Quasi in situ: a bridge between ex situ and in situ conservation of plants

Plant conservation urgently needs a concept that would unify different aspects of population viability as parts of conservation methodology. Such unification is especially lacking for ex situ conservation. We introduce a novel conservation approach in which ex situ collections maintained in natural or semi-natural environment and preserving both neutral and adaptive genetic diversity are a part of a complementary ex situ–in situ conservation strategy. Our approach is the first that explicitly takes into account ecologically significant (i.e. adaptive) variation of plants in both ex situ and in situ conservation actions. Using this approach we provide detailed guidelines for (1) representative sampling of the populations; (2) collection maintenance; and (3) utilization for in situ actions.     

In situ population structure and ex situ representation of the endangered Amur tiger

The Amur tiger ( Panthera tigris altaica ) is a critically endangered felid that suffered a severe demographic contraction in the 1940s. In this study, we sampled 95 individuals collected throughout their native range to investigate questions relative to population genetic structure and demographic history. Additionally, we sampled targeted individuals from the North American ex situ population to assess the genetic representation found in captivity. Population genetic and Bayesian structure analyses clearly identified two populations separated by a development corridor in Russia. Despite their well-documented 20th century decline, we failed to find evidence of a recent population bottleneck, although genetic signatures of a historical contraction were detected. This disparity in signal may be due to several reasons, including historical paucity in population genetic variation associated with postglacial colonization and potential gene flow from a now extirpated Chinese population. Despite conflicting signatures of a bottleneck, our estimates of effective population size ( N _e = 27–35) and N _e /N ratio (0.07–0.054) were substantially lower than the only other values reported for a wild tiger population. Lastly, the extent and distribution of genetic variation in captive and wild populations were similar, yet gene variants persisted ex situ that were lost in situ . Overall, our results indicate the need to secure ecological connectivity between the two Russian populations to minimize loss of genetic diversity and overall susceptibility to stochastic events, and support a previous study suggesting that the captive population may be a reservoir of gene variants lost in situ .     

In situ NMR systems

In situ NMR is becoming an established technology for applications in bioprocessing and metabolic engineering. The in situ NMR biosensor acts as a noninvasive pH, ion, and concentration meter, with 31P and 13C as the two main isotopes of study. A substantial data base now exists for phosphorus and carbon spectra of bacteria and yeast. In situ NMR can provide many of the state variables needed for modeling glycolytic pathway function. NMR micro-reactor technology has improved significantly in the last decade. Several designs for immobilized cell reactors have been tested, and in particular, considerable gains have been made in the feasibility of studying aerobic, chemostat cultures with in situ NMR. Acquisition of 31P spectra from cell suspensions of 3-5% v/v under controlled conditions can be made in 3-7 minute time resolution in several systems.     

In situ chondrocyte viscoelasticity

It has been proposed, based on theoretical considerations, that the strain rate-dependent viscoelastic response of cartilage reduces local tissue and cell deformations during cyclic compressions. However, experimental studies have not addressed the in situ viscoelastic response of chondrocytes under static and dynamic loading conditions. In particular, results obtained from experimental studies using isolated chondrocytes embedded in gel constructs cannot be used to predict the intrinsic viscoelastic responses of chondrocytes in situ or in vivo. Therefore, the purpose of this study was to investigate the viscoelastic response of chondrocytes in their native environment under static and cyclic mechanical compression using a novel in situ experimental approach. Cartilage matrix and chondrocyte recovery in situ following mechanical compressions was highly viscoelastic. The observed in situ behavior was consistent with a previous study on in vivo chondrocyte mechanics which showed that it took 5-7min for chondrocytes to recover shape and volume following virtually instantaneous cell deformations during muscular loading of the knee in live mice. We conclude from these results that the viscoelastic properties of cartilage minimize chondrocyte deformations during cyclic dynamic loading as occurs, for example, in the lower limb joints during locomotion, thereby allowing the cells to reach mechanical and metabolic homeostasis even under highly dynamic loading conditions.     

Non-radioactive in situ hybridization

Summary A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT+CIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetyl-aminofluorene (AAF)) were used. The advantages and dis-advantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.     

Nucleic acidin situ probing

The main principles that underly the use of nucleic acid probes forin situ hybridization are summarized. These include probe design, target preparation, hybridization formats and conditions, and signal generating systems. These principles underly the specific protocol that is described, namely the use of an akaline phosphatase-labeled cloned sequence of the alphoid repeated DNA family as a centomere probe for human chromosomes.     

