Transfer of defined numbers of chloroplasts into albino protoplasts by subprotoplast/protoplast microfusion: chloroplasts can be "cloned", by using suitable plastome combinations or selective pressure.
Defined numbers (1-5) of (donor) chloroplasts were transferred into (acceptor) protoplasts of plastid albino mutants by subprotoplasts/protoplast microfusion. Single transferred plastids gave rise to new organelle populations in the progeny of the fusion products when suitable combinations of plastomes were used or when selective pressure for the plastome transferred was applied. This process is termed "chloroplast cloning" and is the first reported case of "cloning" a cell organelle. The plastome combination and the presence or absence of selective pressure were found to influence the frequencies with which cell lines, containing both plastomes or acceptor or donor only, were obtained, and the number of cell generations needed for complete segregation - as measured by the duration of culture before the green donor plastome could be detected. The high frequency of cell lines and regenerated shoots recovered with donor plastome only, even when only a single chloroplast was transferred, leads to the conclusion that all organelles present in the fusion product contribute to the organelle population of the progeny, i.e. organelle death or loss are not regularly occurring events during plant regeneration from protoplasts in Nicotiana tabacum.