The Effect of Carvacrol, Thymol, BA and NAA on in vitro Regeneration in Fritillaria imperialis L

Document Type : Research Article

Authors

• Esmaeil Chamani ¹
• Zahra Eftekhari ¹
• Alireza Ghanbari ¹
• Hamid Reza Heydari ¹
• Mousa Arshad ²

¹ University of Mohaghegh Ardabili

² Islamic Azad university of Mahabad

https://doi.org/10.22067/jhorts4.v32i2.31740

Abstract

Introduction: Fritillaria imperialis L. is an ornamental and medicinal plant native to mountainous regions of Iran. This plant genetic resource is in danger of extinction, Because of grazing livestock and pest outbreaks. Therefore, micro propagation of Fritillaria through in vitro regeneration is essential for conservation and commercial production. Thymol and Carvacrol are one of the main essential oil compounds in family Lamiaceae.
Material and Methods: Fritillariaimperialis L. bulbs in dormancy stage obtained from mountainous regions of Lorestan in Iran and were placed in cold room at +4 °C for 4-6 weeks. Then, Bulbs were surface-sterilized with 70% ethanol for 45 s followed by immersion in 5% (v/v) NaOCl solution for 20 min with gentle agitation, and then rinsed three times in sterile double distilled water. Present study was conducted in two separate experiments. In first experiment, effect of different concentration of Thymol and Carvacrol and in second experiment, different concentration of NAA and BA on in vitro characteristics of Fritillaria was evaluated. Explants (1× 1 cm) prepared from the lower third of scales with basal plate and were placed in MS basal medium supplemented with different concentrations of Thymol (50, 100, 150 and 300 ppm), Carvacrol (10, 100, 500 and 100 ppm), BA (1, 2 and 4 mg/l) and NAA (1, 2 and 4 mg/l).All cultures were incubated in a growth chamber at 24±2°C, and a photosynthetic photon flux of 40-60 μmol m–2 s–1 was provided by cool white fluorescent lamps with a 16-h photoperiod. This experiment wascarried out in completely randomized designs with fivereplications.
Results and Discussion: Analysis of variance showed that Thymol and Carvacrol were not effective on number of new bulblets but had significant effects on bulb diameter, number and length or roots, number and length leaves and callus induction and diameter of callus obtained from scales (P< 0.05). The highest rate (3 bulblets) of bulblets formation was obtained fromMS medium supplemented with 50 ppm Thymol that showed significantly difference from other treatments. Medium containing 10 ppm Carvacrol gave the highest Bulblet formation (2.5 bulblets) between Carvacrol treatments. Investigation of rooting was done by assessment of the number and length of roots. Mean comparison of the effect of cultivar type on root number showed that the largest number of roots per explant was obtained fromMS medium containing 50 ppm Thymol. Lowest number of roots observed in mediums supplemented with 300 ppm Thymol and 100 ppm Carvacrol. The best medium for increasing the root length per explant (10.90 cm) was MS medium supplemented with 100 ppm Carvacrol, while the least increasing in root length per explant observed from culture mediums contained 300 ppm Thymol and 100 ppm Carvacrol. Also, the largest number of leave formation obtained from culture medium supplemented with 50 ppm Thymol that significantly higher than other treatments. Statistical analysis (ANOVA) of the data showed that high frequency callus induction and formation occurred in MS mediums contained 50, 100 and 150 ppm Thymol and 10 ppm Carvacrol and culture mediums supplemented with 300 ppm Thymol and 1000 ppm Carvacrol showed least callus induction. In contrast, largest callus diameter observed in culture mediums supplemented with 300 ppm Thymol and 500, 100 ppm Carvacrol.
Statistical analysis of results showed that different concentrations of BA and NAA had significant effects on bulblets number and bulblets diameter (P

Keywords

• Bulblet
• Callus
• Essential oil
• In vitro culture
• Plant growth regulator

20.1001.1.20084730.1397.32.2.1.2

References

1. Alizadeh Ajirlo S., and Kargar M. 2016. Effect of Clone and Growth Regulators on Callus and Bulblet Formation Using Daughter Bulb Explants of Fritillary (Fritillaria imperialis L.). Plant Production, 39(2): 81-94.

2. Arora R., and Bhojwani S. 1986. In vitro propagation and low temperature storage of Saussurea lappa C.B. Clarke-anxiety endangered medicinal plant. Plant Cell Reports. 8: 44- 47.

