EMS MUTAGENESIS 
of Medicago truncatula

(Cook lab-U.C. Davis; drcook@ucdavis.edu)

Mutagenesis:

Seeds of genotype A-17 are scarified by immersion in concentrated sulfuric acid for 5-10 min, rinsed in distilled water 5 times, surface sterilized in commercial bleach (~5% NaOCl) for 3 min and rinsed extensively (6-8 times) in sterile deionized water.

For Ethyl Methyl Sulfonate (EMS) treatment, scarified and surface sterilized seed (as described above) are soaked for 15 hours in the appropriate concentrations of EMS on a rotary shaker set at 30 r.p.m. Treated seed are rinsed extensively (12 times, 30 minutes each) in sterile deionized water to remove residual EMS. (Spent EMS solution should be decontaminated by adjusting solutions to 1N NaOH). Rinsed seed are suspended in 0.1% agar (to aid even distribution) and aliquoted on to water soaked soil in troughs of size 35 x 50 x 10 cm at a rate of 500 seeds/trough, and covered with a very thin layer of soil. Troughs are covered with saran wrap and placed at 4°C for 36 hours before transfer to the greenhouse. Once seedlings have emerged from the soil the saran wrap is punctured to allow humidity to decline gradually; saran wrap is removed completely after 3 days. Flats are fertilized with Osmicote. Pods from each trough (500 treated seed) are harvested as M2 bulks.
 

Development of the mutagenized population: optimizing EMS dose.

Our initial goal was to generate a highly mutagenized population of Medicago truncatula as a resource for identification of plant nodulation mutants. Based on EMS dosages commonly used for mutagenesis in plants, we selected EMS concentrations ranging from 0.025% to 2.025% and quantified putative somatic and germinal effects; the objective of this analysis was to maximize EMS dose while retaining a high level of gametic transmission to the subsequent (M2) generation. Thus, we assayed seedling survival, frequency of fertile individuals, and seed production in treated seed (M1) as a measure reproductive capacity, and we assayed the frequency of arrested embryos in the M2 population as a measure of mutagenicity. The analysis was conducted on six lots of 500 seed each, treated in parallel with different EMS concentrations. Fertility of treated seed became a limiting factor at 0.225% EMS despite a seedling survival rate of near 40%. Lower doses of EMS had a relatively small effect on seedling survival and the frequency of fertile individuals, although fecundity (seed per pod) decreased significantly as EMS dose increased from 0.025 to 0.075%. The frequency of arrested embryos was also correlated with EMS dose, increasing roughly four-fold from 0.025% to 0.075% EMS. EMS dose did not significantly effect on seed size or weight.

Based on results of the preceding analysis, we selected EMS concentrations of 0.1% and 0.15% for large scale seed treatment. Typically five to ten thousand seed were treated simultaneously, and subdivided into lots of 500 seed each for growth and seed collection. Seed bulks derived from individual lots represent unique mutant populations, and comprised the basic unit used in subsequent mutant screens. To assess the efficacy of EMS treatment, we analyzed seedling survival, plant fecundity, and the frequency of arrested embryos in a single M2 seed bulk (bulk "C", 0.15% EMS treatment) derived from 354 M1 individuals. The results are consistent with our previous dose response data; in particular, the diminished fecundity of the 0.15% EMS treated population indicates that 0.15% EMS is probably close to the maximum dose. Nearly 25% of the bulk C population was altered in seed or seedling pigmentation, including seed coat coloration and chlorophyll phenotypes.

Plant embryos contains two to three cells that serve as germline precursors; therefore, each M1 individual should give rise to two or three unique cell lineages, each carrying a distinct set of mutant alleles. As most of our 0.15% EMS treated seed bulks were derived from an average of 280 surviving M1 individuals, the corresponding M2 populations should contain up to 840 unique lineages. The limit of confidence (LOC) for recovery of a given recessive phenotype from a population of 840 unique M1 lineages is 76% if 4,200 M2 are screened, 94% if 8,400 M2 individuals are screened, and >99% if 16,800 M2 individuals are screened.
 
 

Aeroponic chamber|Cultivation|DNA extraction|EMS Mutagenesis|
Freezer Medium|PCR Amplification|Seed Germination|Seed Mill|
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