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 O 2 (dO 2 ) tensions, osmotic potentials, and pH at several locations in cotton ovules during embryony. Clark O 2 microelectrodes were micromanipulated into intact ovules at an angle lateral to the developing embryo, and dO 2 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 O 2 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 O 2 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 O 2 tensions. Growth and pigmentation were most normal for embryos simultaneously exposed to O 2 tensions and osmotic potentials that best simulated the observed in ovulo conditions.

Ultrastructural aspects of the embryo and different endosperm compartments, inEruca sativaHill cv. Nemat (Brassicaceae) during Heart and Torpedo stages

“…Even these plastids have a well developed thylakoid system, even if not so well developed as in the other two endosperm compartments. ROLLETSCHEK et al (2003) and LIDDIARD and CARMAN (2010) suggested that O2 production by photosynthetic activity in the seed could be necessary to provide O2 to the developing embryo, particularly if the seed coat acts as a barrier against the diffusion of this gas. Moreover the amount of functional chloroplasts in the seed of Eruca suggests that the photosynthetic product could be a relevant part of the carbohydrates necessary for the anabolic activity in the developing embryo.…”

Improving cotton embryo culture by simulating in ovulo nutrient and hormone levels

“…Low O 2 tensions next to cultured embryos were established and maintained as in Liddiard and Carman (2010). In brief, this included the use of an autoclavable polycarbonate plastic culture chamber with gas inlet and outlet ports and an E-444 O 2 sensor (Remming Bio Instruments Co., Redfield, NY) that was attached to a CR-10 data logger (Campbell Scientific, Inc., Logan, UT).…”

“…The latter approach is becoming increasingly feasible because of advancements in chemical and biophysical measurement instrumentation of small samples (Murray 1963;Carman 1995;Otegui et al 2002;Carman et al 2005;Fuller et al 2009;Liddiard and Carman 2010). For the present study, we hypothesized that the routine regeneration of isolated cotton proembryos by direct embryogenesis in vitro could be accomplished by simulating the nutritional and hormonal requirements of embryony in situ.…”

“…Additionally, we used enzyme-linked immunosorbent assays to determined levels of indole-3-acetic acid (IAA), six cytokinins, and abscisic acid (ABA) in ovule fluids during embryony (Hess et al 2002;Carman et al 2005;Fuller et al 2009). We also used microelectrodes, psychrometers, and osmometers to monitor in ovulo pH and osmotic potentials during embryony (Liddiard and Carman 2010). Finally, we used microelectrodes to monitor in ovulo O 2 tensions during embryony (Carman and Bishop 2004;Liddiard and Carman 2010).…”

“…We also used microelectrodes, psychrometers, and osmometers to monitor in ovulo pH and osmotic potentials during embryony (Liddiard and Carman 2010). Finally, we used microelectrodes to monitor in ovulo O 2 tensions during embryony (Carman and Bishop 2004;Liddiard and Carman 2010). Based on these analyses, we have constructed chemically defined media and unique culture equipment that better simulate the physicochemical environment of the cotton ovule.…”