Transformed cells are reluctant regenerators
Although the first successful laboratory use of
Agrobacterium tumefaciens to bring about plant genetic transformation occurred over 20 years ago, many economically important crops remain recalcitrant. There are several key factors in successful genetic manipulation. One of these is susceptibility of the host to the
A. tumefaciens-mediated gene transfer system: extensive research has extended the list of susceptible species way beyond the natural range, including several
Citrus species, as described by
Peña et al. (Valencia, Spain, pp. 67–74). Further, even if a particular species cannot be transformed
via A. tumefaciens, then direct DNA delivery systems, developed over the past 15 years, may be used. The other key step is the regeneration of plants from the transformed cells and, as the Valencia group point out, transformation-competent cells may not be the same as regeneration-competent cells. This group has used epicotyl explants from a hybrid citrus to study the location of the transformed cells (detected by assay of the beta-glucuronidase reporter gene), under different culture conditions. The data clearly show that a medium containing auxin was by far the most efficient for transformation. This was associated with the induction of DNA replication and cell division in the cambium of the explants, as demonstrated by flow cytometry; this cell division activity led to the establishment of a cambium-derived callus. Such data are consistent with observations made by several authors (including Alicja Ziemienowicz in my lab): efficient integration of foreign DNA requires the host’s DNA replication ‘machinery’. However, de-differentiation and cell division are not good attributes for morphogenesis and, in fact, relatively few shoots were formed from the auxin-incubated callus. Nevertheless, the transformation frequency was so much higher than in other media, that it still gave the most transformed shoots. This illustrates nicely that well-worn English expression: ‘What you lose on the swings you gain on the roundabouts’. Whether this is the most primitive form of SI however remains unclear.
j.a.bryant{at}exeter.ac.uk
