Improved Survival of in Vitro-stored Rubus Germplasm

Medium-term in vitro cold storage of Rubus germplasm was investigated using various temperatures, photoperiods, and storage containers. Shoot cultures of several Rubus taxa were grown either in tissue-culture hags or 20 × 150-mm glass tubes. Cultures stored at 10C in darkness were in poor condition after 6 months. Overall survival and condition ratings were significantly better for bags than tubes when cultures were kept at 4C. Contamination was present in 14% of the tubes, but only 3% of the bags. Addition of a 12-hour photoperiod to 4C storage significantly improved both condition ratings and survival percentages of many individual genotypes. Evaluation of the 250-accession germplasm collection after 12 months at 4C (dark) showed 92% of accessions in bags and 85% in tubes in suitable condition to remain in storage. Storage of cold-sensitive genotypes in tissue-culture bags at 25C with a 16-hour daylength was extended to 9 months when the MS-medium nitrogen level was reduced to 25% of standard concentration. Survival of `Mandarin' raspberry stored for 9 months improved from 40% at 4C (100% N) to 90% at 25C (25% N). Results of these studies suggest that most Rubus germplasm can be stored safely at 4C with 12 hours of light. Plastic tissue-culture bags are preferred over tubes due to higher survival and lower contamination rates. Storage at 25C on reduced-nitrogen medium is an alternative method for cold-sensitive genotypes.

COLD STORAGE OF IN VITRO RUBUS GERMPLASM

In vitro cold storage of Rubus germplasm was investigated using several environmental conditons and types of storage containers. Shoot cultures of Rubus species and cultivars were grown in either tissue culture bags or 20 × 150 mm glass tubes and compared for plant condition and survival under various storage conditions. Cultures stored at 10 C in the dark were in poor condition after 6 months. Cultures kept at 4 C were in much better condition and had higher survival rates after 18 months when stored with a 12 h daylength rather than total darkness. Overall there were no differences in survival or condition between cultures in tubes and bags. Contamination rates were 15% in tubes and 0% in bags. Plants in tissue culture bags could be stored for 9 months at 25 C with 16 h light when the nitrogen level of the MS medium was reduced to 25% and the medium volume was increased from 10 to 20 ml per bag. Genotype differences were apparent under all conditions tested. The best storage condition for Rubus germplasm was 4 C with 12 h light. Plastic tissue culture bags were preferred over tubes due to lower contamination rates.

Viremic Phase in a Leukemic Child After Live Varicella Vaccination

Live varicella vaccine has been shown to be efficacious for prevention of varicella even in immunocompromised children.1-4 Although the site and extent of viral replication in vaccinated hosts have not been fully defined, vaccinees have developed humoral and cellular immunity.1-5 A viremic phase has been demonstrated in immunocompromised and normal children with natural varicella.6,7 However, a viremic phase has not been reported in vaccinated children. We report our observation of viremia in a leukemic child in whom vesicles and hepatitis developed after live varicella vaccination. MATERIALS AND METHODS Human embryonic lung fibroblasts (HEFs) were used for virus isolation. The cells were grown in 25-cm2 plastic tissue culture flasks containing Eagle's minimal essential medium with 7.5% fetal bovine serum.

Dexamethasone accelerates differentiation of A6 epithelia and increases response to vasopressin

When seeded heavily on a porous tissue culture dish, A6 cells, derived from the kidney of Xenopus laevis, form a highly differentiated epithelium within 4-6 days. When dexamethasone is added to the culture medium, morphological differentiation is completed by day 2, a time at which the control (untreated) is still a disorganized multilayer of cells. In addition to the morphologically evident monolayer of cuboidal cells, the accelerated differentiation is expressed as high transepithelial electrical resistance, short-circuit current, and adenylate cyclase response to vasopressin. When grown on impermeable plastic tissue culture dishes, A6 epithelia are less differentiated and do not respond to vasopressin. With the addition of dexamethasone at the time of seeding on a plastic tissue culture dish, vasopressin responsive adenylate cyclase activity is expressed, albeit at a slower rate than when grown on a porous surface. In addition, dexamethasone treatment of mature epithelia grown on a porous surface results, in hours, in an increase in the adenylate cyclase response to vasopressin. These results reveal two previously unrecognized interactions between adrenal steroid hormones and vasopressin, namely, accelerated differentiation and increased responsiveness of adenylate cyclase.

Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.

Transient correction of genetic defects in cultured animal cells by introduction of functional proteins

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.

Porous-bottom dishes for culture of polarized cells

Porous-bottom dishes offer several advantages for growing and studying epithelia in culture. Many epithelia differentiate more on porous surfaces than on plastic tissue culture dishes. In addition, separate solutions can be maintained on each side of the epithelium and can be sampled easily for studies of transport and other polarized functions. We describe the fabrication of dishes with a cellulose ester filter, a collagen-coated polycarbonate filter, or a collagen membrane forming the surface for cell attachment at the bottom of the dish.