Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

The role of ethylene and reducing agents on anther culture response of tetraploid potato (Solanum tuberosum L.)

Summary The role of ethylene in embryogenesis of cultured potato anthers was studied indirectly by testing various substances known to affect ethylene formation. The reducing agents ascorbic acid and L-cysteine prevented browning of anther cultures and significantly stimulated embryogenesis. Embryogenesis was also promoted by the use of the ethylene inhibitors AgNO₃ and n-propyl-gallate and by the polyamines spermidine and putrescine. The use of the ethylene releasing compound ethrel significantly inhibited embryogenesis.Abbreviations MS Murashige & Skoog - PVP polyvinylpyrrolidone - MW molecular weight - ACC 1-aminocyclopropane-1-carboxylic acid - ethrel 2-chloroethylphosphonic acid (ethephon)     

Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Associative N2-fixation in plants growing in saline sodic soils and its relative quantification based on15N natural abundance

The interactive effect of low P supply (0, 10, 20 and 40 M) and plant age on nodule number, mass and functioning (ureide analysis technique), vegetative growth and pod production were investigated in glasshouse-grown nodulated cowpea (Vigna unguiculata L.cv. Kausband) in sand culture. Compared with 40 M P, P stress (0 M P) or very low (10 M P) supply markedly impaired nodulation, allantoin and amino-N concentrations and weight of N solutes in xylem exudates. Consequently, P stress reduced top growth and pod yields by 48 and 90%, respectively. N solutes in xylem exudates and total plant N assayed by Kjeldahl technique (as estimates of N₂ fixation) responded similarly to P supply. However, the relative ureide index [(ureide-N/ureide N+amino-N)×100] remained constant (99%), irrespective of P supply, indicating the plants' complete dependency on symbiosis for growth, without implying that growth was markedly increased by N₂ fixation. Although P concentrations in plant tops, roots and nodules increased with P supply, N concentrations in these plant tissues were unaffected by P supply. The concentrations of N and P in the nodules were 2–2 1/2 times higher than in plant tops. P application interacted strongly with plant age, with the largest P effect evidently achieved at the early podding stage. The significance and implications of these results are discussed.     

Calcium (45Ca) accumulation and transport in chicory (Cichorium intybus L.) root during bud development (forcing)

The lower part (4 cm) of the witloof chicory tap-root (15 cm) was immersed in a complete nutrient solution for 21 days, in the darkness at 18°C and at high RH. This process of forcing which leads to the emergence of an etiolated bud (chicon) was associated with a decrease in root dry weight. Although the amount of calcium in the root and the root cationic exchange capacity remained constant during forcing, the net uptake of calcium, negligible at the onset of forcing, progressively increased to a rate after ten days of 45 mol day^–1. Absorption of ⁴⁵Ca remained at a constant high rate, while the initially low upward migration of ⁴⁵Ca within the root and the chicon accelerated markedly. This upward migration was associated with a progressive decline in the release of newly absorbed ⁴⁵Ca. The data support the hypothesis that calcium acquisition by witloof chicory root is predominantly determined by calcium efflux. As the forcing progressed, the influx remained almost constant while a large decrease in the efflux led to a net uptake of calcium. Upward translocation was probably linked to the formation of new negative exchange sites within the growing chicon. The hypothesis that calcium movement occurred along a preferential pathway (xylem vessels) or involved a mass movement through the root is discussed.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

In vitro micropropagation and plant establishment of muscadine grape cultivars (Vitis rotundifolia)

Shoot apical meristems were used to establish regenerative axillary bud cultures of 9 muscadine grape cultivars. Meristems taken from 10 cm long shoots had less contamination (3%) and a higher survival rate (94%) than those from shorter or longer shoots. Of media tested, MS, 1/2 MS, and C₂D resulted in equivalent shoot proliferation rates, whereas, WPM produced stunted shoots. When pooling results for 3 cultivars, 5, 10 and 20 M BA and 5 M TDZ produced the highest average number of shoots per cultured apex (3.4–3.8). However, shoots produced with TDZ were stunted and did not root well. For rooting of shoots directly in potting mix, a rooting powder pretreatment significantly increased the number of roots per shoot but did not affect percent rooting or root length. For rooting in vitro, 1 M NAA significantly increased all parameters measured. Although more shoots rooted in vitro than in vivo (77% vs. 46%), the latter was judged preferable since acclimatized plants were produced in less time and a major culture step was eliminated. Significant differences among cultivars were noted for measured responses in all experiments.Abbreviations BA benzyladenine - Kin kinetin - MS Murashige & Skoog (medium) - NAA naphthaleneacetic acid - TDZ thidiazuron - WPM woody plant medium     

The frequency of marker changes in sugarcane plants regenerated from callus culture

This study investigates the frequency of apparent and permanent expression of marker change following two types of tissue culture, conventional callus and direct regeneration cultures, and for two markers it relates this frequency to that following breeding. Each clone was used for only one marker. After conventional callus culture, plants of the sugarcane clone Arundoid B, a clone having a growth habit with shortened internodes and leaves, were freed of this marker at a rate of 1 in 172 plants. Marker remission in a second clone with a leaf blotch was enhanced in the presence of a mutagen. Callus culture alone gave a remission rate of 1/280 plants, while treatment of callus with ethyl methanesulfonate gave a remission rate of 1/42 plants. Of two markers subjected to vegetative and sexual transmission, the first, a leaf marker, was stable in callus culture with no remissions; crossing with non-marker parents produced progeny with 54% lacking the marker. The second, a stalk marker (multibud), showed epigenetic effects during two generations of vegetative propagation; plants lacking the multibud marker produced vegetative progeny in which the marker reappeared. Nine crosses to nonmarker parents produced progeny of which an average of 29% had the marker. The use of stalk chimeras as markers demonstrated that passage through conventional callus or direct regeneration culture resulted in the loss of the donor phenotype in all plants regenerated. Phenotypic variation in plants derived from callus culture appears to arise from several sources; chimeral segregants, epigenetic transients, and mutational variants.