Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants

Abstract

Somatic hybridization using protoplasts with whole genomes has often resulted in complex hybrids with many unwanted chromosomes or genes. Several researchers have attempted to reduce the number of undesired chromosomes through irradiation of the donor protoplasts, but so far without much success. Alternatively, micropro‐toplasts containing one or a few chromosomes can be used for partial genome transfer, as has been demonstrated in human and other mammalian cell systems using microcells. Recently, we have optimized the ‘microprotoplast system’ for several donor cell lines (potato, Nicotiana, sugar beet) carrying various genetic markers, such as kanamycin resistance, β‐glucuronidase, nitrate reductase deficiency, hormone autotrophy, opines, etc. Protocols were developed to obtain higher yields of micro‐protoplasts as well as to enrich sub‐diploid microprotoplasts containing one or a few chromosomes. These microprotoplasts were fused with whole mesophyll protoplasts of recipient lines using polyethylene glycol. Various requirements for lines used as donor and recipient partners in microprotoplast‐protoplast fusions are described. The results are discussed in the context of partial genome transfer.