Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle.

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were >or=25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15 min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15 min, followed by wash), a horseradish peroxidase-streptavidin solution (15 min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15 min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90 min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8 +/- 0.2%), specificity (91.7 +/- 1.4%), and negative predictive value (99.7 +/- 0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5 +/- 1.4% vs. 90.5 +/- 1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3 +/- 3.0%), positive predictive value (95.0 +/- 2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

Comparison between vestibular and subcutaneous insertion of deslorelin implants for oestrus induction in bitches.

Investigations using sustained-release deslorelin implants at various insertion sites have shown that this method consistently induces oestrus in anoestus bitches. However, fertility comparisons between implant insertion sites have not been performed. Anestrous beagle bitches received one 2.1 mg deslorelin implant beneath the vestibular mucosa (VM group; n = 6) or in the subcutaneous tissue between the shoulder blades (SubQ group; n = 8). Vestibular implants were removed when serum progesterone concentrations first exceeded 1.5 ng/ml. Vaginal cytologies and blood samples were collected daily and bitches were inseminated during oestrus. Serum progesterone and deslorelin concentrations were measured and pregnancy status was determined using ultrasonography. There were no differences between groups in the intervals between implant administration and the onset of proestrus, the time of the luteinizing hormone surge and the onset of cytologic diestrus. There were also no differences in the number of corpora lutea or foetuses. However, conception rate was significantly lower in the SubQ group. The pregnancy rate did not differ significantly between the VM and SubQ groups [4 out of 6 (66.7%) and 3 out of 8 (37.5%), respectively]. One bitch (16.7%) in the VM group and three bitches (37.5%) in the SubQ group suffered distinct, premature declines in serum progesterone concentrations starting 1-4 weeks after cytologic diestrus. Serum progesterone concentrations did not recover (premature luteal failure), resulting in abortion. Bitches with premature luteal failure in the SubQ group still had serum deslorelin concentrations >100 pg/ml 20 days after implant insertion, suggesting a possible association between prolonged deslorelin release and luteal failure.

Hormone profiles of mares affected by the mare reproductive loss syndrome.

While searching for the cause of the Mare Reproductive Loss syndrome (MRLS), we postulated that 1 of 3 tissues in 40-120 D pregnant mares was the likely primary target of the noxious factor that caused early abortions: The corpora lutea (CL), the endometrium or the fetus and/or its membranes. At this stage of gestation, progesterone (P4) is solely produced by luteal tissue, eCG by endometrial cups in the endometrium and oestrogens by the feto-placental unit. We determined whether concentrations of P4, eCG and/or total conjugated oestrogens (CE) would indicate which tissue was targeted during the MRLS. P4, eCG and CE were measured in single serum samples collected from 216 mares, 60-110 D after ovulation during the 2001 MRLS outbreak. All mares had previously been confirmed pregnant by ultrasonography. The following data was obtained from each mare: Interval from ovulation, pregnancy status and normalcy of fetal fluids at the time of sampling, and pregnancy status 3 weeks after sampling and at term. There were no meaningful differences in hormone concentrations between pregnant mares that had normal and excessively echogenic fetal fluids at the time of sampling. CE were lower (p < 0.05) in mares that aborted after sample collection than in mares the carried to term. In 8 mares from which multiple samples were obtained, CE consistently decreased prior to any decreases in P4 or eCG. Arguments are presented that lead to the hypothesis that the fetal trophoblast was the primary target of the MRLS agent.

The use of deslorelin implants for the synchronization of estrous in diestrous bitches.

A novel approach to estrous induction in diestrous bitches is described. Twelve spontaneously cycling anestrous bitches served as controls. Thirteen anestrous and 15 diestrous bitches were induced to come into synchronous estrous using prostaglandin (diestrous bitches only) and deslorelin implants (Ovuplant). Implants contained either 2.1 or 1.05 mg deslorelin and were administered beneath the vestibular submucosa. All treated bitches came into estrous, regardless of implant size. Whereas all anestrous bitches ovulated, one of six diestrous bitches treated with the larger implant and three of nine treated with the smaller implant failed to ovulate. Induced bitches generally produced fewer corpora lutea than controls. Sixty-seven percent of control bitches became pregnant, with 0.63 fetuses per corpus luteum, whereas the pregnancy rate and fetuses per corpus luteum were 67 and 70% and 0.42 and 0.55 in the anestrous bitches induced with 1.05 and 2.1 mg deslorelin implants, respectively (not different from controls). Only 2 of 15 induced diestrous bitches conceived a detectable pregnancy, one of which was resorbed. In conclusion, although ovulatory estrous can be induced in bitches that had their most recent ovulation 40-100 days ago, these bitches are very unlikely to become pregnant during the induced estrous. The reason for the poor fertility in these diestrous bitches requires further study.

