Femorotibial cartilage morphology: reproducibility of different metrics and femoral regions, and sensitivity to change in disease.

This study was designed to characterize the reproducibility and sensitivity to change of magnetic resonance imaging-based cartilage morphology metrics and femoral regions of interest (ROIs), in order to provide preferable outcome measures in longitudinal studies of cartilage morphology. Test-retest acquisitions were obtained at 3 tesla (T) in 33 subjects with and without radiographic signs of osteoarthritis (OA) (reproducibility study) as well as baseline and 2-year follow-up acquisitions in 28 subjects with radiographic signs of advanced OA (sensitivity study). Cartilage was segmented in the tibia and two distinct anatomical femoral ROIs, a 'long' ROI extending 60% from the trochlear notch to the posterior end of the condyles, and a 'short' ROI extending to the intercondylar bone bridge. Coefficients of variation (reproducibility study) and standardized response means (SRMs, sensitivity study) were obtained for different morphology metrics and anatomical regions. The subchondral bone area of the long ROI was 20% greater and less variable than that of the short ROI; cartilage morphology metrics were generally more reproducible in the long ROI. Normalized cartilage volume (VCtAB) and mean cartilage thickness (over the entire subchondral bone area; ThCtAB.Me) tended to be more reproducible and more sensitive to change (SRM up to -0.62) than cartilage volume (SRM up to -0.44), cartilage thickness over the cartilaginous area (ThCcAB; SRM up to -0.48) or maximum cartilage thickness (ThCtAB; SRM up to -0.35). The long femoral cartilage ROI provided more reproducible measurements than the short one. VCtAB and ThCtAB.Me may be preferable metrics in longitudinal studies of articular cartilage adaptation or OA.

Elevated expression and release of tissue-type, but not urokinase-type, plasminogen activator after binding of autoantibodies to bullous pemphigoid antigen 180 in cultured human keratinocytes.

In bullous pemphigoid (BP), the binding of BP180-specific antibodies to their hemidesmosomal target antigen is not sufficient for blister formation, but must be accompanied by the release of proteases. Using plasminogen activator (PA) knock-out mice, the PA system has previously been shown to be a prerequisite for blister formation in experimental murine BP. Here, we found elevated levels of plasmin and tPA, but not of uPA, in blister fluid from BP patients (n = 7) compared to blisters from patients with toxic epidermal necrolysis (n = 4) and suction blisters in healthy controls (n = 7). Subsequently, we addressed the question whether keratinocytes release PA in response to the binding of anti-BP180 antibodies. Treatment of cultured normal human keratinocytes with BP IgG, but not with control IgG, led to both increased protein and mRNA levels of tPA, but not of uPA, as determined by ELISA and RT-PCR, respectively. The specificity of this finding was confirmed using BP180-deficient keratinocytes from a patient with generalized atrophic benign epidermolysis bullosa, where no tPA release was observed after stimulation with BP IgG. Our results show the elevated expression and release of tPA from normal human keratinocytes upon stimulation with antibodies to human BP180. Keratinocytes, by secreting tPA, may thus play an active role in blister formation of BP.

Production of haploids from anther culture of banana [Musa balbisiana (BB)].

We report here, for the first time, the production of haploid plants of banana Musa balbisiana (BB). Callus was induced from anthers in which the majority of the microspores were at the uninucleate stage. The frequency of callus induction was 77%. Callus proliferation usually preceded embryo formation. About 8% of the anthers developed androgenic embryos. Of the 147 plantlets obtained, 41 were haploids (n=x=11). The frequency of haploid production depended on genotypes used: 18 haploid plants were produced from genotype Pisang klutuk, 12 from Pisang batu, seven from Pisang klutuk wulung and four from Tani. The frequency of regeneration was 1.1%, which was based on the total number of anthers cultured. Diploid plants (2n=2x=22) were also observed in the regenerated plants. The haploid banana plants that were developed will be important material for the improvement of banana through breeding programmes.

Localization of quantitative trait loci (QTL) for agronomic important characters by the use of a RFLP map in barley (Hordeum vulgare L.).

Two hundred and fifty doubled haploid lines were studied from a cross between two 2-row winter barley varieties. The lines were evaluated for several characters in a field experiment for 3 years on two locations with two replications. From a total of 431 RFLP probes 50 were found to be polymorphic and subsequently used to construct a linkage map. Quantitative trait loci (QTLs) were determined and localized for resistance against Rhynchosporium secalis and Erysiphe graminis, for lodging, stalk breaking and ear breaking tendency, for the physical state before harvest, plant height, heading date, several kernel parameters and kernel yield. The heritability of the traits ranged from 0.56 to 0.89. For each trait except for kernel thickness, QTLs have been localized that explain 5-52% of the genetic variance. Transgressive segregation occurred for all of the traits studied.

Progeny tests of barley, wheat, and potato regenerated from cell cultures after in vitro selection for disease resistance.

