Basal leaf segments of 3 to 4 week old maize (Zea mays L.) seedlings plated on SH medium with 30 μM dicamba produced embryogenic callus and/or somatic embryos. Histological evidence showed that some of the embryos arose directly from the explant. When leaf segments with embryos were transferred to MS medium with 1.0 μM NAA, 1.0 μM IAA, 2.0 μM 2iP, and 60 g/l sucrose, the embryos germinated and the resulting seedlings could be established in culture tubes. These responses were obtained from three inbred lines, CHI31, S615, and S7.

Somatic embryogenesis from cultured leaf segments of Zea mays.

Transgenic soybean plants have been produced using an Agrobacterium-mediated gene transfer system. This procedure relied on a regeneration protocol in which shoot organogenesis was induced on cotyledons of soybean genotypes selected for susceptibility to Agrobacterium. Cotyledon explants were inoculated with Agrobacterium tumefaciens pTiT37-SE harboring pMON9749 (conferring kanamycin resistance and β-glucuronidase “GUS” activity) or pTiT37-SE∷pMON894 (conferring kanamycin resistance and glyphosate tolerance) and cultured on shoot induction medium containing kanamycin. Plantlets were tested for gene insertion 3–4 months post-inoculation. Approximately 6% of the shoots (8 plants to date) produced on the kanamycin-selected cotyledons were transgenic based on assays for GUS expression, kanamycin resistance or glyphosate tolerance. Progeny from two of these plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.

Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer.

Plant cell and organ cultures have emerged as potential sources of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, coloring agents, biopesticides, and food additives. In recent years, various strategies have been developed to assess biomass accumulation and synthesis of secondary compounds in cultures. Biomass accumulation and metabolite biosynthesis are two-stage events, and the parameters that control the growth and multiplication of cultured cells/organs and biomass accumulation are controlled in the first stage. Parameters that assist with the biosynthesis of metabolites are controlled in the second stage. The selection of high-producing cells or organ clones; optimization of medium parameters such as suitable medium, salt, sugar, nitrogen, phosphate, and plant growth regulator levels; and physical factors such as temperature, illumination, light quality, medium pH, agitation, aeration, and environmental gas (e.g., oxygen, carbon dioxide, and ethylene) are controlled in the first stage of the culture process. Elicitation, replenishment of nutrient and precursor feeding, permeabilization, and immobilization strategies assist with the accumulation of metabolites and can be applied in the second stage of the culture process. By following stage-specific strategies, it is possible to produce large amounts of biomass with an increase in the accumulation of secondary compounds.

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2008.03418.x

Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

The composition of a medium is described that proved useful to culture callus of a variety of monocotyledonous and dicotyledonous plants. Growth on the medium was often better than on some other ex...

Roy U. Schenk

Papers

Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures