Effect of Cultivar and Plant Growth Regulators on Invitro Regeneration of Lilium Spp. Utilizing Thin Cell Layer Explants

Document Type : Research Article

Authors

• Ahmad Sharifi ¹
• Fatemeh Keykha Akhar ¹
• Mahboobeh Yazdi ²
• Abdolreza Bagheri ²

¹ Academic Center for Education, Culture and Research, Branch of Khorasan Razavi

² Ferdowsi University of Mashhad

https://doi.org/10.22067/jhorts4.v31i3.55475

Abstract

Introduction: Lily (Lilium spp.) is a genus of herbaceous flowering plants, which consisted of many beautiful ornamental species with large prominent flowers. Most species are native to the Northern hemisphere temperate, though their range extends into the Northern subtropics. Some specific hybrids of Lilium spp. have been developed as cut flower in controlled conditions and in some cases can be grown as pot plant. Propagation rate of lily in natural clonal propagation methods is very low and one year produces of 1-2 bulblets per bulb scale. There is also possibility of disease transmission; so that, tissue culture techniques has provided an efficient method for its micropropagation.
Materials and Methods: In this study, two separate experiments under In vitro conditions the bulblet regeneration from thin cell layer (TCL) explants of Lilium spp. was investigated. In the first experiment, after two months the effect of TCL explants with 1, 3 and 5 mm thickness on MS medium containing 1 mg/l BA, 2ip and kin in combination with 0.5 mg/l NAA on regeneration parameters were assayed. In the second experiment to determine the effect of cultivar and cytokinin types, 3mm thickness TCL explants of five cultivars (Robina, Donato, Nymph, Lessoto and Roxana) were tested on MS medium containing different plant growth regulator (PGR) compounds including BA, kin, 2ip and TDZ at concentration of 1 mg/l in combination with 0.5 mg/l NAA. The regeneration parameters were assayed after four months. In all experiments, the medium was adjusted to pH 5.8 and autoclaved at 121°C for 15 min. All cultures were incubated at 25 ± 2°C with a 16 h photoperiod under cool white flourescent lights (30 µmol/m2).
Results and Discussion: According to the first experiment results, plant growth regulator of BA in all of surveyed parameters except root number was better than other PGRs and explants with 3 mm thickness was the best in all of parameters. The interaction of PGR and explants was significant, however maximum bulblet regeneration was observed in TCL explants with 3 mm thickness in all of PGR treatments (100%). While 1 mm thickness TCL in 2ip and 1 and 5 mm thickness TCL in Kin had the least regeneration percentage. Results revealed that the interaction of explants and medium is a key factor for suitable establishment, regeneration and growth of TCLs. Bulb dormancy is one of the limiting factors in regeneration of bulbous crop species. It seems under In vitro condition explants size and PGR combination of media especially cytokinin affected on breaking of dormancy. Maximum number of leaves and dry weight of bulblets in medium containing BA was significantly higher compared with other treatments. Most of studies confirmed the positive effect of BA on regeneration of lily. The function of cytokinin in plant promoted cell division and differentiation, which lead to growth and maintaining cells in meristematic status.
Result of second experiment showed that cultivar was one of the effective factors on regeneration trait. Oriental lily cultivar "Roxana" had the highest number of roots, bulblets, dry weight and length of plantlets and "Nymph" cultivar showed the lowest percentage of regeneration, dry weight, length of plantlets and rooting obtained. In all of cultivars BA induced more organogenesis percentage and plantlet dry weight, while TDZ induced more rooting percentage.The interaction of cultivar and PGR treatments on percentage of regenerated bulblets and rooting were significant. "Nymph" cultivar had minimum percentage of regeneration and rooting in medium containing TDZ and Kin. Furthermore, "Roxana" cultivar in medium containing BA showed the best dry weight comparison to other treatments.
Conclusion Lily has widely used in the floral industry as a cut flower or potted plant. In recent years, tissue culture was developed as reliable and highly effective method to overcome its limitations of vegetative propagation. The most advantage of this method is high multiplication rate and disease free propagation. In this study, bulblet regeneration of lilium Spp. from TCL explants under in vitro condition was considered as a highly efficient procedure for its micropropagation. With optimization of TCL system some parameters such as exogenously applied plant growth regulators, cultivar, explants types were investigated. Favorable conditions for bulblet regeneration were achieved with 3 mm thickness TCLs in MS medium containing 1 mg/l BA with 0.5 mg/l NAA. This protocol can be used for rapid micropropagation of many cultivars.

