A high-throughput flow cytometry method for ploidy determination in oil palm
2013
Forster, Brian P. | Sitorus, Andrew Christian | Caligari, Peter D. S. | Nasution, Olviyani | Nelson, Stephen P. C.
Non-euploid seedlings occur naturally in many plant species including oil palm but at frequencies usually considered too low for practical purposes. A flow cytometry method is described that overcomes the difficulty of exploiting such low frequency events and provides a practical plant breeding methodology to identify noneuploids/ haploids within large sample sizes. In addition, the efficiency of detecting non-euploid seedlings can also be increased greatly by a pre-screen for abnormal phenotypes. Oil palm is relatively difficult to analyse via flow cytometry as tissue disruption initiates secondary metabolite production which interferes with the analyte and data capture. The addition of dithiothreitol and polyvinylpyrrolidone during sample preparation, followed by cold incubation, prior to analysis overcomes these problems. The high-throughput method developed allows the analysis of 1000 samples per day per flow cytometer. The number of haploids produced by this method rivals that of other haploid production systems and is currently the only known method of generating haploids in oil palm. Additionally, the method for oil palm is not season dependent and may be performed all year round. The method can be applied to other species and provides a practical means of harvesting naturally occurring non-euploid. The seedlings selected using this methodology can be grown, thus making the method applicable to a range of species and disciplines including evolutionary studies of speciation of polyploids, reproductive biology, embryology and the production of haploids and doubled haploids for genetic studies and plant breeding of oil palm.
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