Morphological and phenological comparisons of two Hopi maize varieties conserved in situ and ex situ

Over the last twenty-five years, crop genetic resources (CGR) have been preserved in genebanks around the world for use by formal plant breeders. Recently conservation of folk crop varieties for direct use by the farmer-breeders of traditional agricultural communities has been suggested as another purpose for CGR conservation. While both in and ex situ CGR conservation programs have been proposed to meet the needs of formal plant breeders and farming communities, the needs and goals of the two groups are different. Formal breeders seek maximum allelic diversity while farmer-breeders are interested in both diversity and population structure that provide local adaptation. Based on the morphological and phenological data analyzed for this study of two Hopi maize varieties conserved in and ex situ, it appears that both genetic shift and genetic drift have occurred ex situ, and that populations conserved ex situ are different from those maintained in situ. These findings suggest that CGR conservation strategies must be re-evaluated in light of the specific conservation goals that are sought.     

In situ phage screening. A method for identification of subnanogram tissue components in situ

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.     

A comparison of in situ and simulated in situ methods for estimating oceanic primary production

Primary production data measured by in situ (IS) and ‘simulated’in situ (SIS) incubations were compared. To minimize differencesbetween the two types of incubations, SIS experiments were conductedin temperature-controlled incubators in which the spectral distributionand irradiance were adjusted to approximate IS conditions. ISavailable irradiance (I_Is) was computed from vertical attenuationof integrated surface irradiance. Vertical attenuation was estimatedusing a spectral irradiance model, validated by measured profilesof the vertical attenuation coefficient. IS incubations werecarried out using two methods. The first involved deploymentof bottles on a drifting array for whole-day (dawn to dusk)incubations. The second method employed an autonomous submersibleincubation device that performed short term (<1 h) incubationsat multiple depths. Differences between whole-day IS and SISincubation estimates were attributed partially to differencesbetween I_IS and SIS-available irradiance (I_SIS). Photosynthesis-irradiance(P-I) properties of IS and SIS populations from the whole-dayincubations were not significantly different. P-I propertiesof the short-term IS and SIS populations were significantlydifferent, although estimates of P^B (mg C mg Chl^–1 h^–1)from contemporaneous IS and SIS incubations did not differ by>40%. Integrated water-column primary production (IPP) estimatedusing P-I models derived from SIS data were within 15% of ISestimates of IPP.     

Measurement of mitochondrial pH in situ

In this article, we describe the advantages and disadvantages of procedures for monitoring mitochondrial pH in situ using optical microscopic techniques. The first method employs the combination of the fluorescent pH-sensitive indicator carboxy-SNARF and laser scanning confocal microscopy. Manipulation of the loading and post-loading conditions enables relatively specific accumulation of carboxy-SNARF into mitochondria. With the use of a mitochondrial-specific marker, mitochondrial pH can be accurately monitored. More recently, mitochondrial-targeted, pH-sensitive probes have been used to monitor mitochondrial pH. In particular, mitochondrial targeting of the yellow fluorescent protein (YFP) mutant of green fluorescent protein (GFP) combines the advantages of specific mitochondrial localization, high-fluorophore quantum yield, and extinction coefficient with an appropriate pKa for measuring mitochondrial pH. The use of dual-excitation ratiometry with mitochondrially targeted YFP increases the dynamic range of mitochondrial pH measurements and corrects for differences in the amount of expression of mitochondrially targeted YFP at the level of individual mitochondria.     

In situ synthesis of protein arrays

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.     

Quantification of mitochondrial morphology in situ

The structural organization of mitochondria reflects their functional status and largely is an index of cell viability. The indirect parameter to assess the functional state of mitochondria in cells is the degree of their fragmentation, i.e., the ratio of long or branched mitochondrial structures to round mitochondria. Such evaluations requires an approach that allows to create an integral pattern of the three-dimensional organization of mitochondrial reticulum using confocal images of mitochondria stained with a fluorescent probe. In the present study, we tested three approaches to analyzing the structural architecture of mitochondria under normal conditions and fission induced by oxidative stress. We revealed that, while the most informative is a three-dimensional reconstruction based on series of confocal images taken along the Z-dimension, with some restrictions it is plausible to use more simple algorithms of analysis, including one that uses unitary twodimensional images. Further improvement of these methods of image analysis will allow more comprehensive study of mitochondrial architecture under normal conditions and different pathological states. It may also provide quantification of a number of mitochondrial parameters determining the morphofunctional state of mitochondria—primarily, their absolute and relative volumes—and give additional information on threedimensional organization of the mitochondrion.