The Effects of Amino Acids and Ammonium on the Growth of Plant Cells in Suspension Culture

Abstract

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in whichi the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The source (3, 4, 8, 10, 16). Supplementing the media with mixtures of amino acids or commercial preparations of hydrolyzed proteins frequently enhances the growth (2, 14), although amino acids also may inhibit growth (5,8).The concentration of nitrogen and the relative amounts of ammonium and nitrate may be critical for growth and morphogenesis of plant cells (10,15).In an earlier report from this laboratory it was shown that a soybean cell culture growing on a defined medium required ammonium or glutamine in order to grow on nitrate (8). Growth ceased when nitrate was used without a source of reduced nitrogen. A series of amino acids has now been examined to determine their effectiveness as a replacement for ammonium sulfate. Furthermore, the effect of various nitrogen sources on the growth of soybean cells and cells of five other plants was investigated. Table I (8). The cells were grown in 250-ml DeLong flasks in a total volume of medium and inoculum of 40 ml. The flasks were incubated at 27 C in continuous light on a gyrotory shaker (6). METHODS AND MATERIALSThe amino acids used in the experiments were filter-sterilized and added to the autoclaved medium.Analytical Methods. For determining dry weight, the cells were collected on Miracloth, washed with water, and dried in a vacuum oven at 60 C for 20 hr. Total protein was determined by a micro-Kjeldahl method. RESULTSThe results in Table II serve to illustrate that the lack of growth of soybean cells in nitrate alone is not influenced by the concentration of nitrate. The other important observation is the profound influence of ammonium on the utilization of nitrate reflected in the increased growth rate. Ammonium is only one form of reduced nitrogen, and other sources were subsequently tested. In a series of experiments a number of amino acids were added one at a time at a concentration of 1, 2, and 4 mm. These concentrations were selected since those were the levels at which ammonium sulfate was used. Cell growth was reduced in the presence of both isomers of most amino acids at the three concentrations. The data in Figure 1 show the effect on growth of the soybean cells when various amino acids were substituted for ammonium sulfate in the medium. The medium contained 25 mm nitrate, and the concentration of the amino acids was 4 mM. Addition of L-glutamine resulted in growth equal to that obtained with ammonium sulfate. The L-isomers of alanine and arginine could not replace ammonium, but the cell yield was higher than for cells grown in nitrate alone. Chromatographic analysis showed that the majority of the amino acids disappeared from the medium and were presumably absorbed.The results in Figure 2 show the growth achieved by substituting glutamine, urea, and amino acids related to the urea cycle for ammonium sulfate. The L-glutamine was fully equal to ammonium sulfate in supporting growth. On ...

Involvement of G‐proteins in chitosan‐induced Anthraquinone synthesis in Rubia tinctorum

“…Briefly, cells were cultured in B5 medium (Gamborg 1970) containing 2 g l ÿ1 sucrose, 2 mg l ÿ1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l ÿ1 1-naphthaleneacetic acid (NAA), 0.5 mg l ÿ1 indoleacetic acid (IAA) and 0.2 mg l ÿ1 kinetin. After the pH was set at 5.7-5.8, the medium was sterilized by autoclaving (1 bar, 20 min).…”

Development of the Duckweed (Lemna Minor) Genetic Transformation System

“…С целью подбора оптимальных условий для культивирования рас-тений ряски in vitro были протестированы следующие питательные среды: Мурасиге и Скуг (Murashige et al, 1962), Хоагланд (Hoagland et al, 1938), Шенк и Хил-дебрандт (Schenk et al, 1972), Гамборг (Gamborg, 1970). Опыт проводили в 4-кратной повторности, повторность представляла собой чашку Петри с 25 листецами.…”

“…Опыт проводили в 4-кратной повторности, повторность представляла собой чашку Петри с 25 листецами. Пи-тательные среды готовили с добавлением витаминов B 5 в концентрации 0,1 г/л (Gamborg, 1970). рН среды до-водили до значения 5,7 перед автоклавированием.…”

Effect of exogenous auxin on root morphology and secondary metabolism in Tagetes patula hairy root cultures

“…Roots were cultured in Gamborg's B5 medium (Gamborg 1970) supplemented with 3 % sucrose and 100 ¡ig L 1 biotin. The pH was adjusted to 5.75 with NaOH before autoclaving.…”