Failure of hCG to support luteal function in bitches after estrus induction using deslorelin implants.

Induction of estrus with deslorelin implants was followed by abortions in bitches that conceived during the induced estrus. Lowering the deslorelin dose and choosing a better implantation site prevented the abortions. This study investigated the hypothesis that induction of estrus with deslorelin is followed by reduced serum progesterone concentrations (SPC) during the ensuing diestrus. Assuming that reduced luteal function resulted from reduced LH secretion due to hypophyseal down-regulation of GnRH receptors, the effect of human chorionic gonadotropin (hCG) treatment on the SPC of diestrous bitches was also investigated. In Experiment 1, 10 spontaneously cycling bitches served as controls, whereas estrus was induced with deslorelin implants in 24 others. In Experiment 2, six diestrous bitches were treated with a single dose of hCG between Days 39 and 45 of diestrus. The SPC was lower in deslorelin-induced bitches from Days 35 to 56 of diestrus and hCG increased SPC during the first 24 h after treatment, followed by a dramatic decline thereafter. Although SPC recovered in pregnant bitches, it remained much lower (< or = 1 ng/mL) than in untreated, non-pregnant bitches. The suppression of progesterone secretion after hCG treatment suggested that decreased luteal activity in deslorelin-induced bitches may not be a simple consequence of down-regulation of hypophyseal GnRH receptors.

Motile plant cell body: a "bug" within a "cage".

Analysis of the cytoskeleton in morphogenetically active plant cells allows us to propose a unified concept for the structural organization of eukaryotic cells. Their cytoarchitecture is determined by two principal structural complexes: nucleus-microtubule-based cell bodies ("bugs") and plasma-membrane-F-actin-based cell periphery complexes ("cages"). There are dynamic interactions between each of these entities in response to extracellular and intracellular signals. In the case of the cell body, these signals determine its polarization, rotation and migration. Interactions between cell body and cell periphery complexes determine cell growth polarity and morphogenesis throughout the eukaryotic kingdom.

Nuclear components with microtubule-organizing properties in multicellular eukaryotes: functional and evolutionary considerations.

The nucleus and the microtubular cytoskeleton of eukaryotic cells appear to be structurally and functionally interrelated. Together they constitute a "cell body". One of the most important components of this body is a primary microtubule-organizing center (MTOC-I) located on or near the nuclear surface and composed of material that, in addition to constitutive centrosomal material, also comprises some nuclear matrix components. The MTOC-I shares a continuity with the mitotic spindle and, in animal cells, with the centrosome also. Secondary microtubule-organizing centers (MTOC-IIs) are a special feature of walled plant cells and are found at the plasma membrane where they organize arrays of cortical MTs that are essential for ordered cell wall synthesis and hence for cellular morphogenesis. MTOC-IIs are held to be similar in origin to the MTOC-I, but their material has been translocated to the cell periphery, perhaps by MTs organized and radiating from the MTOC-I. Many intranuclear, matrix-related components have been identified to participate in MT organization during mitosis and cytokinesis; some of them also seem to be related to the condensation and decondensation of chromatin during the mitotic chromosome cycle.

Impacts of aluminum on the cytoskeleton of the maize root apex. short-term effects on the distal part of the transition zone

Using monoclonal tubulin and actin antibodies, Al-mediated alterations to microtubules (MTs) and actin microfilaments (MFs) were shown to be most prominent in cells of the distal part of the transition zone (DTZ) of an Al-sensitive maize (Zea mays L.) cultivar. An early response to Al (1 h, 90 µM) was the depletion of MTs in cells of the DTZ, specifically in the outermost cortical cell file. However, no prominent changes to the MT cytoskeleton were found in elongating cells treated with Al for 1 h in spite of severe inhibition of root elongation. Al-induced early alterations to actin MFs were less dramatic and consisted of increased actin fluorescence of partially disintegrated MF arrays in cells of the DTZ. These tissue- and development-specific alterations to the cytoskeleton were preceded by and/or coincided with Al-induced depolarization of the plasma membrane and with callose formation, particularly in the outer cortex cells of the DTZ. Longer Al supplies (>6 h) led to progressive enhancements of lesions to the MT cytoskeleton in the epidermis and two to three outer cortex cell files. Our data show that the cytoskeleton in the cells of the DTZ is especially sensitive to Al, consistent with the recently proposed specific Al sensitivity of this unique, apical maize root zone.

