CYTOGENETIC ANALYSIS OF SEX DETERMINATIlON IN SPINACIA OLERACEAl

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1 CYTOGENETIC ANALYSIS OF SEX DETERMINATIlON IN SPINACIA OLERACEAl MUNEO IIZUKA* AND JULES JANICK Department of Horticulture, Purdue University, Lufayette, Indiana Received May 18, 1962 PINACIA is a dioecious genus in which a wide range of sex expression may soccur including a complete gradation of monoecious types. Extensive studies including polyploid analyses indicate that the genetic mechanism operating in strictly dioecious types acts as an allelic pair termed X and Y. This switch gene works on the Melandrium rather than the Drosophila or Rumex pattern for the Y allele is male determining or female suppressing (JANICK and STEVENSON 1954,1955a; JANICK 1956; SUTO and SUGIYAMA 1957; DRESSLER 1958; KUCKUCK 1960). The monoecious character has been interpreted genetically as either due to a gene or closely linked genes which are distinct from the XY factors (SUGI MOTO 1948; BEMIS and WILSON 1953) or as due to a gene which acts as an allele of the XY pair ( JANICK and STEVENSON 1955a). Trisomic analysis has placed the XY factor on the longest chromosome (chromosome 1) of the complement (ELLIS and JANICK 1960; SUTO and SUGIYAMA 1961a,b). Although in most cases the chromosome 1 pair is cytologically homomorphic in both staminate and pistillate types there have been a number of reports of heteromorphism involving this chromosome (ARARATJAN 1939; ZOSCHKE 1956; DRESSLER 1958; BOSE and JANICK 1961). The discovery of a cytological marker on chromosome 1 facilitated the present cytogenetic analysis of the sex determining mechanism in spinach. MATERIALS AND METHODS Materials used in this study were lines and varieties of Spinacia oleracea as shown in Table 1. Chromosome observations were made on root tip and pollen mother cell squashes. On root tips, a one hour pretreatment of a saturated solution of percent colchicine and/or 0 C cold treatment were used. After pretreatment, root tips were fixed for 24 hours or more in 3:l acetic alcohol, hydrolyzed for 0.5 to two minutes in N HC1 at 58 to 60 C and stained in Feulgen reagent for two to three hours at room temperature. Root tips were squashed in one percent acetocarmine after staining. Materials for observation on meiosis 1 Journal paper No of the Purdue University Agricultural Experiment Station, Lafayette, Indiana, and contribution No. 43 of the Laboratory of Applied Genetics, Research Institute for Food Science, Kyoto University, Japan. This study was supported by the National Science Foundation (Grant 14150). 2 Present address: Research Institute for Food Science, Kyoto University, Kyoto. Japan. Genetics 47: September 1962

2 1226 MUNEO IIZUKA AND JULES JANICK TABLE 1 Origin of material Variety or arcession number P.I Spica Universal Jiromaru Early Hybrid i ([99 x 951 x Virginia Savoy) Sonll c Turkish origin, Regional Plant Introduction Station, Ames, Iowa Institut fur Gartnerishe Pflanzenziichtung der Tezhnischen Hochschule, Hanover, Germany Institute for Pflanzenziichtung Quedlinburg. Deutsch Akademie der Landwirtschaftswissenshaften zu Berlin, Germany Research Institute for Food Science, Kyoto University, Kyoto, Japan U.S. Department of Agriculture were fixed for 24 hours or more in 3: 1 acetic alcohol and squashed in one percent acetocarmine (with iron alum) after passing the slide over an open flame. Pollen fertility was determined after staining two days with 0.1 percent acetocarmine. All plants were grown in the greenhouse. Plants flowered under natural daylength in spring and summer and under longday (16 hours) treatment in autumn and winter. The greenhouse temperatures from late spring to the beginning of autumn were variable but were often as high as 32 C. Greenhouse temperatures during the rest of the year were maintained at 18" to 23 C. Plants were classified for sex by estimating the percentage of pistillate flowers per plant (JANICK and STEVENSON 1955a). Seven classes were used, viz., 0, 5, 25, , and 100 percent female of which zero percent represents a staminate plant, 100 percent represents a pistillate plant, and 595 percent represents various types of monoecious plants. The chromosome designations correspond to those of ELLIS and JANICK (1960). RESULTS Cytological behavior of a morphological variant of chromosome I: A line derived from a single sib cross from P.I was found to segregate for a morphologically distinct variant of chromosome 1 (BOSE and JANICK 1961). The standard chromosome 1 is heterobrachial, one arm being twice as long as the other. The nonstandard chromosome is longer and homobrachial, apparently due to the addition of a segment to the short arm of the standard chromosome (Figure 1). The original line in which this homobrachial chromosome was observed, segregated into four equal classes with respect to sex and the constitution of the chromosome 1 pair, viz., monoecious and heteromorphic, monoecious and homomorphic for the standard chromosome, pistillate and heteromorphic, and pistillate and homomorphic for the standard chromosome. Crosses and selfs involving these types were made to determine the transmission of the homobrachial chromosome and its relation to sex determination.

3 SEX IJETERMINATION 1227 FIGURE 1.Somatic chromosomts of spinach (magnification 5000~). A. Homomorphic for the heterobrachial or stantlartl chromosom? 1. B. Heteromorphic for chromosome I. standard and homohrachial. C. Homomorphic for the homobrachid chromosome 1. As the heteromorphic plants found in the sib cross were monoecious and pistillate, transmission of the homobrachial chromosome could be followed through the pollen and egg. The results of many crosses with lines derived from the original P.I did not show any evidence of differential transmission of the homobrachial chromosome as shown in Table 2. In progeny segregating individuals which were homomorphic standard, heteromorphic, and homomorphic homobrachial for chromosome 1, no obvious morphological differences in plant characters could be detected. In order to determine the origin of the additional segment, meioses in pollen mother cells were examined (refer Figure 2). In homomorphic standard plants the chromosomes pair normally to form six bivalents. Heteromorphic types show four bivalents and one quadrivalent in most cells. While the quadrivalents are generally in chains, rings appeared in about 14 percent of the cases. These types also show some abnormalities following the I1 meiotic division and tetrad stage. These include lagging chromosomes which result in the formation of microcytes. Pollen analysis indicated a lower fertility associated with the heteromorphic chromosome 1 pair (Table 3). The equal transmission through pollen and egg, the lack of morphological effects attributed to the nonstandard chromosome, the reduced pollen fertility, and above all, rings of four chromosomes at meiosis associated with the heteromorphic pair, indicate that a reciprocal translocation is involved. However,

4 ~ 1228 MUNEO IIZUKA AND JULES JANICK TABLE 2 Transmission of homobrachial chromosome I Cross Pistillate Pollen parent parent Morphology of chromosome 1 in progeny Homomorphic Heteromorphic Homomorphic standard homobrachial cm CzzLII L Number of Plants although the segment added to chromosome 1 was large enough to mark the mitotic chromosome easily, a heteromorphic donor chromosome pair could not be distinguished. The reason for this is baffling. However, indirect evidence indicates that the donor chromosome involved in the translocation is chromosome 2. (This identification is not certain, however, because chromosome 2 and 4 are very similar in appearance.) Crosses involving plants heteromorphic for the translocation produced trisomics in about 1.4 percent of the progeny. Of a total of 17 trisomics, three were found to be trisomic for chromosome 1 and the other 14 were trisomic for chromosome 2 (Table 4 and Figure 3). One might expect a translocation occasionally to give rise to 31 chromosome distribution at anaphase I and in fact the results of this irregularity were observed in metaphase I1 plates in a few cases. The two types of trisomics should represent the chromosomes involved in the reciprocal translocation. The differences in the percentages of the two types of trisomics is explainable because a pollen grain or an egg carrying an extra chromosome 1 is especially low in viability compared to other extra chromosome types (JANICK, MAHONEY and PFAHLER 1959). The external appearances of the two trisomics derived from line P.I do not correspond to any of the trisomics derived from Long Standing Bloomsdale by ELLIS and JANICK (1960). Studies are in progress to associate the additional segment translocated onto chromosome 1 with the trisomic set obtained from Long Standing Bloomsdale.

