The ontogenetic developmental stages of saffron somatic embryogenesis have been studied and characterized using light microscopy and the biochemical determination of the antioxidant enzymatic system. The embryogenic callus underwent internal segmented divisions with the formation of globular embryos that were attached to the callus surface by a broad multicellular structure. Further development of the embryoids was characterized by the emergence of a shoot apical meristem and cotyledon (monopolar stage) with the subsequent differentiation of a minicorm at the basal part of the somatic embryo (dipolar stage). During the morphological differentiation of the somatic embryos changes in the antioxidant enzymatic system with increased superoxide dismutase (SOD) and catalase (CAT) activities were detected at the initial stages of somatic embryogenesis. The isoforms of SOD, including two Mn-SODs and four Cu, Zn-SODs, were also detected. Although all the isoforms were expressed during the successive stages of somatic embryogenesis, an increase in Mn-SODs and a decrease in Cu, Zn-SODs during the last two stages was observed. Significant changes were also detected in the antioxidant activities ascorbate peroxidase, dehydroascorbic acid reductase and glutathione reductase.

Saffron embryogenic calli increased fresh weight four times when cultured in a temporary immersion system compared to those cultured in solid medium. Paclobutrazol was effective reducing hyperhydricity in the explants; the best results were obtained with 1mg/l. The development of somatic embryos on solid medium was significantly improved with the addition jasmonic acid (0,5 mg/l). Saffron somatic embryos contained more conjugated than free polyamines. This was true for all stages of embryogenesis except for initial stage. The establishment of polar embryos determined a significant increase in the level of polyamines mainly due to a drastic rise in the conjugation of the diamines diaminepropane and putrescine. This stage was also characterized by the depletion of free diaminepropane. In dipolar embryos a decrease in the concentration of conjugated polyamines was observed. The last stage of regeneration, in which corms are well formed at the base of each plantlet, coincided with further reduction in total polyamines; the almost complete depletion of most free polyamines (only traces of putrescine and spermine are detected), being conjugated spermidine the most predominant amine.

The ontogenic developmental stages of saffron somatic embryogenesis have been studied and characterized using light and electron microscopy. The embryogenic callus underwent internal segmented divisions with the formation of globular embryos that were attached to the callus surface by a broad multicelular structure. Further development of the embryoids was characterized by the emergence of a shoot apical meristem and cotyledon (monopolar stage) and subsequent differentiation of a minicorm in the basal part of the embryo (dipolar stage). During the morphological differentiation changes in the antioxidant enzymatic system of the somatic embryos were detected with increased SOD and catalase activities during the initial stages of the process. The fact that MDHAR activity was markedly higher than DHAR could point towards a regeneration of ascorbic acid mostly dependent of NADH. The isoforms of SOD were studied being detected 2Mn-SODs and 4Cu,Zn-SODs. Although all the isoforms were expressed during the successive stages of somatic embryogensis an increase in the levels of Mn-SODs and a decrease in Cu,Zn-SODs during the last two stages was observed.

The allohexaploid Aegilops species (2n = 6x = 42), Ae. neglecta 6x (UUX^tX^tNN), Ae. juvenalis (D^cD^cX^cX^cUU), and Ae. vavilovii (D^cD^cX^cX^cS^sS^s) regularly form bivalents at metaphase I. However, in Ae. crassa 6x (D^cD^cX^cX^cDD) 0.27 quadrivalents per cell were observed probably as a consequence of the partial homology displayed by the D and D^c genomes. Likewise, the synthetic amphiploid Ae. ventricosa-Secale cereale (DDNNRR) is fertile and displays a diploid-like behavior at metaphase I, despite its recent origin. The pattern of synapsis at late zygotene and pachytene in the natural and artificial allohexaploids was analyzed by whole-mount surface-spreading of synaptonemal complexes under an electron microscope. It revealed that chromosomes were mostly associated as bivalents in all cases, the mean of multivalents per nucleus ranging from 0.17 (Ae. neglecta 6x) to 1.03 (Ae. crassa 6x) in the natural species and 1.05 in the Ae. ventricosa-S. cereale amphiploid. It can be concluded that the mechanism controlling bivalent formation in these species and also in the synthetic amphiploid acts mainly at zygotene by restricting synapsis to homologous chromosomes, but also acts at pachytene by preventing chiasma formation in the homoeologous associations. These observations are discussed in relation to the origin and evolution of the mechanism of diploidization in the allopolyploid species of the Poaceae family.

To initiate the evaluation of gene expression patterns in saffron, 80 random chosen clones from a cDNA library prepared from corms were immobilized in amino-silane coated slides and hybridised with labelled cDNA from in vitro embryos at different developmental stages. Constitutive genes showed similar expression patterns at all stages, while developmentally regulated genes changed their expression. We detected strongly regulated expression of a xyloglucan endotransglycosylase (a cell wall loosening activity enzyme involved in cell growth), formaldehyde dehydrogenase and an abscisic stress ripening protein. Some unidentified genes also showed high hybridisation signal. In addition, the expression pattern of the major storage protein, a mannose-binding lectin, was studied by means of RT-PCR. The expression level is higher during summer, when the corm is at a dormant stage, and this lectin accumulates in big storage vacuoles. Once the corm sprouts and blooms, the expression of this protein decreases to minimum levels.