AN ALBINO MUTANT IN P L A N T A G O ZNSULARZS REQUIRING THIAMINE PYROPHOSPHATE. I: GENETICS SANDRA M. MURR2

AND

G. LEDYARD STEBBINS

Department of Genetics, Uniuersity of California, Dauis, Calif. 95616 Received October 5, 1970

ALBINO mutants are one of the most common hereditary defects resulting from mutagens such as gamma rays. Most of the mutations induced by ionizing radiations in higher plants involve single recessive genes but even these do not always segregate in monogenic ratios (MOHand SMITH1951). A recessive albino mutant requiring thiamine pyrophosphate has been induced by gamma irradiation of the seed in Plantago insularis. Heterozygotes for this mutant give a segregation ratio of 1 normal to 1 mutant when they are selfed. The purpose of this study was to clarify the genetics of this apparently aberrant segregation. The involvement of thiamine pyrophosphate in the physiology of the mutant and the effects of its deficiency on the ultrastructure of chloroplast development will be considered in later publications. MATERIALS A N D METHODS

Seeds were sterilized by immersion in a 1 : 1 solution of absolute ethanol and 6% hydrogen peroxide (after LANGRIDGE 1957) for 5 min, then rinsed in sterile distilled water. They were germinated at 27°C under continuous light for 12 hr in a solution containing 50 ppm potassium gibberellate (85%) and 0.4% sucrose. The seedlings with radicles from 1 to 2 mm long were transferred with a Pasteur pipette to flats containing fine sand. The flats were watered at weekly et d . (1957) as modified by STEBBINS and DAY intervals with a nutrient solution of JOHNSON (1967). To prevent moisture loss the flats were covered with transparent plastic for 3 days or until the cotyledons were fully emerged from their seed coats. Plants were maintained in a growth chamber on a 12 hr light regime of 1500 ft-c of mixed incandescent and fluorescent lamps with temperatures of 27°C during the light and 18°C during darkness. Slides for pachytene analysis of pollen mother cells (PMC's), mitotic analysis of root tips, and and pollen viability were prepared according to STEBBINS and DAY (1967) and WHITTINGHAM STEBBINS(1969). The thiamine pyrophosphate requirement for greening and normal growth was not recognized until after the genetic analysis was started. Since the mutant did not survive to flowering, plants heterozygous for the mutant character were crossed with homozygous translocation lines to localize the mutant gene. Two somatically distinct homoszygous translocation lines were used: T3-122.3 involving chromosomes 3 and 4 and T4-73.41 involving chromosomes 1 and 4 . RESULTS

Characterization of the mutant: The albino mutant of Plantago insularis (2n = 8 ) was discovered among the X, progeny of a y-irradiated seed (WHITTFrum a thesis submitted by S.M.M. in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Genetics. This investigation was supported by NIH Training Grant GlM701 and in part by NSF grant GB5713X. Present address: Dept. of Animal Science, University of California, Davis. Calif. 95616. Genetics 68: 231-258 June 1971.

232

S. M. MURR AND G. L. STEBBINS

INCHAM and STEBBINS 1969). The mutants were completely white and survived only until the 13th day from planting when grown under normal greenhouse conditions. The characteristics of the standard pachytene karyotype (Figures 1A, B) are discussed in detail by WHITTINGHAM and STEBBINS ( 1969). The XI heterozygote was found to have a heterozygous reciprocal translocation between chromosomes 3 and 4 at the centromere region (Figures lC, D) .

B 3-4 I C

..

'L

0

FIGURE 1 .-A: Pachytene chromosomes of standard karyotype. B: Interpretive tracing of A. C: Pachytene chromosomes of translocation between chromosomes 3 and 4 (T,-103). D: Interpretive tracing of c.