Because plant cells cultured in vitro express genetic variability and since they can be regenerated into functional plants, procedures have been designed to use this system for the production of plants with new important agronomic characteristics, particularly for disease resistance. For barley, wheat, and potato somaclones have been found that were less susceptible to a toxin of Helminthosporium, fusaric acid, Fusarium coeruleum, F. sulphureum, or Phytophthora infestans, when screened in the first in-vitro-derived generation. Here the progeny of such somaclones is evaluated after natural and artificial infection, using greenhouse-grown or field material. The progenies of the same somaclones did not express detectable differences, which indicated that no heterozygous mutations occurred. Most lines and clones differed in their level of susceptibility to the pathogen compared to the level of the starting material, but these data were in no instance significant. It is discussed here whether this lack of significance is due to a lack of genetic differences or whether the test procedures are in adequate for detecting and securing the slight, probably quantitative, alterations.

Recurrent selection alternating with haploid steps - a rapid breeding procedure for combining agronomic traits in inbreeders.

Doubled haploid lines are increasingly used in practical breeding programs. Depending on the nature of the starting material as well as on the breeding aims, one or more haploid steps are recommended, and F1 hybrids or selfed progenies in later generations serve as starting material for the haploidization. It is demonstrated that one haploid step followed by selection in the greenhouse and in the field during the first androgenetic (A1) and two subsequent selfed generations (A2; A3) is the most efficient procedure, if characters from related varieties are to be combined. For breeding programs that include wild types or unrelated genotypes, recurrent selection alternating with several haploid steps is the most efficient. A prerequisite for successful application of this combination of repeated haploidization and selection steps alternating with backcrosses is an effective selection system. The different approaches are demonstrated on a winter barley breeding program, with the central aim of combining the qualitatively inherited character resistance to barley yellow mosaic virus with quantitatively inherited complexes, particularly yield.

In vitro microspore reaction of different German wheat cultivars.

The in vitro microspore androgenesis reaction of 25 commercial German spring (including 4 Triticum durum) and 50 winter wheat cultivars was investigated. Tremendous genotypical differences were found in microspore response. The best-responding winter wheat cultivai, "Florida", is characterized by the presence of a 1B/1R wheat-rye translocation chromosome. The significance of this finding and other genetic systems for future use of haploids in plant breeding is discussed.

Production of doubled haploid lines in frequencies sufficient for barley breeding programs.

Winter barley (Hordeum vulgare L.) anthers were cultured on different liquid and on starch-solidified media. The optimal embryo and callus formation with different F1-lines and the cv. 'Igri' was obtained on a liquid medium with 20% Ficoll, 20 g/l maltose and barley starch. But the influence of the growth conditions of the donor plants and the genotypical differences are still enormous. The procedure has been optimized to such an extent that it can be used economically.

Rapid production of recombinant barley yellow mosaic virus resistant Hordeum vulgare lines by anther culture.

In a winter barley breeding program for barley yellow mosaic virus (BaYMV) resistance, the resistant six-rowed cv. Franka was crossed to 17 susceptible and two resistant cultivars, three of which were tworowed. A total of 233,445 anthers of the 19 hybrids and their parents were cultured and 831 green plants regenerated. Anther culture responsiveness varied greatly between genotypes, and the responsiveness of F1hybrids was generally related to that of the more responsive (high) parent. On average, 3.6 green plants were recovered from 1,000 cultured anthers, almost twice as many as in comparable spring barley experiments. Androgenetic green plants were tested for their reaction to mechanical inoculation of BaYMV. In crosses of resistant parents, all the cross progeny proved to be resistant, which indicates that both parents carry identical gene(s). In the crosses of the resistant cv. Franka to susceptible parents, an average of 62% of the androgenetic progenies were resistant, which indicates that probably more than one gene is responsible for Franka's BaYMV-resistance. From the crosses of Franka to two-rowed cultivars, 282 androgenetic plants were produced. When 132 of these were tested for their reaction to BaYMV, 79 (59.8%) were resistant, and 30 of the latter were shown to be two-rowed recombinant lines. Doubled haploid lines are field-tested for other agronomic characters including grain yield and its components.

On the genetic improvement of androgenetic haploid formation in Hordeum vulgare L.

Anthers of 55 different spring barley (Hordeum vulgare) hybrids and four varieties were cultured in vitro. Microspores of each hybrid gave rise to calluses and subsequently plantlets, from all hybrids, except one. As criteria of microspore responsiveness, callus formation and plant regeneration frequencies were studied in detail. Large differences with regard to these criteria were found, which were traced back to the genotype of the anther donor plant. Callus formation varied between 3.3 and 73.2 per 1,000 anthers plated, whereas green plant regeneration ranged from 0 to 12.7 per 1,000 anthers cultured.Comparisons of microspore regeneration frequencies of hybrids and their parents indicated that culture responsiveness is a heritable, complex character involving at least two different and separately inherited mechanisms: 1) the ability of microspores within anthers to divide and give rise to calluses and subsequently 2) the ability of calluses for morphogenesis, to yield green or albino plants. Because it is heritable, anther culture responsiveness can be transferred to breeding material which is initially non-responsive. This genetic way of improving success in androgenetic haploid production appears to be more realistic than the search for optimum culture conditions.