Fruitful EMS mutagenesis: search for suppressors of lg1 and discovery of an adh1 trans-acting mutant
--Lisa Harper, Barbara Kloeckener-Gruissem, and Michael Freeling

EMS mutagenesis of pollen, rather than of kernels, permits the recovery of high numbers of independent new mutations. We followed the method of Dr. Gerry Neuffer (Mutagenesis, The Maize Handbook, Freeling and Walbot, Eds, Springer-Verlag New York, 1994, pp. 212-219) to generate a mutagenized seed stock to search for second site suppressors of lg1 and trans-acting mutants affecting adh1 expression. We found that 0.1% to 0.2% EMS resulted in approximately 50% pollen death as monitored by germination on agarose media (see below for recipe). Germination of mutagenized pollen was monitored before and after actual pollinations. Germination on agarose media proved to correlate with seed set, and was an important step in predicting the success of the experiment.

Pollen from plants homozygous for lg1-R was EMS-treated and crossed to lg1-R testers. 6725 kernels on 242 ears were generated from 515 crosses. These M1 seeds were planted, observed, pollen tested, and self pollinated. Twenty-five kernels from each of 1520 M2 families were screened for lg1 revertants. Of 5 families segregating 25% wild-type, no wild-type individuals were found to give 100% lg1 progeny upon test crossing to lg1. Thus, a simple recessive suppressor was not found. We discovered that the lg1 reference allele contains a large deletion; hence, a wild-type can possibly be explained by gene conversion, the reactivation of a pseudogene, the co-optation of a similar gene from another pathway, or contamination. A second suppressor screen is currently being set up with a partially functional lg1 allele.

The spectrum of plant and kernel phenotypes found in the M2 screen was similar to what Neuffer has reported.

We screened a subset of M1 plants (4000) for deficient ADH1 enzyme activity in pollen, thereby avoiding the screening of 25 x 4000 M2 plants. Pollen was frozen, dialyzed, and stained by ethanol-dependent NBT reduction. Eight independent mutants were recovered which lack ADH1 enzyme activity. A cis-trans test was performed by crossing heterozygous M1 plants to a tester with an electrophoretically distinguishable adh1 allele. Pollen from progeny was tested for ADH1 enzyme activity by in situ and starch gel staining. As expected, the in situ staining showed that the pollen of half of the progeny segregated 1:1 for ADH1 activity and no activity. If the new mutation acts in cis, plants heterozygous for adh1 deficiency are expected to show only the tester allele homodimer on starch gel staining. If the new mutation acts in trans, those pollen samples are expected to show both the tester and mutant ADH1 homodimers. According to this cis-trans test, one of us (BKG) recovered the first adh1 trans-acting mutant and 5 new cis-acting mutants. The test has not yet been completed on two of the new mutants.

While screening for the absence of ADH1 activity we were also able to score partial enzyme activity. In this initial screen, approximately 6% of the M1 plants produced pollen segregating 1:1 for various levels of reduced activity. In addition, we discovered that 4.6% of all M1 plants generated pollen that segregated 1:1 for small pollen grains, which occasionally correlated with changes in the ADH1 phenotype. Pollen abortion was found in 2.8% of M1s.

Although cloning of EMS induced alleles is not easy, the mutation frequency is at least one order of magnitude higher than that of Mutator-induced mutants in our lab (our standard Mu-active line, called mum9, has about 5 MuDR elements). This high frequency, and the easy monitoring of mutagenized pollen death to predict the success of the experiment, makes EMS an especially good mutagen for two types of experiments: the recovery of low-activity alleles, and the identification of new classes of mutants such as suppressors. As shown for adh1, partial phenotypes are observed quite frequently (6%) which most likely reflects the nature of the molecular lesion. With such low-activity alleles, new genes with multiple functions can be discovered.

The pollen germination medium was essential in this experiment. This recipe was modified by Steve Modena and Ed Coe from: F.S. Cook and D.B. Walden, Can. J. Botany 43:779, 1965.

To prepare the plates:
(Note: This medium is not sterile. Empirically, autoclaved OR FILTER-STERILIZED sucrose in this media results in very low pollen germination.)
    1. Make three solutions: A: 3.0g CaCl2.2H2O in 100 ml; B: 1.0g H3BO3 in 100 ml; C. 30g sucrose in 100 ml.
    2. Add 0.7g agarose to 50 ml water.
    3. Heat to a boil in the microwave to melt the agarose.
    4. Transfer to a cold stirrer and add: 1 ml solution A; 1 ml solution B; 50 ml solution C.
    5. Quickly pipette 8-10 mls to each of 10 petri plates.
    6. Let cool with lids cocked half off.
    7. Replace lids when cool. Plates can be stored inverted at room temp for a few days or at 4C for a month or so. Before using plates, make sure there is absolutely no water condensation on them. This will instantly burst pollen.

To use the plates:
    1. Sprinkle fresh pollen, or brush pollen in oil onto room temperature plates.
    2. Allow at least 15 minutes for the pollen to begin germination. For monitoring death over time, pollen can be applied at appropriate time points, and scored all at once some time later. Plates can even be left at room temp overnight and scored the next day.
    3. Observe through a dissecting scope at 20X. Plate can be illuminated from below on a clear background or from above on a black background.
    4. If desired, 10 ml of 50:30:10:10 sucrose:water:EtOH:HOAc can be applied to the plates after one hour. This will kill and fix the pollen, allowing clear viewing of the field due to removal of refraction from the meniscus surrounding each grain. 

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