Maize plants were subjected to 600Kpa of nitrous oxide (NO2) gas from 30-50 hours after pollination. At the end of the time period, the plants were taken out of the NO2 chamber and grown until 11 days after pollination. By using the chromosome counting technique described by A. Kato (Biotechnic & Histochemistry 73:160-165), slides of endosperm tissue were obtained and the chromosomes were stained with 2.5% acetic orcein. Normal endosperm is a triploid tissue (3x=30; Figure 1). Endosperms treated with NO2 gas from 30-50 hours after pollination can be successfully doubled creating a hexaploid endosperm (6x=60; Figure 2). In some rare cases, the endosperm genome quadrupled, creating a dodecaploid endosperm (12x=120; Figure 3). At 30 hours after pollination, the endosperm contains four nuclei (Mol, R et al., Plant Journal 5:197-206). In the multinucleated endosperm, it is possible to double one nucleus and not the others. When this occurred, mosaic endosperms were created. We have observed mosaic endosperms that contain some of, or all of the three types of nuclei above. So far, dodecaploid endosperm nuclei have only been observed in mosaic endosperms. It is possible to avoid mosaics by treating with NO2 gas starting at 14 hours after pollination, which is prior to the first endosperm nuclear division. Hexaploid endosperms have been seen with NO2 treatment from 14-34 hours after pollination.
In summary, using NO2 gas from 30 to 50 hours after pollination, it is possible to double the endosperm of maize. Quadrupling the genome also occurred in some few cases. To our knowledge, this is the first time a dodecaploid endosperm cell has been documented in maize.
Figure 1. Normal maize endosperm cell containing 30 chromosomes (400X)
Figure 2. Maize endosperm cell with 60 chromosomes (400X)
3. Maize endosperm cell with 120 chromosomes (400X)
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