Effect of Temporary Immersion System on in vitro development of Himantoglossum robertianum (Loisel.) P. Delforge (Orchidaceae)

Orchids reveal to be amazing plants when their life cycle, reproductive strategies and ecological relationships are investigated. They go through different and peculiar steps, from seed germination to adult plant, in which they change their trophic status. In the first phase of germination they can establish a specific relationship with symbiotic fungi, which supply nutrient to embryos – seeds have not storage tissues - and protocorms. In these early developmental stages, orchids are myco-heterotrophic, i.e. totally dependent by fungi for sustenance. This condition is particularly binding for terrestrial orchids, which also in the adulthood maintain a symbiotic relationship with fungi at root level. To grow orchids out of their natural habitat is possible by applying in vitro technics, providing them inorganic macro- and microelements, source of carbon and nitrogen, often in organic form. Despite appropriate media and favourable culture conditions are now available, the seeds of some orchid species require a long time to germinate and to develop protocorms, and a lot of months to grow plantlets. The main aim of this work has been to evaluate if the application of a Temporary Immersion System (TIS), in which plants are periodically immersed in liquid medium following optimized cycles, is effective to improve orchids growth in comparison with traditional in vitro culture systems. In this study, we selected three accessions of seeds of Himantoglossum robertianum, maintained in conservation in the Unimore Seed Bank. The seeds, after scarification treatment with NaOCl solution, were sown on BM2 medium with some modification. Only one accession of seeds gave high percentage of germination and a sufficient number of protocorms to set up all the following tests. In a first test, 50 little protocorms (diameter 2-3 mm) were taken from agarized medium and transferred both on the same medium without activated charcoal in Microbox vessels, and in Plantform Bioreactors (TIS). in this last using BM2 medium without activated charcoal and Plant Agar. Air inside Plantform was renewed for 10 min every 4 hours, whereas the condition of temporary immersion were 10 min every 8 hours. This first test has been repeated twice, within two months of each other. A second test has been carried out with the same experimental design using larger protocorms (about 5 mm diameter), which already presented a shoot apex. All cultures were maintained in a growth chamber at 23 °C, 12-h photoperiod for 33-36 days. The growth of protocorms were evaluated as Relative Growth Rate (RGR) index, based on the initial and final fresh weights of the plant material and the time of culture. The comparison of protocorm growth on agarized medium and in TIS in first test did not show any difference. Interestingly, when this first test was considered by maintaining separate each of two repetitions, a significant difference emerged. This result could be related to the characteristics of a natural population of seeds, as protocorms firstly used are those that firstly developed in culture, likely individuals with higher growth potential. During the second test an evident and definite protocorm development was observed, as many plantlets with green leaves and roots occurred both in Microbox and in Plantform within one month. However, a noticeable increase of growth was obtained in TIS in comparison with that observed agarized system. TIS is known to be a useful device to obtain a rapid and effective propagation in vitro, until now widely applied in micropropagation. A lot of advantages, i.e. better nutrients supply and more effective dispersal of toxic metabolites can be obtained with bioreactors based on TIS. In the light of these first results, Plantform system may be considered a good biotechnological device to grow terrestrial orchids in comparison with traditional systems, mainly to increase plant biomass.