Effect of Plant Growth Regulators on Callogenesis and Regeneration Of Fritillaria imperialis L

Crown imperial (Fritillariaimperialis L.) is an ornamental and medicinal plant native to mountainous regions of Iran. This plant genetic resources is in danger of extinction, because of grazing livestock and pest outbreaks. However, due to slow reproduction in natural conditions and traditional multiplication methods such as scaling and Bulb division, many species of this genus are endangered. Using of biotechnology, namely in vitro plant propagation, is a solution to the problems of reproduction of rare and endangered plant species with difficult propagation and mass production of valuable genotypes. Therefore, micropropagation of F. imperialis through in vitro regeneration is essential for conservation and commercial production.
Material and The bulbs of F. imperialis in dormancy stage obtained from Ilam mountainous regions in Iran and theywere placed in wet vermiculite at 4 °C for 4-6 weeks. Then, Bulbs were surface-sterilized with 70% ethanol for 60s followed by immersion in 5% (v/v) NaOCl solution for 20min with gentle agitation, and they rinsed three times in sterile double distilled water. Explants prepared from the lower third of scales with basal plate and were placed in MS basal medium supplemented with different concentrations of NAA and 2,4-D for callus induction. Test tubes with bulb segments were maintained within 25±2°C in growth chamber at 16 hours light period by the illumination from white florescent tube light and 8 hours dark. After two months callus were transferred to MS basal medium without PGRs. Then, callus excised to 0.5 cm pieces and were transferred to MS basal medium supplemented with NAA in 0, 0.3 and 1 mg/l concentration.Three types of cytokinins with different concentrations were arranged in three seperated experiments. Thefirst experiment medium contained NAA with BA (0, 0.3, 0.5 and 1 mg/l), the second experiment NAA combined with 0, 0.1, 0.3 and 0.5 mg/l TDZ and the third experiment MS basal medium included NAA with Kin (0, 0.5, 1 and 1.5 mg/l). After three months, percentage of callogenesis, diameter of calli, percentage of regeneration, number of leaves and roots and length of leaves and roots were measured. This experiment were carried out in completely randomized design with 4 replications.
In the first experiment application of NAA and BA on in-vitro multiplication of F. imperialis were evaluated. Highest callogenesis and formation (100 %) was observed in mediums contained 0.3 mg/l NAA 1 mg/l BA, 0.6 mg/l NAA (0.3, 0.5 and 1 mg/l) BA. Also, callogenesis was obtained in medium contained 0.5 mg/l BA without NAA. This result showed that only in medium supplemented with 1 mg/l BA provided highest (100%) callogenesis, when NAA concentrations were low. However, high levels of NAA (0.6 mg/l) in all concentrations of BA were obtained maximum callogenesis. We concluded that NAA is essential for callogenesis and enhancing its levels can increase callogenesis. Also, application of low levels of BA (0.4 µM) in callogenesis mediums of Cynodon dactylon contained Auxins resulted in increment of embryogenetic calli formation. In the other hand, presence of BA is essential for plantlet regeneration, however NAA is not necessary. Plantlet regeneration was obtained in PGRs free medium. Statistical analysis of results showed that different concentrations of BA and NAA had significant effects on percentage of callogenesis, diameter of calli, percentage of regeneration, length of leaves and roots (P