Effects of liquid, temporary immersion bioreactor and solid culture systems on micropropagation of Lilium ledebourii via bulblet microscales— An endangered valuable plant with ornamental potential

Document Type: Original Research Papers

Authors

1 Department of Plant Physiology, College of Sciences, University of Tehran, Tehran, Iran

2 Department of Horticultural Sciences, Faculty of Agriculture, University of Tehran, Karaj, Iran

Abstract

Lilium ledebourii (Baker) Boiss. (Liliaceae) is a critically endangered lily species native to
northern Iran, where it is protected by law. In order to develop a cost effective method for largescale
propagation, the effects of three culture systems (solid, liquid and temporary immersion)
and two types of cytokinins [6-Benzyladenine (BA) and Thidiazuron (TDZ)] were studied on
the in vitro plant regeneration of L. ledebourii. To establish the protocol, we used in vitro
regenerated bulblets obtained from bulb scale segments that were cultured on solid Murashige
and Skoog (MS) media as starting material. The bulblet microscale transverse thin cell layers
were cultured on MS solid medium containing 3% sucrose and different combinations of plant
growth regulators. Choice of both, the culture system and the type of cytokinin, affected the
differentiation of explants. Two types of calli formed on explants: type I callus was
embryogenic, while type II callus was shoot organogenesis. The highest percentage (94%) of
embryogenic callus was obtained when calli were transferred on MS solid media supplemented
with 0.54 μM α-Naphthaleneacetic acid (NAA) and 0.44 μM BA. In addition, it was also
observed that the use of temporary immersion bioreactor resulted in a significantly lower
amount of shoot organogenesis rather than solid culture systems. Seventy percent of the
plantlets were successfully acclimatized to ex–vitro conditions and were phenotypically similar
to the mother plants.

Keywords

Main Subjects


1. Azadi, P. and Khosh-Khui, M. (2007) Micropropagation of Lilium ledebourii (Baker) Boiss as
affected by plant growth regulator, sucrose concentration, harvesting season and cold treatments.
Electron J Biotechnol, 10:582–591. doi:10.2225/vol10-issue4-fulltext-7.
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 Tiss Org. 74:37–44.
3. Bakhshaie, M., Babalar, M., Mirmasoumi, M. and Khalighi, A. (2010a) Somatic embryogenesis
and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species. Plant Cell Tiss
Org. 102:229–235. doi: 10.1007/s11240-010-9726-4.
4. Bakhshaie, M., Babalar, M., Mirmasoumi, M. and Khalighi, A. (2010b) Effects of light, sucrose,
and cytokinins on somatic embryogenesis in Lilium ledebourii (Baker) Bioss. via transverse thin
cell-layer cultures of bulblet microscales. J Hortic Sci Biotech, 85:491–496.
5. Chen, J. and Ziv, M. (2001) The effect of ancymidol on hyperhydricity, regeneration, starch and
antioxidant enzymatic activities in liquid-cultured Narcissus. Plant Cell Rep., 20:22–27.
doi:10.1007/s002990000283
6. Escalona, M., Lorenzo, J.C., Gonzalez, B., Daquinta, M., Gonzalez, J.L., Desjardins, Y. and
Borroto, C.G. (1999) Pineapple (Ananas comosus L. Merr) micropropagation in temporary
immersion systems. Plant Cell Rep., 18:743–748. doi:10.1007/s002990050653.
7. Ghahreman, A. (1997) Flora of Iran. Research Institute of Forests and Rangelands, Iran. Volume
16, No. 1944. Code 137, 001, 001.
8. Godo, T., Kobayashi, K., Tagami, T., Matsui, K. and Kida, T. (1998) In vitro propagation
utilizing suspension cultures of meristematic nodular cell clumps and chromosome stability of
Lilium × formolongi hort. Sci Hortic-Amsterdam, 72: 193–202. doi:10.1016/S0304-
4238(97)00126-X.
9. Godo, T., Matsui, K., Kida, T. and Mii, M. (1996) Effect of sugar type on the efficiency of plant
regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of
Lilium × formolongi hort. Plant Cell Rep., 15:401–404. doi:10.1007/BF00232063.
10. Han, B.H., Yae, B.W., Hee, J.Y. and Peak, K.Y. (2005) Improvement of in vitro
micropropagation of Lilium oriental hybrid ‘Casablanca’ by the formation of shoots with
abnormally swollen basal plates. Sci Hortic-Amsterdam, 103: 351–359.
doi:10.1016/j.scienta.2004.07.003
11. Han, B.H., Yu, H.J., Yae, B.W. and Peak, K.Y. (2004) In vitro micropropagation of Lilium
longiflorum ‘Georgia’ by shoot formation as influenced by addition of liquid medium. Sci Hortic-
Amsterdam, 103:39–49. doi:10.1016/j.scienta.2004.04.020.
12. Ho, C.W., Jian, W.T. and Lai, H.C. (2006) Plant regeneration via somatic embryogenesis from
suspension cell cultures of Lilium × formolongi Hort. using a bioreactor system. In Vitro Cell
Dev-PL 42:240–246. doi:10.1079/IVP2006756.
13. Jin, S., Wang, J., Wang, X., Sun, D., Li, G., Genovesi, A.D. and Liu, S. (2014) Direct and indirect
shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum, an endangered
species. In Vitro Cell Dev-PL 50:69–75. doi: 10.1007/s11627-013-9573-1. 

