Characterisation of plants regenerated from barley (Hordeum vulgare L.)-H. bulbosum L. hybrids

Abstract

Hordeum bulbosum L. is an obligatory outcrossing perennial grass and is considered to be the closest relative of cultivated barley, H. vulgare. H. bulbosum has many useful agronomic traits such as winterhardiness and drought tolerance. The resistance it has to many pathogens, including powdery mildew, yellow rust, net blotch, and scald would be worthwhile transferring to H. vulgare.

Although hybrids between H. vulgare and H. bulbosum are easily obtained and homoeologous chromosome pairing occurs in H. vulgare x H. bulbosum hybrids, successful gene transfer is rare. Possible reasons for this include: 1) the diploid constitution of H. vulgare may not tolerate introgressions from an alien species; 2) the existence of certation, where the recombinant male gametes may not be able to compete with non-recombinant gametes to effect fertilisation; 3) the pairing between H. vulgare and H. bulbosum chromosomes may not result in crossing over; 4) the recombinant pollen grains may be sterile and 5) the recombinant chromosomes may be unstable depending on the size of the introgression.

In an attempt to overcome the problem of certation, plants were regenerated directly from pollen grains, of H. vulgare-H. bulbosum hybrids, by anther culture. Embryo-like structure (ELS) formation was initially optimised and generally it was found hybrids responded best to anther culture when grown in either a growth chamber at 15°C or a glasshouse at 22°C, when spikes were cold pretreated for 28 days and when ELS were induced on either of the two medium tested (modified medium 1 and BAC3 medium). The two hybrid parents most responsive to anther culture, genotypes 3028 and VG2 558/9, were triploid hybrids and contained 14 H. vulgare and 7 H. bulbosum chromosomes. Both ELS induction and green plant regeneration was genotype dependent.

A total of 43 green plants were obtained, three of these were triploid, three were tetraploid, one was aneusomatic and the remainder were haploid or diploid.

The most responsive genotype to anther culture, 3028, was subjected to spike inflorescence culture, backcrossing to a diploid H. bulbosum genotype Cb2920/4, and selfing. Regenerated plants produced by anther culture, backcrossing and selfing were analysed at the morphological, cytological and molecular level (by RFLP analysis). It was found that each plant regeneration system produced genetically unique plants. Great variation was observed in the locations of H. bulbosum introgressions, which were detected using single or low copy telomeric probes. The reactions of regenerants to three fungal diseases, powdery mildew, scald and net blotch, were scored. Some regenerants were more resistant than the hybrid parent from which they were derived, reflecting DNA rearrangements had occurred resulting in increased resistance.

RFLP analysis of green and albino anther culture-derived plants using the methylsensitive endonucleases MspI and HpaII, showed albino plants had much higher levels of methylation than the corresponding green regenerants. These findings supported the hypothesis that changes in DNA methylation could affect the frequency of green plant regeneration.