5 SEX DETERMINATION 1229 A 4 L B C "Is FIGURE 2.Meiotic chromosomes of spinach (magnification 5000~ ). A. Metaphase plate in PMC's of plant homomorphic for the standard chromosome 1; note 611. B, C, D. Metaphase plate in PMCs of plant heteromorphic (standard and homobrachial) for chromosome 1. B. 41, C. 4,, + I,,(Nchain). D.4,, + l,,.(ringof4). Plants grown from remnant seed of the original P.I packet were examined cytologically to determine the frequency of the homobrachial chromosome. All of the 74 plants examined were found to be homomorphic for the standard chromosome. The frequency of the homobrachial chromosome in this line must be very low or its occurrence may have been due to a fortuitous event in the parents of the original sib cross. Inheritance of sex in P.I : At the same time the original monoecious plants heterozygous for the translocation involving chromosome 1 were selfed. they were used as pollen parents with pistillate plants homomorphic for the standard chromosome. The summarized results of these crosses are presented in Table 5. The quarter of the self progeny that were homomorphic for the standard chromosome 1 were either classified as staminate (0% female) or highlystaminate monoecious (5 % female). The heteromorphic progeny were all mon

6 1230 MUNEO IIZUKA AND JULES JANICK TABLE 3 Chromosome pairing. abnormal tetrads. and pollen sterility in spinach homomorphic standard and heteromorphic (standard/homobrachial) for chromosome 1 Chromosome pairing or fertility Chromosome pairing at metaphase 1' Morphology of Chromosome 1 czzil2 cii=l Per cent 6n =+ 2 I 4=+ lm+ 11: ,A v /\ \/ Abnormal tetrads tt Abnormal pollen t Based on 500 cells observed for each type. tt Based on 1200 tetrads observed for each type. $ Based on 1800 pollen grains observed for each type. oecious with the two Ixgest classes being 25 or 50 percent female. Fortyone of 42 plants which were homomorphic for the homobrachial chromosome were pistillate ( 100% female). The single monoecious segregate of this type, designated 343, was selfed and at the same time crossed with pistillate plants which were homomorphic standard for chromosome 1. When the original heteromorphic monoecious plants were used as the pollen parent in crosses with homomorphic standard pistillate plants, 239 of the 241 homomorphic standard progeny were monoecious, and two plants were pistillate. All of the 256 plants that were heteromorphic were pistillate. These results can be explained by assuming that the monoecious factor is linked tightly to the translocated segment. This agrees with the hypothesis of JANICK and STEVENSON (1955a) that the monoecious factor is controlled by a gene that acts as an allele of the XY gene pair and that the monoecious factor Xm

7 SEX IjETERMINATION 1231 acts as an incomplete dominant to X with XmP types being more highly male than Xrj*X plants. Assuming that the heteromorphic monoecious plants from P.I were X"/T (translocation) X, the self progeny would be expected to segregate into three classes: 1 Xm/Xm (staminate or highlystaminate monoecious) : 2 Xm/T X (monoecious): 1 T X/T X (pistillate). Under this assumption, the exceptional homomorphic homobrachial plant, 343, represents a crossover between the monoecious gene and the translocated segment (i.e., T X"/T X). If this were true the progeny of this selfed plant should segregate three monoecious to one pistillate and when crossed to pistillate plants should segregate one monoecious to one pistillate. These progenies were grown in the summer when temperatures were high and in the winter when cool temperatures could be maintained. The results shown in Table 6 are in agreement with the hypothesis. However, the data obtained in the summer is muddled by a shift toward maleness in the monoecious progeny brought about by high temperature (THOMPSON 1955; JANICK and STEVENSON 1955b; KATAYAMA and SHIDA 1960; SUTO and SUGIYAMA 1960, 1961a,b). Definite proof that a crossover has occurred between the X" factor and the translocation is obtained from selfing plants of the recombinant genotype T X"/X obtained from the XX x T Xm/T X cross. The TABLE 4 Frequency of occurrence of trisomies from diploid x diploid crosses Cross No. of Type of Trisomic Pistillate Pollen plants Percent parent parent analyzed trisomics Number K=3Ix X X c=n2 5 X LII x X X

8 1232 MUNEO IIZUKA AND JULES JANICK FIGURE 3.Somatic chromosomes of trisomics derived from diploid crosses involving the heteromorphic chromosome 1 pair (magnification 5000~). A. Plant trisomic for chromosome 2. Note both chromosome 1 s are homobrachial and one chromosome 3 is satellited. B. C. Plants trisomic for chromosome 1; Reler to case B of Figure 6 for explanation of B, and to case C of Figure 6 for explanation of C. results agree with expectations, uiz., 1 T X /T X (staminate or highlystaminate monoecious, 0 or 5% female) : 2 T X l/x (monoecious, 2595% female) : 1 X/X (pistillate, 100% female) as shown in Table 7. The exceptional pistillate plants that were homomorphic for the standard chromosome 1 were tested in a similar manner. The data indicate that only one of them was a true XX type. In order to prove that the monoecious gene X is allelic to Y, crosses were set up to test the linkage between the Y gene and the translocated segment. Pistillate plants that were homomorphic for the homobrachial chromosome (T X/T X) were crossed to staminate plants of the constitution X/Y. Heteromorphic staminate progeny (T X/Y), were then crossed to homomorphic standard pistillate (X/X) plants. The results of this cross as shown in Table 8 indicate complete linkage between the Y factor and the normal chromosome 1. Of special interest

