Analysis of Genes for Stigma Coloration in Rice - IRRI books

ice callus plates was very poor (maximum 34). The number of expression units in
different experiments varied perhaps because of the inability to obtain uniform
dispersion patterns and precise targeting of the microprojectiles. A few potentially
transformed calli exhibiting GUS activity were regenerated into plants, and their
genetic analysis is under way. This technique was also utilized in unsuccessful attempts
to introduce DNA vectors into highly embryogenic calli of wheat anthers. The failure
with wheat was probably due to the very compact and brittle texture of the anther callus,
which is apparently refractive to microprojectile entry. Studies are in progress to
optimize the bombardment conditions and to alter the wheat callus texture so that it is
more conducive to microprojectile entry.
Although the microprojectile-mediated DNA transfer technique suffers from some
disadvantages, i.e., low transformation frequency and expensive instrumentation, it is
relatively easy to perform and does not involve any tedious cell culture or protoplasting
steps. This approach has already yielded some stable transformants in dicots (Christou
et a1 1988, Klein et a1 1987, McCabe et al 1988) and could also be a promising approach
for monocots, especially for those plants where a protoplast regeneration system is not
available.
Pollen tube-mediated DNA introduction
In another attempt to transform rice and wheat plants, the pollen tube pathway
technique described by Luo and Wu (1988) was employed. Stigmas of rice and wheat
flowers pollinated for 6-8 h were cut with a sterile razor blade, and a DNA solution
containing the GUS reporter gene was applied to the decapitated florets. Histochemical
staining for GUS activity (Jefferson et a1 1987) in seeds obtained from the DNA-treated
rice florets showed that in some instances seed tissues could be transfected with
exogenous DNA. Approximately 10% of the seed samples exhibited GUS activity, but
only in small patches distributed over the seed coat. Less than 1% of the seeds showed
stain in the embryo region. The potentially transformed rice seeds are being analyzed
for the stable integration of plasmid DNA in the F 1 and F 2 . Despite several attempts,
this technique has not been successful with wheat, as no-trace of GUS activity was
evident in the embryo regions.
Thus it appears that this approach, though simple and easy, may not be applicable
to all monocots. Although the pollen tube method does not involve tissue culture
expertise or expensive instrumentation, the frequency of seed set on decapitated
stigmas is very poor (~10%). Moreover, only about 10% of the progeny display any
evidence of DNA uptake, as judged by histochemical staining methods, and an even
smaller percentage of seeds exhibits GUS gene expression in embryonic tissues. The
stable integration of DNA in these seeds is yet to be confirmed. However, if the DNA
introduced by this technique were stably integrated and expressed faithfully in
subsequent generations, it would be an inexpensive method for obtaining transgenic
rice plants, especially with indicas and those varieties where protoplast regeneration
is too cumbersome.
600 Aryan et al