Use of the gene pools of Triticum turgidum ssp. dicoccoides and Aegilops squarrosa for the breeding of common wheat (T. aestivum), through chromosome-doubled hybrids

Euphytica 59: 197-212, 1992. (~) 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Use of the gene pools of Triticum turgidum ssp. dicoccoides and Aegilops squarrosa for the breeding of common wheat (T. aestivum), through chromosome-doubled hybrids I. T w o strategies f o r the p roduc t i on o f the amph ip lo ids

W. Lange & G. Jochemsen DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, NL-6700 AA Wageningen, The Netherlands

Received 5 December 1991; accepted 21 February 1992

Key words: Triticum turgidum ssp. dicoccoides, wild emmer wheat, Aegilops squarrosa, interspecific hybridisation, synthetic hexaploid wheat, chromosome doubling, embryo rescue, meiosis

Summary

Triticurn turgidum ssp. dicoccoides (wild emmer wheat, AABB, 2n = 28) and Aegilops squarrosa (goat grass, DD, 2n = 14) comprise a rich reservoir of valuable genetic material, which could be useful for the breeding of common wheat (T aestivum, AABBDD, 2n = 42). Many accessions of both wild species, most of them selected for resistance to stripe rust, were used to make amphiploids. Two strategies were applied: (1) the production of autopolyploid cytotypes of the wild species, followed by hybridisation, and (2) the production of allotriploid interspecific hybrids, followed by doubling of the number of chromosomes. The first route was unsuccessful because of failure of the crosses between the autopolyploid cytotypes, possibly due to incongruity between the two species and to reduced fertility in the autopolyploid cytotypes. The second route yielded the desired synthetic hexaploids. However, the rate of success of the crosses was low and there were great differences between years, and within years between crosses. Embryo rescue was applied to obtain the primary hybrids (2n = 21), which were highly sterile and had on average 0.3 bivalents and 20.4 univalents per pollen mother cell. Various abnormalities were recorded. Doubling of the number of chromosomes sometimes occurred spontaneously or was brought about by colchicine treatment. The large scale of the interspecific hybridisation programme ensured that one-third of the female and one-sixth of the male accessions were represented in the synthetic hexaploids.

Introduction

According to Miller (1987) the evolution of common wheat (Triticum aestivum (L.) Thell.; AABBDD) consisted of three steps: (1) the formation of T. turgidum (L.) Thell. ssp. dicoccoides (Kfrn.) Thell. (AABB, wild emmer wheat) from hybridisation between T. urartu Thum. (AA) and an un- known diploid, similar to the present members of

the section Sitopsis of the genus Aegilops; (2) the domestication of tetraploid wheat (T. turgidum ssp. dicoccum (Schrank) Thell.; AABB) from wild emmer; and (3) the hybridisation between the cultivated tetraploid and the wild diploid Ae. squarrosa L. (DD). Thus T. turgidum ssp. dicoccoides and Ae. squarrosa can be considered as the nearest wild progenitors of common wheat. (Nomenclature according to Kerby & Kuspira, 1987).

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In a review on the use of the wild progenitors in the breeding of common wheat and barley, Lange & Balkema-Boomstra (1988) stated that the gene pools of the wild progenitors of wheat comprise a rich reservoir of valuable genetic material (see also Nevo, 1988; Jaradat et al., 1988). T. turgidum ssp. dicoccoides is a valuable source of resistance to stripe rust (Puccinia striiformis Westend.) and powdery mildew (Erysiphe graminis DC. ex Merat f. sp. tritici E. Marchal), has a high protein content of the grain, and shows extensive variation for high molecular weight glutenins and isozymes (see references in Lange & Balkema-Boomstra, 1988; as well as: Van Silfhout & Gerechter-Amitai, 1988a, b; Van Silfhout et al., 1989a, b; Gerechter-Amitai et al., 1989a, b; Reader & Miller, 1991; Kolster et al., 1988; Levy et al., 1988a, b; Levy & Feldman, 1989b, c; Nevo & Beiles, 1989). T. turgidum ssp. dicoccoides also shows much variation for morphological traits (Poyarkova, 1988; Levy & Feldman, 1989a), and contains resistance to leaf blotch, glume blotch, and perhaps leaf rust and head blight, as well as tolerance to wheat streak mosaic virus and drought (see references in Lange & Bal- kema-Boomstra, 1988; as well as Gill et al., 1983; Blum et al., 1983; Groenewegen & Van Silfhout, 1988; Raupp et al., 1988).

