Development of an in vitro culture technique for conservation of Saccharum spp. hybrid germplasm

An in vitro method for the establishment and storage of over 200 Saccharum spp. hybrid clones was developed that involved only 1 medium for shoot development and multiplication, and no decontamination procedures. Apical buds, from the leaf axils of developing leaves surrounding the apical meristem, were cultured on medium containing the plant growth regulators 6-benzylaminopurine (BAP) and 6-furfurylaminopurine (kinetin), and regenerated multiple shoots. Shoots transferred to medium containing naphthaleneacetic acid (NAA) developed roots. In vitro plants transferred to a medium containing half strength salts and vitamins without plant growth regulators were placed in storage at 18°C. After 12 months of storage plants were transferred to fresh medium and returned to storage. The genetic integrity of clones (based on phenotype assessment) was not affected by the in vitro culture method and up to 14 months of low-maintenance storage conditions. These in vitro plants will be further tested for genetic stability using biochemical and molecular techniques.     

A genomic approach to identify hybrid incompatibility genes

Uncovering the genetic and molecular basis of barriers to gene flow between populations is key to understanding how new species are born. Intrinsic postzygotic reproductive barriers such as hybrid sterility and hybrid inviability are caused by deleterious genetic interactions known as hybrid incompatibilities. The difficulty in identifying these hybrid incompatibility genes remains a rate-limiting step in our understanding of the molecular basis of speciation. We recently described how whole genome sequencing can be applied to identify hybrid incompatibility genes, even from genetically terminal hybrids. Using this approach, we discovered a new hybrid incompatibility gene, gfzf, between Drosophila melanogaster and Drosophila simulans, and found that it plays an essential role in cell cycle regulation. Here, we discuss the history of the hunt for incompatibility genes between these species, discuss the molecular roles of gfzf in cell cycle regulation, and explore how intragenomic conflict drives the evolution of fundamental cellular mechanisms that lead to the developmental arrest of hybrids.     

Ear culture as a technique to overcome hybrid necrosis in wheat

Hybrid necrosis in wheat is a problem for gene transfer in wheat breeding. Hybrid necrosis occurs due to dominant complementary interaction of two genes, Ne1 and Ne2. A cross between wheat (Triticum aestivum L.) varieties C306 (drought tolerant, Ne1 carrier) and WL711(high yielding, Ne2 carrier) produced necrotic F1 hybrids, which died before ear emergence and produced no seeds. To overcome the problem of hybrid necrosis, ears enclosed in the leaf sheath were taken and cultured to maturity in liquid medium containing 5% sucrose and 0.04% glutamine. The necrotic hybrids produced only a few seeds per ear compared to parents, but individual grain weight was similar in the hybrid and the parents. The F1 ear culture study has been repeated for three years and F2 seeds obtained. In 1996–97, the cultured ears of F1 hybrids produced 62 seeds, of which only 52 showed germination and were grown under normal field conditions. Out of the 52 seeds, 50% were non-necrotic and showed segregation for various physiological traits. The results reveal that hybrids ears had the potential to form viable seeds. Culturing of wheat ears before ear emergence and production of viable F2 seeds from necrotic hybrids is a simple and efficient method for overcoming the problem of hybrid necrosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

A new adhesive technique for internal fixation in midfacial surgery

Background  

The current surgical therapy of midfacial fractures involves internal fixation in which bone fragments are fixed in their anatomical positions with osteosynthesis plates and corresponding screws until bone healing is complete. This often causes new fractures to fragile bones while drilling pilot holes or trying to insert screws. The adhesive fixation of osteosynthesis plates using PMMA bone cement could offer a viable alternative for fixing the plates without screws. In order to achieve the adhesive bonding of bone cement to cortical bone in the viscerocranium, an amphiphilic bone bonding agent was created, analogous to the dentin bonding agents currently on the market.     

A saltwater flotation technique to identify unincubated eggs

ABSTRACT.   Field studies on nesting birds sometimes involve questions related to nest initiation dates, length of the incubation period, or changes in parental incubation behavior during various stages of incubation. Some of this information can be best assessed when a nest is discovered before the eggs have undergone any incubation, and this has traditionally been assessed by floating eggs in freshwater. However, because the freshwater method is not particularly accurate in identifying unincubated eggs, we developed a more reliable saltwater flotation method. The saltwater method involves diluting a saturated saltwater solution with freshwater until a salt concentration is reached where unincubated eggs sink to the bottom and incubated eggs float to the surface. For Laughing Gulls ( Leucophaeus atricilla ), floating eggs in freshwater failed to identify 39.0% ( N = 251) of eggs that were subsequently found by candling to have undergone incubation prior to collection. By contrast, in a separate collection of gull eggs, no eggs that passed the saltwater test ( N = 225) were found by a later candling to have been incubated prior to collection. For Double-crested Cormorants ( Phalacrocorax auritus ), floating eggs in freshwater failed to identify 15.6% ( N = 250) of eggs that had undergone incubation prior to collection, whereas in a separate collection, none of the eggs that passed the saltwater test ( N = 85) were found by a later candling to have been incubated prior to collection. Immersion of eggs in saltwater did not affect embryo survival. Although use of the saltwater method is likely limited to colonial species and requires calibrating a saltwater solution, it is a faster and more accurate method of identifying unincubated eggs than the traditional method of floating eggs in freshwater.     

