relation with photosynthesis. As a study object, the bromeliad hybrid Guzmania ‘Hilda’ is used. This ornamental plant is commercially produced by tissue culture and its in vitro part consists of three phases: multiplication, elongation and rooting. For the experiments the rooting medium is supplied with four different sucrose concentrations: 15, 37, 73 and 117 µmol g-1medium. After an 8 week culture period on this medium, plants are transferred to the greenhouse for further growth.
 
When sucrose is added to the culture medium, almost all sucrose is depleted after four weeks, both by uptake and hydrolysis in glucose and fructose. The remaining part of the culture period, G. ‘Hilda’ plants are depending for sustaining growth on hexose uptake (preferably glucose), photosynthesis and degradation of storage (starch) molecules. 
It is clear that increasing the sucroselevel results in a higher carbohydrate uptake from the medium and a stimulated heterotrophic growth (bigger plants containing a higher number of roots and shoots) at the end of the in vitro culture. About 50% of thesugars available at the start of the rooting phase remain in the mediumduring eight weeks of culture. Only for plants grown on the lowest sucrose concentration a more efficient use of the available resources is observed, since 65% was metabolized. 
Autotrophic behavior is mainly dependent on the accumulated CO2 levels in the headspace of the culture vessel, originating from dark respiration. Plants grown on increased sucrose release more CO2 during night and improved CO2 uptake rates are noticed. This way autotrophic behavior is indirectly influenced by sucrose and the negative effects of sucrose on the chlorophyll levels and activity of Rubisco are covered by the accumulated CO2 levels in the headspace of the culture vessels. At the onset of the day CO2 is depleted (by photosynthesis) in less than 90 minutes, resulting in only limited CO2 concentrations during the remaining part of the day. If photosynthesis of the plants is measured under a constant CO2 flow (no accumulation), the inhibitory effect of sugars on the photosynthetic capacity of G. ‘Hilda’ emerges as lower CO2 uptake rates are measured in plants grown at increased sucrose in their medium. So it is suggested that quality improvement of the in vitro plants is possible by a combination of reducing sucrose and CO2 enrichment to stimulate a more efficient carbon utilization. 
 
The leaves act as storage organs since the carbohydrates (mainly starch) are degraded when sucrose is no longer available. After three months of transfer to the greenhouse, plants supplied initiallywith lower sucrose concentrations resembles the starch content of the in vitro leaves. On the other hand, plants cultivated in vitro on increased sucrose show a starch breakdown during these first three months ex vitro. This indicates the plants grown on lower sucrose need to engage more in photosynthesis for further growth, which is confirmed by higher Rubisco activities. After four months of ex vitro culture, the observed differences in plant growth at the end of the in vitro period (bigger plants when more sucrose was supplied) are no longer noticed ex vitro as the lower treated plants equals the growth of plants cultivated with increased sucrose levels. These are indications that reducing the sucrose concentration in the medium improves carbon utilization after transfer to thegreenhouse.
 
It is believed that improvement of G. ‘Hilda’ tissue culture is possible by optimizing carbon utilization. The photosynthetic capacity of in vitro plants is not fully exploited and can be enhanced by CO2 enrichment. Under these conditions it is recommended to reduce the sucrose concentrations, because they show a negative effect on the photosynthetic capacity of the plants. Lowering sucrose in the medium is also recommended since a more efficient use of the available resources occurs.