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233

When the albino mutants first appeared in a planting of seeds from T,-103, the X, progeny segregated 37 green to 13 albinos closely approximating a 3 : 1 ratio as would be expected if the albino mutant were a single recessive gene. Later, however, when selfed seeds were collected from each of the 5 branches of the XI plant, only branches 1 and 2 segregated both green and albino offspring in a 1 : 1 ratio, while branches 3,4, and 5 produced only green progeny (Table 1) . Chromosome analyses of PMC’s in each branch indicated that only branch 3 had the 3;4 translocation. The original 3 : 1 segregation was obviously due to a mixture of seed from segregating and nonsegregating branches. The differences in the 5 branches indicate that the original plant was a chimera as a result of gamma irradiation. Pollen viability was 50% in branches I and 2 which produced albinos, while viability in branch 3 was 68% as might be expected for a heterozygous translocation. Branches 4 and 5 which produced only green progeny upon selfing had essentially complete pollen fertility. Cytological observations of all stages of PMC meiosis in branches 1 and 2 revealed no abnormalities in chromosome behavior. When the progeny of each of the 5 branches was selfed to the X, generation, progeny from branches 1 and 2 continued to produce green and albino plants in a 1 : 1 ratio while progeny from branches 3,4, and 5 continued to yield only green plants. In all cases, the observed deviations from this ratio had a probability higher than 0.40. In reciprocal crosses between branches 1 and 2 (Table 2 ) , the 1 : 1 ratio of green to albino offspring was continued through Fa, indicating that the gene causing albinism is the same in both branches. The goodness-of-fitprobability for a 1 : 1 ratio in these crosses is better than 0.40. The green progeny from these crosses continued to have about 50% pollen viability. Abnormal transmission of the albino gene: Reciprocal crosses were made between branch l of T,-103 and a plant giving only green progeny and having no chromosomal abnormalities. When T,-103.1 was used as the female parent, all of the F, progeny were green and half of these had 50% sterile pollen (Table 3A). The F, progeny of plants with 50% pollen sterility were all green with half of these having 50% pollen sterility. The plants with 50% pollen sterility continued to produce in F, all green plants with half having 50% pollen sterility. Plants with full pollen fertility yielded green plants with all fertile pollen. The selfed F, plants having all fertile pollen produced 3 green : 1 albino in the F, generation. All of the green F, plants had fertile pollen. In the F, generation, 1/3 of the green F, plants gave all green plants with fertile pollen, while the remaining % produced a ratio of 3 green : 1 albino with all the green plants having fertile pollen. When T,-103.1 was used as the male parent (Table 3B) in a cross with a normal green plant, all of the F, offspring were green and had fully fertile pollen. Progeny in the F, generation were in a ratio of 3 green : 1 albino with all the green plants having fully fertile pollen. Two-thirds of the F, green plants repeated the 3 : 1 ratio in the F, generation while the remaining 1/3 green F, plants yielded all green plants with fertile pollen.

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S. M. MURR A N D G . L. STEBBINS

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A N A L B I N O M U T A N T I N PLANTAGO

TABLE 2 Phenotype and pollen viability of progeny from reciprocal crmses between branches I and 2 F, Crosses involving branches of 'l'*-l03

54 48

19 X 2 6 29 X 1 8

Fa

F2

Percent pollen Green Albino viability

46 52

Percent pollen Green Albino viability

49.3 50.7

Percent pollen Green Albino viability

51

44

52.7

47

55

45.

49.7

48

52 52

48.6 51.5

The reciprocal crosses revealed that the albino gene is transmitted through both the pistillate and the staminate parent thereby ruling out maternal inheritance. No appreciable female gamete abortion occurred in this or other crosses since the plants had a full seed set. Although zygotic elimination could result in defective seeds, this did not occur because all separate collections of seeds showed at least 98% germination. Since the progeny from branches 1 and 2 (Table 1) selfed to the X, generation showed a 1 : 1 ratio of viable to inviable pollen, some type of gamete elimination might be expected in this material. It has long been known that among higher plants the susceptibility of the male gamete to,abortion is determined by the genotype or by chromosomal abnormalities of the gametes. The phenotypic information in Table 3 permitted the determination of the genotypes of the plants involved in the reciprocal crosses (also Table 3 ) . If the genetic TABLE 3 Phenotype, genotype, and pollen viability from reciprocal crosses of T,-IOS.I F3

