Karyotypes, nucleoli, and amphiplasty in hybrids between Hordeum vulgare L. and H. bulbosum L

Genetica 46: 217-233 May 15, 1976

KARYOTYPES, NUCLEOLI, AND AMPHIPLASTY IN HYBRIDS BETWEEN HORDEUM VULGARE L.

AND H. BULBOSUM L.

W. LANGE and G. JOCHEMSEN Institute de Haaff, Foundation for Agricultural Plant Breeding,

Wageningen, The Netherlands Received and Accepted June 13, 1975

Chromosome measurements were carried out in Hordeum vulgare, H. bulbosum, and their diploid, triploid, and tetraploid hybrids. The chromosomes were classified by using relative values, and thus karyotypes were established. For comparison of these karyotypes both relative and absolute values were used. It was concluded that differential amphiplasty occurred, whereas neutral amphiplasty could not be demonstrated. In the hybrids the relative length of the parts of the chromosomes (long arm, short arm, satellite) was not changed in comparison with these lengths in the pure species. The karyotypes of both species had considerable similarities. From comparing the mean absolute genome lengths, it was, however, concluded that in the pure species, as well as in all hybrid types, the chromosomes of H. vulgate were longer than those of H. bulbosum. In the diploid and tetraploid hybrids the mean genome lengths were shorter than those in the pure species and the triploid hybrids. The differential amphiplasty was such that the secondary constriction of chromosome 6 of H. bulbosum, did not show up in the hybrids. This could be related to the suppression of nucleolar formation in the genome of 11. bulbosum, because the maximum number of nucleoli in root tip cells equalled the number of satellite chromosomes. Finally it was found that the pattern of nucleolar fusion in diploid and triploid hybrids deviated from the expectation. The results were discussed in relation to chromosomal disturbances that occurred in the hybrid tissues and that resulted in elimination of chromosomes and other effects.

Introduction

A programme of interspecific hybridizat ion between barley (Hordeum vulgare L.) and bulbous barleygrass (H. bulbosum L.) has led to the discovery of a process of chromosomal e l iminat ion in hybrid tissues (LANCE, 1969, 1971a, b), which at abou t the same time was described by others (SYMKO, 1969; KASHA & KAO, 1970; KAO & KASHA, 1970). In addi t ion the morphological characters of the chromosomes have been studied, which provided interesting insights into intergenomic relationships, especially amphiplasty. This phenomenon was first described by NAVASHIN (1928, 1934) in some interspecific hybrids of Crepis. This au thor dist inguished neutral amphiplasty and differential amphiplasty.

218 W. LANGE AND G. JOCHEMSEN

Neutral amphiplasty is expressed in mitotic metaphases in interspecific hybrids, when the chromosomes of one of the species involved are longer, and those of the other species shorter than is normal for the pure species. This seems to be a chromosomal interaction which takes place in reci- procal crosses as well. HENEEN (1962) described a similar situation in interspecific hybrids in Agropyron. He referred to a hypothesis in DAR- LINGTON (1937) which stated that chromosomes in a hybrid should adjust their thickness to each other, which could result in changes in their lengths. From a study of the literature HENEEN (1962) concluded that Darlington's hypothesis had no general validity. LANGRIDGE et al. (1970) confirmed the results observed in Crepis. These authors related the phenomenon to the regulation of the cell cycle. When the sets of chromosomes need different times for (S-[-)G 2 d- Prophase, the set which needs the longer time will at metaphase not be fully contracted, while the set that needs the shorter time will be more contracted than usual.

The second form of amphiplasty, differential amphiplasty, occurs in certain interspecific hybrids where the secondary constriction(s) of one of the participating species is (are) lacking. The satellite is retracted on to the chromosome and is therefore not distinguishable. NAVASHIN (1928, 1934) concluded that the effect resulted from the interaction of alien chromosomes. McCLINTOCK (1934) introduced the term nucleolar organizer to name the site on the chromosome at which the nucleolus is formed and noted that this organizer is often attached to the secondary constriction. She also noted that differential amphiplasty in Crepis was related to the formation of nucleoli in such a way that only chromosomes with a secondary constriction were active in the formation of nucleoli. WALLACE ~¢ LANGRIDGE (1971) confirmed the earlier results in Crepis and presented examples in other Crepis hybrids. They clearly related differential amphiplasty to the formation of nucleoli, thus demonstrating a genetic control of the nucleolus formation. It was concluded that the segment of the chromosome carrying the nucleolar organizer contracts normally at prophase unless prevented from doing so by the mechanical interference of a nucleolus. The interaction might be considered as a case of allelic repression.

Differential amphiplasty has been reported in many genera. In addition to the above mentioned situation in Crepis it occurs in interspecific hybrids of Salix (WILKINSON, 1941, 1944), Ribes (KEEP, 1960, 1962), Agropyron (HENEEN, 1962), Hordeum (LANGE, 1969; KASnA & SADASI- VAIAH, 1971), Arena (SADASIVAIAH ~¢ RAJHATHY, 1969), and Secale (Sybenga, pers. comm.). BHATTACHARYYA et al. (1961) demonstrated that differential amphiplasty occurred in Triticale, although PIERITZ (1970) reported for this hybrid that the satellite of rye could vaguely be dis-

KARYOTYPES OF H O R D E U M HYBRIDS 219

tinguished in some cells. The intergenomic relations between wheat and rye were also studied in rye addition lines to wheat (BHATTACHARYYA et al., 1961), where again the secondary constriction of rye was not visible. Differential amphiplasty also occurs in the natural trispecific hybrid Triticum aestivum (CRoSBY, 1957; LONGWELL & SVIHLA, 1960; GIORGI & BOZZINI, 1969a, b; DARVEY & DRISCOLL, 1972a). The phenomenon seems to be restricted to interspecific and intergeneric hybrids, which led WALLACE & LANGRIDGE (1971) to suggest that it is consequence of an evolutionary divergence.

In this paper a description will be given of the karyotypes of H. vulgate, H. bulbosum and diploid, triploid and tetraploid hybrids between these species. Furthermore results will be presented of studies concerning the occurrence of nucleoli in somatic tissue. The results will be discussed, with special attention to neutral and differential amphiplasty.

Material and Methods

The plant material was described in earlier papers (LANGE, 1969, 1971a). It consisted of diploid and tetraploid varieties of H. vulgate, one haploid plant of H. vulgate obtained through hybridization between 1-1. vulgate and H. bulbosum followed by elimination of the chromosomes of H. bulbosum, diploid and tetraploid clones of H. bulbosum, and diploid, triploid and tetraploid hybrids between H. vulgare and H. bulbosum. In the diploid and tetraploid hybrids the genomes of the parental species were present in equal numbers, while the triploid hybrid had one genome of H. vulgate and two genomes of H. bulbosum. The haploid as well as the hybrid plants were obtained through embryo culture.

Mitotic chromosomes and nucleoli were studied in root tip squashes, using material which had as similar developmental origin and charac- teristics as possible. The roots were pretreated with 8-hydroxyquinoline (0.002 M in water), according to TJIo & LEVAN (1950), for 4--16 hours at about 4°C. The roots were fixed in acetic alcohol (1 : 3), hydrolized in 1 N HC1 at 60°C for 10 min., and stained with leuco-basic fuchsin. After the staining, the roots were mostly macerated in a solution of Pectinol (a pectinase-containing powder which is used in the fruit processing industry). The root tips were squashed in acetic acid (45~) and mounted in euparal according to BRADLEY'S (1948) vapour exchange method.

The measurements of individual arms of chromosomes were carried out in cells in which the chromosomes were as straight, flat and non- overlapping as possible. The measurements were made directly with an eyepiece screw-micrometer (I 5 x ) and an oil immersion objective (90 × ).

220 W. L A N G E A N D G . J O C H E M S E N

The arms of each chromosome as well as the satellites were measured in duplicate and the mean was taken as the final value. When the two measurements of an arm or satellite differed by more than 10~, two new measurements were carried out. It was aimed at measuring more than 25 cells per plant type. For comparison, the absolute values had to be transformed to relative values. This was done by expressing the lengths of all chromosome arms and satellites in a cell as percentages of the sum of the total lengths of all chromosomes in that cell, and by calculating the arm ratio (short arm/long arm) for each chromosome. The comparison was further facilitated by displaying the results of each cell in two graphs. One in which the ordinate and abscissa were the values for short and long arms, and the other in which they were the values for arm ratios and total lengths. Each graph represented the total amount of information in a different way.

The number of nucleoli per cell was counted in root tip squashes. In these preparations the cavities in which the nucleoli are situated appear as unstained globular structures in the interphase nuclei. To find the frequency distribution of the nucleolar number 400 cells per slide and 10 slides per plant type were analyzed.

In this paper the following abbreviations have been used: HV = H. vulgare, HB = H. bulbosum, and HY = interspecific hybrid.

Results

THE KARYOTYPES

To establish karyotypes of the different plant types the chromosomes had to be classified. First the two parental types were studied (Figs 1-2). For H. vulgare, 11 cells of diploid plants and 24 cells of a haploid plant were analysed. Within both groups of data it was possible to distinguish a certain pattern in each cell. The two satellite chromosomes and the longest and shortest of the remaining five chromosomes could easily be distinguished, and the other chromosomes were classified on basis of the measurements. Per cell the chromosomes were numbered, and per chromosome type the mean values for long arms, short arms, total length and ratios were calculated, which was done for diploid and haploid cells separately. Both groups of data were in good agreement with each other, so they were pooled (see the columns HV in Tabs. 1-4). The result was very like the karyotype of H. vulgare as known from the literature (TJIo & HAGBER6, 1951; BURNHAM & HAGBERC, 1956), which supported the decision to handle the other plant types in the same way.

KARYOTYPES OF HORDEUM HYBRIDS 221

Figs. 1 4 . Hordeum chromosomes in mitosis: (1) H. vulgare, 2x; - (2) H. bulbosum, 2x; - (3) and (4) diploid and tr iploid hybrids, respectively, between H. vulgate and H. bulbosum.

For H. bulbosum, the chromosomes of 26 diploid cells were measured. In this plant type, one pair of chromosomes was marked with a satellite and another pair could be easily distinguished by the sub-median position of the centromere. The remaining five chromosome pairs, all with centro- meres in the median region, could be grouped according to a recurring pattern. Thus the chromosomes were numbered and the mean values of each type were calculated. In Tables 1-4 (columns HB) the results are summarized and it should be noted that the sum of the lengths of the seven chromosome types, as they are presented in the tables, is not 100~o, but about 88~ . This has been achieved by multiplying the original relative values for long and short arms and for total chromosome length (with grand total = 100~o) by a factor 0.88, i.e. the ratio HB/HV as it was calculated from the means of the absolute values of the genome lengths in both pure species (Tab. 5). This recalculation was carried out,


TABLE 1

RELATIVE LENGTH OF LONG ARMS OF CHROMOSOMES OF/-/ . vulgare (HV), 1-1. bulbosum (HB), AND THEIR DIPLOID, TRIPLOID, AND TETRAPLOID HYBRIDS (HY)

The length is expressed as percentage of total length of all complete chromosomes, including satellites. One genome HV = 100K, and in the pure species one genome of

HB = 88 ~ . Figures in brackets refer to number of cells.

Chrom. HV HB HY 2x HY 3x HY 4x No. (35) (26) (28) (26) (12)

HV 1 9.03 8.80 8.98 8.77 2 8.26 8.10 8.31 8.02 3 7.56 7.23 7.61 7.20 4 7.89 7.69 7.56 7.82 5 7.23 7.21 7.05 7.13 6 6.94 7.10 7.08 6.92 7 9.05 8.76 8.74 9.11

HB 1 7.59 7.76 7.19 7.63 2 8.29 8.29 7.75 8.15 3 7.22 7.10 6.87 6.98 4 6.60 6.76 6.22 6.40 5 6.52 6.57 6.12 6.44 6 6.04 6.02 5.77 5.82 7 8.56 8.44 8.07 8.23

TABLE 2

RELATIVE LENGTH OF SHORT ARMS OF CHROMOSOMES OF H. vulgare (HV), H. bulbosum (HB), AND THEIR DIPLOID, TRIPLOID AND TETRAPLOID HYBRIDS (HY)

Conventions as in Table 1. N.B. 6 + and 7 + mean chromosomes 6 and 7 plus respective satellites.


HV 1 7.00 7.01 7.11 6.94 2 6.97 7.04 7.08 7.18 3 6.77 6.83 6.95 6.90 4 6.03 6.14 6.04 6.06 5 5.06 4.99 4.81 4.93 6 4.24 4.19 4.30 4.33 6 + 6.56 6.87 6.63 6.92 7 4.04 4.31 4.14 4.25 7 + 5.60 6.23 6.06 6.10

HB 1 6.59 6.59 6.36 6.49 2 5.66 5.80 5.45 5.75 3 5.70 5.89 5.53 5.71 4 5.38 5.33 4.92 5.22 5 4.38 4.61 4.22 4.35 6 3.23 6 + 5.63 5.36 5.10 5.26 7 3.83 4.29 4.06 4.16


TABLE 3

RELATIVE TOTAL LENGTH OF CHROMOSOMES OF H. vulgare (HV), H. bulbosum (HB), AND THEIR DIPLOID, TRIPLOID, AND TETRAPLOID HYBRIDS (HY)

Conventions as in Tables 1 and 2.


HV 1 16.04 15.81 16.08 15.71 2 15.24 15.15 15.39 15.21 3 14.34 14.05 14.56 14.11 4 13.92 13.82 13.60 13.88 5 12.29 12.20 11.86 12.05 6 11.19 11.29 11.38 11.25 6 + 13.50 13.98 13.71 13.85 7 13.10 13.07 12.88 13.37 7 + 14.65 15.00 14.80 15.21

HB 1 14.19 14.35 13.55 14.13 2 13.95 14.09 13.20 13.91 3 12.92 12.99 12.40 12.68 4 11.99 12.09 11.14 11.62 5 10.89 11.18 10.34 10.80 6 9.27 6 + 11.66 11.39 10.87 11.08 7 12.38 12.73 12.13 12.39

TABLE 4

CHROMOSOME ARM RATIOS (SHORT/LONG ARM) OF H. vulgare (HV), H. bulbosum (HB), AND THEIR DIPLOID, TRIPLOID, AND TETRAPLOID HYBRIDS (HY)

Conventions as in Tables 1 and 2.


HV 1 0.78 0.80 0.80 0.79 2 0.84 0.87 0.85 0.89 3 0.90 0.94 0.91 0.96 4 0.77 0.80 0.80 0.78 5 0.70 0.69 0.68 0.69 6 0.61 0.59 0.61 0.62 6 + 0.95 0.97 0.94 1.00 7 0.45 0.49 0.48 0.47 7 + 0.62 0.71 0.70 0.67

HB 1 0.87 0.85 0.89 0.85 2 0.68 0.70 0.71 0.70 3 0.79 0,83 0.81 0.82 4 0.82 0.79 0.79 0.82 5 0.67 0.70 0.69 0.68 6 0.54 6 + 0.93 0.89 0.89 0.91 7 0.45 0.51 0.51 0.51


T A B L E 5

MEAN ABSOLUTE LENGTHS OF GENOMES OF H. vulgare ( H V ) ANI:

H. bulbosum ( H B ) , IN ARBITRARY UNITS, IN BOTH SPECIES AND 1N

THEIR DIPLOID, TRIPLOID, AND TETRAPLO1D HYBRIDS ( H Y )

V a l u e s w h i c h d o n o t d i f fe r s i g n i f i c a n t l y a r e m a r k e d w i th t h e s a m e le t te r .

Genome length Genotype Number

of cells HV HB

HV 35 50.6 a HB 26 44.6 b HY 2x 28 44.6 b 39.6 c HY 3x 26 53.9 a 45.2 b HY 4x 12 46.5 b, d 40.4 e, a

as it appeared to facilitate the comparison between the chromosomes of H. bulbosum as they showed up in the parent and in the hybrids.

Next the triploid plant type was analysed, by measuring the chromosomes of 26 cells. This plant type had in each cell only one genome of H. vulgare and two genomes of H. bulbosum, which was used as genome marker. The number of satellite chromosomes in the triploid hybrid was two, although four such chromosomes were expected. The two satellite chromosomes were clearly unequal in form and appearance and they resembled very much the satellite chromosomes of H. vulgare. Thus it was concluded that in this hybrid the satellites of the two genomes of H. bulbosum were suppressed. The easy recognition of the chromosomes 6 and 7 of H. vulgare, together with the fact that the three longest chromosomes of the complement appeared in singular and resembled the chromosomes 1, 2, and 3 of H. vulgare, allowed the assumption that in this hybrid the relative values of all chromosomes of H. vulgare, when compared with the parental karyotype, were not changed in relation to each other. This meant that the chromosomes 4 and 5 could be reconstructed, and the actual chromosomes, which equalled the reconstructed values mostly, were given the corresponding numbers. Thus in the triploid hybrid the chromosomes of H. vulgare could be distinguished with fair certainty. The remaining fourteen chromosomes constituted the two genomes of H. bulbosum. In this group of chromosomes the two with the centromere in sub-median position were easy to distinguish, and the group as a whole fitted the pattern which was established in the parental plant type. The conclusion that in the hybrid the relative values of the chromosomes did not change in relation to each other, therefore appeared to be valid for the chromosomes of H. bulbosum as well. The chromosome


type that in the parental plant type had the secondary constriction was such that the length of the satellite simply was added to the length of the short arm, making this arm nearly as long as the long arm. The mean values were calculated (see the columns HY 3x in Tabs. 1-4). For the sake of easy comparison a recalculation was carried out, such that the sum of the seven chromosomes of H. vulgare equalled 100~, without changing the mutual relations within the hybrid.

In the diploid hybrid plant type, which was analysed by measuring the chromosomes of 28 cells, each chromosome type was present as a single chromosome. The morphology of the easily recognizable chromosomes, as well as the pattern of all chromosomes were such that the basic con- clusions that were drawn for the triploid hybrid, applied to the diploid hybrid, too. Thus the satellite of the genome of H. bulbosum was suppressed and the relative values of the chromosomes within each genome, when in turn compared with the parental karyotypes, were not changed in relation to each other (see also Figs. 3-4). The mean values for each chromosome type were calculated, and again a recalculation was carried out, whereby the same principle as in the triploid was applied (see the columns HY 2x in Tabs. 1-4).

Finally 12 cells of the tetraploid hybrid plant type were analysed. The number of cells was low, because of the elimination of chromosomes from many cells, and because complete cells often had too many chromosomes overlapping. The results of measurements in this plant type were much like those in the diploid hybrid plant type, with the difference that all chromosome types were represented in pairs. Therefore an analogous calculation procedure was carried out to obtain the figures as presented in the last columns of Tables 1-4.

In summary, it was concluded that there was a good general agreement between the observed relative values of the morphological characters of the chromosomes in the parental species and the hybrids. The genomes of H. vulgare and H. bulbosum each had in turn no drastic change in their appearance, when combined in diploid, triploid, and tetraploid hybrids. The only exception was the suppression of the satellite of the genomes of H. bulbosum in all hybrid types, revealing a good example of differential amphiplasty.

To study the possible occurrence of neutral amphiplasty there was a need to look into the absolute values, rather than the relative ones. Thus for each cell the absolute genome length was calculated in arbitrary units, of one genome of H. vulgare and one genome of H. bulbosum. The means of these values are presented in Table 5.

An analysis of variance showed that for the pure species as well as for the diploid and triploid hybrids the mean genome length of H. vulgate


was significantly longer than the mean genome length of H. bulbosum. In the tetraploid hybrid, however, this difference in genome length was not statistically significant. In comparing the means of the genome lengths of H. vulgate with each other it appeared that there were no significant differences between the means of the pure species and those of the triploid hybrid, while also the means of the diploid and tetraploid hybrids did not differ significantly. But between those two groups the differences between the means were significant. The same results were obtained in comparing the means of the genome lengths of H. bulbosum with each other. It should be mentioned that the variances in the diploid hybrid were much less than for all other items, which could not be explained.

When for the hybrid plant types the individual values, from which the means were calculated, were plotted against each other, it appeared that the regression lines of the three hybrid types did not differ significantly and therefore were pooled (Fig. 5). In this way a regression line was obtained of which the coefficient of regression was 0.864 + 0.034, which was significantly different from 1.0. The value was in conformity

Genome length HB o

in o diploid hybrid, mean • / o in tr ip loid hybrid, mean • /

60 ~ in tet raplo id hybr id,mean , . ~ o

/

50 o

o0~ o o o

° 0 o o ° u

40 o •°~ o o

o

~" ~o go 6o io 8o Genome length HV

Fig. 5. The relation between the genome lengths of 1t. vulgare (HV) and H. bulbosum (HB) as measured in the diploid, triploid and tetraploid hybrids (in arbitrary units). Coefficient of regression, 0.864 + 0.034.

KARYOTYPES OF HORDEUM HYBRIDS

TABLE 6

MEAN NUMBER OF NUCLEOLI PER CELL IN SOMATIC TISSUE OF VARIOUS

CYTOTYPES OF H. vulgate (HV), H. bulbosum (HB), AND wrtEm HYBRIDS (HY)

The numbers of varieties or clones analysed are in brackets.

227

Ploidy HV HB HY

haploid 1.04 (l) diploid 1.88 (3) 1.08 (1) 1.30 (3) triploid 1.29 (2) tetraploid 3.83 (3) 2.01 (3) 2.02 (3)

8 0 - H V x

60-

4o 1 2 0 -

0 - 2

BO - 2x

6 0 -

4 0 -

2 0 -

HV 2x HV 4x

I 2 3 4 1 2 3 4 5 6 7 8

I

HB 4x HY 3x HY 4x

-7

\

HY 2 x

7

) O - 1 2 1 2 3 4 2 "~ 2 3 2 3 4

Fig. 6. Frequency distr ibut ion o f cells with different numbers o f nucleoli in root tips o£ various cytotypes o f H. vulgare (HV) , H, bulbosum (HB) and their hybrids (HY) .


with the genome length ratio HB/HV = 0.88, as it was calculated from the means of the absolute values in both pure species.

THE NUCLEOLI

In Table 6 a summary of the analyses concerning the number of nucleoli in somatic tissue is presented, and Figure 6 shows the frequency distribution and the observed maximum number per cell. This maximum number nearly always equalled the number of satellite chromosomes. The only exception was found in the triploid hybrids where a few cells had three nucleoli, the frequency was, however, only 0.25~. It was therefore concluded that the differential amphiplasty showed up not only as the suppression of satellite formation, but also as suppression of the formation of certain nucleoli.

In the parental species the doubling of chromosome number resulted more or less in doubling of the mean number of nucleoli per cell, and there was good agreement between the types with equal numbers of satellite chromosomes (viz. haploid H. vulgare with diploid H. bulbosum and diploid H. vulgate with tetraploid H. bulbosum). In the hybrids the picture was slightly different. The tetraploid hybrid showed good agreement with the parental types with four satellite chromosomes, but in the diploid and triploid hybrids the average number of nucleoli was considerable larger than that in the parental types with two satellite chromosomes.

Discussion

From Tables 1-5 it is clear that there exists a significant difference between the lengths of a genome of H. vulgare and of a genome of H. bulbosum, which difference also becomes manifest in the fact that the coefficient of regression of the line in Figure 5 differs significantly from 1.0. This observation is striking because of the fact that H. vulgare and H. bulbosum have the same amount of DNA per cell (BENNETT & SMITH, 1971). Because the widths of the chromosomes were not measured, it is not possible, however, to discuss this point in more detail.

Except for this difference in genome length and for the clear manifestation of differential amphiplasty in the hybrids, the morphology of the chromosomes in H. vulgate and H. bulbosum had considerable similari- ty. Also KASHA t~ SADASIVAIAH (1971) observed such similarities, which made them suggest that, if such similarities could be taken as a measure of homology, the relationship between the species might be considered


rather close. Such a statement, however, needs to be verified with other investigations, which are outside the scope of this study.

In an attempt to relate the observations of this study with the phenomenon of neutral amphiplasty Figure 5 may be interpreted as follows: the absence of significant differences between the regression lines of the three hybrid types, which led to the pooling of the data of all hybrid types into one regression line, suggests that in the diploid, triploid, and tetraploid hybrid types chromosomal contraction proceeds similarly. However, the same data suggest that on an average the chromosomes in the diploid and tetraploid hybrid types were more contracted than the chromosomes in the triploid hybrids and the pure species (Table 5). LANGRIDGE et al. (1970) related changes in chromosome length in interspecific hybrids in Crepis, the neutral amphiplasty, to the regulation of the cell cycle. But in Hordeum interspecific hybrids the effect is different from the one described for Crepis hybrids (l.c.) and for Triticum-Aegilops amphidiploids (LI & Tu, 1947). In Hordeum the stronger chromosomal contraction seems to occur in only two out of three hybrid types, and the chromosomes of the two species reacted similarly.

The question arises whether these phenomena have any relation to several disturbances which were observed in the hybrids (LANGE, 1969, 1971a, b; KASHA et al. 1970; KASHA 8£ SADASIVAIAH, 1971). The most remarkable of them was a chromosomal instability, which occurred rarely in the triploid hybrids, but was pronounced in the other hybrid types, giving rise to chromosomal elimination (see also SVMKO, 1969; KAO & KASHA, 1970; KASHA & KAO, 1970). In the endosperm also nuclear abnormalities occurred, like the formation of giant nuclei or multinucleate cells. Finally meiosis was irregular, especially in diploid and tetraploid hybrid types.

LI & Tu (1947) and LANGRIDGE et al. (1970) speculated that a loss of chromosomes could be a manifestation of the differences in chromosomal contraction. The latter authors supposed the contraction to be an essential preparation for the separation of chromatids, so that an early anaphase could exclude "lagging chromosomes" from both daughter nuclei. This explanation, however, does not fit the elimination of chromosomes in hybrid tissues between H. vulgate and H. bulbosum, because in those genome combinations where the contraction was strongest, the elimination also was most pronounced. Furthermore the behaviour of the chromosomes in metaphase and anaphase cells, in which irregularities were observed, was such that the separation of chromatids did not seem to be disturbed much more than did the orientation in the metaphase plate and the synchronization of the anaphase movement.

All this evidence leads to the conclusion that neutral amphiplasty, as


described by LANGRIDGE et al. (1970), does not occur in the hybrids between H. vulgate and H. bulbosum. The observed changes in chromosome length in some hybrid types might be one more manifestation of the disharmony between the genomes of the two species. It is unclear whether there exists a causal relation between the changed chromosomal contraction and the other disturbances.

TSUCHIYA (1960, 1964) concluded from his studies with trisomics, that the nucleoli in H. vulgate were exclusively attached to the satellite chromosomes. The nucleolar activity was largest in chromosome 6, somewhat lower in chromosome 7, and minute in the other chromosomes. In the hybrids between H. vulgate and H. bulbosum the two satellite chromosomes of H. vulgare showed up in all hybrid types, but the secondary constriction in the satellite chromosome of H. bulbosum (No. 6) was invisible. The maximum number of nucleoli per cell fitted the number of satellite chromosomes, thus exhibiting a good example of differential amphiplasty. A minor exception was observed in a few cells in the triploid hybrids, which had three nucleoli instead of two.

WALLACE • LANGRIDGE (1970) concluded that the disappearance of the secondary constriction was the result of the suppression of nucleolus formation, which might be brought about by genic repression between the genomes. According to this hypothesis the interaction between the genomes of H. vulgare and H. bulbosum leads to suppression in hybrid tissues of the nucleolus formation in the genome(s) of H. bulbosum, which in turn results in the disappearance of the secondary constriction. To explain the minor exception, which was observed in the triploid hybrids, one might expect that in these hybrids, cells with three satellite chromosomes would occur rarely, which in fact was the case. In studying the chromosomal instability in some triploid hybrids (LANGE, unpubl.) the chromosome number in somatic tissue was found to vary between 19 and 23, and indeed cells that were disomic for chromosome 6 or chromosome 7 of H. vulgate were found. In contrast to this the chromosomal instability in diploid and tetraploid hybrids was nearly completely restricted to elimination, and accordingly no deviations above the maximum number of nucleoli were observed in these hybrid types.

The distribution pattern of the nucleoli was supposed to be a reflection of their fusion. The question arises why this pattern, and with it the average number of nucleoli per cell, in the diploid and triploid hybrids deviated from the pattern in haploid H. vulgate, because the satellite chromosomes were supposed to be the same. DARVEY & DRISCOLL

(1972a) concluded that in wheat enforced proximity of nucleolus organi- zing regions resulted in more frequent fusion of nucleoli. The same authors (1972b) also stated that the location of nucleolar organizers in


somatic cells was such that fusion between "homologous" nucleoli did not occur more often than fusion between "non-homologous" nucleoli. This second statement seemed to be confirmed in this study, as far as the parental plant types were concerned. The pattern of fusion in haploid H. vulgare (two non-homologous nucleoli) was not different from the one in diploid H. bulbosum (two homologous nucleoli), and the same situation was more or less apparent in diploid H. vulgare versus tetraploid H. bulbosum, where the four homologous nucleoli even showed slightly less fusion than the two pairs of non-homologous nucleoli. However this situation is confused by the fact that the nuclear volumes may have been different, resulting in differences in nucleolar proximity. Therefore the nucleolar behaviour in diploid and triploid hybrids cannot easily be explained by assuming that the distance between the nucleolar organizers in the genome of H. vulgare is enlarged by the presence of one or two genomes of H. bulbosum. It must be concluded that another, unknown interaction between the genomes is causing the deviations in the pattern of fusion.

Finally the possibility of a relation between the differential amphiplasty and the chromosome elimination has to be considered, especially with respect to why the chromosomes of H. bulbosum are more often elimi- nated. To this the study only allows the supposition that the suppression of the nucleolar activity in the genome of H. bulbosum enlarged its "susceptibility" to the unknown factor causing the elimination of chromosomes in the hybrid tissues.

Many thanks are due to Mr. J. Post for carrying out the statistical analyses, to Mrs. W. VAN DE HAM-VAN ELDEr~ and Miss H. HAASE for their technical assistance and to Dr. R. A. FINCH (Cambridge, U.K.) and Dr. J. SYBENOA (Wageningen, Netherlands) for correcting the manuscript and giving very valuable advice.

References

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Karyotypes, nucleoli, and amphiplasty in hybrids between Hordeum vulgare L. and H. bulbosum L