ISSN 1021-4437, Russian Journal of Plant Physiology, 2006, Vol. 53, No. 2, pp. 252–256. © MAIK “Nauka /Interperiodica” (Russia), 2006.
Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284.
L.), as a world-
wide vegetable plant, needs continuous efforts in tradi-
tional breeding and genetic transformation in order to
improve its quality and yield. Since the ﬁrst commer-
cial genetically modiﬁed (GM) tomato plant was pro-
duced in 1994, 105 events of commercial GM plants
have been released, in which 6 events of tomato are
included by 2005 (Essential Biosafety, Crop Database).
When dicot plants are the targets for genetic transfor-
-mediated technology is nor-
mally used. This method has a number of advantages
such as high and stable gene insertion efﬁciency, trans-
ferring relatively long DNA fragment, low copies of
gene insertion . Many transgenic tomatoes have been
produced with this method [2–8]. Although
-mediated transformation already becomes practi-
cable, the factors inﬂuencing transformation efﬁciency
still need to be optimized. In this paper, the elements
inﬂuencing the transformation efﬁciency will be dis-
MATERIALS AND METHODS
cv. Lichun) seeds were rinsed with 70% ethanol for
1 min. Then, the seeds were surface-sterilized in 10%
(v/v) sodium hypochlorite solution with 2 drops of
Tween-20 for 5–8 min followed by ﬁve rinses in sterile
water. Seeds were germinated on 0.5 MS medium 
with 16-h light and 8-h dark regime. Cotyledons and
hypocotyls from 10-day-old seedlings were cultured on
a preculture medium (table) for 48–72 h.
Bacterial strains and plasmids.
strain LBA4404 with pTOK233  and
GDD  were employed.
inoculated overnight in LB medium containing 50 mg/l
kanamycin and 100 mg/l rifampicin at
rotatory shaker. LB medium was made of 5 g/l yeast
extract, 10 g/l tryptone, and 10 g/l NaCl.
ture was precipitated, and the pellet was resuspended
in sterile H
O containing 50
M acetosyringone until
optical density at 600 nm becomes equal to 1.0. At the
end of the one-day preculture, the explants were dipped
suspension for various durations.
The excessive bacterium culture was absorbed by ster-
ile ﬁlter paper. Then, the explants were placed on the
preculture medium covered by a piece of sterile ﬁlter
paper for a three-day cocultivation (Zhiming Wei, per-
An Experimental Assessment of the Factors
-Mediated Transformation in Tomato
Y. F. Wu*, Y. Chen*, X. M. Liang**, and X. Z. Wang*
*Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China;
fax: +86-431-5099822; e-mail: email@example.com
**Department of Biochemistry, Hulunbeir College, Inner Mongolia, Hailaer, 021008, China
Received August 23, 2005
strain LBA4404 carrying a binary vector pTOK233, which contained
reporter gene and a kanamycin-resistance gene
, was employed for optimizing the transformation
efﬁciency evaluated by a
gene transient expression level. Eight factors including explant types, explant
size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media,
bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail.
This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic
virus (CMV) mediated by
, strain LBA4404, carrying a binary vector pR-
containing the kanamycin-resistance gene and CMV replicase gene with GDD deletion. The presence of the
gene was conﬁrmed by genomic DNA Southern blot analysis in all transformants analyzed. Field
spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin.
Key words: Lycopersicon esculentum - Agrobacterium tumefaciens - transformation - transgenic tomato
: AS—acetosyringone; BA—benzyladenine;
CMV—cucumber mosaic virus; GM—genetically modiﬁed;
MS—Murashige and Skoog nutrient medium; PCR—polymerase
The text was submitted by the authors in English.