9 ~~ SEX DETERMINATION 1233 TArBLE 5 Sex distribution of progeny of selfed and crossed monoecious selections inuoluing heteromorphic chromosome 1 Distribution of progeny in Morphology of percent femaleness classes chromosome 1 Total Parentage in progeny plants at Only one proved to be an XX recombinant TABLE 6 Distribution of progeny of the suspected monoecious recombinant(343) Distribution of progeny in percent femaleness classes Total Parentage plants Percent pistillate T xm/ T x self Summer Winter XX x TXm/TX Summer Winter

10 1234 MUNEO IIZUKA AND JULES JANICK TABLE 7 Distribution of progeny of T X",/X self Distribution of progeny in percent femaleness classes Chromosome Total morphology plants 4 4 W were the small percentage of monoecious plants in line 90. Of the seven monoeious plants. four were heterozygous for the translocation while three were homozygous normal. These exceptional plants are under study. Preliminary data show a peculiar type of inheritance and indicate that an allelic effect is involved in some of the monoecious plants. DRESSLER (1958) reported linkage between the Y factor and a satellite appearing on the longest chromosome of the complement in lines derived from the variety Spica. While his lines were not available, the original variety Spica from which these types were isolated was received from DR. KUCKUCK from which BOSE and JANICK (1961) also reported a satellited chromosome 1. A similar satellited chromosome was also found in the variety Universal. However, in contrast to DRESSLER'S report, both staminate and pistillate plants were found to be heteromorphic for the satellited chromosome and a single staminate plant homomorphic for the satellited chromosome was of the genotype XY for it produced a 1: 1 sex ratio when crossed with pistillate (XX) plants. Progeny analysis of these types in the present study indicated no linkage between the satellited chromosome and sex. Careful examination of the material, however, indicated that the satellited chromosome was not chromosome 1 but chromosome 3. This is indicated in the photograph of the report of BOSE and JANICK (1961). Chromosome 3 and chromosome 1 are very similar in morphology except that chromosome 3 is slightly shorter (ELLIS and JANICK 1960). To determine, the identity of the satellited chromosome found in Spica and Universal, crosses were made between pistillate plants which were homomorphic for the homobrachial chromosome 1 with staminate plants which were heteromorphic for the satellited chromosome found in Spica and Universal. Crosses were then made between staminate and pistillate plants which had both one satellited

11 SEX DETERMINATION TABLE 8 Distribution of progeny from crosses of X/X x T X/Y 1235 Morphclogy of Chromosome 1 Distribution of progeny in percent femaleness classes Line in progeny plants L n 33 Total m Total chromosome aid one homobrachial chromosome. The results of this cross are presented in Table 9. The isolation of plants having at least three of the four nonstandard chromosomes in question indicates that these chromosomes are not homologous (Figure 4). An analysis of this cross indicates that sex is determined by chromosome 1. In these crosses there was a deficiency of plants homozygous for the homobrachial chromosome and an excess of plants with the normal chromosome 1. This is reflected in the excess of staminate plants in the progeny (70 males: 52 females), for all plants homozygous for the standard chromosome 1 are

12 1236 MUNEO IIZUKA AND JULES JANICK TABLE 9 Distribution of progeny from crosses between plants heteromorphic for chromosome 1 (standard and homobrachial) and chromosome 3 (standard and satellited) Chromosome Distribution of progeny in morphology percent femaleness classes Total plants 7 R F 15 at I f I "I Total Included as staminate in analysis Chisquare analysis Segregation df X? P Chromosome 1 (1:l:l:l) chromosome 3 (1:e: 1) o Interaction &.70 Total cl:% 1 : 1 :2: 1 : 1 :2: 1: 1:2: 1) eo.10 staminate and all plants homozygous for the homobrachial chromosome are pistillate. The total sex ratio of a number of similar crosses was 174 males: 121 females and agrees with these results. Because the sex ratio of the plants heterozygous for the translocation was equal, it is not clear if this selective advantage of the normal chromosome 1 occurs through the male or female gametes, or if it is zygotic. In the light of DRESSLER'S data there must be karyotypic variation in the kinds

