Columbia, Missouri

University of Missouri - Columbia

A safe procedure for EMS treatment of pollen in maize --Kato, A EMS (ethyl methanesulfonate, methanesulfonic acid ethyl ester) treatment on maize pollen in paraffin oil is known to be very effective to induce mutants. The treatment was originally developed by Neuffer and Coe (Maydica 1978. 23:21-28), and several researchers followed the procedure with successful results (Harper et al. 1995. MNL 69:22 ; Zhao et al. 1998. MNL 72:15-16). Detailed descriptions by Neuffer of the treatment are found in Mutants of Maize (pp.397-398) and in The Maize Handbook (pp.212-218.).

Extreme precaution is necessary to handle EMS because the chemical is a volatile carcinogen. Because EMS treatment of pollen is done in an open field, specific safety procedures must be employed to conduct the treatment in a way that protects the experimenter as well as personnel working in the field. Nevertheless, there are no descriptions of safety procedures for this treatment. In the summer of 1999, I had to develop safety procedures for this treatment. The following is a detailed description of the safety procedure for EMS treatment of maize pollen in the field.

Before starting the experiment the following items should be in hand.

A gas mask (Willson chin-style gas mask, and a canister for organic vapors)

Coveralls (Tyvek/Saranex 23-P Coveralls with hood and booties)

Gloves (disposable rubber gloves, size M and L)

Gloves (long gloves that cover the sleeves)

Disposable soft plastic pipets (e.g., Graduated pipets 5ml Fisher Cat. No. 13-711-9A)

Plastic tubes (45 ml disposable plastic tubes with a screw cap)

A pipetter (e.g. Pipetman 20, Rainin)

Spray bottles

Polyethylene bags with zipper

Sodium thiosulfate (500g)

Tween 20

A timer

The mask fit well and was easy to wear and take off. The canister of the gas mask was extended with a rolled towel, and the tip of the towel was sprayed with thiosulfate solution (10%). The coveralls were disposable, and are thin, lightweight and convenient for body movement.

Before doing the following procedures with EMS, it is better to practice without EMS.

Preparation of EMS - oil mixture. In a fume hood deliver 20 ml of paraffin oil (Sigma, mineral oil M8410) to 20 tubes. Pre-mark the 12 microliter volume level on three or four dry 20 microliter tips. This is easily done by pipetting 12 microliters of water with one tip and marking the water level line on the other tips. The wet tip is disposed of. The marked dry tips are attached to a disposable soft plastic pipet. Wearing the gas mask and double disposable rubber gloves, EMS is moved into the hood from a refrigerator, and EMS delivery starts. The materials should be brought into the hood and placed on disposable paper as follows.

An EMS bottle enclosed in a protective can.

Tipped disposable pipets (three to four).

300 ml 10% sodium thiosulfate solution (addition of 1% Tween 20) in a 500 ml plastic beaker.

10% sodium thiosulfate solution (addition of 1% Tween 20) in a spray bottle.

Twenty plastic tubes (45 ml) containing 20 ml paraffin oil.

A thin metal blade to open the lid of the can and the inner cap of the EMS bottle.

Several sheets of paper towels.

The caps of the plastic tubes are removed. The lid of the can and the cap of the EMS bottle are removed very carefully. Twelve microliters of EMS are delivered to each tube using the tipped disposable pipet. The used pipets are dipped into the sodium thiosulfate solution and the inside of the pipet is washed with the solution after delivery. All contaminated materials are dipped into the sodium thiosulfate solution. Sodium thiosulfate reacts with EMS quickly to produce harmless products. Any undesirable EMS contamination is sprayed with the sodium thiosulfate solution and wiped thoroughly with a piece of paper towel. The EMS bottle is capped and sealed in the can and stored in a refrigerator. The EMS containing tubes are capped, numbered and stored in the hood. Materials that have been decontaminated with the sodium thiosulfate solution can be disposed of as regular waste the next day.

Preparation of maize plants for EMS treatment. An inbred with high fertility should be used (e.g., Oh43). The field should be isolated and have warning signs. Husks of the plants are covered with a polyethylene bag (5 x 15 cm) before silk emergence. A paper bag (5 x 19 cm, Lawson 217) was inserted between the ear and the stalk. Also a paper tag was attached to every plant (Fig. 1).

