Plant Cell Reports

, Volume 30, Issue 8, pp 1541–1553 | Cite as

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

  • Josh Rosnow
  • Sascha Offermann
  • Joonho Park
  • Thomas W. Okita
  • Nathan Tarlyn
  • Amit Dhingra
  • Gerald E. EdwardsEmail author
Original Paper


To study the developmental transition of chloroplasts from C3 to C4 photosynthesis in the terrestrial single-cell C4 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 CO2 enrichment (1.2% CO2). When calli without RNS were cultured under Stage 3 conditions with 1.2% CO2, there was an increase in growth, protein content, and photosystem II yield, while structural and biochemical analyses indicated the cells in the calli had C3 type photosynthesis. CO2 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% CO2, showed leaves developed C4 type chlorenchyma cells, including expression of key C4 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.


Callus C4 photosynthesis CO2 enrichment Halophyte Regeneration 



This material is based upon work supported by the National Science Foundation under Grant IBN-0641232. Seeds of Bienertia sinuspersici were kindly provided by Dr. Abdulrahman Alsirhan, Kuwait. We thank the WSU Plant Transformation Center, the Franceschi Microscopy and Imaging Center of Washington State University for use of facilities and for staff assistance, C. Cody for plant growth management, and N. Koteyeva for assistance with graphics.

Supplementary material

299_2011_1067_MOESM1_ESM.doc (1.2 mb)
Supplementary material 1 (DOC 1218 kb)


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Josh Rosnow
    • 1
  • Sascha Offermann
    • 1
  • Joonho Park
    • 2
  • Thomas W. Okita
    • 3
  • Nathan Tarlyn
    • 4
  • Amit Dhingra
    • 5
  • Gerald E. Edwards
    • 1
    Email author
  1. 1.School of Biological SciencesWashington State UniversityPullmanUSA
  2. 2.Department of BiochemistryQueen’s UniversityKingston, ONCanada
  3. 3.Institute of Biological Chemistry, Washington State UniversityPullmanUSA
  4. 4.Plant Transformation CenterWashington State UniversityPullmanUSA
  5. 5.Department of HorticultureWashington State UniversityPullmanUSA

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