A freeze-fracture study on the differentiation of Golgi and plasma membranes in plant cells.

Dictyosomes, Golgi vesicles, and plasma membranes were investigated after freeze-fracture in cells from growing root tips of cress (Lepidium sativum L.), that are distinguishable by different cellulose content of the cell wall, into (i) meristematic cells during early formation of the cell plate, (ii) statocytes of the root cap, and (iii) cortex cells of the differentiation zone. The results of this study show that the number of intramembrane particles (imps) is high in dictyosome cisternae, but low in membranes of budding or dictyosome-derived vesicles. Imps are disperse in the vesicle membranes of meristematic cells (i), but are often grouped into clusters in vesicle membranes of statocytes (ii), and of cortex cells (iii). For the number of particle aggregates in vesicle membranes, the following relation holds: (i) < (ii) < (iii). The number of particles on both fracture faces (PF and EF) of the plasma membrane differs widely between the cell types investigated. There are approximately 250, 1400, and 3100 imps microns-2 on the PF and 50, 500, and 300 on the EF of (i), (ii), and (iii), respectively. The structural complexity of the plasma membrane as judged by the degree of particle aggregations on the PF and the number of cellulose microfibrils in the cell wall show the same relationship: (i) < (ii) < (iii). Thus, the strong correlation between the distribution of imps in vesicle membranes, the structural complexity of the plasma membrane, and the content of cellulose microfibrils indicate that selection of imps during vesicle formation at dictyosome cisternae is an integral component of biogenesis and structural differentiation of plant plasma membranes.

Monoclonal antibody CRA against a fraction of actin from cress roots recognizes its antigen in different plant species.

The globular cytoskeletal protein G-actin was isolated from the crude extract of soluble proteins from cress (Lepidium sativum L.) roots. The crude extract was loaded onto a deoxyribonuclease (DNase) I-affinity chromatography column and subsequently eluted with EGTA and urea. The fraction eluted with 2 mM EGTA was characterized by molecular weight determination, binding to DNase I, isoelectric focusing, and immunoblotting. These samples clearly showed one main 43,000 dalton protein with a pI value between 5.5 and 5.7. This polypeptide is an isoform of actin. It was stained using commercially available monoclonal and polyclonal actin antibodies. We used the EGTA fraction as plant actin antigen to produce a monoclonal cress root actin antibody. Antibodies (CRA) showed specific labelings on Western immunoblots against a 43,000 dalton protein of the cress root crude extract. Under the fluorescence microscope CRA detected actin in fixed statenchyma cells of cress roots. This antibody also demonstrated intact bundles of actin filaments in unfixed internodal cells of Chara australis. On the basis of these results we concluded that we had obtained a new monoclonal antibody (CRA) against actin from cress roots. We also found a cress root actin-binding protein antibody (CRAB) showing a filament staining pattern in internodal cells of Chara.

Rearrangements of F-actin arrays in growing cells of intact maize root apex tissues: a major developmental switch occurs in the postmitotic transition region.

Immunofluorescence labeling, using a monoclonal antibody developed against actin, revealed the relative abundance and rearrangements of F-actin arrays which occur in cells of the maize root apex as they make the developmental transition from proliferative growth in the meristem to a non-proliferative state in more mature root parts, and during the concomitant process of tissue differentiation. Cells in both the root cap and the quiescent center are depleted of F-actin, whereas it is abundant in cells of the central cylinder but less so in the cortex. The cortical cytoplasm associated with the endwalls of both mitotic and postomitotic cells is characterized by a more intense reactivity to the actin antibody than the longitudinal side walls. A major change in F-actin arrangement occurs in the transitional growth region interpolated between the meristem and the zone of rapid cell elongation. The location and nature of these F-actin rearrangements within the root suggest that the F-actin system might be involved in generating a force associated with the developmental transition of cells from their slow near-isotropic mode of growth close to the base of the meristem, to rapid anisotropic growth which is characteristic of the zone of cell elongation. This attraction notion was strongly supported using specific inhibitors of F-actin and myosin.

Profilin is associated with the plasma membrane in microspores and pollen.

