Optimization of in vitro conditions for stigma-like-structure production from half-ovary explants of Crocus sativus L.

Studies were conducted to optimize the in vitro production of stigma-like-structures (SLS) that yielded the important biochemical constituents responsible for the color, taste, and aroma naturally found in the stigmas of autumn crocus. Immature half-ovary explants were evaluated for the frequency of proliferation of SLS by culture on five basal media supplemented with different combinations of plant growth regulators,rs and vitamins. The optimum proliferation of SLS was observed on B5 basal medium containing alpha-naphthalene acetic acid (NAA) (5.4 mu M), N-6-benzyladenine (BA) (44.4 mu M), MS organics, casein hydrolysate (0.05%), and L-alanine (11.2 mM) 50-60 d after inoculation. Some explants formed other structures (roots, corms, petals, leaves), the growth anti development of which substantially reduced the development of SLS. Removal of brown tissues and other tissues during subculture (3-wk intervals) allowed continuous culture of half-ovary explants for 9-10 mo. SLS that had deep red pigment and reached more than 0.5 cm length were removed from the explants and with concomitant reculturing of the ovaries, enabled SLS to be harvested three times. Activated charcoal (1%) added to B5 basal containing NAA (5.4 mu M), BA (44.4 mu M), and sucrose (3%) was found to be a helpful addendum to prevent browning of explants and accelerate the initiation, growth, and development of SLS. The amounts of crocin, crocetin glucosyl esters, picrocrocin, and safranal in SLS, as determined Ly high performance liquid chromatography analysis, were found similar to those in natural saffron.