Rhipsalidopsis × graeseri

The hybrid and the material studied

POWO lists Rhipsalidopsis × graeseri as an artificial hybrid with the formula R. gaertneri × R. rosea. Hatiora × graeseri and Schlumbergera × graeseri are synonyms. The multiplication sign remains part of the name because it identifies hybrid origin. A cultivar name additionally identifies particular horticultural material [2-WWW].

The title of the 2006 paper by Al-Ramamneh and colleagues names Rhipsalidopsis gaertneri. In the methods, however, the authors identified the cultivar used, ‘CB5’, as a hybrid between R. gaertneri and R. rosea. The results below concern that cultivar and material derived from its stem segments. Assignment to R. × graeseri follows the stated parentage and the current taxonomic treatment [Al-Ramamneh et al. 2006a] [2-WWW].

From stem segment to transformed callus

Stem pieces were used to produce callus, tissue multiplied on a growth medium. It was subcultured for 9–12 months and then exposed to Agrobacterium tumefaciens. Strain LBA4404 transferred a construct carrying the reporter gene uidA and the selectable marker nptII. The former enabled detection of introduced gene activity through blue staining in the GUS assay. The latter allowed tissue selection in the presence of kanamycin [Al-Ramamneh et al. 2006a].

Selection was prolonged because untransformed tissues showed substantial tolerance to kanamycin. The resulting callus lines were maintained, among other treatments, at 600 mg/l. Bacterial suspension density also affected the outcome. Excessively dense suspensions damaged callus, whereas successful treatments used optical densities OD₆₀₀ of approximately 0.4–0.6. These parameters describe a particular laboratory procedure and its starting material [Al-Ramamneh et al. 2006a].

Shoot regeneration and transformation evidence

After prolonged selection, reducing or removing kanamycin and allowing the culture medium to become nutrient depleted favoured adventitious shoot formation. The best reported treatment yielded 10 GUS-positive shoots from 44 initial callus explants. The authors expressed this relationship as a transformation efficiency of 22.7%. The denominator was the number of callus explants entering the procedure, which defines the meaning of this measure [Al-Ramamneh et al. 2006a].

GUS staining assessed reporter gene activity. An immunoassay detected the NPTII protein, while Southern blot analysis supplied evidence that introduced DNA was present in the genomes of selected callus lines. The methods therefore addressed different aspects of the procedure's outcome. Transformation and the regenerative capacity of some tissues were established, while inheritance in a subsequent sexual generation remained outside the study [Al-Ramamneh et al. 2006a].

Distinguishing a method from an improved horticultural trait

The authors also tested another bacterial strain carrying a construct with etr1-1, a gene associated with altered ethylene response. That treatment produced no transformed callus or shoots. The successful part of the work involved the reporter and selectable marker genes. The publication therefore provides a method and evidence of transformation in ‘CB5’, rather than a demonstrated increase in flower longevity [Al-Ramamneh et al. 2006a].

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