F2

Fl

Cross

47 green, f

al+ pe+ 1 al+ pe+ 0

T3-103.1, green, ss*

&I&

al+ pe+

I .I+

T1-122.1, green, ft .l+

pe+

1 a1+

pe+

1

48 green, f

.I+ pe+ / a1 pe+

I

25 green, f

al+ pe+

1

a1 pe+ / a1 pe+

.1+

pe+

I .I+

gmete + etimimted semisterile t fertile

pe+

I--

pe+

28 albinos

0 T3-122.1, green, f

100 green, f 50 green, f 49 green, PS

pe

A. d

1-

52 green, 8s

99 green, f

22 albinos

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S . M. M U R R AND G . L. STEBBINS

constitution of branches 1 and 2 were such that a recessive gene for albinism ( a l ) were linked in repulsion with the normal allele for a pollen-eliminator gene ( p e + ) , i.e., al+ pe / al pe+, then the ratios and inheritance patterns observed would be explained. With the heterozygous mutant as the female parent, half of the F, offspring are homozygous for the wild-type albino alleles but heterozygous for the polleneliminator alleles. The other half are heterozygous for the albino allele and homozygous for the wild-type allele of the pollen eliminator gene. The F, plants heterozygous for the pollen-eliminator gene when selfed continued to give progeny half heterozygous and half homozygous for the normal allele of the eliminator gene. This is caused by survival of both kinds of female gametes ( p e t and p e ) but survival of only one kind of male gamete ( p e + ). The abortion of p e gametes would account for the 50% pollen sterility. The green plants with fertile pollen are heterozygous for the albino gene but homozygous for the normal allele of the pollen eliminator. Since the eliminator was no longer present in these crosses. a normal 3 : 1 ratio of green to albino progeny was observed from heterozygotes in the F, and F, generation. With the heterozygous mutants in repulsion in the male parent (Table 3B), the eliminator gene is not transmitted through the male parent, so that all F, offspring are heterozygous for albinism but homozygous wild type at the polleneliminator locus. A 3 : 1 ratio of green to albino progeny occurs in the F, generation and the F2heterozygotes continue to produce green and albino offspring in a 3 : 1 ratio. That does not imply that it is a recessive gene. Since the gene apparently operates in the haploid male gamete, it is not possible to use the terms recessive or dominant. Evidence for pollen elimination: To determine whether the elimination of the male gamete carrying the al+ gene was the cause for a 1 : 1 ratio of green to albino progeny from selfed heterozygotes and not the result of nonrandom segregation in the formation of the female gametes, pairs of seeds (Plantago insu!aris has two seeds per capsule) from the same capsule were germinated and the seedlings scored for color. The two types of female gametes, al+ and al. would be expected to be associated at random in the formation of the two seeds from the same seed capsule in a ratio of 1 al+, al+ : 2 al+, a1 : 1 al, al. Since all male gametes presumably will be carrying only the a1 gene, plants from the seed pairs should also occur in a ratio of 1 green-green : 2 green-albino : 1 albino-albino. If some abnormality in the formation of the female gametes resulted in the 1 : 1 ratio, then seeds from the same capsule m;ght produce either progeny of like phenotype (green-green or albino-albino) always associated with each other or progeny of different phenotypes (green-albino) always associated with each other. When 400 such seed pairs from 10 different selfed heterozygotes were germinated, the phenotypes of the offspring fitted the expected 1 : 2 : 1 ratio (P > .60, Table 4). The overall total ratio of green to albino progeny is 392 : 408 (P > .60). The P values for individual ratios of total green to albino progeny range from 1.O to .30. This evidence along with that presented earlier makes developmental abnormalities or meiotic drive on the female side an extremely un-

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A N A L B I N O M U T A N T I N PLANTAGO

TABLE 4 Frequency of phenotype classes of seed pairs from selfed heterozygous mutants Plant number

Green-green

T,- 103.1- 1 2 3 4 5 T,-I03.2-1 2 3 4 5 Total

pairs

Greenlalbino pairs

A1bino:albino pairs

Total green

Total albino

8 7 10 12 9 11 12 10 11 10 100

18 20 18 19 20 21 15 22 18 21 192

14

13 12 9 11 8 13 8 11 9 108

34 34 38 43 38 43 39 42 40 41 392

46 46 42 37 42 37 41 38 40 39 408

likely mechanism for obtaining the observed 1 : 1 ratio of green to albino progeny. Localizat.’on of mutant genes: Normally when mutant genes are mapped using aneuploids or translocations, the mutant is crossed in the homozygous recessive form. but since the albino seedlings do not normally survive to flowering, another method of mapping was devised using homozygous translocations. Figure 2 shows pachytene ideograms of the two homozygous translocation stocks used in locating the mutant genes. These translocation lines were chosen because they are very easy to recognize at mitosis as well as meiosis. The first translocation line, T,73.41 (Figure 2A) is a whole-arm translocation involving chomosomes I and 4 TABLE 5 Phenotype and karyotype of progeny when heterozygous mutant crossed with homozygous translocation lines Cross

F2

F1

Phenotype

G / a1+ pe+

y

9 homozygous translocation

2 9 green

homozygous translocation A.

