Glutamine enhances storage protein synthesis in Medicago sativa L. somatic embryos

Abstract

The vigor of seedlings from dry somatic embryos is considerably less than the vigor of seedlings from seeds, perhaps due to the low levels of storage proteins and low levels or different species of carbohydrates deposited in the dry somatic embryos. The effects of sucrose, abscisic acid and glutamine applied during maturation on the accumulation of storage proteins in the dry somatic embryos of Medicago sativa L. were investigated in an attempt to improve vigor. A cell suspension containing 2,4-dichlorophenoxyacetic acid (2,4-D) was sieved and the somatic embryos were developed to the torpedo stage on hormone-free BOi2y medium for 7 days. For experimentation, embryo maturation was divided into two phases of 10 days duration each: phae II and phase II maturation. The components of the cell culture medium were varied during the first 10-day period of maturation (phase I). This was followed by phase II maturation on standard BOi2y medium with 20 μM abscisic acid (ABA) to induce desiccation tolerance. Fully matured and air-dried somatic embryos were sampled for analysis. Increasing sucrose from 30 to 50 g l −1 and adding a 50-mM glutamine supplement to the phase I maturation medium increased somatic embryo dry weight from 1.5 to 2.5 mg. Sucrose did not have a significant effect on synthesis of either the low salt soluble (S-1) protein fraction containing water soluble enzymatic proteins, high and low molecular weight and alfin (7S) storage proteins, or the high salt soluble (S-2) fraction proteins containing mainly medicagin (11S) storage proteins. The presence of 2 μM ABA depressed total protein accumulation, whereas 50 mM glutamine increased the synthesis of the S-2 relative to S-1 proteins. In the presence of 50 mM glutamine the quantity of high salt soluble S-2 proteins, consisting of mainly medicagin (11S) storage proteins, increased 2-fold in somatic embryos. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that glutamine increased the polypeptides of the storage proteins, especially medicagin (11S). This enhancement may represent improved nutrition of the somatic embryo or enhanced transcription or translation of the storage protein genes.