Micronucleation by mitosis inhibitors in developing Spathiphyllum wallisii Regel microspores for microprotoplast preparation

Asymmetric protoplast fusion through microprotoplast mediated chromosome transfer (MMCT) is a known method for overcoming sexual breeding barriers between distantly related plant species. To obtain microprotoplasts, it is necessary to induce mass micronucleation either in somatic or gametic cells. We chose to use pollen mother cells as starting material. We have tested the efficiency for micronuclei induction of 5 mitosis inhibitors, amiprophos-methyl (APM), butamiphos (BUT), chlorpropham (CIPC), oryzalin (ORY) and propyzamide (PRO), on developing microspores of diploid Spathiphyllum wallisii Regel. Dissected full anthers of genotype ‘6526’ were treated in a culture room with spindle toxins, at concentrations of 10, 20, 50 and 100 µM in half-strength liquid MS media under normal day-light condition. Observations were made at 24, 48, 72 and 96 hour intervals after treatment. The observations were made using a fluorescence microscope after staining the anthers with 4’ 6-diamidino-2-phenylindole (DAPI). The micro-nucleation index (percentage of cells with additional micronuclei) was measured. We observed micronuclei (MNi) in pollen mother cells, dyads and tetrads. The highest micronucleation index of 86% was obtained in microspores treated with 10 µM ORY during 72h. Next to ORY, also CIPC (MNi index of 74%) was a very efficient MNi inducer, but other toxins were less effective. The optimal concentration for all the toxins tested was situated between 10 to 50 µM. At a higher concentration of toxins, the efficiency decreased. The average number of MNi found in micronucleated cells varied between 1.67-6.44 for CIPC and 0.83-5.50 for ORY. The maximal number of MNi observed was 12 for CIPC and 9 for ORY. Our results demonstrate that CIPC and ORY can be applied for mass micronucleation on developing microspores of S. wallisii as a first step of MMCT in aroid interspecific or intergeneric breeding.