14. Kakani, A., Li, G.S. and Peng, Z. (2009) Role of AUX1 in the control of organ identity during in
vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in
Arabidopsis. Planta 229:645–657. doi:10.1007/s00425-008-0846-6.
15. Lian, M.L., Chakrabarty, D. and Paek, K.Y. (2003a) Bulblet formation from bulbscale segments
of Lilium using bioreactor system. Biol Plantarum, 46:199–203.
16. Lian, M.L., Chakrabarty, D. and Paek, K.Y. (2003b) Growth of Lilium Oriental Hybrid
'Casablanca' bulblet using bioreactor culture. Sci Hortic-Amsterdam, 97:41–48.
doi:10.1016/S0304-4238(02)00086-9.
17. LingFei, X., FengWang, M. and Dong, L. (2009) Plant regeneration from in vitro cultured leaves
of Lanzhou lily (Lilium davidii var. unicolor). Sci Hortic-Amsterdam, 119:458–461.
doi:10.1016/j.scienta.2008.08.026.
18. Malik, M. (2008) Comparison of different liquid/solid culture systems in the production of
somatic embryos from Narcissus L. ovary explants. Plant Cell Tiss Org., 94:337–345.
doi:10.1007/s11240-008-9415-8.
19. Mii, M., Yuzawa, Y., Suetomi, H., Motegi, T. and Godo, T. (1994) Fertile plant regeneration
from protoplasts of a seed-propagated cultivar of Lilium × formolongi by utilizing meristematic
nodular cell clumps. Plant Sci., 100:221–226. doi:10.1016/0168-9452(94)90079-5.
20. Mirmasoumi, M., Azadi, P., Sharafi, A., Ntui, V.O. and Mii, M. (2013) Simple protocol for plant
regeneration of Lilium ledebourii using transverse thin cell layer. Progress in Biological Sciences,
3: 117-122
21. Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol Plantarum, 15:473–497. doi:10.1111/j.1399-
3054.1962.tb08052.x.
22. Nakano, M., Sakakibara, T., Suzuki, S. and Saito, H. (2000) Decrease in the regeneration
potential of long-term cell suspension cultures of Lilium formosanum Wallace and its restoration
by the auxin transport inhibitor 2,3,5-triiodobenzoic acid. Plant Sci., 158:129–137.
doi:1016/S0168-9452(00)00313-7.
23. Nhut, D.T., Bui, V.L., Minh, N.T., Teixeira da Silva, J.A., Fukai, S., Tanaka, M. and Tran Thanh
Van, K. (2002) Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of
Lilium longiflorum. Plant Growth Regul, 37:193–198. doi:10.1023/A:1020532511081.
24. Nhut, D.T., Bui, V.L., Tanaka, M. and Tran Thanh Van, K. (2001a) Shoot induction and plant
regeneration from receptacle tissues of Lilium longiflorum. Sci Hortic-Amsterdam, 87:131–138.
doi:10.1016/S0304-4238(00)00163-1.
25. Nhut, D.T., Bui, V.L. and Tran Thanh Van, K. (2001b) Manipulation of the morphogenetic
pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin. In vitro
Cell Dev-PL., 37:44–49. doi:10.1007/S11627-001-0009-y.
26. Nhut, D.T., Hanh, N.T.M., Tuan, P.Q., Nquyet, L.T.M., Tram, N.T.H., Chinh, N.C., Nguyen,
N.H. and Vinh, D.N. (2006) Liquid culture as a positive condition to induce and enhance quality
and quantity of somatic embryogenesis of Lilium longiflorum. Sci Hortic-Amsterdam, 110:93–97.
doi:10.1016/j.scienta.2006.05.015. 

