Gyratory shakers are commonly employed to maintain oxygen levels in plant suspension cultures. Growth of plant cells in semi-permeable polypropylene bags is a potential alternative to this technique. Carrot and tobacco cell cultures were grown in stationary autoclavable polypropylene bags (AgriStar, Inc.) and 125 ml Erlenmeyer flasks on a shaker for up to 56 days. Experimental cultures were inoculated from a common source of exponentially growing cells. Measurements of cell growth were determined by measuring absorbance at 550nm and correlated with actual cell counts. The number of cells grown in polypropylene bags increased at a similar rate to cells grown in flasks during the first 35 days. After 35 days, polypropylene bag cell cultures out-performed flask grown cultures. Flask grown cultures were highly pigmented, an indication of culture senescence.
Daniel C. Wright and Linda Franzen
Kenneth W. Mudge and Chin-Chang Chu
In vitro asymbiotic seed germination, subculture, and outplanting of orchids is presented as a laboratory exercise suitable for students of plant propagation or tissue culture. Dendrobium antennatum (Lindley), Phalaenopsis (Blume) white hybrid, or both, are used in this exercise because they flower predictably in the greenhouse, are reliable for seed production, and germinate and grow rapidly in vitro. The exercises can be used to instruct students in the skills involved in orchid seed sterilization, sowing, and culture, as well as instruct students in the unique features of orchid reproductive biology and symbiosis. A schedule is suggested for stock plant flower pollination, capsule harvest, seed sowing, and seedling subculture so that the necessary plant material is available for students to sow, subculture, and outplant seedlings during a single laboratory session.
Enio Tiago de Oliveira, Otto Jesu Crocomo, Tatiana Bistaco Farinha, and Luiz Antônio Gallo
disinfection. Shoot proliferation. After 30 d in the SPM medium, the best 136 green apical shoot explants bearing axillary buds (GAS) were selected, longitudinally divided, and transferred from the test tubes to 350-mL glass flasks containing 50 mL of the same
Wei-Ling Guo, Yao-Chien Alex Chang, and Chien-Yuan Kao
dissected longitudinally. The seeds were spread on the surface of autoclaved basal culture medium in a 500-mL flask. The medium consists of 100 mL of Murashige and Skoog (MS) salts ( Murashige and Skoog, 1962 ) with 2 g·L −1 Bacto-tryptone (BD Biosciences
Teryl R. Roper and Armand R. Krueger
Cranberry plants exclusively utilize ammonium forms of nitrogen. Nitrification of applied ammonium and subsequent leaching through sandy soils is a potential problem for growers. Peat, sand, and striped soils were collected in cranberry beds in central Wisconsin and soil pH was adjusted to 3.5, 4.5, or 5.5. Twenty-five grams of dry soil was placed in flasks and half the flasks were sterilized. Distilled water was added to half of the samples, and the other half received 15N-labeled ammonium. Flasks were incubated at 20°C for up to 70 days. Striped soils showed no nitrification at pH 3.5 or 4.5 during the 70 day incubation. At pH 5.5, nitrification began at 20 days and was almost complete at 70 days. Nitrification did not occur at any pH in sandy soils. This research suggests that ammonium fertilizer applied to cranberry is likely taken up before nitrification would occur.
Rochele C. Strachan, E.G. Rhoden, and G. W. Carver
Growing crops using poor quality Later can result in poor germination and seedling survival. Low germination rates of various crops in the Bahamas result from the high salinity of the irrigation water. This study investigated the effects of using varying levels of sea water on germination and imbibition rates of lupine (Lupinus albus) seeds. In separate completely randomized design experiments, 100 lupine seeds were placed in conical flasks and either de ionized distilled water (DDW). 100%, 75%, 50% or 25% sea water added to each flask. Seeds ware removed from each flask every hour for the first 8 hours and every six hours thereafter for 48 hours. lmbibition rate is expressed as mg/ghr using the formula: (original weight - weight at y hr) × 1000)/(original weight x y hrs). Germination of seeds was measured beginning 3 days after imbibition began and the experiments were terminated after 10 days. The highest rate of imbibition (178.8 mg/g/hr) was recorded for lupine seeds placed in 25% sea water and the lowest of 152.8 mg/g/hr for seeds placed in 100% sea water after two hours. Germination ranged between 49% (100% sea water) to 94.7% for seeds placed in DDW. It would appear that if lupine seeds were primed with 25% sea water (approximately 150 ppm, NaCI) there would be no significant reduction in either the imbibition or the germination rates.