Transportation of the tubes containing EMS. One to three EMS tubes are transported to the field every day during the treatment. Tubes are covered with a zipped polyethylene bag containing a piece of paper towel soaked with sodium thiosulfate solution. The covered tubes are stood upright in a Styrofoam box and the box is sealed with sticky tape.

Mixing pollen with paraffin oil containing EMS. Wind direction is determined before mixing. A pole with long tape is helpful. Smoke will also work. Once the wind direction is determined, all the materials necessary for the treatment are placed upwind from the EMS field.

Each tube contains 20 ml of paraffin oil and 12 microliters of EMS. EMS is observed at the bottom of the tube as a translucent droplet. The tube is shaken five to ten times vigorously and placed in the shade. Maize pollen is collected and filtered to eliminate anthers. Collected pollen is poured into a disposable cylinder and 2 ml of pollen is measured. Wearing gloves and the gas mask, the cap of the tube which contains oil and EMS is opened very carefully. The pollen is poured into the tube and the cap is screwed tightly. This tube is shaken vigorously every 3-5 minutes for 30-50 minutes. During shaking it is not necessary to wear the coveralls and the gas mask.

EMS pollination. Near the end of shaking, preparation for EMS-pollination starts, i.e. taking off shoes, putting on coveralls, triplicate gloves (medium disposable glove, long sleeve glove, and large disposable glove), and the gas mask (be sure your chin is fitted to the mask).

After putting on protective clothes, EMS pollination can begin. The polyethylene bag is removed from the ear and 0.5 - 1.0 ml of the EMS-oil/pollen mixture is dropped onto the silks using the disposable pipet (Fig. 2). The pollinated silks are covered with the shoot bag which is already inserted between stalk and the ear (pin or stapler is not necessary). Continuous agitation is necessary during pollination to prevent maize pollen accumulation at the bottom of the tube. 20 ml of oil containing EMS is enough to pollinate 20 - 40 ears and it takes about 10 - 15 minutes. Silks can be cut with a blade at 2 cm height above the husk just before pollination. For this purpose a knife blade can be attached to the wrist with a string.

After each pollination the paper tag on the stalk is bent according to a pattern that indicates the number of the treatment. Bending is easier and faster than writing letters on the label, and it can be done with one hand. Consequently EMS pollination can be done without any assistance.

Neutralization of EMS After pollination, 10% sodium thiosulfate solution (addition of 10% Tween 20) is added (1:1 ratio) to the remaining oil-EMS mixture and shaken. The gloves and coveralls are sprayed with the sodium thiosulfate solution (addition of 1% Tween 20). The gloves are disposed in a polyethylene bag. Coveralls can be used several times; tears can be fixed with sticky tape. The gas mask is put in a cardboard box and other materials are also put in another box. The boxes are covered with the coverall kept at the field. All garbage is treated with sodium thiosulfate solution (addition of 1% Tween 20) and stored in the field for thirty days. After storage, they can be disposed of as regular waste.

Dealing with high temperature during treatment. The person who conducts pollen-EMS treatment may have to deal with high daytime temperatures. The coveralls and the gas mask prevent evaporation of perspiration, and it makes pollination work very difficult. However, about 15 minutes of work can be done without the risk of heat exhaustion. If the temperature is very high and sunshine is strong, the following tips may be helpful.

1. Eating a lot of broken ice before pollination.

2. Bagged broken ice is placed in the gas mask. The ice can be eaten during pollination.

3. Other work, such as covering ears and detasseling in the EMS field should be done in the cooler times of the day.

Results of pollen-EMS treatment varies according to the quality of pollen, temperature during mixing, and temperature after pollination (personal communication from Dr. Neuffer). I did the pollen-EMS treatment on the inbred line Oh43 in the summer 1999. The results of the trials I have done varied considerably and 80 % ears resulted in no seed set. The remaining ears with seed set suggested that appropriately treated pollen lost fertilizing ability within 10 minutes after the start of pollination and the viability seems temperature dependent. 30 - 50 minutes of shaking seems appropriate to obtain M1 kernels. Trials involving different shaking times and different concentrations of EMS would be necessary to determine the appropriate treatment for a particular inbred line.

Fig. 1. Preparation of a shoot for EMS pollination. The silks are covered with a bag and another bag is inserted between the sheath and the stalk. A paper tag is attached.

Fig. 2. EMS pollination with the protective clothes (photo by D. Auger).
 
 


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