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For detailed protocols, volumes, spin speeds, recipes, and Agrobacterium strains see Weigel and Glazebrook, I like to isolate a minimum of five. You have no control over how many insertions occurred. Having too many insertions can cause artifacts from over-expression and it is likely that such a construct will be silenced in subsequent generations. You will know if you obtained a transformant with a single insertion because it will segregate in the T2 generation.
You also have no control over where your construct will be inserted. If your insertion occurs in a coding region, it may cause unexpected phenotypes.
For most phenotypic analysis it is appropriate to begin working in the T2 generation. For a protocol for antibiotic selection, check out Harrison et al. Laura Lee is a graduate student at Stanford University.
She studies cell fate maintenance in the stomatal lineage and has made countless transgenic plants in the process. Emami, S. BioMed Central, 2 1 , p.
Kyndt, T. National Academy of Sciences, 18 , pp. Nakagawa, T.
Weigel, D. Cold Spring Harbor Laboratory Press. Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues. Preparing plants Whatever plants you are transforming into will be your T0 generation. Tips: Use healthy plants: Your plants should show no signs of stress, such as anthocyanin production, or disease, such as fungal growth.
In contrast to animal cells, however, plant cells have a rigid cell wall which poses limitations to the simple utilization of protocols established for animals. In this protocol, I describe the method used in our laboratory to study histone modifications in the plant model organism Arabidopsis thaliana.elebenylox.ml
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This protocol is an adapted version of the original procedure published by Lawrence and co-workers Lawrence et al. PDF version. Plant Biotechnology. Journal home Advance online publication Journal issue About the journal.
Technical Note. The floral inoculating protocol: a simplified Arabidopsis thaliana transformation method modified from floral dipping.
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Keywords: Agrobacterium , Arabidopsis , floral dip , floral inoculation , transformation. Published: September 25, received: December 03, Released: September 25, accepted: April 16, [Advance Publication] Released: - corrected: -. Article overview. References 7. Related articles 0.