Month: November 2018

Transforming Agrobacterium by the freeze thaw method

Plant transformation is frequently carried out with the aid of Agrobacterium. Agrobacterium is a naturally occurring bacteria that genetically modifies plants in the wild. If you have ever seen a tree with a large tumor on it, you have seen the work of Agrobacterium. Currently, scientists exploit the genetic engineering capabilities of a domesticated Agrobacterium to transform plants.

This video shows you how to transform Agrobacterium by the freeze-thaw method. Agrobacterium transformation by the freeze thaw method is cheap, fast and efficient. The transformation and recovery steps can be performed in a single tube. Making Agrobacterium competent cells for the freeze thaw method is easy because it is not necessary to capture the cells in log phase growth. Instead, an overnight culture of Agrobacterium can be used to make these competent cells. The freeze-thaw method does not require electroporation apparatus or expensive electroporation cuvettes. The freeze-thaw transformation method is not as efficient as electroporation, however, it always gives more than enough colonies. A written version of the Agrobacterium freeze thaw transformation can be downloaded here. Once you have your desired binary vector in Agrobacterium, you could easily transform Arabidopsis by Agrobacterium floral dip.

Advantages over electroporation

  1. No expensive equipment required.
  2. Very cheap.
  3. Competent cells are made from an overnight culture rather than a culture in log phase growth.
  4. Transformation and recovery are performed in a single tube.

Sterilizing seeds and selecting for hygromycin resistance

This video protocol shows you how to surface sterilize Arabidopsis seeds and select for hygromycin resistance. Hygromycin selection is one of the fastest and most clear cut selections for transgenic Arabidopsis. Unfortunately, hygromycin does not do a good job killing non-resistant plants. However, the selection outlined in this post is extremely fast and easy to do. The selection takes advantage of the fact that plants elongate in the dark and that hygromycin inhibits this elongation. An Arabidopsis Seed Sterilization Protocol for Screening can be downloaded here. Surface sterilized Arabidopsis seeds that have been cold treated overnight  should be put onto half strength MS plates with 50 µg/mL hygromycin for selection of transgenic plants. Plates are exposed to light for 4 hours followed by 5 days in the dark. Transgenic plants will have elongated hypocotyls. The recipe of 0.5x MS hygromycin is shown in the table below.

0.5x MS from MS powderamountunits
ddH 2 O196.7mL
MS Powder (MP)0.443g
0.5 M MES (pH 5.7)0.8mL
plant agar or agarose (to get 0.5%)1g
add after autoclaving:
40% sucrose2.5mL
desired volume200mL
add ingredients in order except sucrose solution to a flask
autoclave for 5-15 minutes on a liquid cycle
mix agar by swirling after autoclaving is finished
add indicated amount of sterile sucrose
if necessary, cool to 55-60 °C and add desired selection agent like glufosinate, kanamycin, or hygromycin
(ie. 200 µL 50 mg/mL hygromycin that was frozen at -20°C)
mix by swirling then pour plates
Notes:
10x MS salts = 4.43 g MP Biomedicals (Cat. No. 2623122) per 100 mL, store at 4 °C
prepare sterile 40% sucrose by making 40 g sucrose per 100 mL H2O and autoclaving by itself
note: sucrose will break down partially if it is autoclaved with iron, which is a component of MS salts
note: plant agar is cheaper than agarose; do not use bacterial agar

You can also download an Excel sheet that can calculate the components for any volume of 0.5x MS plant agar or agarose from powder or stock MS salts here.

Using top agarose

Optionally you can plate your seeds using top agarose to get a very even spread and even clearer cut selection. This video illustrates the results with top agarose. Seeds were mixed with an equal volume of 70°C 0.5% agarose and 2 mL (1 mL of seeds) was plated.

Definitive and rapid selection of BASTA resistant plants on plates

The method on Jose Alonso’s website for selecting BASTA resistant Arabidopsis is excellent. In 5 days you can get a clear cut selection. The method involves plating seeds in top agar. Compared to all other plate based BASTA selections I have tried, this one is by far the best. Seeds are plated, cold treated, and light treated before putting them in the dark for 3 days. Then the plates are moved to the light for 2-5 days. Only BASTA resistant plants will develop a green cotyledon.

Transforming Arabidopsis by Agrobacterium floral dip

Transforming Arabidopsis is one of the easiest things to do. Easy transformation has certainly helped make Arabidopsis the premier model plant for scientific study. A printable protocol for Arabidopsis Transformation by Agrobacterium floral dip can be downloaded here. A cheap and efficient way to get your binary vector of choice into Agrobacterium is by the Agrobacterium freeze and thaw transformation method.

Notes:

  • Other pages on on this website show how to select for transformants on sterile plates.
  • Healthy plants are really important for the transformation process to work.