This investigation documents the key anatomical features in embryo development of Cypripedium formosanum Hayata, in association with the ability of embryos to germinate in vitro, and examines the effects of culture media and seed pretreatments on seed germination. A better understanding of zygotic embryogenesis for the Cypripedium L. species would provide insights into subsequent germination events and aid in the in vitro propagation of these endangered species. In seeds collected at 60 days after pollination (DAP), soon after fertilization, no germination was recorded. The best overall germination was found at 90 DAP (≈70%), at which time early globular to globular embryos with a single-celled suspensors can be observed. After 135 DAP, the seeds germinated poorly. At this time the inner integument shrinks and forms a tight layer, which encloses the embryo, the so-called “carapace.” Using Nile red stain, a cuticular substance was detected in the carapace, which may play a role in the impermeability of the mature seed and may help the seeds survive in the stringent environment. At maturity (after 210 DAP), the embryo proper has an average size of eight cells along its length and six cells across the width. Lipids and proteins are the main storage products within the embryo. To improve seed germination, experiments were conducted to test the suitability of various media and pretreatments of seeds. When different media were used, except for the Harvais medium at 120 DAP, there was no significant difference in seed germination at three different developmental stages tested. Soaking mature seeds in 1% NaOCl or treating them with ultrasound may slightly increase the germination percentage. For seed germination, our results indicate that the timing of seed collection outweighs the composition of medium and the seed pretreatments.
The sporadic nature of inflorescence production and flower protogyny in caladium (Caladium ×hortulanum Birdsey) makes it desirable to store pollen and to rapidly assess its viability for cross-pollinations in breeding programs. This study was conducted to develop a procedure to determine caladium pollen viability and to use that procedure to evaluate the effect of short-term storage conditions on pollen viability. The sucrose level in the culture medium was found to have a significant impact on the in vitro germination of caladium pollen; a concentration of 6.8% was determined to be optimal for pollen germination. Caladium pollen lost viability within 1 day under room (24 °C) or freezing (-20 °C) temperatures, but could be stored at 4 °C for 2 to 4 days. Pollen stored at 4 °C produced successful pollinations. Data obtained from large-scale greenhouse pollinations supported use of this in vitro germination assay as a convenient way to evaluate caladium pollen viability (and fertility).
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.
Previous greenhouse studies in Raleigh have shown that nighttime cooling increases tomato fruit weights from 11% to 53%, depending on planting dates. The physiological mechanism was unclear, except that temperatures during fruitset were most critical We report here on a phytotron experiment comparing pollen characteristics and in vitro pollen germination of plants grown at night temperatures of 18, 22,24 or 26°C in a 12-hour photoperiod with 26°C day temperature in all treatments. There was considerable variability between sampling dates in pollen characteristics and % germination. The most consistent and significant effects were a decrease in total pollen and an increase in % abnormal pollen at high night temperatures. Number of seed present in the fruit also decreased with increasing night temperatures, indicating that the changes in pollen characteristics adversely affected seedset. Night temperatures of 22C appeared optimal for many of the pollen and growth characteristics measured, but fruit developed most rapidly at the higher night temperatures.
Noni, Morinda citrifolia, is receiving a lot of attention for its potential medicinal effects. Hawaii is an ideal growing environment for this plant, where it has been used for many purposes, including medicinal ones, by ancient Polynesians. Currently, there is a rapidly developing noni industry in the state of Hawaii. Propagation of this plant is almost exclusively by seeds, and germination generally requires a couple of months without preconditioning or about a month if mechanically scarified. We developed an in vitro protocol that significantly improves percent germination rate by altering incubation temperature and the in vitro culture basal medium. Germination time was decreased to 4 days when the embryo was extracted and exposed to 31 °C. A basal medium containing 1/2 Murashige and Skoog (M&S) salts was the most effective in reducing germination time and increasing percent germination. Stem pieces obtained from in vitro-propagated seedlings produced callus when explanted in 1/2 M&S containing various levels of naphthalene acetic acid (NAA). The most effective treatment was 0.5 μm NAA and the least effective treatment was 2 μm NAA. Treatments without NAA did not produce callus. Calli treated with 4.40 μm 6-benzylaminopurine (BA) or 8.80 μm BA were the most effective in promoting caulogenesis. We also demonstrated that the number of first generation seedlings produced from each embryo could be increased by treatment with 8.80 μm BA.
