Effect of osmotic stress on tolerance of air-drying and cryopreservation ofArabidopsis thaliana suspension cells

Summary Arabidopsis thaliana suspension cells were preserved in liquid nitrogen for over three years, using embedding of cells in calcium-alginate prior to subculture in sucrose-enriched medium, air-drying, and direct quenching in liquid nitrogen. Survival of cells reached 34%, yielding regrowth at the surface of all cryopreserved beads in less than 7 days. Following pretreatment and dehydration, the water content dropped from 2300% to 34% with respect to dry weight. Differential scanning calorimetry showed that glass transition occurred on cooling, followed by a slight crystallization event on rewarming. The survival of cells was independent of the cooling rate. The tolerance of the acute dehydration step increased progressively with sucrose pretreatment duration, indicating the requirement for adaptative cellular alterations. Ultrastructural studies revealed several changes in cells after sucrose pretreatment prolonged from 1 to 7 days: reversal of the initially plasmolyzed state, microvacuolation, numerous autophagic structures, scarcity of ribosomes, increase in number and size of starch grains. No cell division seemed to occur during this period. After air-drying and after a freeze-thaw cycle, followed by 24 h rehydration, regenerating cells had recovered a high level of ultrastructural organization and contained numerous polysomes suggesting an intense metabolic activity. Trehalose, a cryoprotective disaccharide not considered to be a metabolic substrate, yielded only 70% regrowth after freezing. Biochemical analysis showed that soluble sugars accumulated during the pretreatment, essentially sucrose or trehalose; the monosaccharide content also increased. In the light of these results, the action of sucrose in inducing freezing tolerance is discussed.Abbreviations HPLC high-performance liquid chromatography - LN liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride     

The chloroplast generps 4 as a tool for the study ofPoaceae phylogeny

Phylogenetic analyses of 28Poaceae species based on the chloroplastrps 4 gene are presented using parsimony and distance methods. Two monocots from other families were used as outgroups. The chloroplast generps 4 was amplified, cloned, and sequenced for each species. The inferred phylogenetic trees were compared to recent classifications and are shown to fit their general features. There is a dichotomy in our tree between the pooid group and the other grasses. This is in contradiction with other molecular phylogenies, where the bamboos appear first within the family. This result led us to discuss some hypotheses about the relationships of the bambusoids with the other groups of grasses, and also about the relative position of rice and bamboo, which are found close to each other in our trees.     

Ectopic expression of Arabidopsis CYCD2 and CYCD3 in tobacco has distinct effects on the structural organization of the shoot apical meristem

Transgenic tobacco lines expressing Arath-CYCD2 or Arath-CYCD3 genes under a cauliflower mosaic virus 35S promoter are modified in the timing of their development, but not in the phenotype of their vegetative organs. They display an increased rate of leaf initiation, which is shown to be associated with distinct changes in the structural organization of their shoot apical meristem (SAM). Constitutive expression of Arath-CYCD2 leads to a progressive modification of the SAM structural organization with predominant periclinal divisions in the L3 layer and to the loss of the classical cytophysiological zonation, the central zone being reduced to the central cells of the L1 and L2 layers. These changes reveal a particular sensitivity of the corpus cells (L3) to Arath-CYCD2 over-expression and suggest a role for CYCD2 in controlling the planes of cell division in these cells. The SAM structural modifications in the Arath-CYCD3 over-expressing lines are less drastic; only an increased cell number together with a reduced cell size, particularly in the L1 layer, characterizes the peripheral zones. This could be related to the shortening of the G1-phase duration that renders cell growth incomplete between successive mitoses. Cell proliferation continues beyond the SAM in the developing internodes and confers a delayed senescence to Arath-CYCD3 over-expressing juvenile tissues. In addition, the ploidy levels of mature stem tissues in both types of transgenic lines are unaffected, suggesting that the studied G1 to S cell-cycle genes have no effect on the extent of endoreduplication in tobacco stem tissues.     

Isolation of an inulinase derepressed mutant of Pichia polymorpha for the production of fructose

Pichia polymorpha has inulinase activity and could be used for the production of fructose syrup from inulin. The application of immobilized P. polymorpha whole cells for the continuous hydrolysis of inulin is, however, limited since the biosynthesis of this enzyme system is repressed by the reaction products, dextrose and fructose. A derepressed mutant hyperproducer of inulinase was isolated after treatment with EMS followed by a selection step with deoxyglucose.     

