Direct organogenesis provides an efficient means of propagating plants in vitro without the necessity of growing callus cultures. Shoot regeneration was achieved from bud explants of Bienertia sinuspersici on Murashige and Skoog's media supplemented with 6-benzyladenine (BA). The frequency of regeneration varied based on the concentration of BA used. The highest rate of new shoot induction was observed when explants were cultured on media containing 4.4 lM BA after three weeks. Shoots were successfully elongated on media containing gibberellic acid with the highest shoot elongation observed at 2.9 lM. Elongated shoots developed adventitious roots in media without plant growth regulator and were subsequently transplanted to soil. Light microscopic analysis of cross sections of leaves from regenerated shoots showed similar intracellular cytoplasmic compartmentalization of organelles in chlorenchyma cells compared to those observed in leaves grown under greenhouse conditions. Moreover, Western blot analysis detected high expression of key C 4 photosynthetic enzymes, suggesting the C 4 cycle is maintained in the single cell system. We have developed a procedure for whole plant regeneration via direct organogenesis in B. sinuspersici.

An efficient in vitro method for rapid vegetative propagation of Bienertia sinuspersici, one of four terrestrial species of family Chenopodiaceae capable of performing C4 photosynthesis within a single cell, was developed. Cuttings of B. sinuspersici were used to examine the effects of Murashige and Skoog (MS) media strength and auxins on adventitious root formation. Half-strength MS medium was determined to be ideal for adventitious root formation in Bienertia cuttings. Although cuttings cultured in medium containing 5.0 mg/L α-naphthalene acetic acid (NAA) promoted the highest number of adventitious roots, cuttings cultured in medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) produced the longest adventitious roots and had the highest survival rate upon transplanting to soil. Histological analysis revealed variations in the root anatomy generated by the various auxins which may affect adventitious root formation and subsequent establishment of cuttings in soil. Overall, the established procedure provides a simple and cost-effective means for the rapid propagation of the single-cell C4 species B. sinuspersici.

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