Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film

Somatic hybridization has been identified as one method for the genetic improvement of roses. The success of somatic hybridization programmes relies to a great extent upon efficient protoplast isolation and culture and selection of heterokaryons. This paper reports the isolation of rose cell suspension protoplasts by direct sucrose flotation and demonstrates their culture using extra thin alginate film. A comparative assessment of the efficiency of conventional culture techniques versus those with extra thin alginate film or thin alginate layer is also presented. A very high plating efficiency (80%) was obtained using thin alginate layer or extra thin alginate film techniques with improved media formulations. Protoplasts of Rosa damascena and R. bourboniana were fused by using polyethylene glycol as fusogen and later immobilized in the thin layer of alginate. The fused protoplasts were tracked on the basis of differential fluorescent staining, and the hybridity of heterokaryons following their development to callus was confirmed by molecular characterization. This novel selection strategy has general applicability and is faster and simpler to perform during somatic hybridization experiments.