2022 Volume 21 Issue 1 Pages 1-10
Nagabo-natsuhaze (Vaccinium sieboldii Miq., 2n = 2x = 24), which is designated as critically endangered (CR), is a Japanese endemic Vaccinium species. In order to use it as a breeding material for blueberries, we carried out in vitro chromosome doubling of multiple shoots in two lines (M3 and M15) of Nagabo-natsuhaze. Furthermore, we investigated the morphological characteristics of tetraploids induced by these treatments. Multiple shoots were aseptically treated with two antimitotic agents, oryzalin and colchicine, at different concentrations and times. The survival rate of colchicine-treated shoots tended to decrease with increasing treatment time, whereas the survival rate on oryzalin treatment differed between M3 and M15. M3 showed high survival rates (over 90.0%) of colchicine-treated shoots regardless of the concentration and time. In those of M15, the survival rate tended to decrease with increasing treatment time. When the ploidy levels of the new shoots obtained from colchicine and/or oryzalin treatments were analyzed by flow cytometry, various polyploid shoots such as tetraploid and ploidy chimera (2x + 4x and 4x + 8x) were obtained in both treatments. Among the treatment conditions tested in the present study, tetraploid induction by colchicine treatment occurred in 3 of 9 treatment conditions in M3 and in only 1 treatment condition in M15. In the case of oryzalin treatment, on the other hand, tetraploid shoots were induced more than with colchicine treatment in both lines. Especially, the tetraploid induction rate in both lines reached its highest (10.0%) on treatment with 0.005% oryzaline for 24 h. Moreover, these tetraploids had large leaves, flowers, and pollen, which are typical traits of other tetraploid plants, and produced pollens that could be stained with acetocarmine.
