The following student awards were presented at the 2003 Congress on In Vitro Biology, Portland, Oregon. Information related to the available specific student awards can be found on the SIVB Website (http://www.sivb.org/meeting_awards.asp) or by contacting the SIVB Business Office at (301) 324-5054, firstname.lastname@example.org, or Dr. Gertrude Buerhing, Chair, Student Affairs & Awards, (510) 642-3870, email@example.com.
Enhancing the Sensitivity of Rainbow Trout Cells in Culture to the Toxicity of Metals
A gill cell line, RTgill-W1, from rainbow trout was used to investigate the toxic actions of 5 metals: Cu, Zn, Cd, Fe and Ni. Cellular viability was measured using three fluorometric assays. These were alamar Blue for metabolic activity, CFDA-AM for membrane integrity and neutral red for lysosomal activity. The toxicity of these metals was affected by exposure medium. Little or no toxicity occurred in fish cell cultures exposed to the metals in basal L-15 medium or in L-15 medium with a fetal bovine serum (FBS) supplement. However, exposure in the minimal medium L-15/ex, which contains the salts, galactose and sodium pyruvate of L-15, resulted in cytotoxicity. In this exposure medium, the rank order of toxicity from least to most toxic was Ni _ Fe _ Cd _ Zn _ Cu. Sensitivity to copper was enhanced by modifying L-15/ex in two different ways. Firstly, toxicity was increased upon removal of pyruvate from the L-15/ex medium. Secondly, a further increase in sensitivity was seen upon treatment with DL-Buthionine-[S,R]-sulfoximine (BSO), which is an inhibitor of gamma-glutamyl cysteine synthetase and decreases glutathione levels. The increased sensitivity depended on the timing of the BSO addition to cell cultures. Cytotoxicity was not enhanced by concurrent exposure of cells to BSO and copper. On the other hand, sensitivity was increased dramatically when the cells were treated with BSO for 24 h followed by the removal of BSO and by the addition of copper. This enhancement of sensitivity opens up the possibility of using fish cells as a bioassay tool to detect toxic metals in industrial effluent.
Vivian Dayeh, University of Waterloo, Department of Biology, Waterloo, Ontario, N2L 3G1, CANADA. In Vitro Cellular and Developmental Biology, 39:5-A, 2003
Expression of a Synthetic Avidin Gene in Maize for Control of Corn Rootworm Diabrotica and Other Insect Pests
Corn rootworms cause millions of dollars worth of damage each year. There are currently two main methods of control that are used: crop rotation and insecticides. Crop rotation works well unless the species of corn rootworm employs extended diapause, which allows the eggs to remain in the soil for two winters and hatch the following spring. The use of insecticides for control creates concerns for environmental safety as well as health safety. Growers are looking for new management options. One potential option is the use of transgenic corn containing a synthetic avidin gene. Avidin, a glycoprotein found in egg whites, has a very high affinity for the vitamin biotin. Several insects including beetles (Coleoptera) and flies (Dipetera) have been reported to show stunted growth and mortality after consuming a diet containing avidin. We have introduced a synthetic avidin gene with maize-preferred codons into maize (HiII background) using three separate plasmids via particle bombardment. The three plasmids include the following: pRUBIAVD -rice ubiquitin promoter, rice chitinase signal sequence and avidin gene, and PinII terminator in pUC18 vector; pACAVD – rice actin promoter, rice chitinase signal sequence and avidin gene, and PinII terminator in pBluescript vector; and pACAVDBAC – rice actin promoter, rice chitinase signal sequence and avidin gene, vacuolar targeting signal and PinII terminator in pBluescript vector. Out of ten separate bombardments, 22 independent events were selected for regeneration. All events were grown to maturity and either selfed or backcrossed. Twenty events were PCR positive for the avidin gene. Further molecular analyses will provide integration and expression information necessary for future bioassay studies.