Whei-Lan Teng, Yann-Jiun Liu, Yu-Ching Tsai, and Tai-Sen Soong
A bioreactor was used to establish a scale-up system for somatic embryogenesis in `Scarlet' carrot (Daucus carota L.). At a cell density of 1–2 × 106 cells/ml, mature and germinating embryos could be observed within 4 to 5 weeks. As cell density exceeded 2 × 106 cells/ml, the culture turned darker yellow, and embryo development was inhibited. Cell densities below 106 cells/ml resulted in abnormal embryos. Bioreactor design had a critical impact on somatic embryogenesis due to various types and the strength of shear forces generated. In this study, an air-lift bioreactor was selected from three different types (spinner flask, screen column bioreactor, and air lift) because it resulted in the highest biomass production and somatic embryogenesis. Foaming was eliminated by preculture of embryogenic cells in flasks; cells were then sieved on a 60-μm polyester screen and thoroughly rinsed with distilled water before being transferred to the bioreactor. Such preculture for at least 10 days significantly increased the regeneration of somatic embryos. During somatic embryogenesis, dissolved O2 concentrations decreased to 33% of saturation, and then increased up to 80% when embryo development approached maturity and mature embryos germinated. Bioreactor-cultured embryos germinated with relatively short cotyledons and long roots, whereas flask-cultured embryos germinated with relatively long cotyledons and short roots.
Enaksha R. Wickremesinhe and Richard N. Arteca
Cephalotaxus harringtonia plants produce alkaloid compounds possessing antitumor properties, the major one being homoharringtonine. The purpose of this study was to produce roots from callus cultures developed earlier. Fast growing callus cultures were placed on MS basal salt medium with B-5 vitamins, 2% sucrose, 10 μM kinetin, 0.45 μM 2,4-D and 0.2% Gelrite. Upon subculture onto basal medium without hormones, we observed organogenesis of both shoots and roots. Roots were excised and established on basal medium without hormones. By subculturing two 2-inch root tips containing numerous visible laterals in liquid medium we were able to harvest 30 g of roots/250 ml flask after 3 weeks and 50 g/250 ml flask after 6 weeks. A 20-fold increase in fresh weight was achieved within 3 weeks when 15 grams of roots were initially seeded into a 3 liter air-sparged bioreactor. However, most of these roots appeared to be fleshy/swollen while root cultures established from half inch root tips grew slower but were normal in appearance. We arc currently in the process of establishing growth characteristics for these roots and assaying roots for the presence of these alkaloids.
Rosilene Barbosa de Franca, Gerson Renan de Luces Fortes, and Adriano Nunes Nesi
The aim of this work was to evaluate the effect of sucrose on the in vitro muliplication of potato, cultivars Baronesa, Macaca, and Cristal. The nutrient medium used was the MS basal salts and vitamins added to 100 mg·L-1 myo-inositol. Four sucrose concentrations (20, 30, 40, and 50 g·L-1) were tested. The pH was adjusted to 5.9 before autoclaving. Each treatment had 15 explants, which were collected from the lower part of the shoot containing two buds. This material was inoculated in a 250-mL flask with 40 mL of nutrient medium. After inoculation the flasks were kept in a growth room under 25 ± 2 °C, 16-h photoperiod, and 19 μMol·m-2·s-1 radiation provided by cool-white fluorescent lamps for 30 days. This trial was designed in a randomized block with three replicates. Every 7 days, the parameters were collected as follows: number of buds, shoot length and number of shoots. It was observed that `Baronesa' presented the highest number of buds and rate of multiplication. `Cristal' had a slightly better performance for these parameters. Plants treated with sucrose at 50 g·L-1 led to a higher number of shoots. However, `Macaca' treated with sucrose at 40 g·L-1 had the highest shoot length.
Gerson R. de L. Fortes, Rosilene França, and Adriana C. M. Dantas
This work was carried out in the Tissue Culture Laboratory of Embrapa Temperate Climate aiming to maximize the protocol for in vitro culture of potato cv. Baronesa. The treatments consisted of multiplication of microcuttings with one, two, or three buds with/without leaves and originated from different regions of the shoot: apical, middle, or basal. Each treatment was repeated five times with each replication composed of five explants that were inoculated in 250-ml flasks with 40 ml of the medium containing MS salts and vitamins added to: sucrose (30 g·L-1), myo-inositol (100 mg·L-1), agar (6 g·L-1). The pH was adjusted to 5.6 before autoclaving. After inoculation, the flasks remained in a growth room at 25 ± 2 °C, 16-h photoperiod, and 19 μmol·m-2·s-1 light intensity provided by cool-white fluorescents lamps. Observations were done every 5 days. Final evaluation was performed after 30 days. It was observed that basal microcuttings provided longer shoots and that microcuttings with leaves bore the best ones. This kind of explant also favored a higher number of shoots, axilary buds, and better multiplication rate. The presence of leaves in the microcutting is important when basal explants are used once it can improve the number of axillary buds and the rate of multiplication. The higher the number of buds in the microcutting the lower the rate of multiplication. The in vitro multiplication of potato could be improved by using one-leaf bud basal microcutting.