X

dal+ pe I a1 pe+

Karyotype

2 0 heterozygous translocation

al+ pe+ / a1 pe+ heterozygous &;i translocation

11 albino

11 homozygous standard

(Segregation type 1)

standard karyotype

yal+ pe+ / al+ pe+ homozygous 2;i translocation B.

X

dal+ p e / a 1 pe+ standard karyotype

a1+ pe+ / a1 pe+

7 homozygous standard 11 heterozygous translocation 9 homozygous translocation

27 green

heterozygous 1;i translocation 13 albinos

4 homozygous standard 6 heterozygous translocations 3 homozygous translocations

(segregation type 2 )

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S. M. MURR AND G. L. STEBBINS

to give rearrangements Is;4, and 1L;4s. I n comparison with chromosomes of the standard karyotype, the Is;4L chromosome is easily recognized because it has a much smaller heterochromatic region. The second line, T,-122.3, (Figure 2B) is a rearrangement involving the short arm of chromosome 3 and a portion of the euchromatin of the long arm of chromosome 4 . This exchange produces a distinctive chromosome with a heterochromatic section on the end of the long, mostly euchromatic arm of chromosome 4 and nucleolar organizer on both ends of the chromosome. The other chromosome, 4L;3L,is very small at mitosis and has a heterochromatic region on only one side of the centromere. Since the albino plants could not be analyzed for karyotype at pachytene, the two translocation lines were chosen because they can easily be recognized at mitosis in root tips. Lines that have a homozygous wild-type genotype and a rearranged karyotype involving either chromosomes I and 4 or chromosomes 3 and 4 were crossed with the heterozygous mutant which has a standard karyotype (Table 5 ) . Since the heterozygous mutant was used as the male parent. all male gametes carried only the aZ pe+ genes (assuming no crossing over) making all F, off spring heterozygous for the albino alleles, homozygous for the wild-type allele of the pollen eliminator, and heterozygous for the translocation. I n the F2 generation, the albino progeny may occur either with each karyotype in a ratio of 1 standard : 2 heterozygous : 1 homozygous or with only the homozygous karyotype. If they occur with only the homozygous standard karyotype, then the gene locus is located on one of the chromosomes involved in the rearrangement (segregation type 1) . If the albino offspring occur with all three possible karyotypes,

I

A

'L I

B -m 4L FIGURE 2.-Ideograms

:/a

I

3L

of pachytene chromosomes. A: Rearranged chromosomes of T,-73.41.

B: Rearranged chromosomes of T,-122.3.

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239

then the locus is not on either of the rearranged chromosomes (segregation type

2). When the homozygous translocation line involving chromosomes 1 and 4 is crossed to the heterozygous mutant with standard karyotype, F, segregation of type 1 is produced (Table 5A), while the cross using the homozygous translocation line involving chromosomes 3 and 4 produced F, segregation of type 2 (Table 5B). These segregation patterns locate the mutant genes on chromosome 1. I n actuality this results in the localization of the albino locus since the polleneliminator gene is not transmitted to the F, generation. Since the alb'ino and pollen-eliminator genes are tightly linked because no crossing over has been observed, localization of one gene determines the location for both. The complete association of a1 with the homozygous standard karyotype suggests tight linkage with the locus of pe and the translocation breakpoint (centromere). The standard, heterozygous and homozygous translocations at metaphase in root-tip cells are shown in Figure 3. DISCUSSION

