VOL. 51, 1964 : BRINK, KERMICLE, AND BROWN 1067

A. Ciferri, and R. Chiang, J. Am. Chem. Soc., 83, 1023 (1961). 10 Hoeve, C. A. J., J. Chem. Phys., 35, 1266 (1961). 11 Flory, P. J., V. Crescenzi, and J. E. Mark, J. Am. Chem. Soc., 86, 146 (1964). 12 Jernigan, R. L., to be published. '3 Mark, J. E., in preparation.

TESTS FOR A -DEPENDENT CYTOPLASMIC PARTICLE ASSOCIATED WITH R PARAMUTATION IN * BY R. ALEXANDER BRINK, J. L. KERMICLE, AND D. F. BROWNt

UNIVERSITY OF WISCONSIN, MADISON Communicated April 21, 1964 The change whereby the -producing action of the R gene in maize is heritably reduced on passing the through a stippled (Rat) heterozygote is known to occur at, or very close to, the R locus. Tests previously made for an autonomous cytoplasmic element underlying the paramutant proved negative; the R't, but not the r', female and male gametes arising from R rr in- dividuals (heterozygous colorless aleurone) transmit the capacity to induce paramu- tation.I Furthermore, among the offspring of heterozygotes carrying marked paramutant and nonparamutant forms of R (e.g., Rl'R' or RgRr'), two independently identifiable kernel classes differing in aleurone pigmentation grade occur in Men- delian proportions. These are the results expected, of course, if the R paramutant phenotype has a chromosomal basis. R is changed from the standard to a paramutant form (R') in somatic cells,3 but the manner in which the alteration is mediated is not known. Two kinds of mechanisms may be visualized as possibilities. The change may result from a direct interaction of some kind between R and Rat, within the nuclei of R Ra plants following chance contact of the , or otherwise. Alternatively, paramutation may be effected by particles produced by the Ra' gene that affect R action which are released into the cytoplasm and are then incorporated at the R locus in the homol- ogous chromosome. The present experiments were designed to determine whether an agent of the latter sort can be detected in a free condition in cytoplasm derived from a plant carrying a paramutagenic R factor. If paramutation is mediated by a cytoplasmic element, then transmission of this element might occur, independently of the gene that produces it, via the cytoplasm of the female gametophyte. Since a nonreplicating paramutagenic particle would be subject to dilution and decay in a growing tissue lacking the gene that produces it, and since the changes incited by such an element in a sensitive R factor also would be subject to reversion,1 an efficient test for the transmission of a cytoplasmic paramutagenic agent requires conditions which permit the early de- tection of paramutation after fertilization. It was thought that this requirement might be met in the endosperm, which is the product of secondary fertilization, and matures in the seed. Introduction of a sensitive R gene via the pollen into an endosperm that has re- ceived a strongly paramutagenic, near-colorless allele, r(I), from the pistillate Downloaded by guest on September 26, 2021 1068 GENETICS: BRINK, KERMICLE, AND BROWN PROC. N. A. S.