The goat grass, Ae. squarrosa, is reported to carry genes for resistance to leaf rust, stripe rust, powdery mildew, leaf blotch, glume blotch, eye- spot, Karnal bunt, Hessian fly, greenbug and curl mite (Kerber & Dyck, 1969, 1978; Martens et al., 1984; Valkoun et al., 1985; Gill et al., 1986; Trottet & Dosba, 1983; Multani et al., 1988; Hatchett & Gill, 1983; Raupp et al., 1988; Hammer, 1985; Whelan & Thomas, 1989). In addition there are reports about physiological traits, such as a high photosynthetic rate (Johnson et al., 1987), resistance to low temperature (Limin & Fowler, 1981; Le et al., 1986), tolerance to salt (Shah et al., 1987; Gorham, 1990a, b), and nitrogen use (Henson et al., 1986). Ae. squarrosa was found to be similar in protein content to cultivated wheats (Waines et al., 1987), whereas it exhibited a wide variation in the composition of glutenins and gliadins (Lagudah & Halloran, 1988).

Various strategies have been successfully ap-

plied to transfer desirable traits from either T. turgidum ssp. dicoccoides or Ae. squarrosa to common wheat (for references see Lange & Balkema- Boomstra, 1988). Several authors also reported the production of synthetic hexaploids, using both wild species (McFadden & Sears, 1946, 1947; Kihara & Lilienfeld, 1949; Tanaka, 1961; Bahrman & Theil- lement, 1987; Krolow, pers. comm.). However, in all studies the number of hybrids, and thus the number of accessions of the wild species involved, was very low, and not much has been reported about the use of these hybrids in breeding programmes. Yet such amphiploids, in which the com- plete genomes of both wild progenitors are transferred to the hexaploid chromosome level, might be an intermediate step in the transfer of not only individual genes, but also of more complex genetical systems from the progenitors to common wheat. Furthermore, the amphiploids can be used in repeated backcrossing programmes, and the interaction between the genomes A, B and D can be studied in terms of expression of genes (Bahrman & Thiellement, 1987; Ch~vre et al., 1989).

This programme involved the use of many accessions of both T. turgidum ssp. dicoccoides and Ae. squarrosa, and aimed at the production of synthetic hexaploids in which as many of these accessions as possible would be represented. This paper reports on the application of two strategies to produce the amphiploids. A second paper (Lange & Jochem- sen, 1992) will describe the hybrids obtained, as well as the meiotic chromosome behaviour of the hybrids and of the F1 from crosses with common wheat. The results of the transfer of genes for resistance to stripe rust will be published elsewhere (Kema et al., in preparation).

Materials and methods

The plant material of T. turgidum ssp. dicoccoides consisted of 32 accessions, progenies of plants collected in Israel by Dr. Z.K. Gerechter-Amitai (The Volcani Centre, Bet Dagan, Israel). Accessions G7 and G25 were obtained through Dr. A.C. Zeven (Agricultural University, Wageningen, the Netherlands). The other accessions were provided

through a Netherlands-Israeli co-operative research programme, in which Dr. C.H. Van Silfhout (DLO-Research Institute for Plant Protection, IPO-DLO, Wageningen, the Netherlands), Dr. Z.K. Gerechter-Amitai, and their co-workers studied a large collection of T. turgidum ssp. dicoccoides for resistance to stripe rust, and carried out a severe selection for this resistance, using many races of the pathogen (Van Silfhout et al., 1989a, b; Gerechter-Amitai et al., 1989a). Therefore all accessions used in the present programme were resistant to stripe rust.

The 47 accessions of Ae. squarrosa were chosen to comprise a wide range of variation. Accession Ren-33 was obtained from Dr. M. Trottet (INRA, Rennes, France; see also Trottet & Dosba, 1983), the accessions with descriptor Cam were provided by Mr. T.E. Miller (IPSR, Cambridge Laboratory, Norwich, UK), and the accessions with the descrip- tors IVP and Gat were obtained from Dr. A.C. Zeven and Dr. K. Hammer (Gene bank, Gaters- leben, Germany) respectively. The accessions IVP-M and Gat-M were in fact a mixture of several of the other accessions carrying the same descriptor. All accessions of Ae. squarrosa were screened for resistance to eight races of stripe rust by Dr. C.H. Van Silfhout and Mr. G.H.J. Kema (IPO- DLO, Wageningen, the Netherlands). Eighteen accessions were highly susceptible, and the others were either fully resistant to some of the races or showed segregation (Kema et al., in preparation).

Plants were grown in an air-conditioned greenhouse. Crosses were made applying standard methods for emasculation and pollination.

Embryo rescue was carried out using young seeds, three to four weeks after pollination. Seeds were surface-sterilised for a few seconds in 70% ethanol and subsequently for 10 min in sodium hy- pochlorite (1% active chlorine), and were rinsed three times in sterile water. Embryos were isolated and placed on a solid medium in such a position that the scutellum of the embryos was in contact with the surface of the medium. The medium contained nutrients and vitamins according to Mu- rashige & Skoog (1962), in half strength, sucrose (30 g/l) and agar (8 g/l). Embryos were cultured at 25 ° C, first in the dark until either the coleoptile or

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the first leaf were about 2 cm long and root formation had started, and then in the light until the plants could be transplanted into sterilised soil.

Doubling the number of chromosomes of the parental species was achieved through immersing germinated seeds in a solution of colchicine (0.10, 0.15 or 0.20%, during 1.5 h, at 25° C). Those seedlings were selected which showed thickening of organs and stunted growth. Only the larger seeds of their offspring were germinated to determine the number of chromosomes. The autopolyploid C1 individuals as well as their progenies were used for interspecific hybridisations. A colchicine treatment according to Jensen (1976) and Henry & De Buyser (1980) was carried out to double the number of chromosomes of the young hybrids. Plants with five to ten shoots were divided up into two or mostly three parts. After regrowth into plants with about five shoots, the plants were lifted and trimmed by cutting the leaves to about 10 cm and the roots to about 3 cm. Plants were dipped, upside down, for 5 h, in a solution of colchicine (2.5 g/l), with DMSO (20 ml/l) and a few drops of Tween 20, while the roots were kept outside the solution. After treatment the plants were rinsed thoroughly within running tap water, and replanted. Soon after regrowth was established, plants were vernal- ised during 8 weeks at 5 ° C.

For the study of mitotic chromosomes, tips of roots of seedlings or young plants were collected and pretreated during 4 h in a saturated solution of 1-bromonaphthalene. The root tips were fixed overnight in glacial acetic acid and stored in acetic ethanol (1: 3) for at least 24h. Hydrolysis was carried out in 1N hydrochloric acid at 60°C for 10-12min followed by staining with leuco-basic fuchsin and squashing in 45% acetic acid. Meiotic chromosomes were studied in pollen mother cells (PMCs). Young anthers were selected, fixed and stored in acetic ethanol (1 : 3), and washed in 70% ethanol and distilled water. Hydrolysis, staining and squashing was carried out as above.

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Fig. 1. Spikelets of natural (right) and autopolyploid (left) cytotypes of T. turgidum ssp. dicoccoides accessions G7 (A) and G315a-IM (B), and Ae. squarrosa accessions Cam-G (C) and IVP-M (D). Bar = 1 cm.

Results

Hybridisation of autopolyploids ofT. turgidum ssp. dicoccoides and Ae. squarrosa

Seeds of three accessions of T. turgidum ssp. dicoccoides (G7, G7-2M and G315a-lM) and of two accessions of Ae. squarrosa (IVP-M and Cam-G) were treated with colchicine to obtain autopolyploid parental cytotypes for interspecific crosses. In general the autopolyploid cytotypes of both species had wider leaves, shorter haulms and larger flower organs than the natural cytotypes (Fig. 1), and showed a reduced tillering. Table I shows that the grains of the autopolyploid cytotypes were similar in size to those of the natural cytotypes, or were slightly larger and heavier, except for G315a-lM. The grains of autopolyploid plants of this genotype were highly shrivelled, which probably was the main cause of the reduced grain weight.

Seed set, expressed as average number of grains per spikelet, was determined on about three hundred spikelets per accession (Table 1). The polyploidisation led to a reduction of seed set with approximately one seed per spikelet, which reduction was proportionally stronger in T. turgidum ssp. dicoccoides than in Ae. squarrosa. Especially the

Table 1. Seed set and grain characteristics of some accessions of T. autopolyploid cytotypes

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octoploid G315a-1M, which had highly shrivelled grains, showed very poor seed set.

Cytological studies on the autopolyploid cytotypes were carried out in the third generation after the polyploidisation. Mitosis was studied in progenies of euploid individuals, to determine the number of chromosomes (Table 2). In all autopolyploid accessions aneuploid plants were observed. How- ever, the frequency of aneuploids in tetraploid Ae. squarrosa was relatively low, whereas in octoploid T. turgidum ssp. dicoccoides about 18% aneuploids were found. Nearly all aneuploids were hypoploid, having one, or less frequently two to four chromosomes less than the euploid number.

Meiosis in the natural cytotypes of the two species was regular; there were very few desynaptic bivalents, no multivalents, and only 3-6% of the bivalents were rod-shaped (Table 2). Only accession G7-2M was studied at the tetrad stage, which was very regular (data not shown). The autopolyploid cytotypes showed multivalents, an increase in desynaptic bivalents, especially in T. turgidum ssp. dicoccoides, and an increase in the proportion of rod-shaped bivalents to about 18% in T. turgidum ssp. dicoccoides and about 15% in Ae. squarrosa (Table 2; Fig. 2). The observed differences between genotypes within the species were not

turgidum ssp. dicoccoides and Ae. squarrosa, as well as their

Accession No Ploidy Number of Length of 1000-grain grains/spikelet grain (mm) 1 weight (g)2

T. turgidum ssp. dicoccoides

G7 4x 1.90 11.0 + 0.8 40.6 G7-2M 4x 2.14 11.2 + 0.8 46.3 G315a-IM 4x 1.93 11.2 _+ 0.6 45.5

G7 8x 0.87 12.0 + 0.9 47.0 G7-2M 8x 1.07 11.7 + 0.9 46.6 G315a-IM 8x 0.36 11.4 + 0.8 38.8

Ae. squarrosa

IVP-M 2x 2.77 5.7 _+ 0.6 22.9 C a m - G 2x 2.65 5.5 + 0.6 22.2

IVP-M 4x 1.89 5.6 + 0.4 23.8 Cam-G 4x 1.89 5.3 _ 0.5 22.4

+ s .d. , de te rmined on 50 grains. z de te rmined as weight of 50 grains.

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Fig. 2. A. Meiotic metaphase I in autotetraploid Ae. squarrosa (5 IV + 4 II); B. Meiotic anaphase I in octoploid T. turgidum ssp. dicoccoides (two groups of 26 chromosomes and four preco- ciously dividing laggards). Bars = 10/xm.

large. Also in later stages the meiosis in the autopolyploid cytotypes was found to be irregular, and the amount of irregularities was highest in T. turgidum ssp. dicoccoides. At anaphase I, both species contained PMCs with an unequal distribution of chromosomes. In the autopolyploid T. turgidum ssp. dicoccoides, PMCs at anaphase I had one to four lagging chromosomes, which often showed preco- cious division.

In the course of three successive years each of the three accessions of octoploid T. turgidum ssp. dicoccoides was pollinated with pollen of the two accessions of tetraploid Ae. squarrosa. In total, 134 spikes were crossed. On each spike an average number of about twenty florets was emasculated and pollinated. Unfortunately this did not result in the development of a single grain. In a few cases pollination led to the beginning of seed development, indicating that fertilisation may have occurred. However, the seed aborted within a few days, thus embryo rescue could not be carried out. Therefore, it must be concluded that in our hands, and using the present genotypes, this cross cannot be carried out successfully.

Table 2. Percentages of aneuploid plants in C 3 progenies of euploid C2 plants, and meiotic (metaphase I) configurations in pollen mother cells (PMCs) in natural and autopolypioid cytotypes of T. turgidum ssp. dicoccoides and Ae. squarrosa

Accession No Ploidy C 3 plants Number of PMCs

Meiotic (MI) configurations*/PMC

n % aneuploids IV III II (rod) I

T. turgidum ssp. dicoccoides G7 4x 40 G315a-IM 4x 110

G7 8x 45 13 4 5.25 0.50 G7-2M 8x 42 26 6 3.83 0.83 G315a-IM 8x 14 14 6 5.67 0.33

Ae. squarrosa IVP-M 2x 60 C a m - G 2x 110

IVP-M 4x 42 2 70 1.61 0.07 C a m - G 4x 31 3 110 2.71 0.17

14.00 (0.42) 13.97 (0.89) 0.06

16.25 (2.75) 1.00 18.00 (3.50) 2.17 15.33 (2.76) 1.67

7.00 7.00

10.62 8.17

(0.27) (0.20)

(1.46) (1.19)

0.10 0.30

* IV = quadrivalents , III = trivalents, II = bivalents, (rod)= rod bivalents, I = univalents.

Hybridisation of natural cytotypes of T. turgidum ssp. dicoccoides and Ae. squarrosa

The second approach consisted of an extensive crossing programme, carried out during five con- secutive years (1982-1986) to obtain triploid hybrids between T. turgidum ssp. dicoccoides as female parent and Ae. squarrosa as male. Because it is difficult to emasculate Ae. squarrosa, the reci- procal cross was not carried out. Most of the obtained triploids were treated with colchicine to produce synthetic hexaploids.

In total, 470 spikes were emasculated and pollinated, representing about ten thousand florets and 163 different genotype combinations. One-third of these combinations was represented by one spike only, and the others by two or more spikes. In Tables 3 and 4 the results of the crosses per female parent and per male parent have been summarised. The results were grouped to show the striking differences between years. Years 1 and 2 are charac- terised by extremely poor results, consisting of very low seed set and extremely poor seed development. Only one hybrid seed was obtained on 146 spikes crossed. In these two years only a few accessions were used in the crosses, while many more accessions were used in the following years.

In Year 3 by far the best results were obtained: 305 hybrid seeds out of 167 spikes crossed. Sixty- three different genotype combinations were made (individual data not shown): 24 of them yielded no seeds, 17 combinations had less than two seeds per spike and in the remaining 22 combinations seed set per spike was two or more seeds. The latter 22 combinations yielded 267 hybrid seeds and involved 12 of the 29 female parents and 7 of the 17 male parents.

In Years 4 and 5 the range of the crossing programme was widened, especially through a considerable increase of the number of accessions used as male parent. The female and male accessions that had yielded many hybrid seed in Year 3, were not used again. In general the results of the crosses were not as good as in the preceding year. Only 27 hybrid seeds were obtained out of 157 spikes crossed, originating from only 9 out of the 80 genotype combinations. Only three cross combinations

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yielded two or more seeds per spike, resulting in 14 seeds and involving three female and three male parents.

The primary triploid hybrids

The vitality of the hybrid seeds was found to be variable. Many seeds showed an early yellow or brown discoloration, leading to or resulting from retardation or arrest of seed development. Except for the only seed obtained in Year 1/2, which gave rise to a hybrid plant that died before flowering, embryo rescue was used. The 332 hybrid seeds obtained in Years 3 to 5 gave rise to 261 plants. The remaining non-vital seeds had embryos which showed no or abnormal development in vitro, e.g. lack of root formation, disordered differentiation, callusing, vitrification, or a combination of these disorders.

Many hybrid plants were normal and vigorous and their general appearance was most like that of T. turgidum ssp. dicoccoides. The hybrids had the expected number of chromosomes: 2n = 21, and showed much variation for plant length and other morphological characteristics. Some hybrids had dark brown or black chaff, like T. turgidum ssp. dicoccoides. In contrast to the parental species, the rachis of the hybrids was tough, or nearly so.

Various abnormalities were recorded. In sixteen plants from several, unrelated progenies a reduced vitality was observed, leading to poor development and death before flowering. Fifteen plants from several progenies showed cells with aneuploid chromosome numbers (either 2n = 20, 2n = 22, or once 2n = 23) together with euploid cells, two plants were aneuploid (2n = 22) in all cells observed, and in one plant cells with 2n = 21 and with 2n = 42 were found together.

Other abnormalities were only found in the com- plete progenies, or in a major part of them, of certain crosses, as summarised in Table 5. In two crosses the plants were very poor, carrying only a few narrow leaves. As the plants grew older, the colour of the leaves turned to red or purple, and in one of the progenies the leaves became chlorotic at the tip. The plants of both progenies died before

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flowering. Another two crosses yielded plants with a striking grass-clump dwarf phenotype, which in one of the progenies was accompanied by early death of the plants. A third aberration, affecting the whole progenies of three crosses, was the formation of many small yellow spots on the leaves (Fig. 3), ostensibly resulting from chlorophyll defi- ciency or breakdown. The plants appeared not to suffer from this aberration, as they showed normal development and vitality. In four crosses early flowering without vernalisation occurred. The plants

formed very few tillers and it looked as if the gener- ative stage started more or less immediately after germination, leading to flowering in two to three months after the plantlets had been transplanted into soil. In contrast to the three earlier mentioned aberrations, early flowering did not occur in whole progenies but in about half of the plants of the progenies affected. Each of these progenies had G4M-1M as the female parent. The late flowering plants of these progenies showed normal development. It should be noted, however, that in three

Table 3. Results of crosses (sp = number of spikes crossed, and hy = number of hybrid seeds) of individual accessions of T. turgidum ssp. dicoccoides as female parent with Ae. squarrosa as male parent (results of indicated number of accessions were pooled)

Female parent Number of Year 1/2 Year 3 Year 4/5 male parents

sp hy sp hy sp hy

G4M- 1M 4 11 53 G7 9 106 0 G7-2M 8 8 6 G7-2-4M 2 3 0

G7-2-6B-3M 2 3 2 G25 7 40 1 G25-4M 5 6 4

G25-78-27 2 4 16 G29-1M-8-2M 2 2 1 G90-1-1-BM 3 9 27 G148-1-2M 5 10 52

G156-3M 5 2 0 G168-1-2-4BM 7 12 29 G193-1M 3 6 17

G194-3M-6M 7 3 0 G197-2-1M-4M 8 2 0 G213-2M 9 3 0 G280-1-BM 8 7 5 G297-3M 7 5 2 G303-3M 6 3 0 G306-3M 5 4 2 G306-12M 2 4 1 G315a-IM 10 9 8 G326-1-4-5-3M 2 1 1 G327-2BM 5 3 0 G330-1-6B - 1M 2 11 26 G332-1-3-3M 5 10 0 G342-2-2M 5 12 36 G363-4-4BM 2 4 8 G387-2M 5 4 5 G395-7-1M 7 6 4 G487-2M 4

Total 47 146 1 167 305

10 4

8 15

8 7 7

13 5 1

14 1 4

3 2

8 10

8

157

4 0 8 0 2 0

1 0 6 0 6 0

2 0

2 2 6

27

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Table 4. Results of crosses (sp = number of spikes crossed, and hy = number of hybrid seeds) of individual accessions ofAe. squarrosa as male parent with T. turgidum ssp. dicoccoides as female parent (results of indicated number of accessions were pooled)

Male parent Number of female Year 1/2 Year 3 Year 4/5 parents

sp hy sp hy sp hy

Ren-33 13 33 93

Cam-A 3 5 0 4 0 Cam-G 10 12 0 21 30 Cam-K 2 2 0 2 0 Cam-L 9 21 77 Cam-R 3 9 0 2 0 1 0 Cam-S 2 30 0 1 0 IVP-10 5 11 0

IVP-12 3 8 3 IVP-15 1 2 0 IVP-17 1 2 0

IVP-18 10 21 2 IVP-19 1 2 0 IVP-31 4 5 0 IVP-32 8 12 0 IVP-43 6 13 4 IVP-46 2 2 0 IVP-M 10 88 1 22 26 Gat-3 1 2 0 Gat-141 1 1 0 Gat-143 2 7 9 Gat-179 1 2 0 Gat-183 2 3 0 Gat-184 2 2 0 Gat-189 1 1 6 Gat-194 3 5 4

Gat-212 6 7 0 Gat-256 11 18 0 Gat-265 1 3 0 Gat-275 4 6 0 Gat-400 7 9 0 Gat-422 1 1 0 Gat-424 1 2 0 Gat-425 1 2 0 Gat-426 1 2 2 Gat-428 2 5 0 Gat-429 1 2 0 Gat-430 2 5 2 Gat-432 1 2 0 Gat-434 1 2 0 Gat-469 3 5 6 Gat-473 2 8 44 Gat-499 1 1 0 Gat-525 4 11 14 Gat-527 3 10 1 Gat-541 2 4 0 Gat-M 3 11 9

Total 32 146 1 167 305 157 27

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20.42 univalents (range 19.72-20.80). Seventy-

three per cent of the pollen mother cells showed no bivalent at all (Fig. 4A), and the other PMCs contained one (25%), two (1.6%), or three (0.4%) bivalents. Only one ring bivalent was observed, all others were rod shaped (Fig. 4B). All hybrid plants were highly sterile.

Fig. 3. Leaves of primary hybrids (2n = 21) between T. turgidum ssp. dicoccoides and Ae. squarrosa showing no (left), few (middle: G90-1-1-BM x Cam-G), and many (right: G148- 1-2M x Cam-L) small light spots.

more progenies having G4M-1M as female parent early flowering did not occur at all.

First metaphase of meiosis was studied in 25 PMCs each of a random sample of ten hybrids. The average pattern of chromosome association per PMC was 0.29 bivalents (range 0.10-0.64) and

Doubling the number o f chromosomes o f the hybrids

As many plants as possible were used for colchicine treatment, i.e. with the exception of those that died early, but including the two progenies which showed retarded growth and one of the grass- clump progenies, the early flowering plants, and all hybrid plants obtained in Year 4. After the cloning procedure more than five hundred plants, representing approximately 200 hybrids, were treated. About 25% were killed by the treatment and the others showed various degrees of regrowth. After

vernalisation the plants were grown to flowering, forming on average twelve spikes per plant. In 71 plants, i.e. 19% of the individual surviving plants and originating from 50 hybrids, some fertility was

observed, leading to 574 seeds. Not only following colchicine treatment, but also

without colchicine some seed set occurred. The

Table 5. Crosses of T. turgidum ssp. dicoccoides with Ae. squarrosa showing specific aberrant plants in their progenies

Crosses Number of plants Type of aberration

total aberrant

G7-2M x Ren-33 6 G387-2M × Gat-M 5

G280-1-BM × Ren-33 4 G487-2M × Gat-469 5

G90-1-1-BM x Cam-G 5 G148-1-2M x Cam-L 14 G297-3M x Cam-G 2

G4M-1M x Ren-33 11 G4M-1M x Gat-473 8 G4M-1M x Gat-473 5 G4M-1M x Gat-525 4

6 Retarded growth, 'red' leaves 5 Retarded growth, leaf top chlorosis

4 Grass-clump dwarf 5 Grass-clump dwarf

5 Yellow spots on leaves 14 Yellow spots on leaves 2 Yellow spots on leaves

6 Early flowering 5 Early flowering 2 Early flowering 2 Early flowering

above-mentioned early flowering plants (16 hybrids, represented by 23 plants) formed a few small spikes, with in total 260 florets. Six of them contained a viable seed. In Year 4 seven hybrid plants were divided into 69 plantlets which underwent vernalisation and formed 329 spikes, about 4300 florets, and seven viable seeds.

Most of the seeds obtained after colchicine treatment and all seeds that were formed spontaneously were grown to plants, which showed a normal fertility. This observation, together with the results of counts of numbers of chromosomes in a sample of the plants indicated that the hexaploid number of chromosomes (2n = 42) had been obtained. In total 56 hybrids (77 plants) gave rise to hexaploid progenies. The hybrids represent 21 cross combinations, or eleven of the accessions of T. turgidum ssp. dicoccoides and eight accessions of Ae. squarrosa (see also Table 6). More details on the synthetic hexaploids will be presented in the second paper on this programme (Lange & Jochemsen, 1992).

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Discussion

Strategy I

The causes of the failure to obtain allohexaploid wheat from crosses between autopolyploid cytotypes of the wild progenitors, T. turgidurn ssp. dicoccoides and Ae. squarrosa might be two-fold. First there appeared to be a certain degree of incongruity between the two species, as is demon- strated by the low rate of success of the interspecific crosses involving the natural cytotypes (Tables 3 and 4). Except for the cross G7 × IVP-M, which was unsuccessful, the other cross combinations between autopolyploid cytotypes have not been tried with the original diploid accessions. However, Ta- ble 3 shows that the original accessions G7-2M and G315a-IM (both 2n = 28) could be used as female parents in crosses with diploid Ae. squarrosa, although the seed set, one seed in three spikes, was low. In the same way the diploid accessions Cam-G and IVP-M were functional male parents in crosses with natural cytotypes of T. turgidum ssp. dicoc-

Fig. 4. Meiotic metaphase I in primary hybrids (2n = 21) between T. turgidum ssp. dicoccoides and Ae. squarrosa. A: with only univalents; B: with one rod-shaped bivalent. Bar = 10 tzm.

coides (Table 4), the average seed set being one seed in two spikes. Assuming no specific incongruity between the accessions used, the failure of the crosses between the autopolyploid cytotypes therefore might be attributed to the polyploidisation of the parental plant material itself.

In all autopolypoid cytotypes, seed set after self- ing was reduced with about one seed per spikelet, which for T. turgidum ssp. dicoccoides, the female parent in the crosses, is equal to a reduction of at

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least 50%. Such distrubance of natural seed set is likely to be reflected in the results of the interspecific hybridisations, at least to the same extent as in the autopolyploids themselves. From the total number of observations at anaphase I it was esti- mated that in tetraploid Ae. squarrosa about 30% of the microspores would have been aneuploid, whereas in octoploid T. turgidum ssp. dicoccoides this proport ion of the microspores is likely to have been about 70%. The discrepancy between the est imated numbers of aneuploid gametes and the observed numbers of aneuploid individuals (Table 2) indicates a strong selection in favour of euploid gametes. Such selection was also observed in autopolyploids of e .g .T , monococcum L. (Kuspira et al., 1985), where the offspring of such individuals contained no aneuploids at all. In the present study the unability of most aneuploid gametes to function might be the major cause of the reduction of female fertility.

Strategy 2

The present p rog ramme aimed at obtaining a wide range of synthetic hexaploid wheats, based on as many as possible of the selected accessions of T. turgidum ssp. dicoccoides and Ae. squarrosa. Therefore , the process to obtain the doubled hy-

brids has been summarised in Table 6, with empha- sis on the genetic variation involved. The 470 spikes crossed involved a large number of accessions, but only about 11% of the possible cross combinations. All female parents and two-thirds of the male accessions were used in more than one cross combination, but only two accessions were used in more than ten combinations (Tables 3 and 4). A striking difference between the years was observed, which could not be explained or under- stood. In all years the parental plants grew during the same period of the year, in the same conditioned greenhouse, under the same conditions, and nearly all crosses were made by the same person. Within the years, and especially in Year 3, there were also great differences in the rate of success between the cross combinations. Although rele- vant studies were not carried out, it is assumed that genetical influences on crossability may play an important role. The 333 hybrid seeds obtained represent the majori ty of the female accessions and about one-third of the male accessions.

The embryo rescue approach to obtain the hybrid plants was accompanied by a loss of 22% of the potential hybrids, while another 10% of the plants was not viable. Most of these losses occurred at random amongst the cross combinations and did not indicate specificity regarding accessions or cross combinations. Such non-specificity was also

Table 6. The efficiency of the production of synthetic hexaploids between T. turgidum ssp. dicoccoides and Ae. squarrosa

Production step Numbers Accessions used Cross combinations involved

female male

Spikes crossed 470 32 47 163 Hybrid seeds 333 25 17 49 Rescued embryos 261 25 16 45 Inviable plants:

abnormal 16 3 3 3 other causes 16 10 6 10

Viable plants (1): abnormal 40 5 5 7 normal 189 16 14 32

Viable plants (2): colchicine-treated 208 17 12 34 not treated 21 3 5 5

Chromosome-doubled hybrids or synthetic wheats 56 11 8 21

observed regarding the plants which showed small deviations from the euploid number of chromosomes. In addition, 56 hybrid plants showed abnormalities that were specific for certain cross combinations (Table 5), indicating a genetic origin. At least two genetical models may be applicable. The abnormalities might be the result of recessive al- leles, which however, would also lead to segregation among the offspring of the cross combinations. The second genetical model concerns interaction between genes on the parental genomes, which could lead to both uniform and segregating offspring. For three of the abnormalities no segregation occurred, thus pointing to the model of gene interaction. In the case of early flowering the first model cannot be excluded.

The rate of chromosome association at MI of the primary hybrids is very low. On average, 0.29 bivalents per PMC were observed and nearly all bivalents were rod shaped, which confirms the early observations made on a similar amphiploid by McFadden & Sears (1946). However, chromosome association in haploids of common wheat is often reported to be higher (Kimber & Riley, 1963; Riley & Law, 1965), and also in several triploid amphiploids, e.g. between T. turgidum ssp. durum Desf. and Ae. caudata L. (Simeone et al., 1989) or between T. turgidum and three Aegilops species of section Sitopsis (Riley et al., 1958), a considerable higher chromosome association has been observed. Thus either the genetic factors in T. turgidum ssp. dicoccoides preventing homoeologous chromosome association are highly effective in the primary hybrids, or genetic interaction between T. turgidum ssp. dicoccoides and Ae. squarrosa gives rise to the observed suppression of chromosome association in these hybrids. Also in interspecific hybrids of the genus Hordeum the rate of chromosome association at MI was generally found to be low. A genetic regulation of chromosome pairing has been indicated, including a depressing effect of the genome of H. vulgate L. on chromosome pairing in the other genomes of the hybrids studied (Von Bothmer et al., 1983; Fedak, 1985).

The 229 viable primary hybrids represented 18 female and 14 male accessions, combined in 36 cross combinations. At this step a considerable part

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of the original genetical variation had already been lost. Finally also the doubling of the number of chromosomes resulted in a further loss of variation. This doubling was induced by colchicine treatment or it occurred spontaneously. The method of the colchicine treatment was the same as often used for anther-derived haploids of common wheat. Never- theless, the rate of success in the interspecific hybrids was much lower than the 44% (on the basis of plants) observed for the wheat haploids (De Buys- er & Henry, 1986). Although effects of differences in experimental conditions cannot be excluded, it seems also probable that the hybrid condition as such was responsible for the low rate of success. Spontaneous doubling of chromosomes gave rise to 13 seeds, in about 4600 florets, and in most cases as single seeds per spike. Such seeds have also been reported by McFadden & Sears (1946) and Kihara & Lilienfeld (1949), who also studied hybrids between T. turgidum ssp. dicoccoides and Ae. squat- rosa, and by Simeone et al. (1989). In several of these studies 2n-gametes were thought to be the origin of the polyploidisation. In anther-derived haploids of common wheat spontaneous chromosome doubling occurs quite frequently (De Buyser & Henry, 1986), which is not the case if the haploids are produced through crosses with Hordeum bulbosum L. (Inagaki, 1985). Thus the spontaneous chromosome doubling in anther-derived haploids seems to be a special event, which is thought to occur before first mitosis in the microspores and is favoured by the application of cold treatment (De Buyser & Henry, 1986). However, this does not help to explain chromosome doubling in interspecific hybrids, as observed here.

In conclusion Table 6 shows that 21 cross combinations gave rise to synthetic hexaploids, which represent one-third of the female and one-sixth of the male accessions. The production of amphiploids from T. turgidum ssp. dicoccoides and Ae. squarrosa appears not to be very easy, and considerable parts of the original genetic variation have not yet been transferred to the hexaploid chromosome level. On the other hand, no evidence was found for specific crossing barriers, except for a few aberrant primary hybrids. Thus, the present synthetic hexaploids will be used for further studies

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and for breeding common wheat. In addition, the making of new synthetic hexaploids may be labo- rious, but remains promising.

Acknowledgements

Thanks are due to J. Hoogendoorn and A.P.M. den Nijs (CPRO-DLO) for critical reading of the manuscript and useful comments.

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Documents

Use of the gene pools of Triticum turgidum ssp. dicoccoides and Aegilops squarrosa for the breeding of common wheat (T. aestivum), through chromosome-doubled hybrids