The following student awards were presented at the 2005 In Vitro Biology Meeting, Baltimore, Maryland. Information related to the available specific student awards can be found on the SIVB Website (www.sivb.net) or by contacting the SIVB Business Office at (919) 420-7940, email@example.com, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at (508) 831-5196, email: firstname.lastname@example.org.
Production of a Marker-free Wheat (Triticum aestivum) Plants Transformed by Agrobacterium
Wheat (Triticum aestivum) is an important food crop, because of both the nutritional value of its seeds and the unique technological properties of flours prepared from those seeds. The high-molecular-weight glutenin subunits (HMW-GS), members of a family of seed storage proteins synthesized in developing wheat endosperm, are important determinants of the processing characteristic of wheat flours, for this reason, members of this gene family are important candidates for genetic engineering. Selectable marker genes are required to ensure the efficient genetic modification of crops. But agro-economic interests have prompted the development of several strategies to eliminate these genes from the genome after they have fulfilled their purpose. Recently, chemical-inducible Cre/loxP DNA recombination system (CLX) (1) have emerged and seem to provide a highly reliable method to generate marker-free transgenic Arabidopsis plants after transformation. We have used Agrobacterium strain AGL1 harbouring, as control plasmid, pX6-GFP, which contains the CLX system, nptII and GFP. Then, we have used the same strain harbouring the pX6-GFP based plasmid pX6-Glu, which contains also the gene Dx5b (coding for a HMW-GS). Till now, we have obtained 3 transgenic lines (cv. Bobwhite) transformed with the plasmid pX6GFP and 1 with PX6Glu, with a transformation frequency of 3.3-3.4% demonstrated by marker-gene expression and molecular analysis. We are performing chemical-induction tests on the immature embryos from these lines to obtain complete marker free plants: those transformed with pX6GFP expressing GFP and those transformed with pX6Glu expressing an increased amount of HMW-GS.
1. Zuo, J., Niu, Q.-W., Moller, S.G. Chua, N.-H. Chemical-regulated, site specific DNA excision in transgenic plants. Nat. Biotecnol. 19, 157-161 (2001).
Manuela Campa- University of Insubria, Dipartimento ambiente salute sicurezza (DAAS), Via J.H. Dunant 3, Varese 21100, ITALY. In Vitro Cellular and Developmental Biology, 41:52-A, 2005
In vitro cultures of Pueraria lobata (kudzu) were evaluated as a source of isoflavone C-glycosides, which have superior stability relative to isoflavone O-glycosides, and therefore have potential benefits for glucose homeostasis. Sterile P. lobata plantlets were grown in vitro, and leaf- and root-derived solid callus cultures were generated on a B5 medium (supplemented with 1 mg/ml 2,4-D, 1 mg/ml NAA, and 0.5 mg/ml kinetin). The predominant isoflavones accumulated in both callus and cell suspension cultures were puerarin, daidzin, malonyl daidzin, and malonyl genistein. Greater levels of these isoflavones were found in root-derived callus (20.68 mg/g total isoflavones) as compared to leaf-derived callus (3.11 mg/g), and suspension cultures accumulated greater amounts of isoflavones per g cells than the callus. Root-derived suspension cultures accumulated 32.88 mg/g total isoflavones, and leaf-derived suspension cultures had 15.94 mg/g total isoflavones. Untransformed root cultures developed from roots of in-vitro P. lobata plantlets, grown in liquid MS medium (supplemented with 1 mg/ml NAA), produced significantly higher amounts of puerarin and daidzin than the root-derived suspension cultures (14.25 mg/g puerarin in root cultures vs. 1.57 mg/g puerarin in root-derived suspension cultures; 8.17 mg/g vs. 4.90 mg/g for daidzin), but had similar levels of total isoflavones (29.91 mg/g total isoflavones in root cultures). Analysis of root cultures by LC-MS and MS/MS revealed the presence of several novel isoflavone C-glycosides, which may be intermediates in the pathway of isoflavone synthesis. Root-derived P. lobata cell suspension cultures were elicited at day 5 after subculture for 7d with the abiotic elicitor KCN (0.1 micromolar, 1.0 micromolar) to test its effects on isoflavone production, however, elicitation with KCN resulted in no difference in isoflavone production compared to the control.
Nelson Adam Reppert, University of Illinois Urbana – Champaign, College of Natural Resources and Environmental Sciences, 1115 Plant Sciences Lab., 1201 S. Dorner Dr., Urbana, IL 61801. In Vitro Cellular and Developmental Biology, 41:40-A, 2005
Cell cultures from red clover (Trifolium pratense) were investigated as a source for biologically active isoflavones, which have potential benefits for bone density, cardiovascular health, and cancer prevention. In nature, variable levels of isoflavones are produced in certain tissues and in response to growing conditions. Red clover seeds were surface sterilized and 21 d old in vitro germinated seedlings were used as a source of vegetative explants. Leaf, root, and petiole explants were used to initiate callus on Gamborg B5 medium with 2 mg/L NAA, 2.25-mg/L 2,4-D, and 2.12-mg/L kinetin. Callus cultures were maintained by subculturing at 4 wk intervals and then used to induce suspension cultures that were subcultured at 2 wk intervals. Both callus and suspension cultures were grown in darkness or light (100 mol/m2/s). Cells were subsequently harvested, extracted with methanol, and analyzed for isoflavone content using LC-MS and MS/MS. Leaf tissue explants were the most prolific source of callus cells, whereas solution cultures produced the most biomass when root explants were the donor tissue for callus. The predominant isoflavones recovered were formononetin, biochanin A, daidzein, and genistein. On average, 10-fold higher yields of formononetin [0.67 mg/g fresh mass (FM)] and biochanin A (0.13 mg/g FM) were found in petiole-derived solution cultures maintained in darkness.
Nancy Engelmann, University of Illinois – Urbana Champaign, College of Natural Resources and Environmental Sciences, 1024 Plant Sciences Laboratory, 1201 S. Dorner Dr., Urbana IL 61801. In Vitro Cellular and Developmental Biology, 41:40-A, 2005
While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes eliminating the threat of transgene escape through pollen and gene silencing is absent. The border sequences in our vector, pLD-CtV, direct single copy insertion by homologous recombination in the inverted repeat region of the tobacco plastome. Each transformant contains up to 20 000 copies of the transgene in a leaf cell, proteins can accumulate to high levels (up to 50% of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. Two antimicrobial gene constructs, a) a bacterial chloroperoxidase (cpo-p) and b) D4E1, a synthetic, lytic peptide, were ligated into pLD.CtV for transformation experiments. Transformants were identified by selection on regeneration media with 500 mg L-1 spectinomycin, resistance to which was conferred by the aminoglycoside-3′-adenyltransferase (aadA) gene in the plasmid vector. PCR and Southern analysis has confirmed integration in the plastome. Western analysis confirms the presence of the CPO-P in higher abundance in chloroplast transformants than nuclear. Although integration and expression was achieved in these experiments, concomitant increase in the level of protein accumulation was not observed. Northern analysis of primary transformants for cpo-p showed 15 fold higher transcript abundance than nuclear transformants; yet this was not reflected in Western blot, enzyme or bioassay. In an effort to enhance the rate of translation a new construct was developed replacing the single ribosome binding site between aadA and cpo-p with the tobacco full length psbA 5′ UTR.
Tracey Ann. Ruhlman, University of New Orleans, Department of Biological Sciences, SRRC ARS USDA, 1100 Robert E. Lee Blvd., New Orleans, LA 70124-4305. In Vitro Cellular and Developmental Biology, 41:39-A, 2005