parent results in a paramutant aleurone phenotype. Thus, paramutation can occur and can be expressed within the brief life span of this structure. What is the immediate cause of paramutation in the endosperm in such a case? Does the paramutagenic r(I) allele function directly in the developing tissue? Or is the change in R pigment-producing action a response to a particular cytoplasmic ele- ment formed by r(I) in the parent sporophyte which is then passively transmitted in effective concentration through the female gametophyte to the endosperm? If the mechanism is of the latter kind, then it would be expected that the cytoplasm accompanying a nonparamutagenic allele or an R-deficiency from a plant heterozy- gous or hemigygous in this respect also would be paramutagenic in the endosperm. The present experiments were designed to test this possibility, and also to de- termine whether transmission of such an agent to the next sporophytic generation occurs via the egg cytoplasm and embryo. Materials and Methods.-All the stocks used in these experiments carried the residual genotype of W22, a relatively uniform, inbred, dent corn line. The R alleles employed may be characterized as follows: RT--This standard allele gives colored aleurone, red seedling; aleurone pigmentation is mottled in single dose; RTis highly sensitive to paramutation; colored aleurone is dominant to color- less aleurone. R1, R R',R4 , and R9 Colored aleurone, green seedling; each derived independ- ently by mutation from RT, above, and differing from the latter in giving green, rather than red, seedlings. r' and r0-Colorless aleurone, red and green seedling, respectively; nonparamutagenic; red seedling is dominant to green seedling. Rst'Stippled aleurone, green seedling; strongly para- mutagenic; mutates to self-colored (Rac) alleles varying widely in grade of paramutagenic action. RMc, Rsc, Rsc 4, Rsc2, and Rsc4-Self-colored mutants from stippled; give fully colored aleurone in single dose, and nearly green seedlings. R` and R`2 are strongly paramutagenic, Ro and R`4 are weakly paramutagenic, and R`,¶ and R''24 are nonparamutagenic. rr(I)3 and rJ(I)8-Near-color- less aleurone, red and green seedlings, respectively; mutants derived from RRst plants; both alleles strongly paramutagenic. R-def K, and r-xl-Presumed (cytologically nonrecognizable) deficiencies for the R region in chromosome 10; give no pigment in seed or plant, and are not di transmissible; 9 transmissibility of r-x, approximately normal, but that of R-def K is markedly reduced. K rep- resents a large distal knob on chromosome 10. Paramutant alleles are distinguished in the text from their normal counterparts by the prime sign, e.g., Rr and RT'. They differ from the latter in showing reduced aleurone pigmentation in single dose. The pistillate parents in the test crosses in which ev- idence was sought for a cytoplasmic paramutagenic agent were either heterozygotes for a paramuta- genic R allele or corresponding hemizygotes carrying an R-deficiency; the staminate parents were either homozygotes or heterozygotes for a paramutable R allele. The purpose served in using pistillate parents hemizygous for an R-deficiency, of course, was to derive cyto- plasm from the 9 parent free, in an absolute sense, from an R chromosomal factor. Paramutable R factors whose anthocyanin-forming capacities had been slightly reduced by exposure to a paramutagenic R allele were employed in most of the experiments. The use of a slightly attenuated form, rather than the standard form, of a paramutable R allele was shown in earlier experiments to increase the sensitivity of the test for paramutation in the endosperm.I The effect of the pretreatment is probably attributable, not to increasing the inherent sensitivity of the allele to paramutation, but rather to reducing its pigmenting action to a level at which sub- sequent change is more readily observable with the particular scoring method used. The kernel scoring methods used were like those employed in previous R paramutation studies. Random samples of seeds (usually 50) of the appropriate class were removed from the central part of the ear, and then matched individually against a graded set of standard kernels defining seven pigment intensity classes (O = colorless; 7 = fully colored). Unless otherwise indicated, each entry in the tables following is the average aleurone color score for the kernel sample from a single ear. Results.-(1) Tests for a paramutagenic agent transmitted through the cytoplasm of the central cell of the female gametophyte to the endosperm: Rstrr and rrrr plants were pollinated by R9,R9 plants in one experiment, and by R2'Rg' plants in another. Downloaded by guest on September 26, 2021 VOL. 51, 1964 GENETICS: BRINK, KERMICLE, AND BROWN 1069

