Protoplast fusion, chloroplast transfer

The chloroplasts of triazine-resistant weed species are a potential source of herbicide resistance for treinsfer into crop plants. Chloroplast transfer, that is the combination of desirable chloroplasts and nuclei, can be achieved by crossing or by protoplast fusion. Chloroplast transfer by crossing is carried out using the line with the desirable chloroplasts as the maternal parent and the line with the desirable nuclear background as the pollen parent. Repeated crossing results in substituting the chromosomes (nucleus) of the chloroplast source with those of the recurrent pollen parent as chloroplasts in crops are only inherited by the maternal parent. This strategy has been used to introduce the triazine-resistant chloroplasts from bird s rape (a weed, Brassica campestris) into cultivated B. campestris and B. napus... 

Genetic techniques for obtaining chloroplast-encoded herbicide resistance in crop plants are reviewed. These are based on the use of cell culture methods and Include mutant selection in cultured cells, chloroplast transfer via protoplast fusion, and genetic recombination between chloroplast genomes. The first two methods are described in detail using trlazine resistance as an example. 

Cell culture-based methods suitable for obtaining chloroplast-encoded herbicide resistance in crops will be reviewed. These will Include mutant selection in cultured cells, transfer of chloroplasts via protoplast fusion, and selection of recombinant chloroplast genomes. Examples will be limited to resistance to triazine herbicides. Chloroplast transformation involving introduction of novel DNA into chloroplasts, will not be covered. Preliminary data indicate that chloroplast transformation may be feasible (4) but have not been confirmed. The genetics, physiology... 

Protoplast fusion provides ein alternative method for chloroplast transfer (17,18). Transfer is based on the independent segregation of chloroplasts eind nuclei in heterokaryons obtained by protoplast fusion. Efficiency of recovery for specific combinations of chloroplasts and nuclei has been facilitated by using selectable chloroplast markers such as streptomycin or linoomyoin resistance, or by using maternal pigment-deficient mutants to visually differentiate between clones carrying donor and recipient chloroplasts. Elimination of the nucleus of the chloroplast donor is facilitated by irradiation of donor protoplasts prior to fusion. Methods of chloroplast transfer by protoplast fusion have been reviewed (17,18). In this review only transfer of triazine-resistant chloroplasts will be covered. Work in Nicotiana is more recent than in rapeseed ( 9) and potato (, 1 ), but serves to Illustrate the methodology of chloroplast transfer by protoplast fusion. 

Combination of atrazine resistance and cytoplasmic male sterility in rapeseed. Transfer of atrazine resistant chloroplasts from bird s rape (B. oapestris) into oilseed rape has been accomplished by back-crossTng (s this volume). The purpose achieved by somatic cell fusion was to combine the atrazine-resistant chloroplasts carried by a B. napus line with cytoplasmic male sterility (CMS), a mitoohondrlal tralt (l9). The CMS trait was carried by a B. napus line with the cytoplasm of radish (Raphanus sativus). Since both traits, atrazine resistance and CMS, are maternally inherited in sexual crosses, the only way to combine the traits was by protoplast fusion. The desired atrazine-resistant cybrids were identified in a randomly regenerated sample of plants by the presence of the CMS flower morphology and their darker green color at low temperature. Somatic hybrids rather than cybrids were obtained in each clone since no irradiation had been used to facilitate the elimination of one of the parental nuclei. 

Chloroplast transfer into potato. Transfer of atrazine-resistant planum nigrum chloroplasts into S. tuberosum has been attempted by protoplast fusion (20,21 ). In the absence of irradiating the chloroplast donor S. nigrum, only somatic hybrids were obtained... 

Ovcharenko, O., Momot, V., Cherep, N., Sheludko, Y., Komamitsky, 1., Rudas, V., Kuchuk, N., 2011. Transfer of transfwmed Lesquerella fendleri (Gray) Wats, chloroplasts into Orychophragmus violaceus (L.) O.E. Schulz by protoplast fusion. Plant Cell Tissue Organ Cult. 105, 21-27. 

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