Analysis of amino acids in individual wheat embryonic protoplast.
Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl2) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68 x 10(-5) mol/L to 18.18 x 10(-5) mol/L in single protoplast.
Chen
F-G
FG
School of Life Science, Shandong University, Jinan, PR China.
Wang
C
C
Zhi
D-Y
DY
Xia
G-M
GM
D000596:Amino Acids / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007834:Lasers; D011523:Protoplasts / Q000737:chemistry* / Q000378:metabolism; D012639:Seeds / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D046509:Tissue Culture Techniques; D014908:Triticum / Q000166:cytology* / Q000196:embryology*
D000596:Amino Acids; D016650:Fluorescein-5-isothiocyanate