The two experiments differed only in the staminate parents employed. R1' is a "conditioned" allele, obtained by passing the standard RI' factor from a stock culture through an R51 heterozygote, and then selfing the extracted homozygote for a few generations. RI is changed to a weakly pigmenting, but metastable form in R6R8t plants; the result of the subsequent selfing of homozygous R9'R9' individuals is to stabilize Rg' pigment-producing action at a level intermediate between that of the primary Rg' paramutant and R° in its standard form. The rrr and Rstrr pistillate parents employed in each case were derived by selfing a single plant in a W22 rrrr stock culture to obtain the former, and applying pollen from the same individual to a W22 RatR"t plant to produce the latter. An attempt was made to reduce extraneous variability further by pollinating the rrrr and Ratrr plants in pairs, each pair with a single c''. A definitive separation of the RR`tRt and Rgrrrr classes of kernels derived from the Rtrr 9 X RgRgdc matings in each group was made possible by virtue of the fact that the former, when sprouted after scoring for aleurone color, gave green, and the latter red, seedlings. The results from crosses of RIRM c plants on R8trr and rrrr 9 plants are pre- sented in Table 1. It may be seen that in all pairs of matings but three in this group the Rg4rrrr testcross kernels produced on the Rtrr 9 9 were darker than those from the corresponding rY 9 9. The average difference in aleurone color score between the two series of seeds on the 14 pairs of ears was 0.15 units, a value near the borderline of statistical significance at the 5 per cent level. A similar result was obtained from the parallel group of matings involving Rg'Rg' staminate parents. The data from these tests are shown in Table 2. The Rg'rrrr kernels from the Rtrr 9 X R'R'6R' matings were darker than those from the rY 9 X R9'Rg' testcrosses in 14 of the comparisons that can be made, and of equal grade in one. The magnitude of the difference in average aleurone color score of two classes of kernels being compared, however, has increased nearly four- fold as a result of the use of Rg' as the sensitive R factor in the experiment. Not only is this difference, 0.57 units, highly significant statistically, but also it is in the direction opposite to that expected if the Rstrr 9 9 used transmit a cytoplasmic TABLE 1 TABLE 2 R9 rrr' SCORES FOLLOWING R9'R9 MATINGS RgPrrrr SCORES FOLLOWING R2'Rg.' MATINGS ON r~rr AND R8'rr 99 ON r'rr AND R'r' 99 Rg Rg Average Aleurone Color Score Rg-Rgf'Aleurone Color Score - parents rtr' 9 RBrt9 Difference ci parents rtrt 9 R try 9 Difference 45-3614 5.83 6.05 0.22 45-529-1 2.04 3.65 1.61 -5 6.08 6.23 0.15 -2 2.78 3.48 0.70 -6 6.03 6.13 0.10 -3 2.60 3.20 0.60 -7 6.10 5.63 -0.47 4 2.73 3.55 0.82 -8 6.23 6.23 0.00 -6 2.45 3.45 1.00 -9 5.75 5.83 0.08 -8 3.08 3.10 0.02 -10 5.95 6.15 0.20 -10 2.78 3.18 0.40 -11 5.75 6.08 0.33 -11 3.03 3.33 0.30 -12 5.48 5.95 0.47 -13 2.83 3.55 0.72 -13 6.28 6.08 -0.20 -14 2.48 3.45 0.97 -14 6.30 6.73 0.43 -15 2.73 3.20 0.47 -16 6.33 6.40 0.07 -16 3.25 3.63 0.38 -17 6.35 6.63 0.28 -17 2.98 3.33 0.35 -18 6.13 6.55 0.42 -18 3.25 3.25 0.00 Mean 6.04 6.19 0.15 -19 3.20 3.30 0.10 t = 2.206 (where t.os = 2.160 and t-o2 = 2.650). Mean 3.81 3,38 0.57 Statistical significance of difference, therefore, in borderline, Downloaded by guest on September 26, 2021 1070 GENETICS: BRINK, KERMICLE, AND BROWN PROC. N. A. S.

