Effect of Hormonal Combination of Auxin and Cytokinin on Micropropagation of Eastern Lily (Lilium oriental hybrid ‘Casablanca’) Plant Using Bulb Scale Explant

Document Type : Research Article

Authors

• Nima Javaheri
• Behzad Kaviani

Department of Horticultural Science, Rasht Branch, Islamic Azad University, Rasht, Iran

https://doi.org/10.22067/jhs.2021.67900.1007

Abstract

Introduction
 Lily, a member of the genus Lilium, belonging to the Liliaceae family is one of the most important commercial pot and cut flower species and one of the three major bulb crops in the commercial market because of its large, colorful and fascinating flowers. Lily hybrids are the most economically important plants with varied flowers. Hybrid Eastern lily (Lilium oriental hybrid ‘Casablanca’) is a perennial bulbous plant that its propagation by bulb in natural condition is time-consuming, so produces 1–2 bulblets per bulb scale in one years’ time which is not sufficient for large scale cultivation of this plant. One of the most important and best methods for vegetative propagation and breeding of lilies is in vitro bulb scale culture. In vitro adventitious bud regeneration from scales of Lilium rely on many factors like cytokinin and auxin concentrations such as BA and NAA. The successful use of tissue culture techniques for rapid propagation of some species of the genus Lilium including L. ledebourii, L. orientalis, L. longiflorum, L. japonicum, L. speciosum, L. concolor, L. nepalense, L. regale, L. oriental hybrid, L. Asiatic hybrid has been reported. The purpose of the current study was to evaluate the effect of different concentrations of BA and NAA on in vitro proliferation of Lilium oriental hybrid ‘Casablanca’ using bulb scale as explant to establish a suitable protocol.
Materials and Methods
 Effect of various concentrations of 6-benzyle adenine (BA; 0, 0.5, 1 and 2 mg l^–1) and ɑ-naphtaleneacetic acid (NAA; 0, 0.1, 0.2 and 0.4 mg l^–1) were evaluated on in vitro proliferation of L. ‘Oriental’. Bulb scale as explant and MS basal medium as culture medium were used. Activated charcoal was applied to inhibit the browning of the culture medium and explant. The experiments were conducted in completely randomized design (CRD). The 16 treatments were applied, each treatment had 4 replications and each replication had 4 individuals. Therefore, in these experiments, a total of 192 bulbs were used. Traits including total plantlets fresh weight, leaf length, leaf number, bulblet weight, bulblet diameter, bulblet number, survival percentage, root length and root number related to in vitro proliferation were measured. All the statistical analyses were done by using SAS and Tukey’s test. Arcsin software was used for changing percent data.
Results and Discussion
 The interaction effect of BA and NAA was significant for all measured traits. Results showed that the maximum number of bulblet (8.66) and root (5.36) were obtained in culture medium enriched with 0.5 mg l^–1 BA together with 0.4 mg l^–1 NAA. Culture media supplemented with 0.5 mg l^–1 BA together with 0.2 mg l^–1 NAA and 1 mg l^–1 BA together with 0.1 mg l^–1 NAA with induction of 7.33 bulblets per explant were suitable media. The largest number of leaf (4.33) was measured in culture medium containing 1 mg l^–1 BA together with 0.1 mg l^–1 NAA. The highest bulblet weight was measured in culture medium supplemented with 1 mg l^–1 BA along with 0.2 mg l^–1 NAA. The greatest survival rate (100%) was observed in medium enriched with 0.5 mg l^–1 BA together with 0.1 mg l^–1 NAA. Survival rate (90%) in explants treated with 2 mg l^–1 BA along with 0.4 mg l^–1 NAA was high. Obtained results revealed that the presence of both BA and NAA in culture media for enhancement of most traits is necessary and critical. Plantlets were transferred to a growing medium containing cocopeat, peat moss and perlite in identical proportion for acclimatization following proliferation. Approximately, 90% of regenerated plantlets survived and were morphologically similar to the mother stocks. This study will help the producers and breeders for commercial and improvement purposes. The effective role of the simultaneous presence of both auxin and cytokinin in the culture medium in effective organogenesis was shown in the present study. Similar findings were reported for some lilies such as L. ledebourii (Baker) Bioss., L. longiflorum and L. regale. Auxin was effective in stimulating bulb production and growth of the aerial part of the eastern lily, and its presence along with cytokinin is essential for leaf induction. Some studies have reported similar results. The type and optimal concentration of plant growth regulators (PGRs) in the culture medium for suitable in vitro propagation varies in different species. Genetic variations (species type), differences in the amount of endogenous production of PGRs and their interaction with each other are among some reasons for this difference. The proper ratio of auxin and cytokinin in the culture medium is effective for inducing cell division, cell differentiation, organogenesis and finally for achieving a complete plant. Root production with appropriate quantity and quality leads to the suitable survival of seedlings resulting from the growth of cultured explants under in vitro conditions and adapted plants. Current study showed that the presence of both BA and NAA is better than the presence of one of these two PGRs for induction and growth of root. Some similar findings were reported, however in most studies, the presence of auxin as individual PGR has been found to be more suitable for root induction.
 

