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Hauser–Kraus annulation of sulfonylphthalides with simple conjugated nitroalkenes follows the expected pathway to furnish naphthoquinones in moderate yields. However, a strategic variation of this reaction by employing nitroalkenes bearing an additional nucleophilic site results in [4+4] annulation leading to complex fused and spiro heterocycles in high yields with high selectivity through a cascade process involving Michael addition, Dieckmann cyclization, and a series of eliminations and rearrangements.

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An efficient in vitro plant regeneration protocol from protocorms of Dendrobium aqueum was developed. The uniformly developed protocorms (in vitro origin) having shoot initials were cultured on half macro strength MS medium (1/2 MS) supplemented with cytokinins (BA, 2iP, KIN and TDZ) at 1, 3, 5, 7, 10 mg l À1 , natural additives (BP and CW) at 1%, 3%, 5%, 7%, 10% and auxins (IBA, NAA, 2,4-D) at 1, 3, 5, 7, 10 mg l À1 to study their efficacy on complete plant development. A maximum of 9.4 shoots per explant were generated on 3 mg l À1 of NAA followed by 3% of BP (7.0 shoots). Shoot elongation (1.52 cm) was achieved on 1/2 MS medium fortified with NAA 7 mg l À1 followed by TDZ 7 mg l À1 (1.37 cm). Shoots cultured on 1/2 MS medium supplemented with IBA 5 mg l À1 produced an average of 8.75 roots per shoot, however the lengthiest roots (1.48 cm) were noted in NAA 7 mg l À1 . Healthy rooted plantlets successfully acclimatized in ex vitro condition. The role of complete plantlet production by natural additives could be useful for conservation and cost effective commercial production of orchids.ª 2015 Production and hosting by Elsevier B.V. on behalf

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.

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