"Cell grafting": a new approach for transferring cytoplasmic or nuclear genome between plants.

Abstract

A new method based on mixing and wounding of callus tissue was used to transfer plastid or nuclear DNA between cells. Methods alternative to sexual hybridization can be powerful tools for crop improvement. We have developed a new hybridization technology based on wounding a mixed population of cells of two parents growing in vitro as callus ("cell grafting"), and have demonstrated the utility of this system for plastid or nuclear genome transfer. In our proof-of concept experiments, non-organized growing tissue (callus) from tobacco var. Samsun, carrying the nuclear marker genes nptII and uidA (GUS), and tobacco var. Petit Havana, carrying aadA and gfp genes in the plastid genome, were mixed together, wounded with a razor blade and placed for regeneration on selection medium containing both spectinomycin (aadA) and paromomycin (nptII). Plants with aadA and gfp positive plastids and nptII plus uidA positive nuclear background were produced. Molecular analysis confirmed the presence of all four genes in these plants. Morphology and ploidy level analysis confirmed the production of "diploid" plants similar to var. Samsun possessing transformed plastids from var. Petit Havana. Reciprocal crosses between the experimentally produced plants and wild type tobacco confirmed maternal inheritance of aadA and gfp and Mendelian inheritance of nptII and uidA. For transfer of nuclear traits between plants we used two nuclear-transformed parents with different selectable markers; one with nptII (paromomycin resistant), and another with aadA (spectinomycin resistant). Plants resistant to both antibiotics which also had different visible markers were produced.