Avanzamenti nella micropropagazione massale di specie da frutto di elevato interesse economico mediante l'innovativa tecnologia di coltura liquida in immersione temporanea

Responsabili di progetto

Maurizio Lambardi, Efigjeni Kongjika

Accordo

ALBANIA - MoES - Ministry of Education and Sport of the Republic of Albania

Bando

CNR/MOES biennio 2018-2019 2018-2019

Dipartimento

Scienze bio-agroalimentari

Area tematica

Scienze bio-agroalimentari

Stato del progetto

Nuovo

Proposta di ricerca

Great efforts have been recently made in Albania to include in vitro culture techniques in the production and certification system of virus-free fruit tree with the aim to solve disturbing problems of orchard in the conditions of open market and free movement of plant products. Micropropagation, meristem culture, embryonic cultures (in solidified and double-phase media) are used as conventional methods of in vitro propagation in the well-equipped laboratories of Albania. As for Italy, micropropagation is a well-established method of propagation of fruit species and rootstocks, with over 30 commercial laboratories, producing yearly between 40 and 50 millions of high-quality plants; however, micropropagation in Italy is facing today the strong competition of micropropagated plants coming from countries with a much lower cost of handlabour, such as Turkey, Egypt, India, Iran. Hence, in both Albania and Italy there is a high request of innovation in the sector of in vitro propagation.

Commercial micropropagation has always been developed and concentrated on the use of semi-solid culture substrates, i.e. substrates containing a gelling agent to aggregate nutrients and growth regulators necessary to support the growth and morphogenetic processes of shoots culture. This approach to in vitro propagation has its weaknesses in the low proliferation rates often obtained with the use of small-size containers, as well as the necessity of frequent subcultures and, consequently, the high use of labor in the shoot proliferation phase, this step accounting for more than 50% of the final cost of micropropagated plants. For this reason, for over thirty years, attempts have been made to develop bioreactors for liquid culture systems with the aim of developing in vitro mass propagation in large containers, allowing a high degree of automation. Despite the commitment in this direction, to date micropropagation in liquid culture never really exited from the limits of laboratory experimentation. The main applications, in very few cases also of a commercial nature, have been to stimulate and automate the proliferation of callus cultures and embryogenic lines (examples with carrot crops and some conifers), bulbs (Amaryllis, Lilium spp., cyclamen, hyacinth) and potato microtubers. Experiences of micropropagation by liquid culture in bioreactors have been very sporadic in fruit species, and none reached the level of commercial exploitation.

The liquid culture has the advantages to allow the better assimilation of macro- and micronutrients through all the tissue, improving proliferation rates of the cultured shoots. However, fruit species do not adapt well to permanent liquid culture in bioreactors, due to the intensive damages produced by hyperhydricity and continue rubbing of floating material. Recently, the liquid culture in temporary immersion system (TIS) is showing to be a promising technique of in vitro propagation. Its advantages are (i) a more uniform contact between the culture medium and the shoots, in comparison with the culture in gelled medium, (ii) the dilution of toxic compounds released by the shoots (phenols), producing culture oxidation and browning, (iii) the periodic replacement of the atmosphere which limits gas accumulation (mainly CO2 and ethylene), a typical event when working with the traditional glass jars, (iv) the possibility to extend consistently the subcolture time when using large containers, to avoid the careful positioning of shoots in the gelled medium, and to refill easily the liquid medium when necessary, with a consequent marked reduction of labour costs in proliferation, (v) the obtaining, with some species, of rooted shoot clusters which can be maintained in proliferation, or directly transferred to the acclimatization phase. Moreover, optimized protocols can increase the proliferation rates, limit hyperhydricity and, in general, improve the shoot quality.

Innovation in TIS is based on the application of liquid culture by cycles of immersion and dry periods, maintaining the advantages of liquid culture and greatly reducing the damages. New bioreactors for TIS have been recently proposed, differentiating for the size of containers, the technology of immersion and the possibility to produce a periodical change of the internal atmosphere. Evaluation of the effectiveness of this innovative method is based on the determination of the main growth parameters, including the 'Relative Growth Rate' (RGR).

Based on the above considerations, this project aims to identify the effectiveness of large-scale micropropagation by liquid culture in TIS, using innovative bioreactors (Plantform, SETIS, ElecTIS), in comparison to the conventional in vitro propagation method based on the use of gelled (semi-solid) media in glass jars. The collaborative study will be performed working with fruit species (autochthonous cultivars, rootstocks, and minor under-utilized species) of great economic importance in both the countries, such as native and wild cultivars of cherry and almond, peach rootstocks, myrtle and pomegranate.

The results of the project will have an impact on the production of high-quality disease-free fruit tree species of economic importance for Albanian farmers. Results will be also important for the commercial nursery activity in Italy, through the reduction of production costs of fruit rootstocks and varieties. Main achievements will be discussed at workshops and scientific conferences organized by both parts in the frame of the project.

Obiettivi della ricerca

The general goal will be the comparison of different bioreactors for liquid culture in TIS (Plantform, SETIS, ElecTIS) compared to the conventional methods of in vitro propagation in gelled (semi-solid) medium, working with fruit-trees species of high economic importance in the nursery activity of Albania and Italy (native and wild cultivars of cherry and almond, peach rootstocks, myrtle and pomegranate).
The objectives are the following:
1. stabilization of effective in vitro culture protocols by conventional techniques of solidified and "double phase" media;
2. knowledge, application and comparison of innovative bioreactors for liquid culture in TIS (Plantform, SETIS, ElecTIS);
3. selection of the best bioreactor for TIS and comparison with conventional in vitro propagation methods based on the use of semi-solid medium as for: quality of the material, costs of production, implementation in commercial micropropagation laboratory;
4. awareness of specialists, young researchers, farmers and fruit-tree nurseries for the advantages and benefits of liquid culture by TIS by the organization of specific conferences/workshops, and the publication of scientific and technical notes;
5. enrichment of in vitro culture laboratories in Albania (laboratories in FNS, Tirana, and of CATT, Vlora) with the innovative technology for Albania;
6. widening knowledge in the potentiality of liquid culture in TIS by its application to Albanian and Italian valuable fruit germplasm.

Ultimo aggiornamento: 09/12/2024