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Help needed

Wang Haiyang whyang at BIOLOGY.LSA.UMICH.EDU
Tue Aug 20 18:08:30 EST 1996

Hi, Everyone, I got a couple of questions concerning the plant
transformation vectors PBI101 series and PBin19, your information or help
is greatly appreciated.
 1. Any unique restriction sites in the GUS reporter gene or the vector
itself , but not in the NPTII gene?
 2. If I express a cDNA with some promoter using the PBin19 vector, do I
need to add a nos or somekind of terminator sequence?
 3. When I work on plant transformation using the root explant protocol
and Kanamycin selection, I preincubate the roots on CIM for 2-3 days and
then infect with agrobacteria, incubate for another 3 days on CIM, then
washing and transfer to SIM plates, after about 10 days, shoots or
leaf-like structures start to form directly, and I don't see much distinct
green callus first. In the end, most of the transgenic plants are
false-positive. I believe the timing on CIM plates is important for the
root explant to form callus and rediffertiation into embryonic shoots.
Please give me some suggestion on this issue. 

Haiyang Wang
University of Michigan

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