Keywords

• Bulbous plants
• Liliaceae
• Plant growth regulators
• Tissue culture

20.1001.1.20084730.1401.36.1.5.2

Main Subjects

• Breeding and Biotechnology of Plant and Flower

References

1. Azadi P., and Khush-khui M. 2007. Micropropagation of Lilium ledebourii (Baker) Boiss as affected by plant growth regulator, sucrose concentration, harvesting season and cold treatments. Electronic Journal of Biotechnology 10(4): 582–591.
2. Bacchetta L., Remotti P.C., Bernardini C., and Saccardo F. 2003. Adventitious shoot regeneration from leaf explants and stem nodes of Lilium. Plant Cell, Tissue and Organ Culture 74(1): 37–44.
3. Bahr L.R., and Compton M.E. 2004. Competence for in vitro bulblet regeneration among eight Lilium Horticulture Science 39: 127–129.
4. Chang C., Chen C.T., Tsai Y.C., and Chang W.C. 2000. A tissue culture protocol for propagation of a rare plant, Lilium speciosum var. glorisoides Baker. Botanical Bulletin of Academia Sinica 41(2): 139–142.
5. Han B.H., Suh E.J., Yae B.W., and Yu H.J. 2004. Micropropagation of Lilium longiflorum ‘Geogia’ by using bioreactor. Korean Journal of Plant Biotechnology 31(3): 197–201. (In Chinese with English abstract)
6. Han B.H., Yae B.W., Yu H.J., and Peak K.Y. 2004. Improvement of in vitro micropropagation of Lilium oriental hybrid ‘Casablanca’ by the formation of shoots with abnormally swollen basal plates. Scientia Horticulturae 103(3): 351–359.
7. Jeong J.H. 1996. In vitro propagation of bulb scale section of several Korean native lilies. Acta Horticulturae 414: 269–276.
8. Joshi S.K., and Dhar U. 2009. In vitro propagation from axenic explants of Lilium oxypetalum (D. Don) Baker, an endemic bulbous plant of high altitude Himalaya. Acta Physiologia Plantarum 31: 833.
9. Kaviani B. 2020. Conservation of ornamental plants in danger of extinction by biotechnological methods. Publications of Islamic Azad University, Rasht Branch, Iran. p. 187.
10. Kumar S., Chaudhary V., and Kanwar J.K. 2008. In vitro propagation of oriental hybrid lily from root explant. Advances in Horticultural Science 22: 63–65.
11. Kumar S., Kanwar J.K., and Sharma D.R. 2006. In vitro propagation of Lilium. Advances in Horticultural Science 20: 181–188.
12. Langens-Gerrits M.M., and De Klerk G.J.M. 1999. Micropropagation of flower bulbs. Plant Cell Culture Protocols, Springer, New York, pp 141–147.
13. Langens-Gerrits M.M., De Klerk G.J.M., and Croes A. 2003. Phase change in lily bulblets regenerated in vitro. Physiologia Plantarum 119: 590–597.
14. Lian M.L., Chakrabarty D., and Paek K.Y. 2002a. Growth and uptake of sucrose and mineral ions by bulblets of Lilium oriental hybrid ‘Casablanca’ during bioreactor culture. The Journal of Horticultural Science and Biotechnology 77(3): 253–257.
15. Lian M.L., Chakarabarty D., and Paek K.Y. 2003. Bulblet formation from bulbscale segments of Lilium using bioreactor system. Biologia Plantarum 46: 199–202.
16. Lian M.L., Chakarabarty D., and Paek K.Y. 2003a. Growth of Lilium oriental hybrid ‘Casablanca’ bulblet using bioreactor culture. Scientia Horticulturae 97: 41–48.
17. Ling X., Feng Wang M., and Dong L. 2009. Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor) Scientia Horticulturae 119: 458–61.
18. Mir J.I., Ahmed N., Itoo H., Sheikh M.A., Rashid R., and Wani S.H. 2012. In vitro propagation of Lilium (Lilium longiflorum). Indian Journal of Agricultural Sciences 82(5): 455–458.
