Anther Culture Protocols for Barley and Wheat.

Doubled haploid (DH) techniques remain valuable tools for wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) genetic improvement, and DH populations are used extensively in breeding and research endeavors. Several techniques are available for DH production in wheat and barley. Here, we describe two simple, robust anther culture methods used to produce more than 15,000 DH wheat and barley lines annually in Australia.

The Effect of Caffeine and Trifluralin on Chromosome Doubling in Wheat Anther Culture.

Challenges for wheat doubled haploid (DH) production using anther culture include genotype variability in green plant regeneration and spontaneous chromosome doubling. The frequency of chromosome doubling in our program can vary from 14% to 80%. Caffeine or trifluralin was applied at the start of the induction phase to improve early genome doubling. Caffeine treatment at 0.5 mM for 24 h significantly improved green plant production in two of the six spring wheat crosses but had no effect on the other crosses. The improvements were observed in Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Caffeine had no significant effect on chromosome doubling, despite a higher frequency of doubling in several caffeine treatments in the first experiment (67-68%) compared to the control (56%). In contrast, trifluralin significantly improved doubling following a 48 h treatment, from 38% in the control to 51% and 53% in the 1 µM and 3 µM trifluralin treatments, respectively. However, trifluralin had a significant negative effect on green plant regeneration, declining from 31.8 green plants per 20 anthers (control) to 9-25 green plants per 20 anthers in the trifluralin treatments. Further work is required to identify a treatment regime with caffeine and/or anti-mitotic herbicides that consistently increases chromosome doubling in wheat without reducing green plant regeneration.

Genotypic effects on the frequency of homoeologous and homologous recombination in Brassica napus × B. carinata hybrids.

We investigated the influence of genotype on homoeologous and homologous recombination frequency in eight different Brassica napus (AAC(n)C(n)) × B. carinata (BBC(c)C(c)) interspecific hybrids (genome composition C(n)C(c)AB). Meiotic recombination events were assessed through microsatellite marker analysis of 67 unreduced microspore-derived progeny. Thirty-four microsatellite markers amplified 83 A-, B-, C(n)- and C(c)-genome alleles at 64 loci, of which a subset of seven markers amplifying 26 alleles could be used to determine allele copy number. Hybrid genotypes varied significantly in loss of A- and B-genome alleles (P < 0.0001), which ranged from 6 to 22% between hybrid progeny sets. Allele copy number analysis revealed 19 A-C, 3 A-B and 10 B-C duplication/deletion events attributed to homoeologous recombination. Additionally, 55 deletions and 19 duplications without an accompanying dosage change in homoeologous alleles were detected. Hybrid progeny sets varied in observed frequencies of loss, gain and exchange of alleles across the A and B genomes as well as in the diploid C genome. Self-fertility in hybrid progeny decreased as the loss of B-genome loci (but not A-genome loci) increased. Hybrid genotypes with high levels of homologous and homoeologous exchange may be exploited for genetic introgressions between B. carinata and B. napus (canola), and those with low levels may be used to develop stable synthetic Brassica allopolyploids.

Microspore culture preferentially selects unreduced (2n) gametes from an interspecific hybrid of Brassica napus L. x Brassica carinata Braun.

We analysed the products of male meiosis in microspore-derived progeny from a Brassica napus (AAC(n)C(n)) x Brassica carinata (BBC(c)C(c)) interspecific hybrid (ABC(n)C(c)). Genotyping at 102 microsatellite marker loci and nuclear DNA contents provided strong evidence that 26 of the 28 progeny (93%) were derived from unreduced (2n) gametes. The high level of C(n)C(c) marker heterozygosity, and parallel spindles at Anaphase II in the ABC(n)C(c) hybrid, indicated that unreduced gametes were formed by first division restitution. The frequency of dyads at the tetrad stage of pollen development (2.6%) suggested that unreduced gametes were preferentially selected in microspore culture. Segregation of marker alleles in the microspore-derived progeny was consistent with homologous recombination between C(n) and C(c) chromosomes and homoeologous recombination involving A-, B- and C-genome chromosomes during meiosis in the ABC(n)C(c) hybrid. We discuss the potential for using microspore culture of unreduced gametes in interspecific hybrids to map Brassica centromeres through half-tetrad analysis.

Antimicrobial activity of crude extracts from plant parts and corresponding calli of Bixa orellana L.

Ethanol extracts from the different parts of B. orellana showed differential antimicrobial activity. It was found that the extracts of in vitro leaves showed maximum activity against Bacillus pumilus followed by the extracts from the roots and hypocotyls. The callus derived from different explants too showed antimicrobial activity. The leaf callus showed maximum activity. The zone of inhibition for the diluted extracts of in vitro hypocotyls and roots and their corresponding calli showed minimum zone of inhibition at concentration 24 mg/ml, whereas the diluted extract of in vitro leaves and leaf derived callus showed minimum zone of inhibition at 16 mg/ml.