Partial genome transfer through micronuclei in plants

The introduction of gene manipulation techniques has greatly extended the possibilities for plant breeding. New techniques are still emerging, and many are being used, for the introduction of foreign genes, chromosomes and organelles in order to improve the existing cultivars or to develop new varieties, and to localize genes on the chromosomes. The techniques presently available range from the transfer of single, cloned genes via DNA transformation (reviews inNegrutiueta/. 1987; Gasser & Fraley 1989; Potrykus 1990), to the addition of a complete genome of a donor species by somatic hybridization (Negrutiu et al. 1989). Until now, only identified and cloned genes can be transferred through DNA transformation. Traits which are polygenically determined, or with unknown biochemical and molecular background, e.g. many disease resistances, yield, etc. are not yet amenable to this technique. In this regard, somatic hybridization may be a suitable approach, but it involves the fusion of whole protoplasts from two different parental species or genotypes, and thus adds two complete nuclear genomes and all cytoplasmic genomes from the chloroplasts and the mitochondria. This results in the production of highly complex somatic hybrids, with many unwanted additional genes. Moreover, when the genomes of the two parents are incompatible at the somatic level, random loss of chromosomes and organelle segregation or recombination will occur, leading to the formation of chimeric tissues. Several techniques have been used in recent years to eliminate or inactivate the unwanted chromosomes by treatment of the donor protoplasts prior to fusion. Commonly gamma or X-irradiation is used to obtain asymmetric hybrids. Although a large number of experiments have been carried out to transfer a limited number of chromosomes or chromosome fragments for integration into the recipient genome, so far only little success has been obtained through this technique. One of the major problems confronted by using irradiation is the stability of the introduced chromosomes or chromosome fragments, both at the cellular and the plant levels (Famelaer et al. 1989; Wijbrandi et al. 1990). The occurrence of chromosome breaks, deletions and rearrangements after irradiation makes this approach less suitable for transfer of large pieces of DNA with syntenic genes or intact chromosomes. The general application of gamma fusion in plant breeding is also hampered by the low frequency of plant regeneration as well as by the sterility of regenerated plants. Therefore, other alternatives for partial genome transfer were sought in plants. The two most widely used methods for chromosome transfer in mammalian cell biology, i.e. PEG-induced uptake and micro-injection, are being developed in plants. These methods are based on the application of isolated metaphase chromosomes, and are therefore called chromosome-mediated gene transfer (reviews in Klobutcher & Ruddle 1981, De Laat et al. 1989). In the case of mammals, introduction of isolated metaphase chromosomes by PEGinduced uptake into recipient cells resulted in the fragmentation of the donor chromosomes, followed by incorporation of some of the fragments into the recipient genome. The integrated fragments or transgenomes can be maintained stably by applying selection pressure for marker genes. For this method, large numbers of chromosomes were isolated from metaphase arrested cells. By using a GC-specific fluorochrome combined with an AT-specific fluorochrome, it was possible to sort chromosomes by flow cytometry. This method enabled the construction of chromosome-specific transgenomes, which in turn made it possible to map genes on the donor chromosomes (Carrano et al. 1979).