13 SEX DETERMINATION 1237 FIGURE 4.Somatic plate from spinach plant derived from cross of Spica and P.I which is homomorphic for the homobrachial chromosome 1 and the satellited chromosome 3 (magnification 5000x ). of satellited chromosomes present in the complement.. The origin of this satellited chromosome is obscure. No relationship between pollen fertility and the presence of one or two satellited chromosome 3 was found. Lines were isolated which were homozygous for the satellited chromosome 3. The sex ratio of these lines was 1 male: 1 female. DISCUSSION A tentative map of the isobrachial chromosome is presented in Figure 5. The distance from the sex genes to the break of 0.2 map units is calculated only from crosses involving the X"' gene. (Refer Table 5; 2/2( 170)+ 497 = 0.002). It is not possible to determine if the gene is close to the break or if crossingover is CHROMOSOME I+CHROM. 2+ FIGURE 5.Preliminary cytological map of the homobrachial chromosome.

14 1238 MUNEO IIZUKA AND JULES JANICK very much reduced in the whole chromosome by the translocation from these recombination data. Evidence that the gene is actually close to the break comes from estimates of the distance between the sex gene and the centromere estimated from the plants that were trisomic for chromosome 1. The origin and chromosome makeup of the three trisomics involving chromosome 1 are shown in Figure 3. The origin of case A is explainable as a case of nondisjunction. In this instance as the plant was monoecious and contains one homobrachial chromosome the extra chromosome is traced to the pollen parent. In case B the trisomic was staminate but did not contain the homobrachial chromosome. There are a number of explanations: (1) nondisjunction of chromosome 1 in the female, (2) mitotic nondisjunction of chromosome 1 after fertilization, (3) nondisjunction in division I1 of meiosis in the male, and (4) a crossover between the centromere and the sex genes in division I of meiosis followed by nondisjunction in division I and subsequent XYXX distribution in division 11. The net result appears similar to double reduction in trisomic heterozygotes. Of these four possibilities. nondisjunction in the female was felt to be unlikely for trisomics were not observed in 2n 0 x 2n 8 crosses (Table 4). Mitotic nondisjunction and nondisjunction in division I1 of meiosis seems unlikely for the same reason. However, this type of trisomic is to be expected if the distance between the sex gene and the centromere is large when nondisjunction of the heteromorphic pair follows crossingover between the sex gene and the centromere (Figure 6). In case C the trisomic was female and contained only one homobrachial chromosome which indicates that the extra chromosome is probably derived from nondisjunction after crossingover between the gene and the centromere in the pistillate parent. The fact that two out of the three trisomics indicate crossingover between the gene and the centromere is evidence (although meager) that the distance between the sex genes and the centromere is large. These results indicate that the sex determining mechanism in spinach acts as an allelic series. There is considerable evidence however that the sexcontrolling system in spinach is exceedingly complex. For example the monoecious allele described in this report appears to differ from the monoecious factor isolated from the variety Nobel (JANICK and STEVENSON 1955a). While both alleles react similarly to temperature (high temperature producing a decided shift toward maleness). the monoecious allele isolated from P.I when homozygous often produces a completely staminate plant in contrast to the monoecious allele from Nobel. (XmXrn staminate plants can be easily distinguished from XY staminate plants by a genetic test.) This suggests that there is a series of alleles differing in the strength of their maleinducing effects. In addition, the reports of SUTO and SUGIYAMA (1960, 1961a,b) indicate that two kinds of staminate plants, bracted and leafy males (first described by ROSA (1925) as extreme and vegetative males) are due to Y alleles. The extreme or bracted males are characterized by having leaves suppressed entirely or reduced to small scales on the upper portions of the seedstalk. The vegetative male has a leafy stalk and resembles the pistillate types in growth habit.

15 SEX DETERMINATION 1239 CROSS CHROMOSOME CONSTITUTION AND SEX OF TRISOMIC A (NON DISJUNCTION) 9 Q' B 9 (NONDISJUNCTION) d C (NONDIS JUNCTION) 0 d t FIGURE 6.Explanation of the origin of chromosome 1 trisomics.