19. Muhammad N., Ishrat N., Syed M.S.N., and Tariq M. 2013. Standardization of tissue culture conditions and estimation of free scavenging activity in Viola odorata Pakistan Journal of Botany 45(1): 197–202.
20. Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–479.
21. Nhut D.T. 1998. Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture. Plant Cell Reports 17(12): 913–916.
22. Nhut D.T. 2003. The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture and rapid propagation by using in vitro stem nodes. Plant Growth Regulation 40(2): 179–184.
23. Nhut D.T., Huong N.T.D., Le B.V., Da Silva J.T., Fukai S., and Tanaka M. 2002. The changes in shoot regeneration potential of protocorm-like bodies derived from Lilium Iongiflorum young stem explants exposed to medium volume, pH, light intensity and sucrose concentration pretreatment. The Journal of Horticultural Science and Biotechnology 77(1): 79–82.
24. Nhut D.T., Le B.V., Fukai S., Tanaka M., and Thanh V.K.T. 2001. Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture. Plant Growth Regulation 33 (1): 59–65.
25. Paek K.Y., and Murthy H.N., 2002. High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii. Plant Cell, Tissue and Organ Culture 68(3): 247–252.
26. Pelkonen V. 2005. Biotechnological approaches in lily (Lilium) production. University of Oulu, Oulu. Available from: http://herkules.oulu.fi/isbn9514276590/. ISBN 951-42-7659-0 (PDF). ISSN 0355-3191 http://herkules.oulu.fi/issn03553191.
27. Podwyszyńska M. 2012. The mechanisms of in vitro storage organ formation in ornamental geophytes. Floriculture and Ornamental Biotechnology 6: 9–23.
28. Robinson K.E., and Firoozabady E. 1993. Transformation of floriculture crops. Scientia Horticulturae 55: 83–99.
29. Roh M.S. 2011. Controlled flowering in the genus Lilium-review of the past achievements and the future direction of research. Acta Horticulturae 900: 189–203.
30. Taha S.L., Sayed S.S., Farahat M.M., and El-Sayed I.M. 2018. In vitro culture and bulblets induction of Asiatic hybrid Lily ‘red alert’. Journal of Biological Science 18: 84–91.
31. Varshney A., Dhawan V., Srivastava P.S. 2000. A protocol for in vitro mass propagation of Asiatic hybrids of lily through liquid stationary culture. In Vitro Cellular and Developmental Biology Plant 36(5): 383–391.
32. Wawrosch C., Malla P.R., and Kopp B. 2001. Clonal propagation of Lilium nepalense Don, a threatened medicinal plant of Nepal. Plant Cell Reports 20(4): 285–288.
33. Wu Y., Li Y., Ma Y.D., Zhang L., Ren Z.M., and Xia Y.P. 2016a. Hormone and antioxidant response of Lilium Oriental hybrid ‘Sorbonne’ bulblets to humic acid treatments in vitro. Journal of Horticultural Science and Biotechnology 92: 1–13.
34. Wu Y., Sun M.Y., Zhang J.P., Zhang L., Ren Z.M., Min R.H., Wang X.Y., Xia Y.P. 2019. Differential effects of paclobutrazol on the bulblet growth of Oriental lily cultured in vitro: growth behavior, carbohydrate metabolism, and antioxidant capacity. Journal of Plant Growth Regulation 38: 359–372.
35. Younis A., Hwang Y.J., and Lim K.B. 2014. Classical vs. modern genetic and breeding approaches for lily (Lilium) crop improvement: a review. Flower Research Journal 22: 39–47.
36. Youssef N.M., Shima A., Shaaba, Z.F.G., and Lobna S.T. 2019. In vitro bulb formation of direct and indirect regeneration of Lilium orientalis “Starfighter” plants. Bulletin of the National Research Centre 43: 211.