27. Paek, K.Y., Chakrabarty, D. and Hahn, E.J. (2005) Application of bioreactor systems for large
scale production of horticultural and medicinal plants. Plant Cell Tiss Org., 81:287–300.
doi:10.1007/s11240-004-6648-z.
28. Paek, K.Y., Hahn, E.J. and Son, S.H. (2001) Application of bioreactors for large-scale
micropropagation systems of plants. In vitro Cell Dev-PL., 37:149–157. doi:10.1007/s11627-001-
0027-9.
29. Ptak, A. and Bach, A. (2007) Somatic embryogenesis in tulip (Tulipa gesneriana L.) flower stem
cultures. In vitro Cell Dev-PL.,43:35–39. doi:10.1007/s11627-006-9004-7.
30. Rechinger, K.H. (1989) Flora Iranica. No. 165. Liliaceae, Akademische Druck-u, Verlgsantalt,
Graz, Austria. pp. 58–59.
31. Sage, D.O., Lynn, J. and Hammatt, N. (2000) Somatic embryogenesis in Narcissus
pseudonarcissus cvs. Golden Harvest and St. Keverne. Plant Sci., 150:209–216.
doi:10.1016/S0168-9452(99)00190-9.
32. Sankar-Thomas, Y.D., Saare-Surminski, K. and Lieberei, R. (2008) Plant regeneration via
somatic embryogenesis of Camptotheca acuminata in temporary immersion system (TIS). Plant
Cell Tiss Org., 95:163–173. doi:10.1007/s11240-008-9428-3.
33. Shen, X.L., Chen, J.J. and Kane, M.E. (2007) Indirect shoot organogenesis from leaves of
Dieffenbachia cv. Camouflage. Plant Cell Tiss Org., 89:83–90. doi:10.1007/s11240-007-9214-7.
34. Snyman, S.J., Nkwanyana, P.D. and Watt, M.P. (2011) Alleviation of hyperhydricity of
sugarcane plantlets produced in RITA® vessels and genotypic and phenotypic characterization of
acclimated plants. S Afr J Bot., 77:685–692. doi:10.1016/j.sajb.2011.03.004.
35. Takayama, S. and Akita, M. (2005) Practical aspects of bioreactor application in mass
propagation of plants. In: Hvoslef- Eide AK, Preil W (eds) Liquid culture systems for in vitro
plant propagation, pp. 61-78. Springer, Dordrecht. doi:10.1007/1-4020-3200-5_4.
36. Teng, W.L. and Ngai, Y.W. (1999) Regeneration of Oxalis triangularis ssp. triangularis from
suspension cells cultured in three different systems (solid, liquid-flask and bioreactor cultures).
Plant Cell Rep., 18:701–706. doi:10.1007/s002990050646.
37. Thakur, R., Sood, A., Nagar, P.K., Pandey, S., Sobti, R.C. and Ahuja, P.S. (2006) Regulation of
growth of Lilium plantlets in liquid medium by application of paclobutrazol or ancymidol, for its
amenability in a bioreactor system: growth parameters. Plant Cell Rep., 25:382–391.
doi:10.1007/s00299-005-0094-1.
38. Tribulato, A., Remotti, P.C., Loffler, H.J.M. and Van Tuyl, J.M. (1997) Somatic embryogenesis
and plant regeneration in Lilium longiflorum Thunb. Plant Cell Rep., 17:113–118.
doi:10.1007/s002990050362.
39. Wang, W.G., Zhao, X.G., Zhuang, G.Q., Wang, S.H. and Chen, F. (2008) Simple hormonal
regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of
Pogonatherum paniceum (Poaceae). Plant Cell Tiss Org., 95:57–67. doi:10.1007/s11240-008-
9414-9.
40. Wawrosch, C., Kongbangkerd, A., Kopf, A. and Kopp, B. (2005) Shoot regeneration from
nodules of Charybdis sp.: a comparison of semisolid, liquid and temporary immersion culture
systems. Plant Cell Tiss Org., 81:319–322. doi:10.1007/s11240-004-6660-3.

41. Wawrosch, C., Malla, P.R. and Kopp, B. (2001) Clonal propagation of Lilium nepalense D. Don,
a threatened medicinal plant of Nepal. Plant Cell Rep., 20:285–288. doi:10.1007/s002990100322
42. Wendelbo, P. (1977) Tulips and Irises of Iran and their Relatives. Botanical Institute of Iran,
Tehran, Iran. pp. 88
43. Zeng, F.C., Zhang, X.L., Jin, S.X., Cheng, L., Liang, S.G., Hu, L.S., Guo, X.P., Nie, Y.C. and
Cao, J.L. (2007) Chromatin reorganization and endogenous auxin/cytokinin dynamic activity
during somatic embryogenesis of cultured cotton cell. Plant Cell Tiss Org., 90:63–70.
doi:10.1007/s11240-007-9253-0.