During normal meiosis genes and chromosomes are precisely reproduced and the bivalents segregate at random to opposite poles. Among the many apparent exceptions to this 2nd Mendelian law, most do not conflict with the principle of random chromosome segregation, but are due to postmeiotic selection. The segregation of genes linked to a gamete-eliminator gene will be distorted, the degree of distortion depending on the intensity of the linkage. In fact, genes affecting gametophyte development are often discovered as a result of such distortion. The present study shows that segregation of a wild allele at the albino locus is distorted due to complete linkage with a pollen-eliminator gene resulting in a 1 : 1 ratio of normal to mutant progeny. The data indicate linkage between a1 and pe+ with a frequency of crossing over so low that among the 3000 green plants that were grown from selfed heterozygotes and examined for pollen abortion, no homozygous green nor heterozygous green and pollen-fertile crossover individuals were found. Since the PMC's in the heterozygous mutant undergo normal meiosis, the pe gene apparently becomes effective after the second division of meiosis is completed. The pe microspores do not increase in size and the nucleus does not divide. Instead. the microspores undergo progressive degeneration and by anthesis are virtually empty and are only 15p in diameter while viable pollen grains are 27p. Other segregation-distortion mechanisms: Other mechanisms to explain the distorted segregations found in this study have been considered. Low germination of the homozygous wild-type class of seeds must be excluded as a possible mechanism because at least 98% germination was found for all collections of seeds. Since nearly full seed set always occurred in all infloresences examined, elimination of female gametes could not account for the results. Heterosis must also be excluded. This phenomenon is reported by GUSTAFSSON, NYBOM and VON WETTSTEIN (1950) to account for excess heterozygous chlorophyll-deficient mutants. They provided evidence that barley plants heterozygous

240

S. M. MURR A N D G. L. STEBBINS

FIGURE 3.--Metaphase karyotype of root-tip crlls. A: Stantlertl karyotype. D: I-ioniwmgous I ; 4 translocation (TJ3.41). C: Heterozygous I ; 4 translocation ( T , - i 3 . 4 1 ) . D: Homozygous 3;4 translocation (T3-122.3). E: Heterozygous 3;4 translocation (T:,-l22.3).

for the albina-7 factor. the xantha-3 factor. or for both, show heterosis. Although no alf/al+ homozygotes were produced from the heterozygotes in this study, heterosis WAS studied indirectly by comparing heterozygotes to unrelated wildtype plants. They were found to be the same in germination rate, size, spike number. and in seed production. Cytoplasmic male sterility, as reported in maize (RHOADES 1933). onions (JONFS and CLARKE 1943). and in sugar beets (OWEN1945) does not seem to be a likely mechanism since this condition would be expected to affect both pollen carrying the gene for the albino character and pollen carrying the wild-type allele. RHOADES(1942) demonstrated that an abnormal type of chromosome 10 in maize is preferentially segregated during megasporogenesis. More than 70% of

A N ALBINO MUTANT IN PLANTAGO

241

the ovules received the abnormal chromosome instead of the 50% expected with random segregation. Pollen with the abnormal chromosome 10 was only partially successful in competing with pollen possessing a normal chromosome 10. Cytological information concerning preferential segregation is not available in Plantago. It would be impossible to detect such a mechanism without heteromorphic cytological markers. There is no evidence available which would rule out this mechanism as a basis of the apparently non-Mendelian segregation, but the 50% pollen inviability makes it unlikely that this mechanism is occurring. No meiotic abnormalities involving translocations, inversions, large duplications or deficiencies, or nondisjunction were observed which could account for the observed deviations from a 3 normal : 1 albino segregation ratio. However, as pointed out by STADLER (1933), genic changes and certain chromosomal aberrations. especially small deficiencies, cannot always be distinguished from each other. A number of mutant characters in Drosophila, such as Notch and Minute, are known to be the result of segmental deficiencies. The condensed chromosomes of plants are not as favorable for revealing small deficiencies as are the salivary gland chromosomes of the Diptera, so it is possible that the pollen eliminator may not be a gene, but a small undetectable deficiency that causes abortion of the male gamete. Parallel examples of segregation distortion: MOHand SMITH( 1952) described a case in barley in which three coincidental changes occurred as a result of exposure to radiation from an atomic bomb. These were a factor for mutant white seedlings, a chromosomal interchange, and a factor in one of the chromosomes involved in the interchange which is transmissible through the eggs but not through the pollen. The translocated chromosome was transmitted by none of the males, and about half of the female gametes transmitted the mutated gene. In selfed progenies of the irradiated plant, 50% of the seedlings were white, and all of the green plants were heterozygous for the reciprocal translocation and both mutated genes. Another mutant line of barley exposed to atomic bomb irradiation studied by NILANand MOH (1955) was characterized by a high frequency of cream seedlings arising from partially ovule-sterile, green plants. The ovule sterility of the parental plants averaged 40% and no pollen abortion was found. Following selffertilization approximately 39% of the offspring were cream. Ovule sterility and cream seedling were found to be linked in repulsion with approximately 7.4% crossing over. Presumably, the high frequency of cream seedlings was caused by the anomalous female gametic ratios resulting from the partial ovule sterility. Progeny homozygous for ovule sterility did not appear. HOLM(1954) reported a case in barley similar to the finding in this study. When the heterozygous chlorophyll-deficient mutant was selfed, the homozygous normal class was absent or nearly so and the heterozygotes were produced in approximately a 1 : 1 ratio with the mutant. Although the heterozygotes had a degree of semisterility, HOLMdid not hypothesize a mechanism to explain his observations. I n tomato, RICK (1966) found a mutant in which male and female gametes

