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

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 5 mM phosphate, plus 1 mg L ^-1 dichloropenoxy-acetic acid (2,4-D), and 87 mM 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 2 mg L ^-1 6-benzylaminopurine, 43 mM sucrose and 1.5% soluble starch. Stage 3 media had no hormones or organic sources of carbon, and cultures were grown under ambient (~400 ppm) 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 200 mM NaCl, increased tissue osmolality, average plantlet area and regeneration success, but did not affect protein or chlorophyll content.