TABLE 3 factor with the rT gene that reduces aleu- EFFECT OF R6 POLLEN USED ALONE AND IN rone pigmenting action of the R6' allele. R` MIXTURES6 Pollen It was observed a few years ago that if Rg alone Rg` + R8' Mixture - plants homozygous for an R-pale allele, Ear mean Mixture no. Ear mean Mean conditioning very dilute aleurone color, 2.04 1 3.00 2.78 3.28 werepollinated with ItR"t, the background 2.60 2.70 color of the resulting kernels (i.e., in the 2.45 3.28 areas between the solidly pigmented spots 3.08 3.15 3.13 conditioned by R"t) often was enhanced, 2.78 2 3.18 3.03 3.13 as compared with R-pale selfed. It was 2.83 3.10 assumed that the explanation lay in some 2.73 3.15 nongenetic effect of the self-colored sectors 3.25 3.28 on the neighboring pale regions during 3.25 3 2.98 seed development. The possibility that 3.20 3.50 Rat might exert the same effect on a sec- 2.88 ond class of colored kernels on the same 2.85 ear in the present experiments was checked 2.90 3.00 by the use of pollen mixtures in the 2.81 3.09 following experiment. t = 2.863; t.oi = 2.750; t.ooi = 3.646. Equal amounts by weight of pollen from several R"'R"' and RatR"t plants were mixed and then applied to rrrrQ9. Three such mixtures were prepared which, as subsequent counts showed, yielded 45, 37, and 53 per cent stippled kernels on the resulting groups of ears. The R9 rrrr kernels obtained from the R"'R"' matings on sib ?.rrr9 9 (Table 2), made on the same day, served as controls. The results are brought together in Table 3. Each of the three pollen mixtures yielded R6 rrrr kernels whose aleurone pig- mentation level exceeded that of the control seeds. The over-all difference, 0.28 units on the 0-7 color scale, is highly significant statistically. It is clear, therefore, that the presence of kernels carrying the R8t allele raises the average level of aleurone pigmentation of a second class of colored seeds on the same ear. The data presented in Tables 2 and 3, therefore, may be interpreted to mean that if Rstrr plants transmit a paramutagenic cytoplasmic agent through rr female gametophytes to the respective Rgrrrr and R6 rrrr testcross seeds, the action of this agent in reducing aleurone pigmentation is exceeded by an effect in the opposite direction of the R8t carrying seeds on the same ears. (2) Tests for a paramutagenic agent transmitted through egg cytoplasm to the sporophyte: Two chromosomes 10 (R-def K and r-xi), each deficient for a particu- lar, but undefined, segment in the long arm including the R locus, were combined in heterozygotes with normal chromosomes 10 carrying R8t and Rr, respectively. R8t/- and Rr/- hemizygotes in each of the progenies were pollinated with a single collection of RMRM pollen. Testcrosses on rgrg individuals in the W23 inbred line of 10 Rg/- plants of each of the four different origins were then made to test for oc- currence in the two respective R`t/- lines of a cytoplasmically transmitted paramuta- genic agent. The data obtained are summarized in Table 4. It will be noted from the table that the results were negative for both the comparisons that can be made. The aleurone color scores for R9/- plants carrying cytoplasm from either kind of R`t/- parent did not differ significantly from those based on RI/- plants of corre- Downloaded by guest on September 26, 2021 VOL. 51, 1964 GENETICS: BRINK, KERMICLE, AND BROWN 1071

TABLE 4 R2 ACTION AFTER PASSING THE ALLELE THROUGH R-DEFICIENT HEMIZYGOTES CARRYING CYTOPLASM DERIVED FROM R' AND R' PLANTS, RESPECTIVELY Pedigree d Testcross parent no. Origin Mean score P* R2/R-def K D618 Rt/R-def K X R2R2 5.67 + 0.10 0.3 < P < 0.4 R9/R-def K D616 R'/R-def K X R-2R- 5.77 A 0.27 R9/r-xi D621 Rt/r-x, X RR92- 5.77 ± 0.18 0.05 < P < 0.1 R9/r-xi D620 R'/r-xl X RgRg 5.94 i 0.22 * P is the probability of the correctness of the null hypothesis by the t test. sponding RK- parentage. There is thus no evidence from these tests for a cytoplas- mic paramutagenic agent initially formed by the Rat allele and transmitted in the egg cytoplasm to the next sporophytic generation. (3) Effects of cytoplasms derived from plants carrying various R alleles on the pig- menting capacity of conditioned RI' and RI' alleles in RI'/r-xj/r-x1 endosperms: Experimental results demonstrating that the conditioned RI' allele is highly sensi- tive to paramutation are presented in Table 5. The rr and r" alleles used in these tests are nonparamutagenic factors conditioning colorless aleurone and red and green seedlings, respectively. The rr(l)3 and r9(J)8 factors are highly paramutagenic mutants from RTR8t plants, giving near-colorless aleurone and red and green seed- lings. Since RI' conditions green seedlings, the kernels resulting from rTrr 9 X RVRV'a matings of the