In higher plants, a large number of isoforms for the actin monomer-binding protein profilin have been identified, whereas other organisms express only few profilins. Furthermore, plant profilin isoforms are expressed in a tissue-specific manner. These observations raise questions concerning functional and locational differences between isoforms of plant profilins. In this paper, we introduce three polyclonal antisera and one monoclonal antibody developed against purified pollen profilins from Zea mays and against recombinant maize profilin. Immunoblot analyses of native profilins and four recombinant maize pollen profilin isoforms show that three of the antibodies display a preference for certain isoforms. In situ immunofluorescence of pollen of Zea mays and two developmental stages of microspores of Betula pumila indicates that all antibodies label plasma membrane-associated domains. Thus, we show that at least some profilin isoforms are located at a distinct subcellular domain within developing microspores and, less distinctly, in mature pollen. This contrasts previously reported uniform distributions throughout the cytoplasm of mature pollen and pollen tubes. The results are discussed in light of the large number of profilins co-expressed in plants and with reference to accumulating evidence for functional differences between profilin isoforms.

Immunofluorescence detection of F-actin on low melting point wax sections from plant tissues.

We developed a simple and reliable technique for immunofluorescence detection of F-actin on microtome sections of plant tissues. For the first time, large numbers of plant cells from various tissues that pass through their developmental stages could be consistently visualized on one section from plant organs. n-Maleimidobenzoic acid N-hydroxysuccinimide ester-pretreated and formalin-fixed segments of plant roots and shoots were embedded in low melting point ester wax at 37C and sectioned on a microtome. After dewaxing and rehydration, microfilaments were visualized by indirect immunofluorescence technique with a monoclonal anti-actin antibody. The technique has been successfully used for visualization of tissue- and development-specific F-actin arrays in cells of Zea mays and Lepidium sativum root tips and of maize stem nodes.

Actin cytoskeleton in plants: from transport networks to signaling networks.

The plant actin cytoskeleton is characterized by a high diversity in regard to gene families, isoforms, and degree of polymerization. In addition to the most abundant F-actin assemblies like filaments and their bundles, G-actin obviously assembles in the form of actin oligomers composed of a few actin molecules which can be extensively cross-linked into complex dynamic meshworks. The role of the actomyosin complex as a force generating system - based on principles operating as in muscle cells - is clearly established for long-range mass transport in large algal cells and specialized cell types of higher plants. Extended F-actin networks, mainly composed of F-actin bundles, are the structural basis for this cytoplasmic streaming of high velocities On the other hand, evidence is accumulating that delicate meshworks built of short F-actin oligomers are critical for events occurring at the plasma membrane, e.g., actin interventions into activities of ion channels and hormone carriers, signaling pathways based on phospholipids, and exo- and endocytotic processes. These unique F-actin arrays, constructed by polymerization-depolymerization processes propelled via synergistic actions of actin-binding proteins such as profilin and actin depolymerizing factor (ADF)/cofilin are supposed to be engaged in diverse aspects of plant morphogenesis. Finally, rapid rearrangements of F-actin meshworks interconnecting endocellular membranes turn out to be especially important for perception-signaling purposes of plant cells, e.g., in association with guard cell movements, mechano- and gravity-sensing, plant host-pathogen interactions, and wound-healing.

Graviresponse of cress roots under varying gravitational forces.

During the spacelab mission IML-2 threshold values concerning gravity controlled growth processes have been estimated in order to test the reciprocity law (dose = stimulus x time = constant) for the first time under exact physiological conditions. Cress seedlings have been cultivated from dry seeds under conditions of microgravity and on a 1 x g-centrifuge in the ESA-BIORACK. With the help of NIZEMI--the slow rotating centrifuge microscope--these seedlings have been stimulated by different doses ranging from 12 to 60 x g x s. Two different values of acceleration--0.1 x g and 1 x g--have been used. Graviresponses of the roots have been documented by video recording for 60 min under conditions of microgravity. The response of roots to accelerations of 0.1 x g was remarkably less than to 1 x g in spite of the same doses being applied to the seedlings. Roots cultivated under conditions of microgravity showed a higher sensitivity than those grown on the 1 x g-centrifuge. Displacement of statoliths in gravity perceiving cells was mainly less than 1 micron under the different stimulation procedures. These results together with results from former space flights do not confirm the validity of the reciprocity law. They indicate that transformation of the gravistimulus has to occur in close vicinity to the statoliths, probably mediated by the ground cytoplasm and the cytoskeleton suspended therein.