3. Bryan J. 1989. Bulbs. Portlands: Timber press. Oregon, 451pp.

4. Buchanan R.L., and Shepherd A.J. 1981. Inhibition of Aspergillus parasiticus by thymol. Journal of Food Science. 46: 976- 977.

5. Deen W., Thepsithar C., and Thongpukdee A. 2013. In vitro culture medium sterilization by chemicals and essential oils without autoclaving and growth of chrysanthemum nodes. World academy of Science, Engineering and Technology, 7: 6-24.

6. De Hertogh A., Lenard M. 1993. The physiology of flower bulbs. Elsevier. P. 718- 739.

7. Ebrahimie E., Mohammadie-Dehcheshmeh M., Habashi A.A. Ghannadha, M.R., Ghareyazie B., Yazdi-samadi B. 2006. “Direct shoot regeneration from cumin, Cuminum cyminum L”. Embryo, 54-58.

8. Gouran, A., Mozafari A., and Ghaderi N. 2012. The effect of antimicrobial agents on the surface sterilized grape explants in in vitro culture. 6th congress of the Agricultural Research findings. Kordestan University.

9. Kharrazi M., Nemati H., Tehranifar A., Bagheri A., Sharifi A. 2011. In vitro culture of carnation (Dianthus caryophyllus L.) focusing on the problem of Vitrification. Journal of Environmental Biology Sciences. 5 (13): 1-6.

10. Metzger J. 1986. “Hormones and reproductive development”. In: Plant growth and development, ed. Davis, P. J. Martinus Nigh off, Boston.

11. Mohammadi Dehcheshmeh M. 2005. The use of tissue culture technique in Fritillaria endangering species reproduction of native of Iran. MSc Thesis, Tehran University.

12. Nhut D. T. 1998. Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture. Plant Cell Reports. 17: 913-916.

13. Ohkawa M., Kitajima J., Nishino K., and Ohkawa N. 1999. Studies on the Propagation of Fritillaria camtschatcensis Ker-Gawl. by In Vitro Culture II: Effect of Plant Hormones on the Growth, Leaf Emergence, and Rooting of Bulblets. Journal of the Japanese Society for Horticultural Science, 68: 184-188.

14. Paek K.Y., Murthy, H.N. 2002. High frequency of Bulblet regeneration from bulb scale section of Fritillaria thunbergii. Plant Cell, Tissue and Organ Culture, 68: 247-252.

15. Pierik R. L. M. 1998. In vitro culture of higher plants. Ferdowsi university press. 406p.

16. Pierik R. L. M. and Steegmans, H. H. M. 1975. Effect of auxins, cytokinins, gibberellins, abscisic acid and etephon on regeneration and growth of bulblets on excised bulb scale segments of hyacinth. Journal of Plant Physiology. 34: 14-17.

17. Podwyszynska M. 2006. Improvement of bulb formation in micropropagted tulips by treatment with NAA and Paclobutrazol or accymidol. Acta Horticulturae. 725: 679-684.

18. Rix E.M. 1977. Fritillaria L. (Liliaceae) in Iran. Journal of Botany. 1, pp. 75–95.

19. Stimart D. J. and Ascher D. D. 1978. Tissue culture of bulb scale sections for asexual propagation of Lilium longiflorum. Journal of the American Society for Horticultural Science. 103: 182-184.

20. Solg M. 2014. Evaluation of plant-mediated Silver nanoparticles synthesis and its application in postharvest Physiology of cut Flowers. Physiology and Molecular Biology of Plants, 20(3): 279–285.

21. Solgi M., and Taghizadeh M. 2014. The Application of Essential Oils and Silver Nanoparticles for Sterilization of Bermuda grass Explants in in vitro Culture. International Journal of Horticultural Science and Technology, 1(2): 131-140.

22. Takayama S., and Misawa M. 1979. Differentiation in Lilium, bulb scales grown in vitro. Effectof various cultural conditions. Physiologia Plantarum, 46(2): 184-190.

23. Takayama, S. and Misaula, M. 1982. Regulation of organ formation by cytokinin and auxin in Lilium bulbscales growth in vitro. Plant and cell physiol. 23(1): 67-74.

24. Tatari M., Fotouhi Ghazvini R., Hamidoghli Y. 2004. Influence of growth regulators and explants of Chandelie lily scales in vitro culture (Lilium ledebourii). Journal of Agricultural Sciences, 1(2): 19-28.

25. Tefera W., Wannakrairoj S. 2006. Synergistic effects of some plant growth regulators on in vitro shoot proliferation of korarima (Aframomum corrorima). African Journal of Biotechnology, 5: 1894-1901.