In essence, protoplast fusion comprises removal of cell walls and then the amalgamation of cell contents. The first successful protoplast fusion was reported between Nicotiana species over 20 years ago (Carlson et al. 1972). At that stage, the technique was viewed as a means of hybridising sexually incompatible species. Of more recent interest has been the production of asymmetric hybrids - those with fewer genes from one partner than from the other. These may arise from spontaneous chromosome elimination in some distant combinations, or as a result of the inactivation of mitotic capacity in one partner by chemical or radiation treatment (Harms 1992). This is of particular interest where a useful gene is to be introduced to a commercial variety from a wild relative, and offers a rapid alternative to traditional backcrossing programmes.
There have been several successes, notably with species of the Brassicaceae, where new interspecies and intergeneric hybrids have been created (Harms 1992), and the Solanaceae (Puite 1992). Somatic hybridisation between Nicotiana tabacum and N. rustica has produced tobacco plants which varied in nicotine and tar content and in resistance to blue mould and black root rot (Drew 1993). Hybrids between Lycopersicon esculentum and Solanum lycopersicoides have been produced by protoplast fusion, with the intention of introducing cold tolerance into tomato (Handley & Kumashiro 1986). Somatic hybridisation between eggplant, Solanum melongena, and its wild relative S. sisymbrifolium has been effective in introducing nematode and mite resistance into eggplant (Handley and Kumashiro 1986). Both of these hybrids are very difficult to produce sexually. Disease resistance traits have been transferred by protoplast fusion from wild relatives into cultivated varieties also for oilseed rape and potato (Harms 1992). The trait for male sterility (carried in the cytoplasm) has been transferred into tobacco from Nicotiana debneyi, by fusing the tobacco protoplasts with N. debneyi protoplasts previously exposed to X-rays (Handley and Kumashiro 1986). The X-ray treatment inactivates the nucleus without damaging cytoplasmic function. Fusion of haploid protoplasts has been used to combine cytoplasmic male sterility and cytoplasmic atrazine resistance in rape (Drew 1993). Cytoplasmic male sterile cybrids of rice have been produced and used in hybrid seed production (Drew 1993). Successful hybridisation has also been accomplished with Citrus species (Puite 1992).
It has been suggested that protoplast fusion could play an important role in the genetic improvement of many crops for which wild relatives constitute an extensive and untapped disease resistance gene resource, e.g. for Allium species, Citrus, cucumber, melons and squashes, alfalfa, Trifolium, potato, tobacco, rice and wheat (Harms 1992). Nevertheless, protoplast fusion is yet to be applied commercially on any scale in crop improvement programmes. In field testing, a hybrid potato clone derived from protoplast fusion between Solanum tuberosum and S. phureja produced three times the yield of the parent clone (Puite 1992).
Major factors limiting the application of protoplast fusion in crop improvement are:
Most of the successes with protoplast fusion have involved closely related parents. As noted above, spontaneous chromosome elimination may occur in more distantly related hybridisations. In addition, nucleo-cytoplasmic incompatibility has been observed for inter-generic fusions in the Solanaceae, leading to chlorophyll deficiency in cybrids with the Atropa belladonna nuclear genome and Nicotiana tabacum chloroplasts (Puite 1992).
Poor regeneration from protoplasts
Tree taxa for which plant regeneration from protoplasts has been reported include Eucalyptus, Populus alba, P. sieboldii, Picea abies, P. glauca, Pinus taeda and Larix × eurolepis (Puite 1992). In the case of the conifers listed, the protoplasts involved have been isolated from somatic embryos, embryogenic tissue or embryogenic cell suspensions. For most tissues from tree species, regeneration from protoplasts remains problematical.
In spite of continuing good progress in the field, the early expectations of protoplast fusion are yet to be realised, major limitations remain to be overcome, and most of the research is still empirical. Expectations of success with an untested pair of species could be expected to be low.
Many hybrids have been tested for established industrial plantation taxa such as Eucalyptus, Pinus, Picea, and Larix. A number of these hybrids are in commercial use, and it seems likely that hybrids will become of wider importance in industrial plantation forestry. Those currently in use are mostly reasonably easy to produce sexually. To be addressed then is the extent to which sexual incompatibility limits the production of other useful hybrids. This is difficult to assess. The introduction of disease resistance into commercial poplar clones has been limited by interspecific incompatibility, but other solutions to the problem have been identified, e.g. the use of solvents to remove stigmatic recognition factors underlying incompatibility (Knox et al. 1972). There are other instances where transfer of traits among distantly related species would be desirable - e.g. rust resistance in pines. Given the difficulties involved, however, and the fact that protoplast fusion research is empirical and likely to be long term anyway, genetic engineering may offer better hopes for the manipulation of such traits.
Protoplast fusion has little apparent applicability to little known tropical hardwoods and non-industrial species, for which investigations of the possibilities for even traditional hybridization remain minimal.