ISSN 1021-4437, Russian Journal of Plant Physiology, 2007, Vol. 54, No. 3, pp. 420–425. © Pleiades Publishing, Ltd., 2007.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 475–480.
Unicellular plant microspores are single haploid
cells, which can be collected in large amounts and fer-
tilized in vitro. The direct transformation of pollen
grains is a high throughput transformation method. It is
an interesting alternative to transformation procedures
because such tissue culture steps as somaclonal varia-
tion or regeneration are avoided. A variety of methods
have been employed to introduce genes into mature
pollen grains, including the electroporation [1, 2] and
particle bombardment . Because of its relatively high
transformation efﬁciency and simple technology with
lower cost, as well as its ability to transfer large pieces
of DNA with minimal rearrangement, the
-mediated transformation has also been widely
used [4–6]. The transgenic plants generated by this
method were stably inherited , and it was indicated
that the method is very efﬁcient and useful.
-mediated vacuum inﬁltration method
(or ﬂoral-dip method), had a major impact on plant
genetic and molecular research. The method suggested
has been considered as the extension of pollen transfor-
mation method . However, the biological mechanism
of this process remains to be clariﬁed.
Lilies are monocotyledonous ornamental plants
widespread in the market; so they can supply enough
pollen for the investigation. A critical step in the devel-
-mediated transformation is
the establishment of optimum conditions for T-DNA
delivery into tissue. Here, using GUS as a marker, the
effects of various factors on T-DNA delivery into
mature pollen of David lily in vitro were investigated.
It was shown that the medium,
density, duration of co-cultivation, and the combination
of bacterial strains and plasmids should be optimized to
get the highest transformation frequency, but the addi-
tion of acetosyringone (AS) and Pluronic F68 seemed
to have no signiﬁcant positive effect in this model sys-
MATERIALS AND METHODS
Plant materials. Lilium davidii
grown in the garden of Lanzhou University were used
in these experiments. Every May, when lily is ﬂower-
ing, its pollen grains were collected to be further used.
Bacterial strains and plasmids.
strains with the following binary vec-
tors (table) were used: EHA101, LBA4404, and
GV3101. An intron-containing GUS gene (
the control of CaMV 35S promoter and terminated by
the NOS polyadenylation signal was present in the
T-DNA regions of all three binary vectors. This gene is
expressed very efﬁciently in monocots, but not in
Strains were grown at
for 3 days on
AB medium consisting of sucrose 1.25 g/l,
g/l, KCl 0.15 g/l,
Factors Influencing T-DNA Transferring into Pollen of Lily in vitro
, Y. F. Mao
, and W. Li
Marine Biological Engineering Dept., Ocean University of China, Qingdao, 266003 China
Institute of Cell Biology, Lanzhou University, Lanzhou, 730000 China;
Received January 4, 2006
—Direct pollen transformation method improves the classical transformation procedures because
some tissue culture steps and subsequent regeneration can be avoided. A critical step in the development of
-mediated transformation is the establishment of optimum conditions for T-DNA delivery into
tissue. The pollen grains of David lily (
Duchartre) are transformable by
their germination, and extremely high GUS expression frequency of pollen had been achieved (
but not for the ungerminated pollen. The culture medium,
cell density, duration of co-cultiva-
tion, and the combination of bacterial strains and plasmids should be optimized to get the highest transforma-
tion frequency. Thus, a method for pollen monocotyledonous species reproductive tissues transformation by
in monocots has been successfully developed.
Key words: Lilium davidii - Agrobacterium tumefaciens -GUS - transformation - pollen
: AS—acetosyringone; GUS—
MS—Murashige and Skoog nutrient medium.
The text was submitted by the authors in English.