1 Employment of Cytokinin Vectors for Marker-Free… 9

ipt gene in the vector backbone as closely to the left border as

possible. In the event of imprecise cutting at the LB, the ipt
gene will identify the plants with even a small portion of the
backbone integrated.
2. Although the majority of potato varieties regenerate well on
hormone-free medium, the addition of small amounts of
auxin or cytokinin may improve regeneration and transfor-
mation efficiencies. The amount of hormone is not sufficient
to promote cells of untransformed explants to proliferate
and regenerate spontaneously. We found that only one
potato variety does not regenerate on hormone-free medium.
In that case, small amounts of the auxin indole acetic acid
(IAA) were needed to induce regeneration. For all other
varieties tested, however, no addition of hormones is required
to get ipt-driven regeneration of marker-free, backbone-free
shoots.
3. Using the ipt-based method described here, the choice of
explant material has an important effect on marker-free trans-
formation frequencies. When we used tomato cotyledon
explants as starting material, only callus formed on the explants
over the duration of the trial. Shoots never arose from the calli
growing from tomato cotyledon explants. Perhaps the addi-
tion of auxin for some time interval would remedy this prob-
lem, but we have not optimized this method because of the
success using the hypocotyl starting material.
4. Choice of gelling agent in the regeneration medium can prove
important. We discovered that replacing agar with gelzan
accelerated the regeneration time needed for every potato vari-
ety tested. The shoots arising on gelzan tend to be more robust,
and the ipt-positive phenotype is more distinguishable from
the phenotype of shoots without the stable integration of ipt.
This fact provides for a more accurate screening step. After
1 month on hormone-free medium, explants are transferred to
fresh hormone-free medium.
5. To familiarize oneself with the phenotype associated with ipt
overexpression in a particular crop species, it is advised to per-
form a transformation using a binary vector that has an ipt
expression cassette and selectable marker gene within the bor-
ders of the transfer DNA. Use a regeneration medium and pro-
tocol proven to cause regeneration of shoots. The ipt abnormal
phenotype can also be seen by putting young, unrooted shoots
on a medium containing high levels of cytokinin for
2–4 weeks.