Serena B. McCoy, Kansas State University, Plant Pathology, 4024 Throckmorton, Manhattan, KS 66506. In Vitro Cellular and Developmental Biology, 39:54-A, 2003
Vaccinium angustifolium Cell Cultures: An Alternative Method of Studying the Anti-Cancer Potential of Wild Blueberries
Vaccinium angustifolium Ation (wild blueberry) fruits have been intensively researched for potential health benefits and are rich sources of antioxidants. They have been shown to contain compounds that inhibit initiation, promotion, and proliferation stages of carcinogenesis, as shown through cycloxygenase, quinone reductase, ornthine decarboxylase, and hepatocyte bioassays, as well as others. Wild blueberry fruit fractions have demonstrated 77% inhibition against cycloxygenase-2, exhibited doubling of quinone reductase activity at 3.5 _g/mL, and provided 94% inhibition in the hepatocyte bioassay. 1H-NMR spectra and thin-layer chromatography indicate that active fractions are often rich in proanthocyanidins and other flavonoids. Blueberry fruits, however, contain many sugars and pectins, which impede the efficiency of extraction and can introduce artifacts in routine bioassays. Plant cell culture is an alternative method for producing phytochemicals without many of the associated interfering compounds, in a highly controlled environment that can be manipulated to influence the types and amounts of active compounds. Wild blueberry suspension cultures were grown in the dark on a 7 day cycle at 25oC on a Gyrotary shaker set at 150 rpm in a solution culture medium. These cultures have been maintained in culture for 1.5 years and were initiated from seedlings germinated in vitro. Spectrophotometric assays compared 70% aqueous acetone extracts of 10 and 14 day old blueberry cultures to determine the harvest date that optimizes production of bioactive proanthocyanidins and other phenolics. Results of the Folin-Denis and the acid-butanol assays, which test for phenolics and proanthocyanidinsrespectively, revealed significantly higher amounts of these compounds in 10 day old cultures compared to 14 day old cultures. Therefore, 10-day-old in vitro cultures provided the best yield of bioactive chemopreventive phytochemicals from wild blueberry germplasm. Cell culture extracts produced in our lab have demonstrated equal or greater potency than fruit extracts in a range of bioassays and permit quantitative assessment of the phytomedicinal value of these natural components.
Tristan F. Kraft Burns, University of Illinois, Natural Resources and Environmental Sciences, 115 Plant Science Laboratory, MC-634, 1201 S. Dorner Drive, Urbana, IL 61801. In Vitro Cellular and Developmental Biology, 39:44-A, 2003
Insulin-like Peptides Stimulate Midgut Stem Cell Proliferation of Lepidopteran Larvae In Vitro
The mechanisms that control the growth rate of internal tissues during postembryonic development are poorly understood in insects. The midgut is the largest organs in lepidopteran larvae, and histologically studied well compared to other tissues. The cell mass increase during molting period, but the factors remodeling the midgut were difficult to study until in vitro system of midgut cells was established. Using the in vitro system, some factors that promote midgut differentiation were isolated from the conditioned medium. However no peptide factors that promote midgut cell proliferation have been reported. Since insulin-like immunohistochemical reaction was observed in the molting midgut, it was expected that an insulin-like peptide might promote cell proliferation in the midgut. Bombyxin is an insulin-like peptide in lepidopteran insects. To clarify the function of bombyxin and the other insulin-like peptides in midgut stem cell proliferation, these peptides were added to the culture of midgut stem cells and the number of the cells were counted. Insulin, IGF-1, IGF-2 and bombyxin stimulated proliferation of midgut stem cells in vitro, the most effective being bombyxin that induced a maximum effect at 10_12M. The highest number of cells was observed 3 days after the addition of bombyxin. Although one time addition of bombyxin could not keep the maximum effect thereafter, the second addition made 3 days after the first addition retained effect. It suggests that the decline of the effect observed was not due to the loss of sensitivity of the cultured cells but the growth factor added to the culture lost the effect.
Shintaro Goto, Kobe University, Grad School of Science and Technology, 1-1 Rokko-daicho, Nadaku, Kobe 657-8501, JAPAN. In Vitro Cellular and Developmental Biology, 39:34-A, 2003
UV and Visible Radiation Effects on Flavonoid Production in Cell Cultures of Vitis vinifera and Glycine max
Thorough analysis of the contribution of biologically-active natural plant compounds in human health maintenance usually requires that individual phytochemicals must be isolated and tested in controlled bioassays. Epidemiological studies as well as laboratory analyses have suggested that bioactive flavonoid compounds in grape (Vitis vinifera), including anthocyanin pigment complexes and proanthocyanidins, have broad therapeutic benefits. Anthocyanins have been linked to anticarcinogenic, anti-inflammatory, antioxidant, and cardioprotective properties, whereas proanthocyanidins share similar beneficial qualities and are closely linked the anthocyanin biosynthetic pathway. Many flavonoids, however, are unstable and/or easily degraded during the process of isolation, and complex multi-unit proanthocyanidin oligomers and polymers of flavan-3-ol units can deteriorate and therefore are difficult to extract and quantify from fruits. Cell cultures, however, can frequently be induced to produce the same range of bioactive phytochemicals, without interferences to extraction/fractionation that are inherent in fruit. In this study, the flavonoid profiles from continuous cell cultures of two grape genotypes, Bailey Alicant (a hybrid) and Merlot, were compared in order to assess their utility resources for phytochemical extraction. Cell cultures were extracted after 6 days liquid suspension medium and extracted in 50% aqueous methanol. The acid butanol assay was used to quantify proanthocyanidins, based on the oxidative cleavage of proanthocyanidin molecules to produce anthocyanidin chromophores. The anthocyanidin abosorbance levels were measured using a spectrophotometer 550 nm). Whereas cultures of Bailey Alicant produced copious yields of both anthocyanins and complex proanthocyanidins, the Merlot cultures produced only proanthocyanidins, which provided an advantage in terms of clean isolation of only proanthocyanidin oligomers for bioassays. Bailey Alicant had a proanthocyanidin concentration of 0.051 mg/L of proanthocyanidin per 0.5 g/mL of extract. Merlot extract (0.5 g/mL) produced 0.057 mg/L of proanthocyanidin exclusively. Further results with biotic and abiotic elicitors, including sodium acetate (NaC2H3O2), are underway to determine the extent to which flavonoid profiles can be skewed to favor production of specific classes of bioflavonoids.
