DENSITY OF SUSPENSION CULTURE OF SOMATIC EMBRYOGENIC CELLS OF LARIX LEPTOLEPIS DETERMINES THE FATE OF DEVELOPMENT: INVOLVEMENT OF PHOSPHATE UPTAKE AND METABOLISM

Hiroshi Ashihara, Hamako Sasamoto, Shinjiro Ogita

Abstract


Cell density of suspension culture of Larix leptolepis embryogenic cells determined the fate of the development. In a low-density cell culture, somatic embryo development was promoted, but the cells continued to proliferate and formed small spherical cell aggregates in a high-density cell culture. In the present study, we compared the uptake and metabolism of 32P-labelled inorganic phosphate (Pi) by the cells of low- (1 %) and high (10 %)-density cultures during first 30 h. Little difference was found in the uptake of 32Pi by the cells in both the low- and high density-cultures up to 6 h, but Pi uptake in high-density culture almost ceased at 6 hr after inoculation. In contrast, Pi uptake continued linearly in the low-density culture. 32P was distributed in small molecular organic compounds (mainly nucleotides and sugar phosphates), nucleic acids, phospholipids, and phosphoproteins along with unmetabolized Pi. The incorporation of 32P into nucleotide and phospholipid fractions was significantly higher in the cells of low-density culture at 30 h after inoculation. No marked difference on the endogenous levels of purine and pyrimidine nucleotides was observed in both density cultures. From these results it is speculated that the de novo nucleic acids and phospholipids synthesis required in the conversion of embryonic cells into somatic embryos may be caused by sufficient amounts of Pi from the medium occurring only in cells of the low-density culture. The nucleotide levels in both cultures were similar at least up to 72 h.  Therefore, sufficient levels of nucleotides for proliferation may be supplied even in cells of the high-density culture. The involvement of Pi uptake in the phenomena observed in the low- and high-density cultures is discussed.Cell density of suspension culture of Larix leptolepis embryogenic cells determined the fate of the development. In a low-density cell culture, somatic embryo development was promoted, but the cells continued to proliferate and formed small spherical cell aggregates in a high-density cell culture. In the present study, we compared the uptake and metabolism of 32P-labelled inorganic phosphate (Pi) by the cells of low- (1 %) and high (10 %)-density cultures during first 30 h. Little difference was found in the uptake of 32Pi by the cells in both the low- and high density-cultures up to 6 h, but Pi uptake in high-density culture almost ceased at 6 hr after inoculation. In contrast, Pi uptake continued linearly in the low-density culture. 32P was distributed in small molecular organic compounds (mainly nucleotides and sugar phosphates), nucleic acids, phospholipids, and phosphoproteins along with unmetabolized Pi. The incorporation of 32P into nucleotide and phospholipid fractions was significantly higher in the cells of low-density culture at 30 h after inoculation. No marked difference on the endogenous levels of purine and pyrimidine nucleotides was observed in both density cultures. From these results it is speculated that the de novo nucleic acids and phospholipids synthesis required in the conversion of embryonic cells into somatic embryos may be caused by sufficient amounts of Pi from the medium occurring only in cells of the low-density culture. The nucleotide levels in both cultures were similar at least up to 72 h.  Therefore, sufficient levels of nucleotides for proliferation may be supplied even in cells of the high-density culture. The involvement of Pi uptake in the phenomena observed in the low- and high-density cultures is discussed.

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DOI: http://dx.doi.org/10.17628/ecb.2014.3.1027-1031

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