Transent Expression n Protoplasts

This protocol was contributed by fumiaki Katagiri (Torrey Mesa Research Institute) and is based on those published by Damm et al. (1989) and Abel and Theologis (1994) and an unpublished protocol from Jen Sheen (Massachusetts General Hospital). Use a wide-bore pipette for all transfers of protoplasts, to avoid lysing them. Enzyme solution (prepare fresh 1 hour before cutting leaves) Use a 14-ml Falcon tube or equivalent. Incubate for 10 minutes at 50 C. Cool to room temperature and then add...

Info

Students and researchers using the procedures in this manual do so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this manual and has no liability in connection with the use of these materials. All registered trademarks and service marks mentioned in this book are the property of the respective owners. Readers should please consult individual manufacturers and other resources for current and specific product...

Flowering Time

This protocol was provided by Miguel Bldzquez (Salk Institute). Flowering in Arabidopsis is modulated by a number of environmental factors (Bernier et al. 1993). It is therefore important to perform flowering-time measurements under controlled environmental conditions, including light quality and intensity, day length, temperature, watering, and spacing between plants. Ideally, pots of plants are rotated, and individual plants of different genotypes are placed randomly within the experimental...

Cultivation Of Arabidopsis

In most natural habitats, Arabidopsis is a winter annual Its seeds germinate in the fall, the young plants survive the winter, floral meristems emerge in the spring, and only the seeds survive the summer months (Baskin and Baskin 1972). The plants are healthiest and most fertile when grown under the conditions given below. For additional information, see the Web Site of the Arabidopsis Biological Resource Center at Most common laboratory varieties of Arabidopsis flower within 4 weeks of...

Quantitative Gus Activity Assays

This protocol was contributed by Miguel Blazquez (Salk Institute). GUS activity can be accurately determined both in extracts and in intact plant Lssue us ng 4-methylumbellyferyl p-D-glucuronide (4-MUG) as a substrate (Jefferson et al. 1987). Upon hydrolysis by GUS, the fluo-rochrome 4-methyl umbelliferone (4-MU) is produced. Using excitation at 365 nm, and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the...

Morphology And Anatomy

The major anatomical features of Arabidopsis and its key developmental landmarks are briefly summarized here cited references provide a more thorough analysis. Recently fertilized ovules contain small embryos with a suspensor (see Figure 1.1, dark gray) and the embryo proper (light gray). The subsequent developmental phase takes place over approximately 7 days and is divided into the globular, heart-shape, torpedo-shape, and cotyledo-nous stages (Meinke and Sussex 1979 Mansfield and Briarty...

Basta Selection on Soil

The major advantage of Basta selection is that it can be performed on plants growing in soil and does not require the use of sterile techniques. Basta glufosinate ammonium can be purchased as a herbicide solution from garden supply stores. In the United States, it is sold as Finale by Scotts Company, which has a long-term supply agreement with the manufacturer, AgrEvo. It can also be ordered on-line http www.biconet. com lawn finale.html . The pure chemical can be ordered in the United States...

Subcellular Localization Of Gus And Gfptagged Proteins In Onion Epidermal Cells

This protocol was contributed by Albrecht von Arnim The University of Tennessee, Knoxville . An excellent alternative to antibodies for determining the subcellular localization of proteins is the use of recombinant tags, i.e., reporter proteins. Tags offer better specificity than antibodies, but they also carry a higher risk of artifacts due to protein folding and overexpression. These factors must be balanced. The most user-friendly tags are the GUS reporter enzyme from E. coli and the...

References

Ecological life cycle and physiological ecology of seed germination of Arabidopsis thaliana. Can. . Bot. 50 353-360. Dolan L,, Janmaat K., W Hemsen V., Linstead P., Poethig S., Roberts R., and Scheres B. 1993. Cellular organisation of the Arabidopsis thaliana root. Development 119 71-84. Felmann K.A. and Marks M.D. 1987. Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana A non-tissue culture approach. Mol. Gen. Genet. 208 1-9....

Luc

Restriction enzymes for inserting promoter fragments A AurlV, B BamHl C Clal H Hindlll K Kpnl P Fsfl S Sail X Smal or Xmal. Restriction enzymes for inserting promoter fragments A AurlV, B BamHl C Clal H Hindlll K Kpnl P Fsfl S Sail X Smal or Xmal. contain the Q translational enhancer region from tobacco mosaic virus Gallie et al. 1987 and the 3'-un translated E9 polyadenylation region from the pea RBCS-3A gene to enhance expression of the LUC transgene. The binary transformation vectors are...