Somatic embryogenesis from stem thin cell layers of Dendrobium aqueum

Abstract

An efficient in vitro regeneration protocol through somatic embryogenesis was established from stem transverse thin cell layers (tTCLs) of Dendrobium aqueum Lindley, an imperiled orchid. This study outlines the induction and successive maturation stages of D. aqueum somatic embryos (SEs). The tTCLs (~ 0.5 mm thick) cultured on halfstrength Murashige and Skoog (MS) medium containing cytokinins and auxins, either individually or in combination, produced embryogenic callus (EC). Treatment with 0.5 mg dm -3 zeatin induced EC in 41.42 % of tTCLs. As many as 42.66 globular SEs per tTCL were formed in the presence of 1.5 mg dm -3 N 6 -(2-isopentyl) adenine (2iP) but only on 10.33 % of explants. The combined treatment of 2iP (1.5 mg dm -3 ) and 0.5 mg dm -3 6-benzyladenine resulted in 34 globular SEs on 14.7 % of tTCLs whereas the combination of 2iP and 1.0 mg dm -3 indole-3-butyric acid (IBA) induced 7.4 globular SEs on 52.33 % of tTCLs. Supplementation of activated charcoal, amino acids, and antioxidants alleviated browning at all the concentrations tested, but the EC response declined. The addition of 0.5 mg dm -3 polyvinylpyrrolidone to 1.5 mg dm -3 2iP and 1.0 mg dm -3 IBA produced 24 SEs on 19.89 % of tTCLs suggesting that the EC and SEs can be effectively induced by individual cytokinins whereas the synergistic treatments with other compounds can only enhance the induction of EC. Histological observations of EC showed the formation of globular SEs from sub-epidermal regions. Successive developmental stages of globular SEs and the intermediate stage of protocorm like bodies until the formation of plantlets were observed. The plantlets obtained through SEs showed no morphological variations, and inter simple sequence repeat profiles also confirmed the genetic fidelity of in vitroderived progeny with high monomorphism (97.78 %). In conclusion, the use of stem tTCLs is an effective method to produce SEs through indirect somatic embryogenesis in D. aqueum.

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding

“…In Dendrobium aqueum, only the cytokinin 2iP [N-6-(2-isopentyl) adenine] at 1.5 mg L −1 proved it efficiency in production of PLBs (42.7 PLBs per explants) from callus, compared to other cytokinins BA, Kin and Zea, and cytokinin-like compound TDZ. These authors also observed that arginine at 25 mg L −1 increased direct somatic embryogenesis, instead of callus derived PLBs [137]. Meta-Topolins, a natural aromatic type of cytokinin, were also reported used in induction and regeneration of PLBs in D. nobile, which combined with 0.5 mg L −1 NAA resulted in best PLBs formation (92%) and shoots/explants (9.2) [139].…”

“…As example, the combination of cytokinin BA (5.0 mg L −1 ) and auxin NAA (2.5 mg L −1 ) were used to induce PLBs (20.55 PLB per primary protocorm) in Cymbidium mastersii protocorms [134]. Thin cell layers (TCL) from different types of tissues was a technique used to improve the production of PLBs in Cymbidium [135], Dendrobium [136,137], Oncidium [129], and Phalaenopsis [93].…”

Recent advances and novelties in the thin cell layer-based plant biotechnology – a mini-review

“…In at least two cases, tTCLs served as the donor explants for genetic transformation experiments (Croom et al, 2016;Vaidya et al, 2016), and root-derived TCLs continue to serve as basic tools in assessing axillary root development in A. thaliana (Della Rovere et al, 2016;Fattorini et al, 2017Fattorini et al, , 2018. Except for three studies (Bose et al, 2017;Bhattacharyya et al, 2018;Parthibhan et al, 2018), none of the other new studies, similar to the vast majority of older studies on TCLs (Teixeira da Silva and Dobránszki, 2015), assessed the genetic stability relative to plants derived from the conventional culture, and such genetic testing needs to be implemented in future research on TCLs. Parthibhan et al (2018) used inter-simple sequence repeats (ISSR) to confirm the monomorphic nature of the resulting somatic embryos.…”

“…Except for three studies (Bose et al, 2017;Bhattacharyya et al, 2018;Parthibhan et al, 2018), none of the other new studies, similar to the vast majority of older studies on TCLs (Teixeira da Silva and Dobránszki, 2015), assessed the genetic stability relative to plants derived from the conventional culture, and such genetic testing needs to be implemented in future research on TCLs. Parthibhan et al (2018) used inter-simple sequence repeats (ISSR) to confirm the monomorphic nature of the resulting somatic embryos. Bose et al (2017) tested genetic fidelity by flow cytometry and intron splice junction (ISJ) markers, finding some polyploidy (5-11% 8C) in the leaves of the donor and acclimatized plants when the former was used, and 5-17% polymorphism in tissue cultured plants when five of the 15 ISJ primers were used.…”

“…TCL-based research has advanced recently for ornamental plants like including Lilium (Marinangeli, 2016) and three orchids, Dendrobium aqueum Lindl. (Parthibhan et al, 2018), Dendrobium aphyllum (Roxb.) C.E.C.…”