16 1240 MUNEO IIZUKA AND JULES JAI\JICK It would appear most certain that a complex locus is involved. The simplest two gene model that will account for monoecism must assume a tight association between a dominant female suppressor Suf (WESTERGAARD 1958) and a male promoting gene M showing incomplete dominance. Under this scheme: Y = SuiM, X =sufm and X= sufm. The phenotype Sufm/Sufm is presumably neuter or weakly male. The presence of the tightly linked cytological marker offers a solution of the problem. Any monoecious recombinants (sufm) from crosses of X/X x T X/Y (i.e., sufm/sufm 0 X T SufM/sufm 8 ) produced by intraallelic recombination can be detected by selfing. Only a crossover (or mutation at the XY locus) will show linkage with the translocation. Other markers on the other side of the sex genes would facilitate this type of study but at this writing no sexlinked genes are known. A study is underway at this laboratory to induce sexlinked mutants by irradiation. SUMMARY The longest chromosome (No. 1) of Spinacia oleracea contains the sex determining XY factors. This chromosome is normally heterobrachial with one arm twice as long as the other. A homobrachial variant of chromosome 1. found in line P.I , appears to involve a reciprocal translocation with chromosome 2. In this line the X gene was tightly linked to the chromosome break with a map distance of 0.2 crossover units. A cytogenetic analysis of sex determination utilizing the translocation marker indicates that a monoecious factor, X. is allelic to the XY factors. LITERATURE CITED ARARATJAN, A. G., 1939 Heterochromosomes in the wild spinach. Compt. Rend. (Doklady) Acad. Sci. URSS 24: BEMIS, W. P., and G. B. WILSON, 1953 A new hypothesis explaining the genetics of sex determination in Spinacia oleracea L. J. Heredity 44: BOSE, S., and JULES JANICK, 1961 Karyoraces in Spinacia oleracea. Am. J. Botany 18: DRESSLER, O., 1958 Zytogenetische Untersuchungen an diploidem und polyploidem Spinat (Spinacia oleracea L.), 2. Pflanzenbau Pflanzenschutz 40: ELLIS, J. R., and JULES JANICK, 1960 The chromosomes of Spinacia oleracea. Am. J. Botany. 47: JANICK, JULES, D. L.M.~HO&EY, and P. L. PFAHLEFC, 1959 The trisomics of Spinacia oleracea L. J. Heredity 50: JANICK, JULES, and E. C. STEVENSON, 1954 A genetic study of the heterogametic nature of the staminate plant in spinach. Proc. Am. Soc. Hort. Sci. 63: a Genetics of the nicnoecious chaiactcr in spinach. Genetics 40: b Environmental influences on sex expression in monoecious lines of spinach. Proc. Am. Soc. Hort. Sci. 65: 41c4.22. KATAYAMA, Y., and S. SHIDA, 1960 Influences of daylength and temperature difference on sexratio in spinach. Univ. Miyazaki Fac. Bull. 6: KUCKUCK, H., 1960 Problems and results in the breeding of agricultural crops in Germany (Abstr.). Japan. J. Breeding 10:

17 ROSA, J. T., 1925 SEX DETERMINATION 1241 Sex expression in spinach. Hilgardia 1 : SUGIMOTO, Y., 1948 Studies on the breeding of spinach. 11. Sex expression and genetical explanation. Hort. Assoc. Japan J. 17: SUTO, T., and S. SUGIYAM~, 1957 On the genetic modification on the sex ratio in spinach. Jap. Abstracts. Japan J. Breeding 7: (Suppl. 11). SUTO, T., and S. SUGIYAMA, 1960 Sex expression and determination in spinach. I. Growth habit and its sexlimited inheritance. Japan. J. Botany 17: a Sex expression and determination in spinach. 11. Inheritance and breeding of intersexuality. Japan. J. Breeding 10 : b Sex expression and determination in spinach Inheritance and breeding of intersexuality (continued). Japan. J. Breeding 11: THOMPSON, A. E., 1955 Methods of producing firstgeneration hybrid seed in spinach. Cornel1 Univ. Agr. Exptl. Sta. Mem. 336: 148. WESTERGAARD. M., 1958 The mechanism of sex determination in dioecious flowering plants. Advan. Genet. 9: ZOSCHKE, U,, 1956 Untersuchungen uber die Bestimmung des Geschlechts beim Spinat (Spinacia oleracea L.). Z. Pflanzenbau Pflanzenschutz 35: