Elsevier

Plant Science

Volume 166, Issue 6, June 2004, Pages 1443-1449
Plant Science

Callus formation and plant regeneration from callus through somatic embryo structures in Cymbidium orchid

https://doi.org/10.1016/j.plantsci.2004.01.023Get rights and content

Abstract

Embryogenic calli were induced from longitudinally bisected segments of protocorm-like bodies (PLBs) of Cymbidium Twilight Moon ‘Day Light’, a hybrid orchid, on modified Vacin and Went medium [Bot. Gaz. 110 (1949) 605] supplemented with 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) within 1 month. The medium containing the combination of 0.1 mg l−1 NAA and 0.01 mg l−1 TDZ was optimal for callus formation. Calli proliferated very well when being subcultured every 4 weeks on the same medium. The PLB formation from callus was achieved when callus was transferred to the medium without plant growth regulators. Histological observation proved the somatic embryo structure formation. Callus-derived PLBs converted into normal plants with well-developed shoots and roots on the medium without plant growth regulators after about 4 months, which were acclimatized in the greenhouse with 100% survival rate. Among 103 twenty-month-old regenerated plants, no morphological variations were observed.

Introduction

Cymbidium is a genus of about 50 species from Asia. Most of the cultivated Cymbidiums are hybrids [1]. Many attractive hybrids of Cymbidium orchid have become commercially important in cut flower and potted plant industries. Cymbidium was the first orchid genus to be propagated using shoot-tip culture by Morel [2]. Since then, there are many reports on micropropagation of Cymbidium using shoot-tip culture or protocorm-like bodies as explants [3], [4], [5], [6], [7], [8]; however, there are very few reports on callus cultures in Cymbidium as well as other orchids, this might be due to the slow growth and a necrotic tendency of orchid callus [9], [10], [11], [12], [13]. Begum et al. [13] reported that globular compact calli were induced from inner tissue of Cymbidium PLB, but these structures could not be subcultured, turned brown and died after 2 months of incubation. Chang and Chang [14] successfully obtained calli from pseudobulbs, rhizomes, and root explants and maintained these in subculture as well as regenerated plants from callus in Cymbidium ensifolium, a terrestrial orchid species; however, this study did not focus on callus induction from explants. Plant regeneration from callus of orchid is usually achieved through PLB formation, a process that is suggested to involve somatic embryogenesis [13], [15], [16], [17]. However, clear evidence of somatic embryogenesis in callus cultures of orchid is still limited.

This study presented optimal conditions for callus formation from PLB segments, callus proliferation, and plant regeneration from callus in Cymbidium Twilight Moon ‘Day Light’.

Section snippets

Plant material and culture condition

Protocorm-like bodies (PLBs) of Cymbidium Twilight Moon ‘Day Light’ originated from shoot-tip culture were subcultured every 2 months on modified Vacin and Went medium [18] supplemented with 0.1 mg l−1 1-naphthaleneacetic acid (NAA) and 0.1 mg l−1 kinetin, and solidified with 8 g l−1 Bacto agar (Difco Laboratories, USA). PLBs were cut tops and then longitudinally bisected. The PLB segments were used as explants for callus induction.

Modified Vacin and Went culture medium [18] supplemented with 1 ml l−1

Effects of plant growth regulators on callus formation from PLB segments

PLB segments were cultured on media containing different combinations of NAA, 2,4-D, and TDZ for callus induction. The basal medium without plant growth regulators was used as a control. Yellow and granular calli were formed from PLB segments within 1 month (Fig. 1). While there was no callus formation in the control, calli were formed from PLB segments in most treatments with different frequencies (Table 1, Table 2). The highest frequency of callus formation from PLB segments was observed on

Acknowledgements

The authors wish to thank Prof. K. Tran Thanh Van, Institut de Biotechnologie des Plantes, Universite de Paris-Sud, France for critical reading the manuscript and valuable suggestions.

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