Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 4.33

DIRECT GENE TRANSFER FOR NUCLEAR AND PLASTID TRANSFORMATION IN POTATO

CRAIG W., GARGANO D., CARDI T.

CNR-IMOF, Research Institute for Vegetable and Ornamental Plant Breeding, Via Università 133, 80055 Portici

cardi@unina.it

Solanum tuberosum, potato, direct gene transfer, nuclear transformation, plastid transformation

Agrobacterium tumefaciens -mediated transformation is the most widely used method for foreign gene transfer to potato (Solanum tuberosum). However, due to its restriction on the number of transgenes that can be transferred during one transformation event (usually less than 3 per T-DNA, at least one of which should be a selectable marker), particle bombardment has been proposed as the method of choice to transfer multiple genes. Considering that biolistics is not yet a routine transformation procedure for potato, and that new technologies (eg chloroplast transformation of higher plants) are emerging which need a highly regenerative A. tumefaciens -free DNA transfer protocol, it was evident that alternative strategies were required in order to expand the crop base to which plastid transformation could be applied. Therefore, in our laboratory, methods for polyethylene glycol (PEG)-mediated direct DNA transfer into leaf-derived protoplasts, and the particle bombardment of leaf explants, were successfully established for transient and stable nuclear transformation of potato cultivar Desireè. These were based upon efficient in house regeneration protocols developed for a wide range of S. tuberosum genotypes. Together, they formed the basis of our current plastid transformation studies.

A number of parameters that influence transient transformation levels were evaluated using the ß-glucuronidase gene (gus ). Firing gus -coated 0.6 µm gold particles, using a pressure of 1100psi, achieved mean transient expression levels of 53.7-85.6 and 31.0-110.2 blue spots per leaf in explants 6 and 9 cm distant, respectively. For direct DNA uptake studies, transient GUS expression experiments regularly produced levels of 5 pmol 4-MU/µg protein/min by incubating 1.6 x 10⁶ protoplasts/ml with 50 µg/ml plasmid DNA and 50 µg/ml carrier DNA for 15 mins with 12.5 % final concentration of PEG 4000.

Taken together, these levels were considered sufficient to begin stable nuclear transformation investigations, incorporating hygromycin B phosphotransferase gene (hph ) as a selectable marker and using 10-15 mg/L hygromycin during plant regeneration. This resulted in 2.1% (total = 13) of bombarded explants producing GUS positive shoots, and up to 62 % (total = 39) of hygromycin-resistant protoplast-derived calli producing a minimum of one GUS positive shoot. For protoplast-based studies, this is equivalent to stable transformation frequencies of 0.1-9.6 x 10^-5 (based on the number of protoplasts treated) or 8.34 % (based on the number of control regenerating p-calli formed).

These results were confirmed by PCR and Southern analyses and are now being used in chloroplast transformation studies of potato, using appropriate transformation vectors. Green, spectinomycin-resistant calli have been recovered. Some of these have begun to develop shoots, the first of which are being characterised at the molecular level.