three kinds shown in Table 5 can be definitively separated into the Rg'/rr/rr and Rg'/rl/rg classes after classifying for aleurone color by sprout- ing, and then recording seedling color. This experimental procedure provides ker- nels of the two classes to be compared from each ear; and possible bias in the aleurone color scores is obviated by the postclassification identification of the R91/rr/rr and Rg'/rg/rg seeds. It will be seen from the data in the third column of Table 5 that the Rg'rrrr and Rg'r'rg seeds resulting from rrrg 9 X Rg'Rg'6 testcrosses are closely alike in aleurone color. This is the result expected in view of the fact that both rr and rg in this case, are nonparamutagenic. The outcome is different, however, when plants heterozygous for a highly paramutagenic allele (either r(J)8 or rr(1)3) are TABLE 5 Rg' ACTION IN ENDOSPERMS HETEROZYGOUS FOR CERTAIN STRONGLY PARAMUTAGENIC OR TABLE 6 NONPARAMUTAGENIC ALLELES R6 ACTION IN r-Xi ENDOSPERMS CARRYING R°' rtrT Mean kernel Score Minus CYTOPLASMS OF DIFFERENT DERiVATIONS 3-R'1/R9' c 'R9r1r' Mean kernel Score- Genotype of 9 d' plant no. r'/rg(I)s rr'99 r'(I)a/r' 9 4-R"'Rg' --Parents - J726-21 0.71 0.04 -0.47 plant no. R'4/r-zR,7/r-zi7 R1'/4r-x1 -23 0.47 0.18 -0.44 J1277-2 5.14 5.46 5.00 -24 0.53 -0.24 -0.33 -3 4.94 4.98 4.90 -25 0.42 -0.11 -0.73 -4 5.30 5.12 5.24 -26 0.47 -0.13 -0.42 -6 5.10 5.08 5.24 -27 0.49 -0.24 -0.38 -8 4.98 5.00 4.98 -28 0.47 -0.24 -0.60 -9 4.50 4.74 4.90 -29 0.64 0.07 -0.62 -10 4.20 4.56 4.70 -30 0.27 0.02 -0.82 -11 4.38 5.38 5.14 Mean difference 0.50 -0.07 -0.53 Mean 4.82 5.04 5.01 F 17.26 <1 20.12 ' 0<. 001 .0. 001 Downloaded by guest on September 26, 2021 1072 GENETICS: BRINK, KERMICLE, AND BROWN PROC. N. A. S.

used as pistillate parents in the testcrosses. Following the rrrg(1)8 9 X RM'R ' matings (column 2) the Rg'rrrr seeds exceed the Rg'rg(1)8rg(1)8 kernels in aleurone color grade by one half a class interval on the 7-class scale, and the difference is highly significant statistically. A difference in mean color score of the same mag- nitude and, likewise, statisticaliy meaningful, occurs between the Rgrgr and R9'rr(I)3rT(1)3 kernels resulting from the rr(1)grg 9 X R9'R6'c? testcrosses (column 4). Again it is the class of kernels carrying the paramutagenic allele (distinguish- able by the red seedling marker in this instance) that gives the lower score. These data corroborate the early finding of McWhirter and Brink4 that paramutation may take place during endosperm development, and also show that the conditioned R6' allele is a sensitive tester for occurrence of the phenomenon in this tissue. The aleurone color scores for the Rg'/r-xl kernels resulting from the application of pollen from RM'Rg' plants to Rg/r-xl, R"/r-x1, and R'124/r-x1 individuals are summarized in Table 6. The pistillate parents of the three kinds of r-xl hemizy- gotes used in the testcrosses were R /r-x1 sibs. R" is a highly paramutagenic mutant from R8t, R'124 is a nonparamutagenic mutant from the same source, and Rg, used as a control, is a nonparamutagenic, green seedling mutant from standard R . If a paramutagenic cytoplasmic element is transmitted to the endosperm from plants carrying a paramutagenic R allele, therefore, and is not formed in other classes of R plants, it would be expected that the R8/r-x1 9 X Rg'Rg'ci matings, but not those involving R24/r-xi 9 9, would yield RI'/r-x1/r-xl kernels of a lower pigment grade than those borne on the control, Rglr-x1 stock. The data in Table 6 show, however, that the Rg'/r-xl/r-x1 kernels produced on the three kinds of plants are alike in aleurone pigmentation grade, within sampling limits. A parallel experiment was made to test whether R'N2, another highly paramuta- genic mutant from R8t, was effective through the cytoplasm in reducing the aleurone color grade of R '/r-x, seeds. The control in this case, in addition to the R9 allele, was RMC, a different, nonparamutagenic mutant from R8t. Pollen from 16 Rg'Rg' plants was used in matched matings on the three kinds of pistillate TABLE 7 parents, Rg/r-xi, R~c/r-xi, and R 2/r-x1. The aleurone color scores for the re- R' ACTION IN r-xi ENDOSPERM CYTOPLASMS DERIVED FROM 9 PARENTS CARRYING sulting Rg'/r-x,/r-xi kernels are summa- PARAMUTAGENIC AND NONPARAMUTAGENIC R rized in Table 7. Again it may be seen ALLELES