Monitoring of haploid maize cell suspension culture conditions in bioreactors

Maize (Zea mays L.) haploid cells were cultivated in a 1500 ml aerated and stirred batch bioreactor using modified BM medium. Cell growth was highly affected by pH and dissolved oxygen, and we observed two fairly distinct growth phases. During the first two days after inoculation at pH 5.8, oxygen consumption was high and the cells lowered the pH to a value around 4.3. After this period the pH stabilized at 4.5 and the dissolved oxygen reached a steady level. Decreasing dissolved oxygen concentration leads to lower growth rate and to higher pH. Both events mean stress conditions for the cell culture and probably result in increased genetic variability, and the loss of regeneration capacity. The stress condition during the adaptation phase can be eliminated by decreasing the pH of the medium to 4.7 before inoculation and by keeping dissolved oxygen above 40%. These conditions provide prolonged exponential growth dynamics and the cell suspensions could be the basis of large scale cultures also.Abbreviations 2,4-d 2,4-dichlorophenoxyacetitc acid - NAA naphthalene acetic acid     

The effect of drought and heat stress on reproductive processes in cereals

As the result of intensive research and breeding efforts over the last 20 years, the yield potential and yield quality of cereals have been greatly improved. Nowadays, yield safety has gained more importance because of the forecasted climatic changes. Drought and high temperature are especially considered as key stress factors with high potential impact on crop yield. Yield safety can only be improved if future breeding attempts will be based on the valuable new knowledge acquired on the processes determining plant development and its responses to stress. Plant stress responses are very complex. Interactions between plant structure, function and the environment need to be investigated at various phases of plant development at the organismal, cellular as well as molecular levels in order to obtain a full picture. The results achieved so far in this field indicate that various plant organs, in a definite hierarchy and in interaction with each other, are involved in determining crop yield under stress. Here we attempt to summarize the currently available information on cereal reproduction under drought and heat stress and to give an outlook towards potential strategies to improve yield safety in cereals.     

Regeneration capacity of microspore-derived structures produced in anther cultures of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

The regeneration capacity of microspore-derived structures, with various morphological characteristics produced in anther cultures of maize (Zea mays L.) were studied in order to identify the morphotype resulting in the highest yield of spontaneous doubled haploid regenerants. Parallel to the morphological studies the ploidy level of microspore-derived structures and regenerants was analysed by flow cytometry. Neither the growth conditions of the anther donor plants nor the media used in the experiment had any effect on the frequency distribution of different morphotypes. The highest number of spontaneous doubled haploid plants was regenerated from white compact structures 2–3 mm in size, derived from the anthers of phytotron-grown donor plants.     

Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis

Genetic characterisation of Trypanosoma cruzi variants is of foremost importance, due to considerable genetic and biological heterogeneity in the parasite populations. Two major phylogenetic lineages, each highly heterogeneous, have been previously described within this species. Here we characterised a geographically and ecologically diverse sample of stocks representative of the breadth of the known clonal diversity of each major lineage, using random amplified polymorphic DNA with 20 primers and multilocus enzyme electrophoresis at 22 loci. Molecular hybridisation experiments were performed to control the homology of randomly amplified DNA markers. Both sets of data were highly consistent and supported the existence of two major lineages. Additionally, we found that lineage 2 appeared further partitioned into five sharply delineated phylogenetic clusters, each comprising one of the following reference strains: CanIII cl1 (Z3 reference), M5631 cl5, Esmeraldo cl3 (Z2 reference), CL Brener, and MN cl2. The two first clusters were found mainly in sylvatic environments, whereas the three latter were restricted to domestic transmission cycles and were only collected South to the Amazon Basin. In contrast, lineage 1, which included Miles' Z1 reference strain X10 cl1, was not further subdivided and was encountered across the entire endemic area, in both domestic and sylvatic cycles. Thus, T. cruzi appeared to be subdivided into six discrete typing units, or DTUs, exhibiting distinct geographic and ecological ranges. Reliable diagnostic markers for the two major lineages and the five smaller DTUs of lineage 2 are described, and correspondence with previous classifications of T. cruzi genotypes is given in order to help communication on T. cruzi phylogenetic diversity.     

Microstructured liposome array

Conversion of a DNA chip to a nanocapsule array was performed by grafting on a liposome an oligonucleotide complementary to an oligonucleotide bound to the array. Each liposome may be loaded by a soluble molecule or may present a hydrophobic or amphiphilic molecule inserted in its wall. To detect liposomes on the chip, we used fluorescent dyes encapsulated in the liposome internal volume or fluorescent lipids. We observed that an oligonucleotide-grafted liposome containing a defined dye specifically accumulated on the area where its complementary oligonucleotide had been spotted on the array. The virtually unlimited amount of addresses allows the specific binding of large amounts of liposomes in one single batch.     

Analysis of the genotypic effect on different developmental pathways in wheat gametophyte cultures

Seven wheat cultivars and one wild subspecies Triticum aestivum ssp. spelta were compared for their in vitro fertility and androgenetic capacity by studying their anther culture response and in vitro seed production. Both haploid embryogenesis and in vitro seed set showed very wide genotype dependent variability in accordance with previous observations. At the same time, an analysis of the data showed a significant negative correlation between the in vitro androgenetic ability and the in vitro fertilization potential, which was especially obvious in the case of highly embryogenic genotypes. The reason for this inverse correlation might be the different genetic regulation of the two quantitative traits. Presumably, alleles which increase the stability of the gametogenetic developmental programme hinder the initiation of haploid embryogenesis, while alleles with the opposite effect promote the sporophytic type of growth.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid     

Regeneration of fertile wheat (Triticum aestivum) plants from isolated zygotes using wheat microspore culture as nurse cells

A new method for growing isolated wheat zygotes by co-cultivation with microspores isolated from the same species has been developed. Although the mortality of isolated zygotes within 6 h after the transfer into the nurse culture was relatively high (70%) in this method, the majority of the structures which survived developed into fertile plants (61%). Zygotic structures grown in these cultures were morphologically not as regular as embryos grown in planta, but they resembled them closely in cytological properties and developmental pattern.