The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Euphytica 25 (1976) 621 631

T H E P R O D U C T I O N A N D B E H A V I O U R O F H E X A P L O I D H Y B R I D S B E T W E E N H O R D E U M

V U L G A R E A N D H. B U L B O S U M

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

Institute de Haaff. Foundation for Agricultural Plant Breeding, Wageningen, the Netherlands

Received 25 November 1975

INDEX WORDS

Hordeum vulgare, Hordeum bulbosum, barley, bulbous barley grass, interspecific hybrids, colchicine treatments

SUMMARY

To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments gave rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.

The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.

INTRODUCTION

In a series of papers the present authors reported extensive studies on hybridization between Hordeum vulgare - cultivated barley and H. bulbosum bulbous barley grass (LANGE, 1968, 1969, 1971 a & b; LANGE & JOCr~MSEN, 1976a, 1976b). It was confirmed that there is a strong crossing barrier between the two species (see also reviews by SMITH, 1951 ; NILAN, 1964; RAJHATHY et al., 1964; PRICE, 1968; KASHA, 1974; SZIGAT,

1974). This barrier prevented a direct and easy use of H. bulbosum germ plasm in the breeding of barley, and only cytoplasmic effects of H. bulbosum, which had no agro- nomic value, could be transmitted into H. vulgare. The crossing barrier consisted, of seed abortion, chromosome elimination, and very low fertility in hybrids, and per- sisted into the second hybrid generation.

The chromosome elimination in hybrid tissues, by which the chromosomes of H. bulbosum were eliminated more frequently than those of H. vulgare, seemed to play a central role. The origin of this phenomenon, which simultaneously was described also by SYMKO (1969), KASHA & KAO (1970) and KAO & KASHA (1970), is not yet fully understood. LANGE (1969, 1971b) suggested that it might have someting to do with the differential amphiplasty that occurred between the two species (LANGE

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

& JOCHEMSEN, 1976a), and studies of BENNETT et al. (1976) suggest that the dramatic effect of mitotic instability may be brought about by a discrepancy between cell cycle duration and control of protein production. The somatic chromosome elimination also allowed haploid plant production, and as such was the subject of several research studies (see review by KASHA, 1974; and BARCLAY, 1975; KASHA, 1976; JENSEN, 1976; FUKUYAMA & TAKAHASHI, 1976).

The triploid interspecific hybrids, with one genome of H. vulgare and two genomes of H, bulbosum showed a relatively good chromosomal stability (LANGE, 1969, 1971a, b; KASHA & SADASIVAIAH, 1971; SUBRAHMANYAM & KASHA, 1973). This led LANGE (1969) to suggest that doubling of the chromosome number of these hybrids may give rise to plants with good chromosomal stability, and, because of the am- phidiploid nature, with some degree of fertility. Therefore attempts were made to produce such polyploid hybrids by means of two methods: (1) from crosses between colchicine induced tetraploid H. vulgare and hexaploid or octoploid H. bulbosum, and (2) from colchicine treatment of triploid hybrids. This paper reports the results of such studies.

M A T E R I A L A N D M E T H O D S

The plant material consisted of eight diploid barley cultivars, eight lines of auto- tetraploid barley (kindly supplied by Dr H. Gaul, BRD and Ing. G. J. Speckmann, the Netherlands), seven plants of hexaploid H. bulbosum, fifteen plants of octoploid H. bulbosum and 44 clones of triploid interspecific hybrids between diploid H. vulgare and tetraploid H. bulbosum. The species and hybrids are denoted by genome symbols : VV = H. vulgate (2x), BBBB = H. bulbosum (4x), VBB = triploid hybrid from VV × BBBB, BBV triploid hybrid from reciprocal cross, etc.

The polyploid H. bulbosum material was obtained by treating germinated seed of natural tetraploid H. bulbosum for six hours with 0.05 ~ or 0.10 ~o colchicine solution. The plants showing visible reaction to the treatment (retarded or stunted growth) were grown in the field to give selfed and open pollinated seed. A sample of this seed was checked for chromosome number. Most of it was tetraploid, but of the selfed seed some octoploid plants, and of the open pollinated seed both octoploid and hexaploid plants were selected.

