Gymnospermous embryos are nourished by fluids secreted from the megagametophyte. During early embryony, these fluids occupy the newly formed corrosion cavity. We describe a novel method for extracting corrosion cavity fluid and provide chemical analyses based on extractions from approximately 120 000 Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) megagametophytes. Levels of potassium, phosphorus, calcium, zinc, and iron were higher in corrosion cavity fluid than in whole tissue, but levels of sulphur and manganese were lower. Levels of cyclitols, sucrose equivalents, erythrose, and arabinose were many-fold higher in corrosion cavity fluid than in whole tissues. Ala, Ser, Arg, Glx, and NH 3 exceeded 80 mmol/kg dry mass in corrosion cavity fluid. These levels were about 100-fold higher than those found in whole tissues. During early embryony, hormone levels in corrosion cavity fluid were higher than levels observed in whole megagametophytes by 120-fold for indole-3-acetic acid, 53-fold for abscisic acid, and 8-to 10-fold for cytokinins. Nutrient and hormone levels tended to be much higher in the corrosion cavity fluid than would have been predicted based on whole-tissue analyses. Dynamic changes in nutrient and hormone levels occurred over time in the corrosion cavity, and these changes may normalize embryony in situ. Résumé :Les embryons des gymnospermes sont nourris par des fluides sécrétés par le mégagamétophyte. Tôt durant l'embryogenèse, ces fluides occupent la cavité de corrosion nouvellement formée. Les auteurs décrivent une nouvelle méthode pour extraire le fluide de la cavité de corrosion et fournir des analyses chimiques basées sur des extractions faites à partir d'approximativement 120 000 mégagamétophytes de douglas. Les niveaux de potassium, phosphore, calcium, zinc et de fer étaient plus élevés dans le fluide de la cavité de corrosion que dans tout le tissu, mais les niveaux de soufre et de manganèse étaient plus faibles. Les niveaux de cyclitols, d'équivalents en sucrose, d'érythrose et d'arabinose étaient plusieurs fois plus élevés dans le fluide de la cavité de corrosion que dans tout le tissu. Ala, Ser, Arg, Glx et NH 3 , dépassaient 80 mmol/kg de masse sèche dans le fluide de la cavité de corrosion. Ces niveaux étaient environ 100 fois plus élevés que ceux trouvés dans tout le tissu. Tôt durant l'embryogenèse, les niveaux d'hormones dans le fluide de la cavité de corrosion étaient plus élevés : 120 fois pour l'acide indol-3-acétique (AIA), 53 fois pour l'acide abscissique (AAB), et 8 à 10 fois pour les cytokinines, que les niveaux observés dans les mégagamétophytes entiers. Les niveaux de nutriments et d'hormones tendaient à être beaucoup plus élevés dans le fluide de la cavité de corrosion que ce qu'on aurait pu prédire sur la base des analyses de tout le tissu. Avec le temps, des changements dynamiques dans les niveaux d'hormones et de nutriments pouvant normaliser l'embryogenèse in situ sont survenus dans la cavité de corrosion.[Traduit par la Rédaction] Carman et al. 2456

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.

Plant ovules provide zygotes with a physicochemical environment that supports embryo differentiation, growth, and maturation. The exact nature of this embryogenesis-enabling environment is not well characterized, as evidenced by failed attempts to induce normal embryony from zygotes or proembryos (precotyledonary) on defined media. To identify factors required for cotton (Gossypium hirsutum L.) zygotic embryony in vitro, we previously performed chemical and dissolved oxygen tension analyses of cotton ovule fluids and tissues at multiple stages of embryony in situ. Based on these analyses, we report herein the development of procedures that normalize embryo differentiation, growth, maturation, and germination in vitro, starting with proembryos. Our medium differed from Murashige and Skoog (MS) medium as follows (percentage of MS): N (30%, mostly from ten amino acids), P (815%), K (237%), Mg (85%), Ca (267%), S (506%), Fe (88%), and myoinositol (883%). Levels of other MS nutrients and vitamins, except sucrose, were kept at MS levels. Additionally, we included 100 mg L −1 casein hydrolysate plus the following (mmol L −1 ): D-glucose (1.8), fructose (4.7), sucrose (62.0), arabinose (7.1), melibiose (3.5), malic acid (11.6), and citric acid (3.8).Mannitol was added to achieve a medium osmotic potential of −1.10 MPa, and an atmospheric O 2 tension of 3.3 mol m −3 at the surface of embryos was maintained during culture. When cultured on medium containing 8.0 μmol L −1 indole-3-acetic acid, 80-90% of proembryos (as small as 100 cells) of cultivars HS-26 and B-27 increased four-to eightfold in surface area during the first 18 d in culture and germinated thereafter to produce viable plants. Increases in surface area of proembryos cultured on a modified MS medium previously used for somatic embryogenesis were from 0.2-to 0.6-fold. The described embryo culture medium should be useful for studying nutritional and molecular aspects of early embryony and possibly for plant zygote transformation protocols.

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