Cryopreservation at CNR-IVALSA in Florence: reflections upon ten years of good results and some failures with woody plants

The first experiments on cryopreservation at CNR-IVALSA in Florence date back to 1998. They were a response to the necessity to explore new and innovative systems for the ex situ conservation of woody plant germplasm, complementary to the traditional approach of in-field collection (clonal orchards). In Italy, the IVALSA Institute has one of the largest germplasm collections of fruit species (over 1800 accessions of peach, olive, plum, pear, persimmon, cherry, apple and quince) spread over the 60 ha of its experimental farm. Year by year, the maintenance costs of the collections are becoming heavier to bear. Furthermore, in the middle of the 90s the Sharka disease (Plum Pox Virus) spread through the large plum collection, obliging the burning of a clonal orchard of over 150 accessions of Prunus domestica and Prunus salicina. Hence, cryopreservation technology was regarded as a possible complementary strategy for the future, reducing to a minimum the risks of germplasm loss. Taking advantage of the considerable expertise in plant tissue culture, a small cryolaboratory was soon established to begin with. The first experiments were with poplar (Populus spp.) and olive (Olea europaea), two species of great importance in Italy in terms of germplasm preservation. Since then, various cryopreservation techniques, numerous species (mainly, but not exclusively, woody plants) and various kinds of explants have been tested, mostly with the practical aim of developing effective and reproducible protocols for long-term conservation. Among the cryoprotocols which were satisfactorily developed (thanks also to National and International collaborations), the following are particularly worthy of mention: the cryopreservation of shoot tips from white poplar (Populus alba), plum (Prunus domestica) and a non-woody species (Cichorium intybus), of embryogenic lines from olive and horsechestnut (Aesculus hippocastanum), and of seeds from Pistacia and Citrus spp. However, it has not been ten years of only successes, not at all! On the contrary, enthusiasm over some very good results has often been followed by deep frustration deriving from disappointing experiments. One example: after ten years of trials, testing all the different cryotechniques today available (included slow cooling), the cryopreservation of olive shoot tips is still unreliable, a long way from being proposable as a complementary approach to the conservation in field. Other failures have been the shoot-tip cryopreservation of a grape rootstock ( Kober 5BB ) and of redwood (Sequoiadendron giganteum), as well as the cryopreservation of an embryogenic line of ash (Fraxinus excelsior). What have we learnt from our ten-year experience in cryopreservation? What are the possible reasons for species adapting extraordinarily well to -196°C (e.g., white poplar, red chicory), and others being highly reacalcitrant (e.g., olive)? Do we know all the potentialities, as well as all the limits, of the technology? First reflection: before working with plant cryopreservation, I had many years of experience in the field of somatic embryogenesis of woody plants. In my opinion, the two techniques share a practical similarity when approaching the study of a new species, i.e., for both, if after the first preliminary experiments you get nil (i.e., no explant producing an embryogenic callus, as well as no explant surviving to -196°C), there is little hope that it will be possible to develop a succesful protocol, whatever you change in the methodology. If you get at least one positive (i.e., a fully-developed plantlet), it is worthy to go on! That means much more than only a glimmer of hope! Second reflection: working with woody plants, species having structural difficulties in the initial stages of micropropagation (e.g., big problems with the in vitro establishment, low proliferation rates, very slow shoot elongation) will be, with most probability, also difficult in the development of an effective cryopreservation protocol. One example: olive is very difficult and slow during the introduction and the establishment in vitro. So, is the difficulty in developing an effective shoot-tip cryopreservation protocol strictly due to its low tolerance to ultra-rapid freezing? Or, rather, is it due to the accentuation of a basic problem when a sort of second establishment in vitro is required (i.e., the shoot-tip development after thawing and plating)? Third reflection: a big mistake was made in the 80s in the field of micropropagation, i.e., private users were given to believe that every species could be, soon or later, successfully micropropagated, it just required experiments and tests of different medium/growth regulator combinations. In Italy, that idea led to the proliferation of commercial laboratories in the early 90s, which soon found themselves fighting to produce and offer the same relatively-few species which could be actually produced in vitro. The majority of them went broke in a few years! Of course, cryopreservation is a totally different story (first, it is a non-profit activity). However, we must be very careful to avoid the same mistake when promoting cryopreservation. Last but not least: a warm welcome to projects like Crymcept and CryoPlanet !! Certainly, we are all convinced that plant cryopreservation still needs much experimentation and, more important, a continuous confrontation between scientists with different experiences, made with different species and in different scientific and national contexts.vokMyynti MTT, Tietopalvelut 31600 Jokioine