Plant Molecular Biology 37: 549–559, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Investigation of Agrobacterium-mediated transformation of apple using
green ﬂuorescent protein: high transient expression and low stable
transformation suggest that factors other than T-DNA transfer are
Siela N. Maximova
, Abhaya M. Dandekar
and Mark J. Guiltinan
Department of Horticulture, 306 Wartik Lab, Pennsylvania State University, University Park, PA 16802, USA
author for correspondence);
Department of Pomology, 1508 Wikson Hall, University of California, Davis, CA
Received 2 October 1997; accepted in revised form 21 January 1998
Key words: Agrobacterium, apple, GFP, transformation
To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green ﬂuor-
escent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple
cultivars (‘Delicious’, ‘Golden Delicious’, ‘Royal Gala’ and ‘Greensleeves’) were infected with Agrobacterium
EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was ﬁrst
detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate. Plant cells with stably
incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic
shoots at low frequencies. The detection of SGFP expression with an epiﬂuorescence stereomicroscope conﬁrmed
the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening
of putative transformants. The efﬁciency of the transformation and regeneration process decreased ca. 10000-fold
from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium
interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.
Genetic engineering offers an exciting opportunity for
improvementof plants with long generationandbreed-
ing cycles such as apple. Transformation of apple
plants expressing marker genes such as nopaline syn-
-glucuronidase (GUS), and neomycin
phosphotransferase(nptII) have been achieved, butthe
transformation frequencies reported are relatively low
[19, 20]. Regardlessof the highshootregenerationfre-
quencies achieved for many apple cultivars with leaf
explantscultured on non-selectivemedia [1, 2, 11, 19–
22, 26, 31, 36, 38, 39], the regenerationof transformed
plants remains difﬁcult and is genotype-dependent.
Recent reports with Agrobacterium-mediated trans-
formation of the commercial cultivars‘Delicious’ 
‘Royal Gala’ , ‘Golden Delicious’ and ‘Elastar’
Green ﬂuorescent protein (GFP) from the jellyﬁsh
Aequorea victoria is a novel genetic reporter system
 and has become an important in vivo reporter
in plants [13, 16, 29, 30, 35]. When expressed in
either eukaryotic or prokaryotic cells and illuminated
with blue light (395 nm), GFP yields bright green
ﬂuorescence. This ﬂuorescence is stable, species-
independent, and can be monitored non-destructively.
Light-stimulated GFP ﬂuorescence does not require
any co-factors, substrates, or additional gene products.
GFP has now been successfully expressed in a large
the nematode Caenorhabditiselegans, Arabidopsis
thaliana , and Nicotiana tabacum . In these
organisms, GFP accumulation did not appear to have
a toxic effect.