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
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.