Development of Protocols for Short-, Medium- and Long-Term Conservation of Thyme

Abstract

400 Thymus species exist worldwide and are used extensively, for medicinal and non-medicinal purposes. However, natural populations are inadequate to support such a great and growing demand for its products. Recently developed in vitro propagation and conservation techniques provide a unique alternative for the high-rate multiplication of plants of horticultural, economic and medicinal importance, and pave the way to medium- and long-term conservation of such valuable germplasm by means of slow growth storage and cryopreservation. The present study was designated to develop effective protocols for in vitro propagation of two distant Thymus species, i.e., T. vulgaris and T. longicaulis, as well as slow growth storage and cryopreservation protocols for conservation of Thymus germplasm. Optimizations for in vitro shoot proliferation included use of kinetin alone or in combination with auxins and/or gibberellic acid. Shoot rooting was easily obtained on semi-solid MS medium, supplemented with 2,4-D. Rooted plantlets were transferred to 250-ml plastic pots and acclimatized successfully by gradually reducing the relative humidity. Slow growth storage was achieved by keeping in vitro shoot cultures of T. vulgaris at 4 degrees C and in darkness up to 12 months. For long-term conservation, three different vitrification-based one-step freezing techniques of cryopreservation, i.e., PVS2 vitrification, encapsulation-vitrification and droplet-freezing, were tested.