Growth of Triphysaria on nodulated Medicago in nitrogen free media

Biao Wu
Nov 2009

Day 1

Put Medicago truncatula A17 seeds in a 50ml Corning tube and soak in 100% H2SO4 for 5 minutes with gentle agitation;

Wash the seeds with H2O three times;

Sterilize the seeds with 100% Chlorox bleach for 10 minutes, with gentle agitation;

Wash the seeds with sterile H2O seven times;

Imbibe the seeds in water at room temperature for 4 hours;

Remove excessive water and invert tube for seeds to germinate;

Cover the tube with foil and leave at room temperature for 2 days.

 

Sterilize Triphysaria versicolor seeds using standard Yoder lab protocol;

Leave the seeds at 4C for 2 days.

 

Streak out Sinorhizobium meliloti ABS7M on a TY medium plate with 10ug/ml tetracycline;

Incubate at 28C for 3 days.

 

TY Medium

5g Tryptone Peptone

3g Yeast Extract

Add water to 1L

Autoclave 30 minutes

Add 6ml 1M CaCl2 before use

 

Day3

Make soil mix ( vermiculite and sand);

Transfer germinated Medicago seedlings to the soil;

Cover pots with a plastic dome and leave at 20C.

 

Pipet Triphysaria seeds onto Hoagland medium plates;

Incubate at 16C for 12 days.

 

Day4

Inoculate S. meliloti ABS7M into 5ml TY medium with 10ug/ml tetracycline;

Incubate at 28C shaker for 2 days.

 

Day6

Inoculate S. meliloti ABS7M to 500ml TY medium with 10ug/ml tetracycline;

Incubate at 28C shaker for 2 days.

 

Open plastic dome and water plants with nitrogen-free Hoagland medium

 

Nitrogen-free Hoagland Medium

5ml 0.5M K2SO4

2ml 1M MgSO4

1ml 1M KH2PO4

200ml 0.01M CaSO4

1ml 1,000X Fe-EDTA

1ml 1,000X micronutrients

 

Day8

Spin down ABS7M cells and resuspend in nitrogen-free Hoagland medium to an OD600 of 0.1;

Inoculate Medicago plant with 1ml ABS7M per pot.

 

Day15

Add Triphysaria seedlings to the pots with nodulated Medicago plants;

Cover with plastic dome for 3 days.

 

Day50

Harvest roots (Tv6.3h) and shoots (Tv6.1h)

 

Day64

Harvest flowers (Tv6.2h)