Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/1999
Publication Date: N/A
Interpretive Summary: Saffron is the world's most expensive spice, and the United States currently imports all of its saffron. Saffron is found in the female flower parts (stigmas) of the autumn crocus (Crocus sativa L.), which only flowers in the fall. This seasonal flowering and the labor intensive process of collecting the stigmas are two of the reasons this spice is so expensive. However, if stigmas from the autumn crocus could be mass produced by cloning procedures using a technique called tissue culture, saffron might be able to be produced in the USA, thus ending the dependence of importing the spice. We conducted experiments to determine the optimal medium to culture stigmas in the laboratory. A second objective was to determine if the concentration of other chemicals called secondary chemicals that give saffron its taste, aroma and color, were similar to the concentrations found in natural saffron. Of all the growth media examined, activated charcoal (1%) added to B5 basal medium + alpha-napthalene acetic acid (NAA) 1.0 mg/ml + N6-benzyadenine supported the optimum growth of cultures in petri dishes, prevented browning of the tissue mass and accelerated the initiation, growth and development of stigma-like structures (SLS). The concentrations of secondary chemicals found in the SLS grown on the select medium were very similar to the concentrations found in natural saffron. Tissue culture appears to be a successful method to provide a constant supply of autumn crocus stigmas from which saffron can be extracted. A supply of high quality saffron in retail stores would lower the price of this important spice and ensure that consumers are getting the highest quality saffron for the price they pay.
Technical Abstract: Studies were conducted to optimize the in vitro production of stigma- like-structures (SLS) that yield 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 including Murashige and Skoog (1962) (MS), Gamborg et al. (1968) (B5), Heller (1953), Nitsch and Nitsch (1969), and White (1963) supplemented with different combinations of plant growth regulators and vitamins. The optimum proliferation of SLS was observed on B5 basal medium + alpha- naphthalene acetic acid (NAA) 1 mg/l + N6-benzyladenine (BA) 10 mg/l + MS organics + casein hydrolysate 0.5 g/l + l-alanine 1.0 g/l 50-60 days after inoculation. Some explants formed non-SLS-structures (roots, corms, petals, leaves) the growth and development of which substantially reduced the development of SLS. Removal of brown tissues and non-SLS during subculture (3 week intervals) allowed continuous culture of half- ovary explants for 9-10 months. SLS that 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 + NAA 1 mg/l + BA 10 mg/l + 3% sucrose 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 by high performance liquid chromatography analysis, were found similar to those in natural saffron.