Simulating in ovulo osmotic potentials and O2 tensions normalize growth and pigmentation of immature cotton embryos

Abstract

Oxygen tensions and osmotic potentials are important physiological factors of plant growth and development. To optimize these variables for cotton (Gossypium hirsutum L.) embryo culture, we quantified dissolved O2 (dO2) tensions, osmotic potentials, and pH at several locations in cotton ovules during embryony. Clark O2 microelectrodes were micromanipulated into intact ovules at an angle lateral to the developing embryo, and dO2 tensions were determined in integuments, nucelli and embryos. Ovular osmotic potentials and pH were determined from extracted ovule sap using vapor pressure osmometers and pH microelectrodes. Dissolved O2 tensions near or in embryos decreased from 104 mmol m−3 at 5 days post-anthesis (DPA) to 83 mmol m−3 at 18 DPA. Osmotic potentials of ovule sap decreased from −0.70 megapascals (MPa) at 2 DPA to −1.12 MPa at 8 DPA but then increased to −0.84 MPa by 17 DPA. Ovule sap pH at 5–17 DPA varied inconsistently and ranged from 5.4 to 6.5. Based on these results, a factorial experiment with two osmotic and three O2 treatments was designed. Immature embryos of cotton cultivar HS-26 were randomly assigned to the treatment combinations and cultured for 33 days. Oxygen treatments did not affect embryo growth, and there were no differences among treatments with regard to percentage of embryos that progressed to a more advanced stage of embryo development. However, cotyledons of embryos grown without osmotic adjustment were abnormally large, and embryos exposed to these treatments were abnormally brown. Browning was less severe for embryos exposed to low O2 tensions. Growth and pigmentation were most normal for embryos simultaneously exposed to O2 tensions and osmotic potentials that best simulated the observed in ovulo conditions.