Embryo Development in Phaseolus vulgaris II

Endosperm liquid bathes the embryo of Phaseolus vulgaris from the heart stage through the late cotyledon stage. This liquid was aspirated from many ovules of the same stage, pooled, and analyzed for the following constituents and parameters: 1. Osmolarity and pH, using freezing point depression and Hydrion paper. 2. The elements Ca, K, P, and Mg, using the electron microprobe, atomic absorption spectrophotometry, and the Bartlett modification of the Fiske-SubbaRow P determination method. 3. NH4+, using the Conway diffusion procedure. 4. Organic acids, using paper and two directional thin layer chromatography for qualitative measures, and the enzymatic oxidation and reduction of NADH for quantitative measures of citrate and malate. 5. Amino acids, using two directional thin layer chromatography and an amino acid analyzer. 6. Sugar, using thin layer chromatography, the SomogyiNelson method, with and without prior hydrolysis, and the Glucostat reagent. These analyses revealed the following: 1. Osmolarity decreased from 0.7 in the heart stage to 0.5 in the late cotyledon stage and pH remained at 5.2 to 5.4 throughout. 2. Ca was not detected, at 0.5 mM sensitivity, K decreased from 34.4 mM to 20.8 mM, P decreased from 5.3 mM to 3.2 mM, and Mg decreased from 4.1 mM to 2.5 mM. 3. NWIL decreased from 110 mM in the early cotyledon stage to 80 mM in the late cotyledon stage. 4. A pool of five organic acids was dominated by malate, which decreased from 23 mM in the early cotyledon stage to 17 mM in the late cotyledon stage, and citrate, which remained at about 5 mM. 5. At least 18 amino acids were present for a total concentration of about 42 mM, including 3 nonprotein amino acids. 6. Most of the sugar was in the form of sucrose, glucose, and fructose. Sucrose increased, from 20 mM in the early 'This work was supported in part by National Science Foundation and Danforth graduate fellowships and by an American Cancer Society grant to Dr. Peter B. Kaufman. 2 This work was submitted to the Rackham School of Graduate Studies, University of Michigan as part of a thesis, in partial fulfillment of the requirement for the doctoral degree. 'Deceased August 1972. 4 Address request for reprints to Dr. Peter B. Kaufman, Department of Botany, University of Michigan, Ann Arbor, Mich. 48104. cotyledon stage to 60 mM in the late cotyledon stage, whereas the total reducing sugar remained approximately constant. These results are discussed in relation to the culture of bean embryos in vitro. Comparisons are made with the compositions of other media which have been used for plant embryo or tissue culture. The ovule of the snap bean, Phaseolus vulgaris L. , is filled with liquid during much of the development of the embryo, i.e., from the end of the globular stage to the end of the late cotyledon stage (19). This liquid is contained within a thin sack of endosperm cytoplasm and bathes the embryo, separated from it by only a thin layer of cellular endosperm. Bean embryos cultured in vitro should grow and develop best in a culture environment most nearly like that in vivo. Thus, analyses of this liquid were made for several stages, i.e., the heart, early cotyledon, and later cotyledon stages. This paper reports the analyses of K, Ca, Mg, P, and NH4, ions, organic and amino acids, and sugars, which together comprise about 76% of the solutes in the late cotyledon stage on an osmolar basis. It does not cover analyses of protein, hormones, sugar alcohols, and an unknown acid component. Somewhat similar analyses have been reported for the endosperm liquids of coconut (22) and cotton (10). MATERIALS AND METHODS Fresh beans of Phaseolus vulgaris L. cv. "Black Valentine" and "Topcrop" were obtained from plants grown in flats in the greenhouse throughout the year. The mean day temperature of the greenhouse was 26 C, and the mean night temperature was 22 C. Additional material was grown in field plots and the Matthaei Botanical Gardens during the summer or purchased from local stores. Materials purchased commercially were used only for the verification of ionic composition and for the preliminary investigation of organic acid content. Osmolarity and pH. Osmolarity was determined by the freezing point depression method of Prosser (14). Endosperm liquid from globular stage, heart stage, early cotyledon stage, and late cotyledon stage ovules was drawn into melting point capillary tubes. These were frozen in a Dry Ice-salt water slurry and visualized with polarized light, as were NaCl standards. Time to melting was used to compute osmolarity. Three different sets of samples were used, and four determinations were run on each set. Because the volume of endosperm liquid was very small,