protocol of in planta transformation
xt at VISION.POSTECH.AC.KR
Wed Dec 4 01:57:50 EST 1991
I realize that many labs have tried the in planta transformation
protocols developed in my lab,but only a few lab have succeeded.The
protocol certainly works but one has to know what he is doing and has
to try many plants at least until you get the feeling.The following is
the protocol I have distributed for the Arabidopsis traing working in
Japan(Nov.26,1991 which was organized by Dr.Okada at NIBB,Okazaki).I
have a postdoctoral fellow who is very good at the protocol.If one is
interested in using this transformation protocol,I could send him to
your lab for about 6 months or so,(which your support for his living
expenses).My modem is out of order at this moment and I am using
other's computer.It might be easier to contat me by fax for the
My fax # is 82-526-79-2199,and my address is
Dept. of Life Science
Agrobacterium genetically colonizes wounded plant cells by
transferring a defined segment(T-DNA) of its Ti plasmid into the plant
nuclear genome.Although this natural gene -transfer mechanism has been
widely adopted in plant transformation,the possibility of generating
transformed progeny by Agrobacterium infection in planta has not been
successfuly exploited.Here we show stably transformed progenies of a
crucifer plant,Arabidopsis ,are efficiently obtained by simple
incision of primary shoots at their bases and by subsequent
Agrobacterium inoculation at the wound site.Adventitous shoots arising
from the infected wound site were transformed at a high frequency and
produced transformed progenies later.
The Transformation Procedure
1. Seed germination and plant growth
(1) Soak seeds on the fillters wetted in 10 mM KNO3,10 mM NaPO4 pH
7.0 and place at 4 degree C. in the dark for 48 hours for
(2) Sow cold-treated seeds on compounded soil or vermiculite soaked
with Hogland's solution and cover the pots with saran wrap.2)
(3) Transfer germinated seedlings to a ligh controlled(16hr
light/8hr dark) growth chamber.
(4) For healthy growth,water and fertilize sufficiently,and protect
from fungal or insects attack.3),4)
2. Wounding and Agrobacteria infection
(1) When the primary inflorescences grow to 2-3 cm in hight,remove
them by severing the basal part with a scalpel or scissors and remove
auxillary buds as completely as possible with a fine forceps.This step
is most critical.What you want to do is to remove as many preformed
shoot meristema as possible,and to make reorganization of shoot
meristems from infected and transformed cells.5)
(2) Sometimes it helps to make deep wound into the remaining stem
with a needle or toothpick.
(3) Inoculate wounds with Agrobacteria from overnight culture grown
in YEP media.6)
(4) Place inoculated plants in the shade or low ligh intensity for
2-3 days at 28 degree C.
(5)Transfer plants (POTS) to standard growth conditions,until
secondary inflorescences emerge (about 10-14 days).
(6) One or more inflorescences should develop from the wound
site.Remove the early emerging inflorescences and reinoculate
Agrobacteria onto the wound sites by repeating the procedure described
(7) Efficiency of transformation may be tested by assay of leaves
on the newly formed shoots.7)
(8) The later emerging inflorescences are maintained through
maturity for seed generation.
1) Seeds may be directly sown on soil without imbibition and/or
cold treatment,but the germination will not be uniform.
2) Metromix 200 or any proper soil.
3) For efficient transformation,plant materials must be healthy.
4) Hogland's solution or commercial mixture ( such as Hyponex).
5) The timing of infection is important.
6) Overnight culture agrobacteria are concentrated to 1/5 volume by
centrifugation at 600 rpm for 5 min,followed by resuspension in the
7)We used pBI121,a binary vector containing the NPT II and GUS
genes within the T-DNA region.
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