Technological study on freeze preservation in order to store genotypes and chromosome types by shoot primordia of annual plants.

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In annual Plants, it is very difficult to transmit genotypes, namely, first filial generation, heterosis and others, and chromosome types, namely, chromosomal structural change, aneuploidy, triploidy and others to filial genetation. For settling this problem, a shoot primordium method was developed by Tanaka and Ikeda (1983). In comparison with using shoot tips, the freeze preservation using shoot primordia will be more advantageous in storing a large number of specimens. Besides after thawing the shoot primordia can be mass-propagated in a short period and regenerated into many colnal plants whenever necessary.Shoot primordia were yielded in 52 species out of 72 species examined for the materials of freeze preservation. Chromosomal mutants of shoot primordia were also induced to examine if the chromosomes of shoot primordia are stable during and after the freeze preservation: 1. Clonal tetraploid shoot primordia were induced by treating with colcemid in diploid shoot primordia of Hapl … More opappus gracilis and Crepis capillaris. 2. clonal shoot primordia with chromosomal change were induced from suspended cells of Crepis capillaris, in which chromosomal aberrations arose frequently. Besides single cells, to which cryoprotectant permeate effectively, were induced from shoot primordia of Haplopappus gracilis. Single cells from shoot primordia could be differentiated into new shoot primordia.In shoot primordia of annual Haplopappus gracilis (2n=4), the method of freeze preservation was complete, and now this method is applied to the shoot primordia of other plants mentioned above. The method of freeze preservation was as follows. Subcultured shoot primodia were precultured in a MS medium supplemented with 5% DMSO for 3 days. They were cooled at a velocity of -0.5゜C/min down to -40゜C in a programming freezer, and then stored in liquid nitrogen immediately. The frozen shoot primordia were thawed quickly in a water bath at 37゜C, and recultured. The recovered shoot primordia were highly stable in the chromosome number and karyotype. Less