Sinee Pauline Kopsombut, University of Illinois, Natural Resources and Environmental Sciences, 1115 Plant Science Laboratory, 1201 S. Dorner Dr., Urbana, IL 61801. In Vitro Cellular and Developmental Biology 39:44-A, 2003
A Novel Disposable Film Culture Vessel for Photoautotrophic Micropropagation of Epidendrum Orchid
To overcome various disadvantages of conventional culture vessels, Tanaka et al. (1988) first developed a film culture system, the ‘‘Culture Pack’’ (CP) made of fluorocarbon polymer film (Neoflon PFA film), which possesses superior properties such as thermal stability, high light transmittance and gas-permeability. Tanaka et al (1995) later developed the ‘‘Miracle Pack’’ (MP), the practical model of the CP. This MP system is found more suitable for the micropropagation of various plant species when compared to a conventional culture vessel. However, the MP made of PFA film and supported by a polycarbonate frame is still expensive due to the high price of the film and the frame, making it ill-suited for widespread use in commercial plant tissue culture laboratories. In order to reduce the cost of film culture vessels, a multiple-layered super cheap OTP film, made of TPX and CPP, with similar characters as the PFA film has been used to make a novel disposable film culture vessel, namely the ‘‘Vitron’’ (Tanaka et al., 2001). Since the frame of Vitron is made of polypropylene, its cost is remarkably reduced. In this study shoots with three leaves of Epidendrum (E. Rouge Magic x E. Joseph Lii ‘Mother’s Day Koto’) were cultured photoautotrophically under high CO2 conditions at a low photosynthetic photon flux density in three film culture systems: MP using PFA film, MP using OTP film and Vitron, with sugar-free liquid modified Vacin and Went medium and rockwool multiblockTM as substrates. The in vitro and ex vitro growth of Epidendrum plantlets cultured in the three film culture systems were almost equal, producing normal and vigorous plantlets. A 100% survival of plantlets was obtained after transferring to soil without any specialized ex vitro acclimatization treatment. Net photosynthetic rate of in vitro Epidendrum plantlets cultured in the three film culture systems were also equal. This study suggests that the novel Vitron film culture system could be used to replace the conventional culture vessel for photoautotrophic micropropagation of Epidendrum orchids.
Giang Thi Thanh Dam, Kagawa University, Agriculture Faculty, Kitagun, Miki cho, Ikenobe, Kagawa 761-0795, Takamatsu 761-0795, JAPAN. In Vitro Cellular and Developmental Biology 39:20-A, 2003
Esther Uchendu, the 2003 SIVB Philip White Award recipient, attended a workshop at the Kawanda Agricultural Research Institute Tissue Culture Laboratory from 4th August to 27th August 2003 for hands-on training on embryo rescue and culture of Musa spp. Esther works with the Plantain and Banana Improvement Programme, International Institute of Tropical Agriculture, Ibadan, Nigeria and wanted to attend this workshop to enhance her expertise of Musa germplasm.
The KARI tissue culture laboratory is located in Kawanda, Uganda and it is one of the sub-units of the National Agricultural Research Organisation (NARO) of Uganda, which promotes and co-ordinates research in all aspects of crops, fishery, forestry and livestock. In collaboration with NARO, the International Institute of Tropical Agriculture’s East African Regional Centre (IITA-ESARC) carries out research on banana germplasm improvement and breeding. At the KARI tissue culture laboratory, these tissue culture techniques are used: shoot tip culture for the rapid clonal propagation and conservation of Musa germplasm, embryo culture for the germination of hybrid seeds in Musa breeding, culture of immature Musa male flowers and scalps for the regeneration of plants through indirect somatic embryogenesis, and micro-propagation of coffee through direct somatic embryogenesis.
Ms. Uchendu’s training at the KARI tissue culture laboratory was a very successful and she feels that she has acquired knowledge and skills that will equip her to train others in this technique and play a more active role in the enhancement of Musa germplasm.
Esther would like to thank the management of the KARI tissue culture laboratory in Kawanda, Uganda and IITA-ESARC for giving her the opportunity to train with them, and the Society of In Vitro Biology (SIVB) Philip White Memorial Award Committee for helping to fund the workshop.