The Journal of Heredity 71: 280-282 (1980)
Haploid barley regenerated from callus cultures of Hordeum vulgare X H. jubatum
T. J. ORTON

HAPLOIDS have been widely advanced as useful intermediates in plant genetics and breeding3. Primarily, they provide a means of 'instant homozygosity', thus bypassing numerous time- and labor-consuming generations of inbreeding. Moreover, genetic variants are more easily selected in vitro from haploid tissue. Haploids are currently obtained by 1) culture of embryogenic microspores5, 2) chromosomally unstable intertaxon hybrids4, and 3) spontaneous or genetically induced development of uniparental gametes (via twin seedling assay or by use of pseudodominant visual markers)10. All of these methods have limited specific application among species and results with any given systems may be highly variable.

Introgession of genetic material between species is a viable means to broaden the base of genetic variability for studies of basic inheritance and breeding12. The following prerequisites are tantamount to intertaxon gene flow: hybrid intermediates must be both viable and fertile; chromosomes of both parents must be maintained and sexually transmitted; and if specific gene transfer is desired, interspecific chromosome pairing and exchange must occur. Such gene flow has been demonstrated between wild and cultivated species of the Gramineae tribes Hordeae11 and Tripsaceae1, and the genus Lycopersicon9, but other groups are stymied by failure to satisfy one or more of the prerequisites cited above (particularly between progressively unrelated species). For example, hybrids between Hordeum vulgare (HV, 2n = 2x = 14) X H. jubatum (HJ, 2n = 4x = 28) both viable and karyotypically stable, but completely sterile even in the diploid state (2n = 6x = 42)8, thus imposing a block of potential gene flow. Numerous hybrids within the tribe Hordeae (Gramineae) involving H. bulbosum (HB) rapidly and selectively lose all HB chromosomes within a few days after fertilization, thus preventing gene flow4.

Previous studies of Hordeum callus cultures have described the genesis of in vitro chromosomal variability manifested as polyploidy, aneuploidy, and chromosomal rearrangements6-8. Moreover, a restricted range of chromosomal variability in callus cultures transcended embryogenesis and was expressed in regenerated plants. By using isozyme bands as chromosomal markers, a quantitative segregation of parental chromosomes was found among populations of plants regenerated from callus cultures of HV X HJ6. Morphological resemblances both to the original HV and HJ parents were noted at the extremes of the distribution. Certain HV-like (HVL) regenerates were analyzed cytogenetically and biochemically to determine their genetic constitution.


Microhybridity, as indicated by the presence of specific HJ isozymes in HVL haploid regenerates could be a consequence of interspecific chromosome transfer by somatic exchange or translocation (chromosomal rearrangements are frequent in these cultures6); or chromosome addition (in eight and nine chromosome cells) or substitution (in seven chromosome cells). Perennial-like growth habit aside, HV-like morphology suggests that whole chromosome substitution is unlikely and asynapsis in eight and nine chromosome metaphase I microsporocytes indicates that extra chromosomes are of HJ origin. Further analyses of phenotypes and cytogenetic studies of doubled HVL X HV hybrids are in progress to elucidate the basis of microhybridity.

Gene flow between species is usually very tenuous, and often restricted or blocked by hybrid sterility, inviability, or chromosome elimination. Callus or cell culture of intertaxon hybrids may facilitate gene flow by the regeneration of fertile intermediates or those exhibiting microhybridity. Segregation and recombination among somatic cells in vitro by processes generating chromosomal variability escape the constraints of sex (e.g., fertility and viability in vivo) and may hence give rise to novel genetic types.

Summary

Haploids are extremely useful tools in plant breeding, but are generally difficult to obtain. Further, interspecific hybrids are essential intermediates for the bridging of genes between pools, but isolating mechanisms such as invariability or sterility often block gene flow. Chromosomally variable tissue cultures of an interspecific Hordeum hybrid were investigated as a potential source of haploids and fertile hybrid regenerates. Approximately 5 to 10 percent of all regenerates from callus cultures of Hordeum vulgare X H. jubatum (n = 3x = 21) were haploids resembling the H. vulgare parent. Certain of these regenerates expressed activity of specific H. jubatum esterases, suggesting that chromosomal or subchromosomal substitution or addition may have occurred in vitro. Tissue culture of interspecific hybrids is discussed as a means of producing haploids and facilitating gene flow.

Hetero-Fertilization / Endosperm Failure