Saffron breeding and improvement using clonal selection, tissue culture, inter specific hybridization and ploidi variation techniques

Saffron is propagated vegetatively by replanting rest corms from previous growing season, during late summer or early autumn. Propagation by this method is slow and the corms can be infected by pathogens and soil-born diseases which are easily expanded during the replanting process, affecting plant growth and development and consequently considerable reduction of flowering and saffron production potential. In vitro propagation of saffron through somatic embryogenesis or cormogenesis is considered to be an efficient alternative method for large-scale propagation of pathogen-free and safe corms. To develop a protocol for in vitro propagation of saffron, effects of different concentrations of auxins (2,4-D and Picloram) and cytokinins (BA, TDZ and Kinetin) alone or in combinations were investigated in several experiments. Terminal buds of the rest corms were used as explants after surface sterilization using mercuric chloride (0.2 % W/V). Different responses were observed depending on the type and concentration of auxins and cytokinins. Somatic embryogenesis was obtained after 6 months when combinations of 2,4-D and BA (both at 2.5, 5 and 10 mgL-1) were applied. A concentration of 5 mgL-1 2,4-D in combination with 2.5 mgL-1 BA appeared to be the most effective treatment for induction of somatic embryogenesis.These embryos produced microcorm in later stages of development.When various concentrations of BA (0, 1, 2, 4, 8 mgl-1) combined with a low concentration of 2,4-D (0.2 mgL-1) were applied, multiple shoot regeneration was observed around terminal bud after 5 months. The highest number of regenerated shoots per explant was obtained with 2.0 mgL-1 BA and these shoots produced microcorm at the basal part after 2 months. Another experiment revealed that TDZ concentrations affected the induction of somatic embryogenesis significantly, while different types of corm explants showed no significant effect on this process. Among TDZ concentrations, 0.5 mg.L-1 was the most effective treatment for embryogenesis induction. Application of different concentrations of Picloram (0, 1, 3, 6 and 9 mgL-1) did not improve somatic embryogenesis response. Five months after culture in picloram containing medium, few embryogenic points were appeared on some of the cultured explants but the frequency was too low and their development was too slow compared to 2,4-D and BA treatments. When treatments of either BA or Kinetin at various concentrations (0, 2.5, 5 and 10 mgL-1) were applied, only growth of terminal buds and production of leafy organs was observed within 4 weeks of culture but no further development of these leafy organs was observed.