Comparison of dose volume histograms for supine and prone position in patients irradiated for prostate cancer—A preliminary study

Aim

To compare DVHs for OARs in two different positions – prone and supine – for prostate cancer patients irradiated with a Tomotherapy unit.

Background

In the era of dose escalation, the choice of optimal patient immobilization plays an essential role in radiotherapy of prostate cancer.

Materials and methods

The study included 24 patients who were allocated to 3 risk groups based on D’Amico criteria; 12 patients represented a low or intermediate and 12 a high risk group.For each patient two treatment plans were performed: one in the supine and one in the prone position. PTV included the prostate, seminal vesicles and lymph nodes for the high risk group and the prostate and seminal vesicles for the intermediate or low risk groups. DVHs for the two positions were compared according to parameters: Dmean, D70, D50 and D20 for the bladder and rectum and Dmean, D10 for the intestine. The position accuracy was verified using daily MVCT.

Results

Prone position was associated with lower doses in OARs, especially in the rectum. Despite the fact that in the entire group the differences between tested parameters were not large, the Dmean and D10 for the intestine were statistically significant. In the case of irradiation only to the prostate and seminal vesicles, the prone position allowed for substantial reduction of all tested DVH parameters in the bladder and rectum, except D20 for bladder. Moreover, the Dmean and D50 parameter differences for the bladder were statistically significant.No significant differences between positions reproducibility were demonstrated.

Conclusion

In patients irradiated to prostate and seminal vesicles, the prone position may support sparing of the rectum and bladder.The reproducibility of position arrangement in both positions is comparable.     

Megasporogenesis in Arabidopsis thaliana L.: an ultrastructural study

 In this study, megasporogenesis of the plant model Arabidopsis thaliana was investigated by electron microscopy for the first time. The data described here could constitute a reference for future investigations of Arabidopsis mutants. During the beginning of meiosis the megaspore mother cell shows a polarity created by unequal distribution of organelles in the cytoplasm. Plastids accumulate in the chalazal region and long parallel saccules of endoplasmic reticulum, small vacuoles and some dictyosomes are found in the micropylar region. Plasmodesmata are abundant in the chalazal cell wall. The nucleus is almost centrally localized and contains a prominent excentric nucleolus and numerous typical synaptonemal complexes. After the second division of meiosis the four megaspores are separated by thin cell walls crossed by numerous plasmodesmata and do not show significant cellular organization. The young functional megaspore is characterized by a large nucleus and a large granular nucleolus. The cytoplasm is very electron dense due to the abundance of free ribosomes and contains the following randomly distributed organelles: mitochondria, a few short saccules of endoplasmic reticulum, dictyosomes and undifferentiated plastids. However, there is no apparent polarity, except for the distribution of some small vacuoles which are more abundant in the micropylar region of the cell. The degenerating megaspores are extremely electron dense and do not show any substructure. Received: 30 July 1998 / Revision accepted: 3 February 1999     

Switch (swi1), an Arabidopsis thaliana mutant affected in the female meiotic switch

In this paper, we describe a novel plant mutant affected exclusively in the female mitosis-meiosis switch. The major effect of the swi1 mutation in Arabidopsis thaliana L. is to delay megasporogenesis events by inserting additional mitotic divisions of the mega- sporocyte. As a result of this delay, megagametogenesis is also affected. The absence of cellular polarity in the megasporocytes was also observed. Ovule ontogenesis is not affected by the mutation. The swi1 mutant is particularly interesting for studying sporophyt-gametophyte interactions. The swi1 mutation, obtained from a T-DNA tagging experiment, is monogenic recessive and mapped on chromosome five, at 16 cM from the yellow inflorescence marker. Received: 29 June 1999 / Revision accepted: 3 August 1999     

Preliminary screening of some fungal strains for protein upgrading of sugar-beet pulp

Summary Fourteen fungal strains were grown on sugar-beet pulp (SBP) in a semisolid state with the view of evaluating their capabilities in increasing the protein content of SBP. The highest increases were obtained withSporotrichum thermophile no 89 Paprican andThielavia terrestric ATCC 38088, which gave a protein content of 14.6% and 13.4% respectively after a 3-day culture or a net increase of 48% and 36% respectively over that of the unfermented SBP.