Genotype of 9Q9 that the mean aleurone color scores for 4-R9R° - Parentas R9/r-xl/r-x1 kernels produced on parents d' plant no. R" /r-xi R6" /r-xi R83c2/r-x1 k pa J1281-1 4.44 4.54 5.08 carrying a paramutagenic R factor (Rc2, -2 4.76 5.02 4.84 in this case) and on parents carrying -10 4.86 5.20 5.08 nonparamutagenic R alleles (R9 and R") -12 4.64 4.18 4.76 are closely similar. -13 5.08 4.86 4.20 The effects of of various -14 5.02 4.14 4.66 cytoplasms ori- -15 4.88 4.20 4.80 gins on the pigmenting action of a con- -16 4.16 4.44 5.00 * * -17 4.12 4.82 4.10 ditioned Rs allele i the endosperm also -19 4.20 4.22 4.50 were examined in another experiment. -22 4.36 4.10 4.26 Pollen from R9R8' and RgI?"4 plants was -23 4.50 4.24 4.22 placed on the silks of R8t/r-x1, Rg/r-x1, -245 4.10 4.22 4.48 rr/r-xl, and Rr/r-x1 individuals. R't is Mean 4.57 4.51 4.58 strongly paramutagenic; R°, rT, and Rr Downloaded by guest on September 26, 2021 VOL. 51, 1964 GENETICS: BRINK, KERMICLE, AND BROWN 1073

are nonparamutagenic. Rst/r-xi pistillate plants of two kinds were used in the matings: (a) hemizygous for three successive sporophytic generations, and (b) hemizygous for one generation, on the supposition that if Rat produced a paramuta- genic cytoplasmic element slowly in terms of plant generations, the concentration should be higher in the former than in the latter, and so easier to detect. R"9 and R734 are weakly paramutagenic mutants from R8t, and the purpose of passing the paramutable R9 factor through heterozygotes with them was to reduce its pigmenting action slightly to a level more suitable than that of the unmodified allele for observing changes in pigmenting action by the scoring method employed. Due to unfavorable growing conditions, some of the r-x1 hemizygotes used as pistillate parents in the testcrosses were barren and many others gave low seed yields. There were additional losses in the progeny test required for verification of kernel genotypes. As a result, the effective numbers of R9'/r-xj/r-xi kernels obtained, as listed in Table 8, were too few and variable to permit a formal statisti- cal analysis of the data. Inspection of the mean scores in the table affords no indication, however, that the R5'/r-xl/r-x1 kernels borne on the Rt/r-xl plants are any less pigmented, on the average, than those produced on the r-x1 hemizygotes car- rying the nonparamutagenic alleles, R9, rT, or RT. The Rg'/r-xl/r-xl kernels borne by R'/r-xi (a) 9 9 which had been bred as hemizygotes for three generations were lower in pigmentation grade, on the average, than those obtained on Ret/r-xl (b) plants, which had been hemizygous for a single generation only. The small number of kernels upon which this observation is based, however, precludes a clear, assess- ment of its significance. It is evident from the experimental results presented in Tables 6-8 that the level of aleurone pigmentation in the endosperm is not detectably influenced by the genotype of the pistillate plant used in the testcrosses. Discussion.-None of the several experiments described affords evidence for the occurrence of a cytoplasmic element associated with R paramutation. The level of anthocyanin formation in endosperms whose nuclei lacked a paramutagenic gene, following introduction via the pollen of a paramutable R allele, was found to be the same, on the average, whether the endosperm cytoplasm was derived initially from a pistillate parent heterozygous for a strongly paramutagenic factor or from an individual not carrying a paramutagenic allele. The results were alike whether the cytoplasms compared were marked by a colorless, nonparamutagenic r allele or by an R-deficiency. This finding is in accord, of course, with an earlier observation that paramutation

TABLE 8 EFFECT ON Rg' ACTION OF r-xi ENDOSPERM CYTOPLASMS DERIVED FROM 9 PARENTS HEMIZYGOUS FOR CERTAIN PARAMUTAGENIC OR NONPARAMUTAGENIC R ALLELES Rg /Rc ci Rg-.Rgc? - Aleurone Aleurone Pistillate parent No. kernels color score No. kernels color score Rs'1r-xj* 118 4.96 172 4.81 R"'/r-xt 62 5.31 211 5.21 Rg/r-xi 259 5.10 209 5.24 rr/r-x1 109 5.18 158 5.35 R'/r-xi 249 5.01 264 4.94 * R' hemizygous with r-xi for 3 generations. t Ra hemizygous with r-xi for 1 generation. Downloaded by guest on September 26, 2021 1074 GENETICS: BRINK, KERMICLE, AND BROWN PROC. N. A. S.

