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

Document Type : Research Article


University of Mohaghegh Ardabili


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


1- Eslam Zade N., Hosseini S.M., and Moradi H.R. 2010. The Study of Fritillaria imperialis Site by Ellenberg Table. Journal of Sciences and Techniques in Natural Resources, 1:83-93 (In Persian with English abstract).
2- Hamidoughlou S. 2011. Study on explant types and plant growth regulators on in vitro propagation of Fritillaria imperialis L.. MS Thesis, University of Mohaghegh Ardabili.
3- Babaoglu M., and Yorgancilar M. 2000. TDZ-specific plant regeneration in Salad Burnet. Plant Cell Plant Cell Tissue Organ Culture, 440: 31–34.
4- Bagheri A., and Saffari M. 2009. in vitro culture of higher plants. Ferdowsi University of Mashhad, 406.
5- Bonyanpour A., and Khosh-Khui M. 2013. Callus Induction and Plant Regeneration in Punica granatum L. ʻNanaʼ from Leaf Explants. Journal of Central European Agriculture, 14: 75-83.
6- Brasileiro A.C.R., Willadino L., Carvalheira G.G., and Guerra M. 1999. Callus induction and plant regeneration of tomato (Lycopersicon esculentum cv. IPA 5) via anther culture. Ciencia Rural, 29: 619-623.
7- Chaudhury A., and Qu R. 2000. Somatic embryogenesis and plant regeneration of turf-type Bermuda grass: effect of 6-benzyladenine in callus induction medium. Plant Cell Tissue Organ Culture, 60: 113-120.
8- Chengalrayan K., and Gallo-Meagher M. 2001. Effect of various growth regulators on shoot regeneration of Sugarcane. In Vitro Cellular and Developmental Biology-Plant, 37: 434-439.
9- De Hertogh A., and Lenard M. 1993. The Physiology of Flower Bulbs.
10- Dominov J.A., Stenzler L., Lee S., Schwarz J.J., Leisner S., and Howell S.H. 1992. Cytokinins and Auxins control the expression of a gene in Nicotiana plumbaginifolia cells by feedback regulation. Plant cell, 4: 451-461.
11- Fratini R., and Ruiz M.L. 2002. Comparative study of different Cytokinins in the induction of morphogenesis in Lentil (Lens culinaris Medik.). In Vitro Cellular and Developmental Biology-Plant, 38: 46-51.
12- Gulshan T.M., Varghese D.R., and Sharma D.R. 1981. Studies on anther cultures of Tomato Lycopersicon esculentum Mill. Biologia Plantarum, 23: 414-420.
13- Hao Z., Dai D., Wang J., Wu X., and Liu M. 2013. Callus induction and plant regeneration from anther walls in Ziziphus jujuba Mill. Journal of Food, Agriculture and Environment, 11: 405 – 409.
14- Hebert J., Touchell V., Ranney R., and LeBude V. 2010. In Vitro Shoot Regeneration and Polyploid Induction of Rhododendron Fragrantissimum Improved’. Hortscience, 45: 801–804.
15- Hernandez L., Celestino C., and Toribio M. 2003. Vegetative propagation of Quercus suber L. by somatic embryogenesis. Plant Cell Reports, 21: 759-764.
16- Hutchinson M. J., Onamu R., Kipkosgei L., and Obukosia D. 2010. Effect of Thidiazuron, NAA and BAP on in vitro Propagation of Alstroemeria aurantiaca cv. ‘Rosita’ from shoot tip explants. Journal of Agriculture and Crop Sciences, 12: 60-68.
17- Khorrami Raad M., Bohluli Zanjani S., Shoor M., Hamidoghli Y., Ramezani sayyadd A., Kharabian-Masouleh A., and Kaviani B. 2012. Callus induction and organogenesis capacity from lamina and petiole explants of Anthurium andreanum Linden (Casino and Antadra). Agriculture and Crop Science, 6: 928-937.
18- Kondamudi R., Vijayalakshmi V., and Sri Rama Murthy K. 2010. Induction of Morphogenetic Callus and Multiple Shoot Regeneration in Ceropegia pusilla Wight and Arn. Biotechnology, 9: 141-148.
19- Mohammadi Dehcheshme M., Khalighi A., and Naderi R. 2007. Indirect somatic embryogenesis from petal explant of endangered wild population of Fritillaria imperialis. Pakistan Journal of Biological Sciences, 10: 1875- 1879.
20- Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bioassay with Tobacco tissue cultures. Physiologia Plantarum, 15:473-479.
21- Sunderland N., and Wells B. 1968. Plastid structure and development in green callus tissues of Oxalis dispar. Annals of Botany, 32: 327-346.
22- Witomska M., and Lukaszewska A. 1997. Bulblet regeneration in vitro from different explants of Fritillaria imperialis. Acta Horticulturae, 430:331–338.