Ex vitro acclimatization is an important stage during in vitro plant propagation, because it deals with gradual transition from the artificial culture conditions to the natural living environment. In the acclimatization stage it is necessary to ensure optimal culture conditions to obtain high survival rates. The usual methods of ex vitro acclimatization use various types of containers filled with different types of potting mix (peat, perlite, soil, vermiculite individually or in optimized combination) as transplanting substrates as well as artificial culture areas (greenhouses, tunnels, growth rooms) in which air humidity is kept at high levels to prevent the dehydration of the plantlets transferred ex vitro.
For acclimatization, generally, plantlets rooted in vitro on media favorable for rooting are used (Bobrowski et al., 1996; Erig and Schuch, 2005; Fira et al., 2009; Najaf-Abadi and Hamidoghli, 2009; Radmann et al., 2003; Ružic and Laziž, 2006, 2007). Hazarika (2003), in a review on ex vitro acclimatization, mentions that during transition, better plantlet development, stomata functioning, and synthesis of epicuticular wax can be achieved by lowering air humidity in the culture vessels, by the use of growth retardants like paclobutrazol or Cycocel, and the use of antitranspirants. Examples of successful direct ex vitro-rooted plants are citrus, tea, walnut, peach, tobacco, and brinjal. The author further suggests application of direct ex vitro rooting of shoots deriving from the in vitro multiplication stage to reduce costs. Cristea (2010) mentions the usefulness of photoautotrophic micropropagation for the improvement of rhizogenesis and acclimatization process.
Rohr et al. (2003) discussed the morphological and anatomical anomalies of in vitro-cultured plants, like the absence of cuticle and stomata that should protect the plantlets from desiccation, as well as the lack of a properly developed root system. They also mention the two main approaches used for ex vitro acclimation: reducing water stress and helping the plantlets become autotrophic. They further offer examples for the beneficial effect of improved aeration during in vitro culture and the use of mycorrhiza for ex vitro acclimatization.
In this study, the ex vitro acclimatization of in vitro-rooted plantlets was studied in float hydroculture. We also studied the direct ex vitro rooting and acclimatization of in vitro-raised shoots harvested during the multiplication stage. In the latter case, both rooting and acclimatization were carried out ex vitro in the same stage.
Another method for the ex vitro acclimatization of several horticultural species was ex vitro acclimatization in floating perlite beds. This technique derives from the technique of acclimatization in flooded perlite in plastic trays (Tromble, 2011).
Most flotation hydroponics systems are made of long, rectangular cemented or wooden tanks and lined with waterproof material, which contain the nutritive solution. On the surface of the nutritive solution, there are floats made of polystyrene or other materials that sustain the plants (Sheikh, 2006; Sweat et al., 2003).
The technique of rooting ex vitro in floating cell trays was inspired from a method used for tomato and tobacco seedlings and for lettuce (Peek and Reed, 2008; Reed, 2009; Ross and Teffeau, 1995; Tyson et al., 1999). The method of float hydroculture presented in our article is different from the previous protocols because no fertilizers, plant growth regulators, or biostimulators were used and aeration was not provided. In our float hydroculture experiments, no porous substrate like peat, vermiculite, or peat and perlite mixture was used for supporting the plantlets. Air humidity was also not maintained at a certain level mechanically or otherwise.
The aim of this study was to test the efficiency of the novel ex vitro acclimatization techniques set up by the authors at the Fruit Research Station Cluj for the ex vitro acclimatization of several important horticultural species and cultivars. Another objective was to carry out direct ex vitro rooting to provide a means for avoiding the in vitro rooting stage where it was the case, by direct ex vitro rooting in float hydroculture or in floating perlite. Both ex vitro acclimatization techniques (float hydroculture and floating perlite beds) are radically new because they do not require the maintenance of high relative humidity around the plantlets to be acclimatized and the maintenance of the substrate’s humidity by irrigation is not required, because the substrate maintains its humidity by passively absorbing water from below in the case of floating perlite beds and, in the case of float hydroculture, the substrate is the water itself from the vessels used for acclimatization.
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