protocol of in planta transformation

kim ki_jong xt at VISION.POSTECH.AC.KR
Wed Dec 4 01:57:50 EST 1991


 Dear Colleague:
 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 
moment.
 My fax # is 82-526-79-2199,and my address is
    Dr.Hong-Gil Nam
    Dept. of Life Science
    POSTECH,P.O.Box 125
    Pohang, Kyungbuk,790-600
    South Korea


 Background

  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 
vernalization.1)
   (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 
above.
   (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.

  -Notes-

   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 
remaining media.
   7)We used pBI121,a binary vector containing  the  NPT  II  and  GUS 
genes within the T-DNA region.
   
   Sincerely,
   Hong-Gil Nam



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