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A wheat (Triticum aestivumL.) immature spike culture system was used to expeditiously generate mutations for use in wheat improvement programs. Wheat immature spikes in culture were treated with three concentrations of ethylmethane sulphonate (EMS) to generate a spike culture derived variant (SCDV) population. EMS in a concentration dependent manner affected seed development in wheat immature spike cultures. Based on the number of seeds produced, inclusion of EMS (25 mM) for three hours in immature spike culture medium generated variants in wheat cv. AC Nanda. The wheat AC Nanda SCDV population showed variation in several phenotypic characters. Flag leaf (length, angle and sheath length), length of first and second internode, spike length, number of spikes, number of seeds per spike and seed weight, showed variation below and above the non-treated controls. A molecular screening technique combining simple sequence repeat (SSR) oligonucleotide primers with high resolution melt (HRM) PCR with EvaGreen was used to identify the variants. Screening for starch branching enzyme IIb (SbeIIb) revealed 75 lines with point mutations. Combining SSR and SbeIIb, a total of 100 Kbp portion of wheat DNA was screened. The estimated mutation frequency in SbeIIbwas one per 20.8 Kbp. The spike culture system utilizes very small amounts of EMS for a brief period, thus needs minimal handling of EMS and saves one generation of plant growth in a greenhouse. The morphological variants observed are similar to those reported for seed-derived variants using EMS.

The rate of development of disease varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected children. The reasons for these observed differences are not clearly understood but most probably depend on the dynamic interplay between the HIV-1 quasispecies virus population and the immune constraints imposed by the host. To study the relationship between disease progression and genetic diversity, we analyzed the evolution of viral sequences within six perinatally infected children by examining proviral sequences spanning the C2 through V5 regions of the viral envelope gene by PCR of blood samples obtained at sequential visits. PCR product DNAs from four sample time points per child were cloned, and 10 to 13 clones from each sample were sequenced. Greater genetic distances relative to the time of infection were found for children with low virion-associated RNA burdens and slow progression to disease relative to those found for children with high virion-associated RNA burdens and rapid progression to disease. The greater branch lengths observed in the phylogenetic reconstructions correlated with a higher accumulation rate of nonsynonymous base substitutions per potential nonsynonymous site, consistent with positive selection for change rather than a difference in replication kinetics. Viral sequences from children with slow progression to disease also showed a tendency to form clusters that associated with different sampling times. These progressive shifts in the viral population were not found in viral sequences from children with rapid progression to disease. Therefore, despite the HIV-1 quasispecies being a diverse, rapidly evolving, and competing population of genetic variants, different rates of genetic evolution could be found under different selective constraints. These data suggest that the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations are compatible with a Darwinian system evolving under the constraints of natural selection.

Viability and fertility profiles of cryopreserved gladiolus pollen from 7 cultivars have shown that it is possible to use cryogenic methods for conservation and management of the haploid gene pool in this species. There was no decline in pollen viability (in vitro) levels after 1 and 10 years of cryogenic storage. Field pollinations with cryogenic stored pollen induced capsule and seed set in varying capacities. Long-term cryogenic storage of gladiolus pollen could enhance breeding efficiency through better management of the haploid gene pool resources. Pollen parents could be made available throughout the breeding programme, ensuring guaranteed supply at the time of peak stigma receptivity. A ‘pollen cryobank’ facility established for this species would increase genetic diversity conservation at the haploid stage.

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.