Keywords

• Auxin
• Bulb explant
• Cytokinin
• In vitro culture
• Micropropagation

20.1001.1.20084730.1396.31.3.4.0

References

1- Anderson N.O. 2007. Flower Breeding & Genetics (Issues, Challenges and Opportunities for the 21st Century). Springer Science & Business Media, Netherlands.

2- Azadi P., and Mojtahedi N. 2009. Effect of growth regulators, sucrose concentration and scale segment on micropropagation of Lilium ledebourii in winter harvest bulbs. In Agronomy and Horticulture.

3- Bacchetta L., Remotti P.C., Bernardini C., and Sccardof F. 2003. Adventitious shoot regeneration from leaf explants and stemnodes of lilium. Plant Cell Tissue Culture, 74: 37-44.

4- Bui V. L., Nhut D. T.,and Tran Thanh Van K. 1999. Plant production via shoot regeneration from thin cell layer pseudo-bulblet explants of Lilium longiflorum in vitro. Comptes Rendus Acadmic Science Paris, 322:303-310.

5- Hohtola A., Haggman H., Bach A., and Tahvonen R. 2005. Biotechnological Approaches in Lily (Lilium) Production. Oulu University Press.

6- Kapoor R., Kumar S., and Kanwar J. K. 2008. Bulblet production from node explants grown in vitro in hybrid lilies. International Journal of Plant Production, 3: 125- 134.

7- Khanghaei L. 2013. Study of explants position, culture medium type and plant growth regulators effects on lilium’s bulbs (Oriental and OT Hybrids) regeneration In Vitro. Master’s thesis, Islamic Azad University of Mashhad. (in Persian with English abstract)

8- Kumar S., Awashthi V., and Kanwar J.K. 2007. Influence of growth regulators and nitrogenous compounds on in vitro bulblet formation and growth in oriental lily. Horticultural Science, 34:77-83.

9- Lojic M., Vinterhalter B., Subotic A., and Vinterhalter D. 2015. Differences in regenerative capacity of Oriental lily (Lilium spp.) cultivars. Botanica Serbica, 39:159-167.

10- Mengli X., Sun L., Qiu Sh., Liu J., and Xu J. 2012. In vitro mutagenesis and identification of mutant via ISSR in lily (Lilium lingiflorum). Plant Cell Reports, 31: 1043-1051.

11- 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.

12- Mojtahedi N., and Azadi P. 2009. Comparision of in vitro bulblet production in two comercial lilium cultivars, lilium longiflorum cv. Gironde and lilium longiflorum cv. Cassandra. Journal of seed, 24: 721-738 (in persian).

13- Nhut D.T., Van Le B., Fukai S., Tanaka M., and Van K.T.T. 2001a. 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, 33:59-65.

14- Nhut D.T., Le B.V., Teixeira Da Silva J.A., and Aswath C. R. 2001b. Thin cell layer culture system in lilium: regeneration and transformation Perspectives. In Vitro Cellular and Developmental Biology - Plant, 37:516-523.

15- Nhut D.T., Van Le B., Tran Thanh Van K. 2003. Manipulation of morphogenetic pathways of lilium longiflorum transverse thin cell layer explants by auxin and cytokinin. In Vitro Cellular and Developmental Biology-Plants, 37: 44-49.

16- Niimi Y., Han D.S., and Makoto F.2001. Production of virus-free plantlets by anther culture of Lilium x 'Enchantment'. Scientia Hortic, 90: 325-334.

17- Paric A., Cakar J., Muratovic E., and Kralija B. 2011. Induction of bulblets on leaf and bulb explants of endangered lilium bosnicum (G. Beck) ex Fritsch. Botanica Serbica, 35:31-35.

18- Thakur R., Sood A., and Nagar P. 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 Reports, 25: 382-391.

19- Teixeira da Silva J.A. 2003. Thin Cell Layer technology in ornamental plant micropropagation and biotechnology. African Journal of Biotechnology, 12: 683-691.

20- Van Tuyl J.M., and Van Holsteijn H.M.C. 1996. Lily breeding research in the Netherlands. Acta Horticulture, 414: 35-46.