A hybridisation technique to identify anthelmintic resistance genes in Haemonchus

The identification of genes associated with anthelmintic resistance can be facilitated in Haemonchus contortus by the ability of this species to hybridise with Haemonchus placei. Although the hybrid males are sterile, the lines can be rescued by backcrossing the females to either parental species. Resistance genes can be retained in Haemonchus hybrids, while the unwanted contortus background is removed through backcrossing to H. placei and anthelmintic selection of the progeny. Under this selection, genes involved in resistance would retain the H. contortus nucleotide sequence, while those that are not would either be H. placei or a random mixture of both, depending on the amount of backcrossing that had occurred. The first candidate gene to be tested in this system was a Haemonchus P-glycoprotein, hcpgp-1. hcpgp-1 was amplified, cloned and sequenced from H. contortus and H. placei. Two restriction sites were then identified in the sequenced product; one specific to H. contortus hcpgp-1 and the other found only in the H. placei gene. These genes were identified from macrocyclic lactone selected and non-selected worms by restricting PCR products from individual worms. Fitted occurrence of the H. contortus allele was 49% of unselected worms and 69% of macrocyclic lactone selected worms. The probability of this percentage occurring by chance was P=0.006. Thus macrocyclic lactone selection was acting to increase the percentage of hcpgp-1 from macrocyclic-lactone-resistant CAVRS.     

A new technique to reduce internal phosphorus loading by in-lake phosphate fixation in shallow lakes

A new, flexible, fast, robust and economic technique was developed to treat sediment in shallow lakes with phosphate binding chemicals. The upper 0.15 m of the sediment is thoroughly mixed with ferric chloride using a water-jet manifold coupled to a dosing pump and a navigation control system. Its logistics were tried out in a small, shallow and hypertrophic peat lake, Lake Groot Vogelenzang.     

A new resorbable tack fixation technique for endoscopic brow lifts

The endoscopic brow lift is now widely accepted in aesthetic plastic surgery, and various fixation techniques have been described in the literature. New developments and technology have expanded the use of resorbable devices in different surgical specialties, including plastic surgery. The authors present a technique that offers simple, fast, and reliable forehead fixation for endoscopic brow lifts using resorbable tacks. Successful facial rejuvenation was obtained in the majority of the patients without complications, need for follow-up visits to tighten the flap fixation system, or secondary procedures to extract the fixation system.     

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.     

A new technique for the culture of fern gametophytes

Fern gametophytes initiated from spores grew successfully when immobilised in reticulate polyurethane foam in liquid shake cultures. Colonisation of this matrix was complete seven days after medium inoculation and appeared to be a completely passive process. Yield (dry weight) was greater than that from liquid grown cultures.     

丁香（Syringa L.）种间杂交幼胚离体培养研究

采用幼胚离体培养方法克服幼胚败育从而直接获得杂种实生苗,对影响丁香幼胚培养成功的各种因子作了较系统的研究。结果表明,丁香幼胚培养的最适培养基为Monnier;最佳糖浓度为50g·L^-1;幼胚在50-60d胚龄时培养最容易成功;加入适量的椰乳、谷氮酰胺或谷氨酸及活性炭可以促进幼胚的萌发和生长。低浓度（0.01mg·L^-1）BA的加入可以提高幼胚的萌发率;NAA浓度以0.01mg·L^-1为最佳。     

A new technique to correct facial paralysis.

In two cases of unilateral ficial paralysis, the advanced atrophy of the muscles forced us to try a new technique for dynamin reanimation. To our knowledge, this has not yet been reported in the literaturel. The technique combines a free nerve graft across the face with a free muscle graft or a muscle flap. The result in both our cases was good.     

Longitudinal growth of skeletal myotubes in vitro in a new horizontal mechanical cell stimulator

Summary A new computerized mechanical cell stimulator device for tissue cultured cells is described which maintains the cells in a horizontal position during mechanical stretching of up to 400% in substratum length. Mechanical stimulation of myogenic cells in this device initiates several aspects of in vivo skeletal muscle organogenesis not seen in normal static tissue culture environments. Embryonic skeletal muscle cells from avian m. pectoralis are grown in the device attached to the collagen-coated elastic substratum. Dynamic stretching of the substratum in one direction for 3 d at a rate (0.35 mm/h) that simulates in vivo bone elongation during development causes the myoblasts to fuse into parallel arrays of myotubes which are 2 to 4 times longer than myotubes grown under static culture conditions. This longitudinal myotube growth is accompanied by increased rates of cell proliferation and myoblast fusion. Prestretching the collagen-coated substratum before cell plating also results in increased cell proliferation, myotube orientation, and longitudinal myotube growth. The effects of substratum stretching on myogenesis in this model system thus occur by alterations in the cell’s extracellular matrix and not by acting directly on the cells. This work was supported by grant AR36266 from the National Institutes of Health, Bethesda, MD, and research grnat NAG2-414 from the National Aeronautics and Space Administration, Washington, DC. Parts of this work have appeard in abstract form, J. Cell. Biochem. 12C:360; 1988.