242

S. M. MURR AND G. L. STEBBINS

were equally affected by an eliminator gene. The gametes, however, aborted only in certain hybrid combinations. He was able to map many genes by their degree of distortion from the expected F, segregation ratio due to linkage with the gamete eliminator. The recombination distances estimated in this manner corresponded approximately with those estimated by standard methods. The only one of these three examples that is closely similar to ours is the one The case of NILANand MOHinvolves abortion only of female described by HOLM. gametes, while in that described by RICK,both kinds of gametes aborted at approximately equal frequencies. A further understanding of the similarities and differences between these examples could be obtained only after detailed developmental and biochemical analyses of the action of the genes involved. The authors are deeply indebted to Dr. ALVAD. WHITTINGHAM for her continuous help with technique and for making available the stocks that were used. SUMMARY

The albino mutant al in Plantago obtained from gamma radiation acts as a recessive gene giving a segregation ratio of 1 normal to 1 albino when the heterozygote is selfed. Departure from a 3 : 1 ratio is attributed to complete trans linkage with a pollen-eliminator gene (pe). Half of the pollen from alfpe ,/ al pe+ heterozygotes aborts, and the mechanism for elimination of alfpe pollen grains is 100% effective. The albino and pollen-eliminator genes were mapped to chromosome I using translocation markers in somatic cells. Apparently when the seed producing the original X, plant was irradiated with gamma rays? three changes occurred: (1) a reciprocal translocation between chromosomes 3 and 4 ; (2) an albino-causing mutation on one homologue of chromosome I ; and ( 3 ) a mutation causing the elimination of microspores on the other homologue of chromosome I . LITERATURE CITED

GUSTAFSSON, A., N. NYBOMand D. VON WEITSTEIN, 1950 Chlorophyll factors and heterosis in barley. Hereditas 36: 383-392.

HOLM,G., 1954 Chlorophyll mutations in barley. Acta Agric. Scandinavica 4:457-471. JOHNSON, C. M., P. R. STOUT,T. C. BROYER and A. B. CARLTON, 1957 Comparative chlorine requirements of different plant species. Plant and Soil 8 : 337-353. JONES,H. A. and A. E. CLARKE, 1943 Inheritance of male sterility in the onion and the production of hybrid seed. Proc. Am. Soc. Hortic. Sci. 43: 189-194. LANGRIDGE, J., 1957 The aseptic culture of Arabidopsis thaliana (L.) HEYNH.Australian J. Biol. Sci. 10: 243-252.

MOH,C. C. and L. SMITH,1951 An analysis of seedling mutants (spontaneous, atomic bomb radiation-, and X-ray-induced) in barley and durum wheat. Genetics 36: 629-640. -, 1952 Three coincidental changes in atom-bombed barley. J. Heredity 43: 183-188. NILAN,R. A. and C. C. MOH,1955 A mutant line of barley induced by atomic-bomb radiation. J. Heredity 46 : 49-52. OWEN,F. V., 1945 Cytoplasmically-inherited male sterility in sugar beets. J. Agric. Res. 71: 423-440.

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RHOADES, M. M., 1933 The cytoplasmic inheritance of male sterility in Zea mays. J. Genetics 27: 71-93. -, 1942. Preferential segregation in maize. Genetics 27: 395-407. RICK, C. M., 1966 Abortion of male and female gametes in the tomato determined by allelic interaction. Genetics 53 : 85-96. STADLER,L. J., 1933 On the genetic nature of induced mutations in plants. 11: A haplo-viable deficiency in maize. Missouri Agric. Exptl. Sta. Res. Bull. 204: 1-29. STEBBINS, G. L. and A. DAY, 1967 Cytogenetic evidence for long-continued stability in the genus Plantago. Evolution 21: 409-428. WHITTINGHAM, A. D. and G. L. STEBBINS, 1969 Chromosomal rearrangements in Plantago insularis EASTW. Chromosoma 26: 449-468.