1021-4437/03/5002- $25.00 © 2003
Russian Journal of Plant Physiology, Vol. 50, No. 2, 2003, pp. 276–281. Translated from Fiziologiya Rastenii, Vol. 50, No. 2, 2003, pp. 309–315.
Original Russian Text Copyright © 2003 by Malyshenko, Tyul’kina, Zvereva, Raldugina.
Two Brassicaceae species, summer rape (
L.) and oilseed rape (
L.) are important oil and fodder crops. Because of
great agronomic importance, these crops engage numer-
ous researchers who work on breeding new cultivars for
higher resistance to fungal infections , viruses ,
insects [3, 4], and herbicides [5–8], for modiﬁed quality of
oil [9–11] and protein [12–14], and other economic traits.
In many cases these researchers employ biotechnologies,
including plant genetic transformation. Most of studies
were performed with
because of the high regen-
eration capacity of this species, which is most often a crit-
ical factor for successful genetic transformation.
In contrast to oilseed rape, summer rape manifests a
low capacity for regeneration [15, 16]. Presently there
is a single report on transgenic
obtained from seedling hypocotyl explants .
Our objective was to work out an efﬁcient technique
of summer rape transformation using cotyledonary
leaves as explants. Transformation was assessed by the
presence of the reporter
(jellyﬁsh) expressing GFP .
MATERIALS AND METHODS
Seeds of summer rape (
Lura), bred in Finland, were used in experiments.
All experiments with
cultured plant tissues
were performed using an agar-supplemented MS media
 plus sucrose and growth regulators at various con-
centrations (Table 1). Agar was added to 5–7 g/l.
To obtain seedlings
, seeds were treated for
5 min with 70% ethanol, for 30 min with 20% Domes-
tos cleanser (Lever, Hungary) and washed ﬁve times
with sterile distilled water. Disinfected seeds were ger-
minated onto the medium I (Table 1) at 20
C and 16-h
For plant transformation, we used
strain AGL1  comprising the following
binary vector system: the Ti-plasmid pTi Bo542, with
deleted T-DNA region, and the vector pBin m-gfp5-ER
(MRC Laboratory of Molecular Biology); as a select-
able marker; the latter contained the
gene for kan-
amycin resistance ﬂanked with the
nos terminator, and also the modiﬁed
gene from the
under the 35S cauliﬂower mosaic
virus promoter (Fig. 1).
Agrobacteria were grown at 26
C in an LB medium
(1.0 g/l yeast extract, 5.0 g/l peptone, 5.0 g/l sucrose,
0.5 g/l MgSO
O, and 15 g/l agar). To obtain a
suspension culture of agrobacteria, agar was omitted
from the medium.
For regeneration experiments, we used the technique
developed previously for rape cotyledon explants .
Cotyledons of 5-day-old seedlings were trans-
formed by cocultivation with overnight (10–12 h) cul-
ture of agrobacteria. Bacterial suspension was centri-
fuged for 15 min at 3000
, suspended in the liquid MS
medium to optical density D
of 0.5, and 50
l of this
suspension was applied onto the agar-supplemented
medium II (Table 1) and distributed uniformly with a
Plants Expressing the
S. I. Malyshenko*, L. G. Tyul’kina*, S. D. Zvereva**, and G. N. Raldugina**
*Faculty of Biology, Moscow State University, Vorob’evy gory, 119899 Russia
**Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;
fax: 7 (095) 977-8018; e-mail: email@example.com
Received June 28, 2001
—A protocol has been worked out for efﬁcient regeneration and genetic transformation of summer
). Cotyledons of the 5-day-old seedlings were transformed with
strain AGL cells comprising a binary vector with a selectable neomycin phosphotransferase II
gene and a reporter gene encoding green ﬂuorescent protein (GFP). Explants were cultured on a regeneration
MS medium supplemented with ABA, and transformants were selected on the same medium (minus ABA and
plus 70 mM AgNO
and 15 mg/l kanamycin). The frequency of shoot regeneration on explant petioles was 30–
40%. Transgenic plants were identiﬁed by GFP ﬂuorescence and by polymerase chain reaction and Western
blotting analysis. The transformation efﬁciency was as high as 75% of the total number of regenerated shoots.
Key words: Brassica campestris - regeneration - transformation - transgenic plants - gfp gene
: BA—benzyladenine; Cc—carbenicyllin; Cl—
claforan; dNTP—deoxynucleotide triphosphate; GFP—green ﬂu-
—gene encoding GFP; Km—kanamycin;
MS—Murashige and Skoog nutrient medium; nos—nopaline
—gene encoding neomycin phosphotransferase II;
PCR—polymerase chain reaction.