The Induction of Haploidy in Tetrahymena pyriformis Following X-irradiation. 1

SUMMARY. Vegetative clones of Tetrahymena pyrifornzis, variety 2, mating type 11, were the recipients of various dosages of X-rays from 10,000 to 400,000r. One clone from the 400,000 leveI (UM7X), when mated with non-irradiated cells of various mating types in variety 2, failed to produce a migratory nucleus. A one-way transfer from the normal to irradiated mate occurred, resulting in the production of haploid exconjugants. When haploids were crossed with diploids the former failed to produce a migratory nucleus. rZ one-way nuclear transfer from the diploid to the haploid occurred resulting again in the production of haploid exconjugants. When two haploids were crossed a clone resulted which showed 80 to 90 percent amicronucleate forms. All derived haploids manifest physiological and morphological characteristics similar to those of the diploid except that the haploids possess a smaller micronucleus. The mating type of the derived haploid is always that of the diploid parent. HE VARIETIES of T. pyriformis so far examined are normally diploid (2n = 10) (7,13). The task of determining the presence of recessive mutant genes requires several generations, whereas if autogamy, cytogamy, or haploidy could be induced, this information could be acquired in one generation. During routine examination of thousands of clones of T. pyriformis under a wide variety of conditions nuclear reorganization in single cells has never been observed. Exhaustive efforts to induce autogamy in variety 1 have failed. Methods that have proven successful in Paramecium (2,12), have been fruitless with Tetrahymena. One is forced to the conclusion that if autogamy occurs naturally in T. pyrifornzis it is rare and that if the process can be induced some methods other than those which have been successful with Paramecium must be found. Efforts were made to induce cytogamy by separating the two conjugants during the early stages of meiosis and before the interchange of gametic nuclei(8). The use of detergents, proteolytic enzymes, electric and heat shock, all failed to separate the animals. Vigorous mechanical agitatioa, however, did accomplish the separation but the “exconjugants”, instead of continuing in meiosis as was hoped, either remained in the stage they had reached when separated or became amicronucleate. Approximately 40 percent of the cells lost their micronuclei and many of these mated with normal or other micronucleate animals. In any case, none of ,the separated cells survived. Apparently the process of separation inflicted irreparable damage to the cells. The remaining alternative, the induction of haploidy, seemed to be the approach most likely to succeed in determining the genetic constitution of the organisms. Two possible advantages would result: (1) lethal and semilethal recessive genes could be eliminated, and (2) recessive mutations in the diploid could be revealed in crosses with the haploid, provided viable exconjugants could be obtained. This being possible, the genotype of parental stocks might be precisely determined and the analysis of the genetic system could be investigated more effectively than is possible at present. The purpose of this paper is to report the degree of success that has been obtained in producing haploid clones of T. pyriformis using X-radiation techniques and the subsequent fate of these haploids in crosses with other haploids and diploids.