In vitro cultures and regeneration of Bienertia sinuspersici (Chenopodiaceae) under increasing concentrations of sodium chloride and carbon dioxide

Abstract

To study the developmental transition of chloroplasts from C(3) to C(4) photosynthesis in the terrestrial single-cell C(4) species Bienertia sinuspersici, a regeneration protocol was developed. Stem explant material developed callus either with or without red nodular structures (RNS) when cultured on Murashige-Skoog (MS) salts and vitamins, supplemented with 5mM phosphate, plus 1mgL(-1) dichloropenoxy-acetic acid (2,4-D), and 87mM sucrose (Stage 1 media). Only calli having RNS were able to regenerate plantlets. MS media plus phosphate was used throughout regeneration, with the Stage 2 media containing 2mgL(-1) 6-benzylaminopurine, 43mM sucrose and 1.5% soluble starch. Stage 3 media had no hormones or organic sources of carbon, and cultures were grown under ambient (~400ppm) versus CO(2) enrichment (1.2% CO(2)). When calli without RNS were cultured under Stage 3 conditions with 1.2% CO(2), there was an increase in growth, protein content, and photosystem II yield, while structural and biochemical analyses indicated the cells in the calli had C(3) type photosynthesis. CO(2) enrichment during growth of RNS during Stage 3 had a large effect on regeneration success, increasing efficiency of shoot and root development, size of plantlets, leaf soluble protein, and chlorophyll concentration. Anatomical analysis of plantlets, which developed under 1.2% CO(2), showed leaves developed C(4) type chlorenchyma cells, including expression of key C(4) biochemical enzymes. Increasing salinity in the media, from 0 to 200mM NaCl, increased tissue osmolality, average plantlet area and regeneration success, but did not affect protein or chlorophyll content.