Nutrient and hormone levels in cotton ovules during embryony

Abstract

In vitro zygotic and somatic embryogenesis protocols rely on nutrient and hormone levels from media to satisfy the physiological and developmental requirements of embryony. To better understand these requirements for cotton, we quantified levels of major and minor elements, carbohydrates, NH 4 ? , free amino acids and six hormones in whole cotton ovules (with fibers removed), nucelli (ovules with integuments removed), or ovule fluid (extracted from the endosperm region). Samples were collected from fieldgrown cotton at 1-18 days-past-anthesis (DPA) during each of three growing seasons. Replication across 2 years was obtained for carbohydrates, NH 4 ? , free amino acids and hormones from nucellus samples. The year effect was large primarily for hormones only. The most abundant minerals across tissue types and years were K, P, Mg and S. Potassium was the most abundant at 260, 600 and 1,660 mmol kg -1 dry mass (DM) in nucelli, whole ovules and ovule fluid, respectively. Magnesium, Ca, Zn and Mn levels were 2-8-fold higher in ovule fluid compared to whole ovules or nucelli. In the free amino acid plus NH 4 ? category, NH 4 ? , alanine, serine, glycine, asparagine (plus aspartic acid), glutamine (plus glutamic acid), leucine, threonine and arginine predominated in nucelli and ovule fluid, and levels tended to be higher in the older samples across years and tissue types. Fructose and glucose levels also increased with age with very high levels being found in late DPA ovule fluid. Arabinose, inositol and melibiose were also prominent sugars. Indole-3-acetic acid levels were similar between nucelli and ovule fluid and ranged from 10 to 80 lmol kg -1 DM. An abscisic acid spike, from 15 to 400 lmol kg -1 DM, occurred in nucelli and whole ovules from 2 to 8 DPA. Thereafter, abscisic acid levels remained between 5 and 10 lmol kg -1 DM. Zeatin and zeatin riboside were the most abundant cytokinins, and levels of these hormones fluctuated between 1 and 4 lmol kg -1 DM in both nucelli and ovule fluid.

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

“…Detailed data concerning the nutritional and hormonal content of ovule fluid adjacent to embryos is available for only a few species (Murray 1988;Carman et al 1996Carman et al , 2005Hess et al 2002;Fuller et al 2009), and even less is known concerning the dissolved O 2 (dO 2 ) status of ovule fluids that bathe embryos in situ. Some data concerning the osmotic potential of ovule fluid is available but only for late stages of embryony.…”

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

“…Four cotton ovule simulation media (GOS1-4) were developed based on nutrient levels observed in cotton ovules (Mauney et al 1967;Fuller et al 2009). Each medium contained levels of micronutrients as found in MS (Murashige and Skoog 1962) medium (except for Fe), MS vitamins (nicotinic acid, pyridoxine-HCl, and thiamine-HCl), and other components ( Table 1).…”

“…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.…”

“…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. To bridge the gap in understanding what these requirements are, we had previously used various chromatography techniques to quantify mineral, watersoluble carbohydrate, NH 4 + , NO 3 − and free amino acid concentrations in small quantities of fluids extracted from ovules of developing seeds (Carman et al 1996(Carman et al , 2005Fuller et al 2009). 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).…”

“…To bridge the gap in understanding what these requirements are, we had previously used various chromatography techniques to quantify mineral, watersoluble carbohydrate, NH 4 + , NO 3 − and free amino acid concentrations in small quantities of fluids extracted from ovules of developing seeds (Carman et al 1996(Carman et al , 2005Fuller et al 2009). 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).…”