Proceedings of the XLVI Italian
Society of Agricultural Genetics - SIGA Annual Congress
Giardini
Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1
Poster
Abstract - 1.07
CRYOPRESERVATION
OF Citrus SEEDS BY
DEHYDRATION AND DIRECT IMMERSION INTO LIQUID NITROGEN
LAMBARDI
M.*, DE CARLO A.**, LOMBARDO G.***, BIRICOLTI S.**, DE PASQUALE F.****
*)
Istituto sulla Valorizzazione del Legno e delle Specie Arboree, CNR, Via Ponte
di Formicola 76, 50018 Scandicci (FI)
**)
Dipartimento di Ortoflorofrutticoltura, Università degli Studi di
Firenze, V.le delle Idee, 50019 Sesto Fiorentino (FI)
stefano.biricolti@unifi.it***) Dipartimento di Scienze Botaniche
dell’Università degli Studi, Via Archirafi 38, 90123 Palermo
****) Istituto di Ricerca per la Genetica
degli Agrumi, CNR, V.le delle Scienze 11, 90128 Palermo
Citrus,
cryopreservation, seeds, germplasm preservation
The recovery and conservation of a broad genetic pool of new and old varieties, as well as breeding lines and wild genotypes, must be regarded as essential for the maintenance of citrus biodiversity. Until now, traditional conservation systems (seed and in-field genebanks) have been the only reliable options for long-term germplasm preservation of woody species, although these approaches have some drawbacks. Numerous species (among which Citrus spp.) have non-orthodox seeds that easily lose their viability or are subject to a rapid decrease in germinability when stored for a long time. On the other hand, the conservation in clonal orchards of temperate fruit plants occupies large areas of land, due to the huge number of selected cultivars and local varieties which make up each single species. Moreover, in-field repositories are constantly threatened by pests and diseases, for which periodic and careful monitoring of trees is essential to maintain them healthy and genetically stable. Hence, there is no doubt that cryopreservation, a low-input method for long-term germplasm storage, will acquire strategic importance in the near future, as space and maintenance costs are reduced to a minimum. Currently there are no established methods for the long-term preservation of citrus seeds using the cryogenic technique. It is well-known that a partial dehydration of seeds can be a protection against the risks of intra-cellular ice formation during ultra-rapid freezing in liquid nitrogen (LN). However, because of the high susceptibility of citrus seeds to dessiccation, the final moisture content of the seeds should be carefully evaluated prior to plunging them in LN. The present study explores the possibility of Citrus seed cryopreservation, following a partial reduction of their moisture content. Seeds from three different polyembryonic genotypes were utilized: Citrus deliciosa Ten. (cv ‘Avana’), C. sinensis (L.) Osb. (cv ‘Vaniglia’), and C. aurantium L. (cv ‘AACNR1C’). Before the beginning of the cryopreservation trials, seed moisture content was calculated to be 50%, 50% and 33%, respectively. Seed germinability was 100% for all the species. Seed dehydration was carried out under the air-flow of a laminar flow bench, after which the seeds were directly immersed and stored in LN. Following cryopreservation, the seeds were rewarmed at room temperature and germinated in vitro, on a MS solidified medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Best germinability ratios were obtained for both C. sinensis and C. aurantium (93%), after the moisture content of their seeds was reduced to 15% and 10%, respectively, prior to being plunged in LN. A lower percentage (60%) was recorded with 15%-dehydrated seeds of C. deliciosa. It should be noted that, in comparison with the control, a reduction in the average number of seedlings per germinated seeds was always observed after cryopreservation (from 4-5, on average, up to 1-2). However, the same negative effect on germinability was observed in seeds that were germinated immediately after the application of the dehydration procedure, proving that ultra-rapid freezing did not induce further reduction of this parameter. These preliminary results indicate that the cryogenic technique can be regarded as an effective and useful technique for the long-term preservation of Citrus seeds, although a species-by-species optimization of the seed dehydration procedure is always required.