Development of an optimum proliferation medium via the graph kernel statistical analysis method for genetically stable in vitro propagation of endemic Thymus cilicicus (Turkey)

Thymus cilicicus is an endemic Eastern Mediterranean element that has aromatic-medicinal properties. Its natural population spreads across gravelly ground and open rocky areas of South and Southwest Anatolia. The current study on in vitro propagation of T. cilicicus focused deeply on environmental applications such as the development of an optimum medium composition for efficient and genetically stable micropropagation and improved preservation procedures for long-time conservation of elite germplasms for further studies. For this purpose, MS and OM media were used individually and in combination with cytokinins, charcoal, AgNO3, Fe-EDDHA, and H3BO3. The raw data were statistically analyzed via the graph kernel method to optimize the nonlinear relationship between all parameters. The optimal proliferation medium for T. cilicicus was OM supplemented with a combination of 10 g L-1 charcoal and 1 mg L-1 KIN and the calculated averages of the best regeneration rate, the best shoot number and the best shoot length were 96.89%, 3 and 1.24 respectively on this medium. The determination of genetic stability of in vitro grown plants on the optimum medium compositions obtained by the graph kernel method was carried out with the use of the ISSR-PCR technique. All the ISSR primers produced a total of 192 reproductive band profiles, none of which were polymorphic. Furthermore, the micropropagated plants were successfully rooted and acclimatized to greenhouse conditions. In this study, we present a graph kernel multiple propagation index which considers all the possible parameters needing to be analyzed. Such an index is used for the first time for the determination of the optimum proliferation medium

Evaluation of Critical Points for Effective Cryopreservation of Four Different <i>Citrus</i> spp. Germplasm

The different pre- and post-treatments are critical in cryopreservation procedures and affect the shoot tip regrowth after freezing. In the present study, the long-term storage of four citrus cultivars [Bodrum Mandarin (Citrus deliciosa Ten.); Klin Mandarin (Citrus nobilis Lauriro); White grapefruit and Red grapefruit (Citrus paradisi L.)] were carried out by droplet vitrification methods, and the critical points for effective cryopreservation of these species were determined. In this study, we investigated the effect of explant size, cold hardening treatments, sucrose concentrations, and media combinations on shoot regrowth after cryopreservation. The highest shoot tip regrowth, ranging from 13.3 to 33.3%, was achieved when they were obtained from 0.3 to 0.7 mm explants excised from cold hardened seedlings at 4 °C for three days that were then precultured in a medium containing 0.25 M of sucrose and treated with PVS2 at 0 °C for 45 min. In addition, it has been determined that a regeneration medium containing boric acid (H3BO3) or ferric ethylenediaminetetraacetate (FeEDDHA) increased the regeneration up to 33.3% after cryopreservation

Evaluation of Critical Points for Effective Cryopreservation of Four Different Citrus spp. Germplasm

The different pre- and post-treatments are critical in cryopreservation procedures and affect the shoot tip regrowth after freezing. In the present study, the long-term storage of four citrus cultivars [Bodrum Mandarin (Citrus deliciosa Ten.); Klin Mandarin (Citrus nobilis Lauriro); White grapefruit and Red grapefruit (Citrus paradisi L.)] were carried out by droplet vitrification methods, and the critical points for effective cryopreservation of these species were determined. In this study, we investigated the effect of explant size, cold hardening treatments, sucrose concentrations, and media combinations on shoot regrowth after cryopreservation. The highest shoot tip regrowth, ranging from 13.3 to 33.3%, was achieved when they were obtained from 0.3 to 0.7 mm explants excised from cold hardened seedlings at 4 &deg;C for three days that were then precultured in a medium containing 0.25 M of sucrose and treated with PVS2 at 0 &deg;C for 45 min. In addition, it has been determined that a regeneration medium containing boric acid (H3BO3) or ferric ethylenediaminetetraacetate (FeEDDHA) increased the regeneration up to 33.3% after cryopreservation