is not attributable to a self-reproducing cytoplasmic element.2 It could also mean that paramutation is mediated between a paramutable and a paramutagenic allele in the somatic cells of a heterozygote without participation in the process of gene- dependent (nonautonomous) cytoplasmic particles. Such a conclusion would be warranted, however, only if it were certain that the criteria applied were adequate to detect particles of this sort, however transient. Such assurance cannot be given in the present case. What has been shown is that in endosperm cells known to be competent to display paramutation the phenomenon was not observed a few cell generations removed from meiosis, if the cytoplasm entering the endosperm is nnaccompanied by a chromosome-borne paramutagenic factor. Either a gene- dependent, cytoplasmic particle is not produced in the parent sporophyte or its concentration falls below an effective level in the endosperm, by dilution or other- wise, before the aleurone phenotype is determined. Summary.-The hypothesis here considered assumes that in a heterozygote carry- ing a paramutable and a paramutagenic R allele the latter produces and releases into the cytoplasm a particlewhich is then taken up at theRlocus in the homologous chro- mosome where it reproduces in phase during mitosis, and partially represses pig- ment-producing action of the R gene. Evidence for such a particle was sought in endosperms whose cytoplasm was derived from plants heterozygous for a para- mutagenic R allele and either a nonparamutagenic r gene (colorless) or a 9 trans- missible R-deficiency. Pollen carrying a paramutable R gene, especially condi- tioned in some of the experiments to disclose even small subsequent changes in pigmenting action, was applied to heterozygotes of these kinds. The resulting R/r/r and R/R-deficient/R-deficient seeds, along with appropriate control kernels, were scored for aleurone color. No evidence was found, in any of the several tests made, for occurrence in the endosperm of a cytoplasmic element conditioning the change of R to a paramutant form; sensitive color-forming R introduced via the pollen into endosperm cytoplasm unaccompanied by a paramutagenic R factor gave the same aleurone phenotype whether the 9 parent was heterozygous for a paramutagenic R allele or, as in the controls, was free of such a gene. Like- wise, tests for transmission of a cytoplasmic particle affecting R paramutation through the egg and embryo were negative. it is concluded that either paramuta- tion is not mediated by a cytoplasmic factor of the kind in question or that the con- centration of such a particle, in the absence of the allele which produces the ele- ment, falls below the level at which it can be detected by the methods used even in the short span of endosperm development. * Paper no. 957 from the Department of Genetics, College of Agriculture, University of Wis- consin. This study was aided by grants, from the research committee of the Graduate School, of funds supplied by the Wisconsin Alumni Research Foundation, and by grants from the National Science Foundation (G-16320) and the U.S. Atomic Energy Commission (contract AT(11-1)-1300). The writers desire to express their appreciation to J. Axtell and E. C. Ingraham for technical as- sistance. t With Atomic Energy of Canada, Inc., Chalk River, Ontario. 1 Brink, R. A., in Exchange of Genetic Material: Mechanisms and Consequences, Cold Spring Harbor Symposia on Quantitative Biology, vol. 23 (1958), pp. 379-391. 2 Brink, R. A., D. F. Brown, J. Kermicle, and W. H. Weyers, Genetics, 45, 1297-1312 (1960). 3Brink, R. A., Quart. Rev. Biol., 35, 120-137 (1960). ' McWhirter, K. S., and R. A. Brink, Genetics, 48, 189-203 (1963). Downloaded by guest on September 26, 2021