In vitro production of autotetraploid Ponkan mandarin (Citrus reticulata Blanco) using cell suspension cultures

An efficient protocol for colchicine mediated production of in vitro autotetetraploids from Ponkan mandarin using cell suspension cultures is described. Cells were treated with 1 g l(-1) colchicine for 4 or 8 days before transfer into solid EME medium supplemented with 5% maltose. Colchicine treated cells were placed in medium with or without an overlay of 1:2 medium-mixture of liquid 0.6 M BH3 medium and 0.15 M EME + maltose liquid media. It was observed that modifying the immediate cell environment by addition of the liquid overlay played a positive role in cell differentiation and subsequent plant regeneration. Ploidy levels were determined with a flow cytometer and confirmed by chromosome staining using the enzymatic maceration method. A large number of non-chimeric autotetraploids were generated using this method. Such plants have great value in a breeding program for the development of seedless triploid citrus, as very few available tetraploid breeding parents are easy to peel.