Preservation of viable biological samples for experiments in space laboratories.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Graviorientation in protists and plants.

Gravitaxis, gravikinesis, and gravitropism are different graviresponses found in protists and plants. The phenomena have been intensively studied under variable stimulations ranging from microgravity to hypergravity. A huge amount of information is now available, e.g. about the time course of these events, their adaptation capacity, thresholds, and interaction between gravity and other environmental stimuli. There is growing evidence that a pure physical mechanism can be excluded for orientation of protists in the gravity field. Similarly, a physiological signal transduction chain has been postulated in plants. Current investigations focus on the question whether gravity is perceived by intracellular gravireceptors (e.g. the Muller organelle of the ciliate Loxodes, barium sulfate vacuoles in Chara rhizoids or starch statoliths in higher plants) or whether the whole cell acts as a sedimenting body exerting pressure on the lower membrane. Behavioral studies in density adjusted media, effects of inhibitors of mechano-sensitive ion channels or manipulations of the proposed gravireceptor structures revealed that both mechanisms have been developed in protists and plants. The threshold values for graviresponses indicate that even 10% of the normal gravitational field can be detected, which demands a focusing and amplifying system such as the cytoskeleton and second messengers.

Acrosomal damage and progressive motility of stallion semen forzen by two methods in 0.5-milliliter straws.

Post centrifugation and postthaw progressive motility and postthaw acrosomal damage were used to evaluate two different freezing methods (A and B) on 11 ejaculates of one stallion and 6 of another. In Method A, extended semen was centrifuged in 50-ml centrifugation tubes in which 0.25 ml hyperosmotic, glucose-based medium were layered in the apex of the tubes to serve as a cushion. Spermatozoa were resuspended in freezing medium, packaged in 0.5-ml PVC straws and frozen at -60 degrees C/min. Method B differed only in that the hyperosmotic cushion was omitted during centrifugation. Straws were thawed at 75 degrees C for 6 sec and then transferred into a waterbath at 37 degrees C. The postcentrifugation and postthaw progressive motility and postthaw acrosomal damage were similar for Methods A and B. Method B, however, was found to be technically easier to carry out. It also offers less chance of error or contamination because only two diluents are admixed to the semen as opposed to the three diluents used in Method A. Method B is regarded as more practical and thus preferable to Method A.

Failure of luteolysis leads to prolonged gestation in a bitch: a case report.

A healthy, 5-yr-old rough collie bitch exhibited prolonged gestation, characterized by intrauterine fetal death and plasma progesterone concentrations (PPC) above 6 nmol/L/d until Day 65 or 66 of cytologic diestrus, which then only dropped following treatment with dinoprost tromethamine. The bitch showed no signs of parturition prior to laparotomy and hysterotomy on Day 63, which revealed 5 corpora lutea (CL) on the left ovary and 1 on the right ovary; 5 full-term, dead conceptuses without obvious autolysis but with 4 of the 5 placentas detached; and a tightly closed cervix. All conceptuses were in the left uterine horn. Treatment with dinoprost tromethamine was initiated 1 d after hysterotomy and was continued for IO d (120 microg/kg twice daily for a total dose of 240 microg kg/d). The discharge of lochia through the cervix and lactation started 3 d after the onset of treatment. Plasma progesterone concentration was 14.4, 16.2 and 1.6 nmol/L/d on the day of hysterotomy and at 1 and 3 d after the onset of prostaglandin therapy, respectively. The reason for the failure of luteolysis to occur is not known.

Gravisensitivity of cress roots.

The minimum dose (stimulus x time [gs]) eliciting a visible gravitropic response, has been determined using continuous and intermittent stimulation and two different accelerations at 1 g and 0.l g. The minimum dose of 20-30 gs estimated for microgravity roots and of 50-60 gs for roots grown on a 1 g-centrifuge indicated a higher sensitivity of microgravity roots. Applying intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus free interval of 118 s. The curvature of microgravity-grown roots to lateral stimulation by 0.1 g was remarkably smaller than by 1g in spite of the same doses which were applied to the seedlings. Microscopic investigations corresponding to stimulations in the range of the threshold values, demonstrated small displacement (< 2 micrometers) of statoliths in root statocytes. Accepting the statolith theory, one can conclude that stimulus transformation has to occur in the cytoplasm in close vicinity to the statoliths and that this transformation system was affected during seedling cultivation in microgravity.