II. 13. Anther culture of autotetraploid Hordeum vulgare varieties and the production of trisomic individuals.
W. Friedt, B. Foroughi-Wehr, G. Mix and H.M. Wilson. Abteilung fur Pflanzengenetik der Gesellschaft fur Strahlen--und Umweltforschung mbH, 8o59 Grunbach/Erding Obb., West Germany.
The production of haploids from a diploid plant species may be restricted by the fact that such individuals will be hemizygous and thus unable to survive the presence of lethal genes. Chase (1974) has suggested that the haploid potential of a stock will be reduced in direct proportion to the frequency of gametes carrying lethal genes. In the case of an inbreeding and homozygous species like barley, however, a significant reduction would not be anticipated. In order to test these theoretical considerations we cultured the anthers of two autotetraploid varieties and compared the results with those obtained using anthers of diploid varieties.
The diploid and tetraploid plants used as anther donors were all two-rowed spring varieties. They were grown under controlled conditions in the glasshouse and the anthers were taken and cultured as described previously (for details of the anther culture procedures, media, and plant regeneration technique, see Foroughi-Wehr et al. 1976).
The results are presented in Table 1. The frequency of anther response of the tetraploid varieties was similar to that of the diploids with regard to both callus production and subsequent plantlet regeneration, although the general rarity of the latter event makes meaningful comparisons difficult. Chlorophyll deficiency was prevalent amongst the plantlets regenerated from the tetraploid anthers, implying that albinism is not a feature specifically related to a reduction in genotype to the haploid level. Although root development was poor in the white individuals, one of those recovered from anther callus of the variety Weihenstephaner contained 14 chromosomes in the root-tips.
Meiosis was studied in several of the spikes of the green plant regenerated from anther callus of Haisa II (spikes fixed in 3:1 abs. alcohol: glacial acetic acid and stained in acetic orcein). Observations of diakinesis/metaphase I revealed that the plant was trisomic (2n+1=15), with the pairing of the trisome being II+1 or III (Fig's A-D). These configurations were found with approximately equal frequency in the pollen mother cells (Table 2). Classification of the trivalents revealed that 19% were of the panhandle type, 23% were linear chains, and 58% were V-type chains. Anaphase I separation occurred either as 7I+ 8I or as 7I + 7I + 1 laggard. The presence of the laggard would account for the high percentage of tetrads showing micronuclei formation (Table 2). At diakinesis the extra univalent was frequently observed in association with the single nucleolus (Fig. B) present in these pollen mother cells. This may be an indication that the extra chromosome is satellited and would thus be a No. 6 or 7 (Tsuchiya, 1963). Further cytological and genetical studies to identify the extra chromosome are in progress.
TABLE 1. A Comparison of the Anther Responses of
Diploid and Tetraploid Varieties of Hordeum vulgare L.
TABLE 2. Meiotic Data from the Trisomic Plant.
There are two possibilities regarding the origin of the trisomic plant; (1) Aneuploidy arose during the callus phase of an anther callus derived from a diploid microspore. (2) Non-disjunction occurred during meiosis in the tetraploid donor plant and the microspore from which the callus developed contained 15 chromosomes. The latter alternative is considered the most likely, since non-disjunction in meiosis, resulting in microspores with 13 or 15 chromosomes, has been observed with a frequency of 22% in the tetraploid variety Haisa II used in this study (Friedt, unpublished).
Previous studies on the anther culture of Hordeum vulgare have underlined the importance of the physiological status of the donor plants. Field-grown plants have been found much more suitable as a source of anthers for culture. Thus, it may be expected that the use of field-grown tetraploid donor plants will result in higher rates of anther callus production and larger numbers of regenerated green plants than are reported in the present study. Should this prove to be the case, then anther cultivation of tetraploid plants may provide a particularly quick and effective method for generating trisomic individuals.
Acknowledgements
We thank Mrs. R. Seibold, Mrs. R. Stein, and Mrs. A. Stocker for excellent technical assistance.
References:
Chase, S. S. 1974. Breeding diploid species. pp. 211-230 in Haploids in Higher Plants. Ed. K. J. Kasha, Univ. of Guelph.
Foroughi-Wehr, B., Mix, G., Gaul, H., and H. M. Wilson. 1976. Plant Production fram Cultured Anthers of Hordeum vulgare L. Z. fur Pflanzenzuchtg. 77, 198-204.
Tsuchiya, T. 1963. Chromosome aberrations and their use in genetics and breeding in barley - trisonics and aneuploids. Barley Genetics I, Proc. 1st Int. Barley Genetics Symp., Wageningen, 116-150.
