Haploids of Hordeum vulgare and H. marinum from crosses between the two species

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1 Hereditas 108: (1988) Haploids of Hordeum vulgare and H. marinum from crosses between the two species RIKKE BAGGER JBRGENSEN and ROLAND VON BOTHMER I Agricultural Research Department, Ris@ National Laboratory, Roskilde, Denmark Department of Crop Genetics and Breeding, Swedish University of Agricultural Sciences, Svalov, Sweden JldRGENSEN, R. B. and BOTHMER, R. VON Haploids of Hordeum vulgare and H. marinum from crosses between the two species. - Hereditas 108: Lund, Sweden. ISSN Received June 25,1987 Thirteen haploids of cultivated barley (Hordeurn vulgare L.) and three haploids of H. marinum were obtained from crosses between these species. Elimination of H. marinum chromosomes was found in combinations with two varieties and four related translocation lines of cultivated barley. There seemed to be a genetic difference between the two subspecies of H. marinum in control of which genome was eliminated. Crosses with ssp. marinum gave haploids of cultivated barley, whereas in crosses with ssp. gussoneanum the genome of cultivated barley was eliminated. Rikke Bagger Jmrgensen, Agricultural Research Department, Rise Narional Laboratory, D K-4OOO Roskilde, Denmark, and Roland von Bothmer, Department of Crop Genetics and Breeding, Swedish University of Agricultural Sciences, S-268 W Svalov, Sweden Doubled haploids of cultivated barley (Hordeum vulgare L.) are a shortcut in the production of homozygous lines for breeding programs. Haploids of barley can be achieved by way of anther culture (e.g., HUANG et al. 1984), ovule culture (SAN NOEUM 1976), the hap gene technique (HAGBERG and HAG- BERG 1980) or the bulbosum technique, i.e., selec- tive chromosome elimination (e.g., JENSEN 1983). Apart from the crosses with H. bulbosum, haploids of cultivated barley have been reported from the combination with the Asiatic species Psathyro- represented by T1-5v, T2-6y and T3-7d, respectively (FINCH and BENNER 1982). The H. marinum stachys fragifis (Boiss.) Nevski (BOTHMER et al. parents were from ssp. marinurn, 2n =14 (H 508, 1984) as well as from crosses with rye (e.g., FEDAK Spain; H 515, Spain; H 604, Greece), the diploid 1977; PICKERING and THOMAS 1979). cytotype of ssp. gussoneanum (Parl.) Thell, 2n = 14 This paper reports the results from crosses be- (H 588, Greece) and the tetraploid cytotype of ssp. tween the wild barley species H. marinum Huds. gussoneanum, 2n = 28 (H 800, Iran). The H. and two varieties plus six lines of cultivated barley. marinum preferentially used was H 515 since this One of the H. vulgare parents, the translocation line population was the one used by FINCH and BENNETT Tuleen 346 and one of the H. marinum parents, H (1983; FINCH 1983). The H. marinum material was 515 was also used by FINCH and BENNETT (1983; FINCH 1983) in the same combination. They found that the chromosomes of H. marinum were eliminated in the embryo and they obtained a single adult plant, a haploid of Tuleen 346. Previously, JACOBSEN and BOTHMER (,1981) reported haploids of H. marinum from the same interspecific combination. Materials and methods The H. vulgare parents were the varieties Golden Promise, Bonus (Elit A and B) and the lines Strengs Franken 111 4x, T6-7d, T1-5v, T2-6y, T3-7d and Tuleen 346. The latter five are translocation lines all derived from Bonus (HAGBERG 1986; RAM- AGE 1971, 1975). Tuleen 346 is a triple translocation line which carries the three reciprocal translocations collected in nature by R. von Bothmer, Svalov, Sweden and N. Jacobsen, Copenhagen, Denmark. Crosses were carried out as described by BOTHMER et al. (1983). Embryos were excised 14 days after pollination and transferred to hormone-free solidified barley growth medium J-25-8 (JDRGENSEN et al. 1986) and placed in darkness at 24 C. Growing

2 208 R. B.10RGENSEN AND R. VON BOTHMER Hereditas I08 (1988) Table 1. Results of crosses between H. marinum X H. vulgare. The five figures separated by dashes indicate number of crossing attempts, number of flowers pollinated, number of seeds, number of embryos and number of plants, respectively H 515 ssp. H. marinum marinurn H 508 ssp. H 640 ssp. H 588 ssp. gur- H 800. gusmarinum marinum soneanum,2n= 14 soneanum,?.n=28 H. vulgare Golden Promise d Bonus Cr T1-5v 0' 0 T24y d P T3-7d d 0 Tuleen 346 d 0 TG7d d StrengsFranken1114x Cr non-morphogenous callus with the Mdh-zymogram of Golden Promise calli. One of these developed roots with the karyotype of haploid Bonus haploidsof cult. barley from4calli. 11 non-morphogenous calli. Electrophoresis of one callus showed the Mdh-zymogram oft1-5v haploids of cult. barley from 5 calli. 11 non-mor. phogenous calli haploidof cult. barley from callus. 8 non-morphogenous calli haploidsofcult. barley from2ealli. 2 non-morphogenous calli and one callus with albino plantsonly, which were not analysed haploid of H. marinum dihaploids of H. marinum embryos were transferred to continuous light (70 pmol m-2 sec-l) also at 24 C. Chromosome numbers of the regenerated plants were obtained from squash preparations of Feulgen stained root-tip cells. Karyotype observations were obtained by Giemsa C-banding (LINDE-LAURSEN et al. 1980). Electrophoresis of malatedehydrogenase (Mdh) of some calli derived from embryos was performed in starch gels with a histidinekitric acid buffer system, ph 7.5 (SECOND 1982). Staining solution for Mdh has been given by BROWN et al. (1978). Results In all, 16 plants were obtained from the 191 crosses between H. marinum and H. vulgare. In all combinations which yielded plants, cultivated barley was used as pollen donor. When H. vulgare was used as the female parent no seed set at all was recorded. Of the 16 plants reared, 13 were haploids of H. vulgare and three were mono- or dihaploids of H. marinum. The results of the crosses are found in Table 1.

3 Hereditas 108 (198%) HAPLOIDS OF HORDEUM VULCARE AND H. MARlNUM 209 Fig. 1. Plant regeneration through somatic embryogenesis (arrow). H 515 (H. marinurn) X Tuleen 346. Combinations yielding haploids of cultivated barley The morphology of all the embryos was anomalous. They were small (< 1 mm), drop-shaped, with a granulated surface and without visible scutellum. On the hormone-free medium, growing embryos al- Fig. 3. Spikes of 1) H. vulgare, 2) the hybrid H. marinum X H. vulgare (TI-Sv), and 3) H. marinurn. Fig. 2. C-banded karyotype of haploid H. vulgare from H 515 X T1-5~. ways produced calli. Of the 554 embryos only 9 YO were able to develop into calli, one fourth of which were regenerative. When placed in continuous light, plants regenerated from calli through somatic embryogenesis (Fig. 1) and organogenesis. The regenerative calli seldom produced more than one plantkallus and the regenerative capacity was lost by subculturing. The haploid plants of cultivated barley were ob- tained from the combinations between H. marinum ssp. marinum, H 515 and four translocation lines of cultivated barley, T1-5v, T2-6y, T3-7d and Tuleen 346. Embryos from crosses between H 515 and Bonus or Golden Promise produced non-regenerative calli only, but relatively few crossing attempts were carried out with these H. vulgare partners. Two of the H. vulgare haploids were verified as such by way of their karyotypes. Giemsa C-banding of one plant each from the crosses with T1-5v and T2-6y revealed that the plants had the haploid chromosome complement of these lines (Fig. 2). The other haploids of cultivated barley were identified combining observations on chromosome number and morphology. The morphology of the haploids was H. vulgare-like; however, they were smaller in all parts (Fig. 3). The haploids of cultivated barley all had the cytoplasm of H. marinum since plants were obtained only with cultivated barley as the male parent. Thirty-six embryos developed into calli with no plant regeneration. One callus, however, from the crosses H 515 X Bonus (Elit B) produced roots. The C-banded karyotype of these roots showed the haploid chromosome set from cultivated barley (Fig. 4). The single callus from H 515 X Golden Promise and one of the non-morphogenous calli

4 210 R. B. I0RGENSEN AND R. VON BOTHMER Hereditas 108 (1988) Fig. 4. C-banded karyotype of roots from callus of H 515 X Bonus. The haploid chromosome set of Bonus is visualized. Fig. 5 a and b. a Mdh-zymogram of 1) Embryo-derived callus from H 515 X Golden Promise, 2) H 515, and 3) Golden Promise. b Mdh-zymogram of 1) Embryo derived callus from H 515 X T1-5v, 2) H 515, and 3) T1-5v. from H 515 x T1-5v were stained for malatedehydrogenase activity after electrophoresis. In both cases, the Mdh-zymogram of the callus was identical to the enzyme pattern found in the H. vulgare parent (Fig. 5). yielding Of H* Two combinations rendered mono- or dihaploids of H. marinum. One monohaploid (2n = 7) was obtained from the diploid combination, H 588, ssp. gussoneanum X Golden Promise. From the tetraploid combination, H 800 ssp. gussoneanum x Strengs Franken two dihaploids (2n = 14) of H. marinum were produced. Morphologically the haploids looked like H. marinum, but they were more slender and low growing. Discussion In interspecific crosses between lines of cultivated barley and 26 wild Hordeum species BOTHMER et al. (1983) found that irrespective of the crossing combination the seed set was low, when cultivated barley was used as the female parent. Analogously, in this study seed set was registered only when H. vulgare was used as the pollinator. Therefore all the haploids of H. vulgare were alloplasmic. The embryos from the crosses with H. marinum yielding haploids of H. vulgare were small and morphologically abnormal. This was possibly the reason why normal embryogenesis was suppressed and calli produced. Only 2 yo ofthe embryos developed into iegenerative calli. From crosses-with another wild barley species, H. bulbosum, haploids of cultivated bailey are often obtained (e.g., JENSEN 1983). The embryos from this combination vary from globular to well developed with scutellum, when they are excised for embryo culture 13 days after fertilization (PICKERINC 1983). The number of plants obtained is among other factors dependent on this embryo differentiation. Most of the embryos from H. vulgare x H. bulbosum produce plants directly without callus forma- tion (e.g., JENSEN 1974). However, JENSEN (1981) deliberately induced calli from this combination and reported Oh haploid H. vulgare plants regenerated from the number of calli units as explants, which is less than the corresponding percentage-namely 27 %-from the present study. The alloplasmic condition together with the low

5 Hereditas 108 (1988) HAPLOIDSOF HORDEUM VULGARE AND H. MARINUM 211 frequency of H. vulgare haploids makes it unlikely that crosses with H. marinum will be used as an alternative technique for mass production of barley haploids. Crosses between other H. marinum populations and/or lines of H. vulgare might, however, increase yields of haploids. A genotypic effect from the H. vulgare parent is probably reflected in the different number of calli and plants obtained from similar numbers of embryos in the combinations with Golden Promise and Tuleen 346, respectively. A genotypic influence from both parents on the frequency of haploids is known to exist from the H. vulgare x H. bulbusum combinations (e.g., Pic- KERING 1980; JENSEN 1981). In the analysis of cytoplasmatically inherited characters the haploids of barley with cytoplasm from H. marinum might be of interest. Also the alien cytoplasm could contribute characters valuable in barley breeding. The alloplasmic haploids might have a potential also in the production of homokaryotic cytoplasm hybrids by fusion of protoplasts from barley haploids with and without alien cytoplasm, and thereby circumvent enucleation. FINCH and BENNETT (1984) pointed out that the direction of chromosome elimination in H. marinum x H. vulgare combinations, viz. production of H. vulgare alternatively H. marinum haploids is dependent on the subspecies of H. marinum used. They did not present any data to justify this; however, their statement is supported by the work of BOTHMER et al. (1983) as well as the present results. These authors reported haploids and dihaploids of H. marinum, from three genotype combinations. They used two diploid populations and one tetraploid of ssp. gussoneanum, and the lines of culti- vated barley were Ris~ F1 and Kenia 4x. In the present study Bonus, Golden Promise and four related translocation lines of cultivated barley crossed to ssp. marinum (H 515) gave haploid callilplants of cultivated barley. Mono- and dihaploid H. marinum plants were reared from two combinations with ssp. gussoneanum crossed to Golden Promise and Strengs Franken 111, 4x, respectively. The distribution areas of the two subspecies of H. marinum overlap (BOTHMER et al. 1988). Hybrids between the diploid cytotypes of the two subspecies revealed seven bivalents at meiosis, and hybrids between diploid ssp. marinum and tetraploid ssp. gussoneanum had a high frequency of trivalents (BOTH- MER et al. 1988). In the light of this close genomic similarity between ssp. marinum and ssp. gussuneanum it is possible, that the direction of chromosome elimination in H. marinum x H. vulgare combinations is controlled by one or a few genes only differing between the subspecies. Further studies with more genotype combinations will be carried out to confirm or reject this assertion. Another striking trait characterizing the mechanism of elimination in H. marinum X H. vulgare combinations is that elimination is without exception, as hybrid plants have never been reported. Hybrids are common in all the other interspecific combinations with cultivated barley (including H. bulbusum) rendering haploids of this species or of the wild Hordeum species (SUBRAHMANYAM and BOTH- MER 1987). Literature cited BOTHMER, R. VON. FLINK, J., JACOBSEN, N., KOTIMAKI, M. and LANDSTROM. T Interspecific hybridization with cultivated barley (Hordeum vulgare L.). - Hereditas 99: BOTHMER, R. VON, JACOBSEN, N., JORGENSEN, R. and LINDE-LAURSEN. I Haploid barley from the intergeneric cross Hordeum vulgare X Psathyrostachys fragilis. - Euphytica 33: BOTHMER, R. VON, FLINK,J., JACOBSEN, N.,JQ)RGENSEN, R. B Variation and differentiation in Hordeum marinum. - Nord. J. But. (in press) BROWN, A. H. D., NEVO, E., ZOHARY, D. and DAGAN, Genetic variation in natural populations of wild barley (Hordeum spontaneum). - Genetica FEDAK, G Haploids from barley x rye crosses. - Can. J. Genet. Cytol. 19: 15 FINCH, R. A Tissue-specific elimination of alternative whole parental genomes in one barley hybrid. - Chrumosoma (Berl.) 88: FINCH, R. A. and BENNETT, M. D The karyotype of Tuleen 346 barley. -Theor. Appl. Genet. 62: FINCH, R. A. and BENNETT, M. D The mechanism of somatic chromosome elimination in Hordeum. - In Kew Chromosome Conference II. George Allen and Clnwin. p FINCH, R. A. and BENNETT, M. D Wide crosses in the genus Hordeum. - Annual Report Plant Breeding Institute, Cambridge 88: HAGBERG. A Inducedstructural rearrangements. --In Genetic Manipulation in Plant Breeding. Proc. Int. Symp. EUCAR- PIA, Berlin, p HAGBERG, A. and HAGBERG, G High frequency of spontaneous haploids in the progeny of an induced mutation in barley. - Hereditas 93: 341 HUANG, B., DUNWELL, J. 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