The origin of the 44 triploid interspecific hybrids was described previously (LANGE, 1969, 1971a). One plant had the genome formula BBV and the others were VBB. However, in this study both plant types behaved similarly, so the results have been pooled.

Chromosome numbers and morphology were studied in root-tip squashes, using Feulgen staining after pretreatment with 8-hydroxy-quinoline (TJIO & LEVAN, 1950) and fixation in 1 : 3 acetic alcohol. The study of chromosome morphology was restricted to easily recognizable chromosomes; see description of karyotypes of H. vulgare, H. bulbosum and their hybrids by LANGE (1969) and LANGE & JOCHEMSEN (1976a). Root-tip squashes also were used to determine the maximum number of nucleoli in somatic cells. The cavities in which nucleoli were situated appeared as distinct unstained globular structures in interphase nuclei. The number of satellite chromosomes and the maximum number of nucleoli per nucleus are mentioned in

622 Euphytica 25 (1976)

I N T E R S P E C I F I C H Y B R I D S OF H O R D E U M

Fig. 1 . Colchicine treatment of VBB plantlet with the capping method.

brackets after the chromosome number. Chromosome numbers also were checked in growing-point squashes, using a

slightly changed version of a method described by BENNETT (1964). Colchicine treatment of triploid hybrids was carried out on plantlets with one stem

only. In Experiment 1 a slightly changed version of the capping method of BELL (1950) was used (Fig. 1). In the Experiments 2, 3 and 4 a method was used of which the priciple was described by POPE & LOVE (1952). The plantlets were dug up from the soil and after their crowns had been cut back to about 3 cm length, they were inverted in the colchicine solution. The treatment of only the crowns of the plantlets restricted damaging of the roots by the colchicine. Penetration of the colchicine between the leaf-sheaths was promoted by putting the system for some seconds under reduced air pressure. During the treatment the system was aerated with a stream of air bubbles (AALDERS & HALL, 1963) and after the treatment the plants were thoroughly rinsed in water.

Experiment 3 differed from Experiment 2 by an extra pretreatment to induce ac- cumulation of mitotic divisions. First the activity of the plantlets was decreased by exposing them for two months to 2"C, whereafter, prior to the colchicine treatment. the plantlets were placed at 20°C for 15 h. In Experiment 4 the cold treatment was extended to four months, which weakened the plantlets considerably. In addition, all newly grown shoots were removed, so that only colchicine-treated shoots were allowed to develop. This treatment killed many of the plantlets, however.

RESULTS

W. LANGE AND G. JOCHEMSEN

CFosses In Table 1 a summary of the crosses is presented. When H. vulgare was the female parent the seedset was low, and the hexaploid male parent gave better results than the octoploid. In the reciprocal crosses the octoploid female parent did not produce a single seed, and the seedset on the hexaploid H. bulbosum was very low. In general the seeds were small and shrivelled, or sometimes empty, and they turned yellow within two to three weeks after pollination. In those seeds where the embryo was relatively well developed, it was taken into embryo culture when the yellowing of the seed started (for methods see LANGE, 1969, 1971a). Only in the cross VVVV × BBBBBBBB was over half of the seeds allowed to ripen on the plants. Neither the embryo culture nor the ripe seeds yielded viable plants. The seeds did not germinate, and the few embryos which germinated on the agar and started development died before reaching the stage in which they could be transplanted into soil.

Colchicine treatment of triploid hybrids Table 2 summarizes the experiments that were carried out to try to double the chromosome number of the triploid interspecific hybrids. In all experiments many plantlets reacted clearly to the colchicine treatment, by showing no growth or stunted growth after the treatment. When the treated shoot was killed, new shoots some-

Table 1. Crosses between diploid or tetraploid H. vulgare and hexaploid or octoploid H. bulbosum.

Cross combination Crosses Seeds Embryo Plants (spikes) cultures

VV × BBBBBB 10 72 54 0 VV × BBBBBBBB 4 0 0 0

VVVV ~ BBBBBB 13 32 23 0 VVVV × BBBBBBBB 64 73 15 0

BBBBBB × VV 8 4 1 0 BBBBBBBB x VV 10 0 0 0

BBBBBB x VVVV 15 3 3 0 BBBBBBBB x VVVV 61 0 0 0

Table 2. Colchicine treatments of triploid VBB- and BBV-hybrids.

Experiment Colchicine Treatment Number of Total Plant No concentration duration clones number of survival

(%) (h) VBB BBV plantlcts ( ~ )

1 0.20 72 43 1 498 31 2 0'10 8 19 1 1055 20 3 0.10 8 13 1 629 62 4a 0.10 6 1 0 340 29 4b 0.05 6 1 0 285 34

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Table 3. Crosses with colchicine treated triploid hybrids (VBB col., pooled data of Experiments 2, 3 and 4) and seedset on such hybrids after open flowering (pooled data of VBB col. and BBV col.).

Crosses and open Spikes Seeds Plants flowering

VBB col. × VV 71 0 VBB col. × VVVV 16 0

VV × VBB col. 9 0 VVVV x VBB col. 8 0

Open flowering Experiment 1 315 0 - Experiment 2 approx. 2300 19 6 Experiment 3 approx. 12000 150 78 Experiment 4 approx. 4000 22 5

times arose, which, however, mostly showed little or no effect of the treatment. The results of the treatments were checked : 1) by counting chromosome numbers

in randomly sampled root-tips and growing-points in Experiments 1 and 2, and in a few plants of Experiments 3 and 4 that showed some fertility; 2) by testing the fertility through crossing and checking of seedset after open flowering, which should be combined with studying the possible offspring. In Experiment 1 a sample of 190 root-tip squashes yielded no indication of chromosome doubling. In over four hundred growing-point squashes, which were made of nearly two hundred plantlets of Experiment 2, cells with 42 chromosomes were observed in the growing-points of two plantlets. In a later counting this chromosome number could be confirmed in only one of these plants, and in a third counting, after a few months, the number appeared to have decreased to about 28. The same colchicine treatment also yielded seventeen plantlets of which the growing-points had unstable chromosome numbers, ranging between 14 and 21 chromosomes. In five fertile plants of Experiment 3 the chromosome number was 21 (2/2), one such plant had 21 23 (2/2) chromosomes. The two fertile plants of Experiment 4 had unstable chromosome numbers, one of them had 3 3 4 2 (4/4) chromosomes (mostly 40) in the first counting, and two years later the number was 42; the other plant had 42 (4/4) chromosomes at first and 21 (2/2) two years later.

The results of the crosses with treated plants and of checking seedset after open flowering are summarized in Table 3. In Experiment 1 only the seedset after open flowering was tested in the spikes that were formed in the first yerar after the treatment. This yielded no seed, which was in agreement with the negative result of the cytological test.

Experiments 2, 3 and 4 gave similar results, and therefore will be reported together. The reciprocal crosses with diploid and tetraploid H. vulgare gave no seed. After open flowering some seed was formed. The average seedset was 8.4 seeds per 1000 spikes and 42 ~ of the seeds gave rise to viable plants. Both these figures are higher than those from the untreated triploid hybrids, where the seedset was 2.7 seeds per 1000 spikes and only 1 0 ~ of the seeds developed into viable plants (cf. LANGE &

Euphytica 25 (1976) 625


JOCHEMSEN, 1976b). The two types of triploid hybrids were grown in the same locality. It was remarkable that all seeds were formed in the first four years after the treatment in which years about half of the number of spikes mentioned in Table 3 was formed.

Offspring of colchicine treated triploid hybrids The plants could be grouped into three types (Table 4).

The first type consisted of eleven plants, which were annual and morphologically resembled H. vulgate. One of them was haploid and sterile. Nine plants were diploid and of these six were fertile and the others died before flowering Neither the plants themselves, nor the progeny of the fertile ones showed any character of H. bulbosum, except one of them which had some hairs on the older leaves. The spike and progeny of this exceptional plant, however, fully resembled H, vulgare. Finally one plant had an unstable chromosome number, which changed from 22-25 (7/6) to 19 21 (4-6/4-6). This plant was sterile, showed pubescence on both sides of the leaves, and formed spikes that predominantly resembled H. vulgare (Fig. 2).

The second plant type consisted of 46 plants, which morphologically resembled H. bulbosum, were perennial, tetraploid or about tetraploid, and fertile. The plants showed no characters of H. vulgare.

The remaining 32 plants were grouped as a third type, which was characterized by a perennial habit, chromosomal instability, and a morphology like the original interspecific hybrids. In comparison to these hybrids, however, the plant vitality often was inferior, and several plants h~td dwarfing growth habit. The chromosome number and the number of satellite chromosomes per cell showed in general a ten- dency to decrease with time, and in many of the plants micronuclei occurred in the somatic cells. The number of satellites generally equalled the maximum number of nucleoli per cell in the surrounding tissue. It never exceeded four and was never lower

Table 4. Offspring after open flowering ofcolchicine treated triploid hybrids (VBB col., pooled data of Experiments 2, 3 and 4).

Number Chromosome Range of Number of Supposed of plants number chromosome satellites or genomes

number within nucleoli a given plant

1 7 0 2 V 9 14 0 4 VV 1 19-25 6 4-6(-7) VVV ± ?

38 28 0 4(-5) BBBB 4 27 29 1 2 4 BBBBi? 3 25 28 2-3 (3-)4 BBBB - ? 1 25 32 7 4(5) BBBB ~ ?

4 28 34 (Y4 2 ~ unknown 10 24-28 0~3 2-3 unknown 12 19-28(-30) 4-8 2 ~ unknown

5 19 23(24) 0~3(-4) 2 ( 3 ) unknown 1 14-24 10 2(-3) unknown

626 Euphytica 25 (1976)

Fig. 2. Spikes of plants, resembling H. vulgare, of the offspring of colchicine treated triploid VBB- hybrids. A. haploid ( × 1.1). B. diploid ( × 1.1). C. diploid, which had some hairs on older leaves ( × 0.8). D. and E. about triploid ; note leaf pubescence (D. × 0.9; E. x 1.1).

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

than two. Cells with approximately 28 chromosomes had three or four satellite chromosomes, and cells with approximately 21 chromosomes seldom had more than two. Although in Table 4 a subdivision of the plants of this type into five groups is presented, based on number of chromosomes and degree of chromosomal instability, it is doubtful whether such division has a biological meaning or is artificial. It may be that the plants of this type show different stages in a process of gradual somatic chromosome elimination. The plants were highly sterile: after open flowering three of them produced a few seeds. F rom these five plants were grown, which did not differ much from the parental plant type.

D I S C U S S I O N

The results of these studies allow two obvious conclusions. First it has not been possible to obtain hexaploid VVBBBB- or BBBBVV-hybrids, having both reasonable fertility and chromosomal stability. Second these studies have not led to the trans- mission of characteristics of H, bulbosum into barley breeding stocks. This means that the strong crossing barrier, which exists between the two species, was not broken down by working at higher ploidy level.

Furthermore, the studies provided several questions, which could not be fully answered, and which will be discussed below. Why did the crosses between diploid or tetraploid H. vulgare and tetraploid or octoploid H. bulbosum gave such poor results? The seed abortion was probably caused by the same sort of chromosomal disturbances, that occurred in endosperms and embryos of other cross combinations between the two species (for references see Introduction). The complete failure of embryo culture, however, remains unexplained, but could be indicative of a greater chromosomal instability than in the other combinations, leading to weaker embryos. The poorer results of the crosses in which H. bulbosum was the female parent, as compared with the reciprocal crosses, is not understood either. A similar effect, however, occurred at the other ploidy levels.

The results obtained after the colchine treatment of triploid hybrids are difficult to interpret, because only open flowering gave rise to viable plants. The treatment caused an increase in the fertility of the hybrids, although the level of fertility remained very low. Was this increase of fertility connected with the occurrence of hexaploid or near-hexaploid sectors? One plant of Experiment 4, which was selected because it showed remarkable fertility, had a chromosome number of 33 42. It ~'ielded a progeny of five plants (out of 17 seeds). Three of these plants were of the hybrid type, two with over 28 and one with about 21 chromosomes, and the remaining two plants were of the H. vulgare type, with diploid chromosome number. The other plant of Experiment 4, which had 42 chromosomes, yielded only an inviable seed, and the six fertile plants of Experiment 3, in which triploid or near-triploid chromosome numbers were counted, gave either inviable seeds or seeds which developed into plants like H. bulbosum. Therefore the direct evidence for a possible connection between the increased fertility and the occurrence of hexaploid chromosome numbers seems small, although it cannot be excluded that the fertile triploids were hexaploid or unstable in the haulms and triploid in the bulbs and roots. Some in- direct evidence for the connection between increased fertility and hexaploidy may

628 Euphytica 25 (1976)


come from the following observations: l) in Experiment l no hexaploidy was observed and also no increased fertility; 2) in all cases where hexaploid chromosome numbers were recorded this number appeared to be unstable and decreasing, and correspondingly the better fertility lasted for about four years after colchicine treatment and then decreased to the very low level of untreated material. This may suggest that in about four years all hexaploid sectors that originated from the colchicine treatment had lost their hexaploid nature, resulting either in triploid tissue, which seems unlikely, or in a weak tissue with unstable chromosome number, which then was overgrown by the triploid sector of the plant. In relation to this it seemed that the small bulb at the base of the stem formed a reservoir of triploid tissue.

The next question concerns the origin of cells in the colchicine treated triploids with chromosome numbers ranging from 14 to 21. Unfortunately it was not possible to establish the specific nature of the chromosomes, It can therefore only be spec- ulated that these cells were either the ultimate result of chromosome elimination starting at the hexaploid level, or that the colchicine induced an extra instability in the triploid tissue and consequently a reduction of chromosome number.

The offspring of the colchicine-treated triploid hybrids create a series of questions. Because of the open flowering the male parents are unknown. On the female side the increased fertility may be related to hexaploidy. Furthermore it is unknown whether the reduction in chromosome numbers in the offspring resulted from chromosome elimination before or after fertilization or both. In comparison to the untreated hybrids (cf. LANGE • JOCHEMSEN, 1976b) the composition of the offspring is slightly different. One of the untreated plants produced offspring that fully resembled H. vulgare through its male gametes, whereas in the treated material such offspring originated through the female gametes. The hybrid-like plants in the offsprings of treated and untreated triploid hybrids did not differ much from each other, although in the treated material the range of chromosome numbers was extended above 28. Finally a new element in the treated material is the occurrence of fully H. bulbosum types. The explanation of the origin of the various types of offspring is hindered by the occurrence of so many variables, viz" the successfullnes of the colchicine treatment, the degree and specificity of somatic chromosome elimination before meiosis, the occurrence of achiasmate segregation of chromosomes of the two species during meiosis, the occurrence of fertilization, and if so the composition of the male gamete, and finally the occurrence of somatic elimination after fertilization. Thus several hypotheses could be formulated, and not enough evidence is available to presume what could be the right one. In the case of the fully H. bulbosum types, and maybe also in the cases of haploid and diploid H. vulgare formation, part of the hypothesis should concern the loss of H. vulgare chromosomes. This could be through somatic chromosome elimination before meiosis or after fertilization, through achiasmate segregation of parental chromosomes during meiosis, or even through a combination of these events. LANGE (1969~ 1971b) reported a case of somatic chromosome elimination in a tetraploid hybrid between H. vulgare and H. bulbosum, in which chromosomes of both species were eliminated. It might well be that also the hexaploid tissue between the two species represents a genetical environment from which the H. vulgare chromosomes are more easily eliminated than in other situations. Unfortu- nately the evidence is insufficient to exclude other explanations.

Euphytica 25 (1976) 629


ACKNOWLEDGMENTS

Thanks are due to Dr R. A. Finch (Cambridge, England) for critically reading the manuscript.

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Documents

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum