پديد آورندگان :
قمری مرضیه نويسنده دانش آموخته كارشناسی ارشد، گروه علوم باغبانی Ghamari Marzyeh , محب الدینی مهدی نويسنده دانشیار گروه علوم باغبانی Mohoboldini Mahdi , قنبری علیرضا نويسنده دانشیار گروه علوم باغبانی Ghanbari Alireza , حیدری حمیدرضا نويسنده دانشجوی دكتری گروه علوم باغبانی Heydari Hamidreza
كليدواژه :
بنزيل آدنين , سايتوكينين , شرايط درون شيشه اي , گياه زينتي , تيديازورون
چكيده لاتين :
Introduction: 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 Methods: 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.
Results and Discussion: 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 <0.01) and number of leaves and roots (P <0.05). Mean comparison showed that maximum number of leaves (17 leaf in each treatment) and length of leaves (4cm) obtained in media supplemented with 0.3 mg/l NAA + 0.5 mg/l BA. Correlation between the number of leaves and other traits such as diameter of callus and length of leaves (P <0.01) and regeneration (P <0.05) was significant.
In the second experiment, application of NAA and TDZ on in-vitro multiplication of F. imperialis were evaluated. Analysis of variance showed that maximum callogenesis obtained in medium supplemented with 0.3 mg/l NAA + 0.5 mg/l TDZ and 0.6 mg/l NAA in all TDZ levels(0.1, 0.3 and 0.5 mg/l). The combination of 0.6 mg/l NAA with all concentrations of TDZ resulted in maximum (100 %) callogenesis. Highest diameter of calli was obtained in medium contained 0.6 mg/l NAA without TDZ. Best plantlet regeneration was observed in media contained 0.3 mg/l NAA + 0.3 mg/l TDZ.
In the third experiment, application of NAA and Kin on in-vitro multiplication of F. imperialis were evaluated. Statistical analysis showed the highest callogenesis in medium supplemented with 1.5 mg/l Kin, 0.3 mg/l NAA + 1.5 mg/l Kin, 0.6 mg/l NAA + 0.5 mg/l Kin, 0.6 mg/l NAA + 1 mg/l Kin, 0.6 mg/l NAA + 1.5 mg/l Kin.
Conclusion: The results of this study show the similarity of callus proliferation in all experiments. In all experiments, medium supplemented with 0.6 mg/l NAA plus all cytokinin concentrations showed the highest rate of callogenesis. The highest callus diameter (7.5 cm) was obtained in third experiment (0.6 mg/l NAA + 1 mg/l Kin), while callus diameter in the first ( 1 mg/l BA and 0.6 mg/l NAA + 0.3 mg/l BA) and the second ( 0.3 mg/l NAA) experiments was measured 2.5 and 2.87 cm, respectively. Regeneration results indicated that Cytokinin is necessary for plantlet regeneration. Plant regeneration occurred only in medium contained both of kin and NAA, while BA and TDZ could regenerate plantlets from Callus independent of NAA presence.