Tillandsia eizii is an epiphytic bromeliad that due to over-collection, habitat destruction, and physiological constraints has declined to near threatened status. This species exhibits high mortality in the wild, and seed are characterized by low percentages of germination. As a means to conserve this species, in vitro culture protocols were developed to enhance seed germination and seedling growth. A sterilization protocol using 70% ethanol for 2 minutes followed by 2.6% NaOCl for 40 minutes disinfested seed and promoted seedling growth. Sucrose incorporated into the culture medium had no effect on germination or growth, while NAA inhibited growth, but not germination. Cultures maintained under a 16-hour photoperiod at 22 °C exhibited greater growth than those grown at 30 °C. Seed that germinated in the dark remained etiolated and failed to develop even after transfer to light conditions. Plants grown in vitro were successfully acclimatized and transferred to the greenhouse. Over 86% survival and rapid growth were obtained with either an all-pine-bark medium, or a mixture of 2 redwood bark: 2 fir bark: 2 potting mix: 1 perlite. This demonstrated that in vitro culture of seed may be used to rapidly produce large numbers of T. eizii, and thus can be used for the conservation and reintroduction of this species.
The goal of this study was to expedite galax seed germination in vitro. Galax seeds were collected from Yancey County, N.C., at an elevation of about 1100 m. Aseptic cultures were established using the tiny rust-colored seeds. In vitro seed germination was achieved under different pH conditions (4.2, 5.0, and 5.8). Seeds cultured in the medium with pH 4.2 tended to germinate early with a better rate than those cultured with a higher pH of 5.0 or 5.8 at the very beginning. Gradually, seeds from media with pH 5.0 and 5.8 caught up in germination. Eventually, seeds from all pH treatments produced a very similar germination rate. Attempts to use the matted and scaly rhizomes and very tender new growth as explant materials to establish aseptic cultures were not successful, due to severe contamination. However, our observations suggested that the very tender new growth could be a good source of explants once the optimum sterilization time is established.
design, consisting of three replications of a 26 × 5 factorial arrangement with cultivars and salinity levels, respectively. Table 1. Predicted salinity levels to reduce 10%, 25%, or 50% final germination rate in 26 commercial creeping bentgrass cultivars
masculinized female hemp genotypes. Materials and Methods In vitro pollen germination was evaluated for five masculinized female hemp genotypes, Abacus, Cherry Wine, Mountain Mango, Wife, and Youngsim10, and two male hemp genotypes, Kentucky Sunshine and
Pollen germination timing has a paramount role in fertilization of a flower. Rapid germination and outgrowth of a pollen tube that penetrates the stigma is required. Physical and biological factors can affect pollen germination timing. The objective of this study was to determine if ACC oxidase antisense gene expression could influence in vitro pollen germination and in vitro pollen tube length growth. A transgenic (ACC oxidase antisense) `Galia' male parental line had a reduced fruit set compared to its wild type. Likewise, embryo abortion and empty seeds after self-pollination in a `Galia' male parental line were observed. Wild type and transgenic `Galia' male parental line melon plants were grown in a greenhouse according to the practices of Rodriguez (2003). Male flowers were collected from these plants between 10 to 12 am; pollen was obtained by dipping the anther in germination medium (10.25% sucrose, 0.031% calcium nitrate, 0.015% boric acid, 0.0075% KNO3, and 0.016% MgSO4) at 25 °C and analyzed immediately, either for total percentage of germination after 5 minutes of incubation or to measure pollen tube growth rate every 5 minutes during 1 hour. Each flower provided an average of 250 pollen grains. Assays were conducted by using the “Hanging Drop Method” (Okay and Ayfer, 1994). Percentage of pollen germination in WT `Galia' male parental line was greater than the transgenic line. Likewise, in vitro pollen tube growth in wild type `Galia' melon was greater than pollen from the transgenic line. Possibly the ACC oxidase antisense gene expression in `Galia' male parental line may have had an influence on the reduced fruit set observed.