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High-risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV-16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF 32-51 ), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF 32-51 -E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF 32-51 -E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF 32-51 -E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast-targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB-like structures were also seen for the cytoplasmic LG. PB-like structure formation was confirmed for both LG and LALF 32-51 -E7 by TEM. LALF 32-51 -E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB-like structures. This study could open a new avenue for the use of LALF 32-51 as a PB-inducing peptide., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The CRISPR/Cas9 system from Streptococcus pyogenes is an increasingly popular tool for genome editing due to its ease of application. Here we demonstrate genomic DNA fragment removal using RNA directed Cas9 nuclease in barley. The high mutation frequency confirms the exceptional efficiency of the system and its suitability for generating loss-of-function mutant lines that may be used in functional genetics approaches to study endomembrane trafficking pathways and posttranslational protein modifications. The generation of doubled haploids from genome edited plants allows the recovery of true breeding lines that are instantly homozygous for the edited alleles.

The cereal endosperm is a complex structure comprising distinct cell types, characterized by specialized organelles for the accumulation of storage proteins. Protein trafficking in these cells is complicated by the presence of several different storage organelles including protein bodies (PBs) derived from the endoplasmic reticulum (ER) and dynamic protein storage vacuoles (PSVs). In addition, trafficking may follow a number of different routes depending on developmental stage, showing that the endomembrane system is capable of massive reorganization. Thus, developmental sequences involve progressive changes of the endomembrane system of endosperm tissue and are characterized by a high structural plasticity and endosomal activity.Given the technical dexterity required to access endosperm tissue and study subcellular structures and (seed storage protein) SSP trafficking in cereal seeds, static images are the state of the art providing a bulk of information concerning the cellular composition of seed tissue. In view of the highly dynamic endomembrane system in cereal endosperm cells, it is reasonable to expect that live cell imaging will help to characterize the spatial and temporal changes of the system. The high resolution achieved with electron microscopy perfectly complements the live cell imaging.We therefore established an imaging platform for TEM as well as for live cell imaging. Here, we describe the preparation of different cereal seed tissues for live cell imaging concomitant with immunolocalization studies and ultrastructure.

The excessive use of antibiotics in food animal production has contributed to resistance in pathogenic bacteria, thereby triggering regulations and consumer demands to limit their use. Alternatives for disease control are therefore required that are cost-effective and compatible with intensive production. While vaccines are widely used and effective, they are available against a minority of animal diseases, and development of novel vaccines and other immunotherapeutics is therefore needed. Production of such proteins recombinantly in plants can provide products that are effective and safe, can be orally administered with minimal processing, and are easily scalable with a relatively low capital investment. The present report thus advocates the use of plants for producing vaccines and antibodies to protect farm animals from diseases that have thus far been managed with antibiotics; and highlights recent advances in product efficacy, competitiveness, and regulatory approval., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Plants have emerged as commercially relevant production systems for pharmaceutical and nonpharmaceutical products. Currently, the commercially available nonpharmaceutical products outnumber the medical products of plant molecular farming, reflecting the shorter development times and lower regulatory burden of the former. Nonpharmaceutical products benefit more from the low costs and greater scalability of plant production systems without incurring the high costs associated with downstream processing and purification of pharmaceuticals. In this review, we explore the areas where plant-based manufacturing can make the greatest impact, focusing on commercialized products such as antibodies, enzymes, and growth factors that are used as research-grade or diagnostic reagents, cosmetic ingredients, and biosensors or biocatalysts. An outlook is provided on high-volume, low-margin proteins such as industrial enzymes that can be applied as crude extracts or unprocessed plant tissues in the feed, biofuel, and papermaking industries.

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of (OS) GRFT in the best-performing plants was 223 μg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. (OS) GRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified (OS) GRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure (OS) GRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCβ1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An alarming increase in emergence of antibiotic resistance among pathogens worldwide has become a serious threat to our ability to treat infectious diseases according to the World Health Organization. Extensive use of antibiotics by livestock producers promotes the spread of new resistant strains, some of zoonotic concern, which increases food-borne illness in humans and causes significant economic burden on healthcare systems. Furthermore, consumer preferences for meat/poultry/fish produced without the use of antibiotics shape today's market demand. So, it is viewed as inevitable by the One Health Initiative that humans need to reduce the use of antibiotics and turn to alternative, improved means to control disease: vaccination and prophylactics. Besides the intense research focused on novel therapeutic molecules, both these strategies rely heavily on the availability of cost-effective, efficient and scalable production platforms which will allow large-volume manufacturing for vaccines, antibodies and other biopharmaceuticals. Within this context, plant-based platforms for production of recombinant therapeutic proteins offer significant advantages over conventional expression systems, including lack of animal pathogens, low production costs, fast turnaround and response times and rapid, nearly-unlimited scalability. Also, because dried leaves and seeds can be stored at room temperature for lengthy periods without loss of recombinant proteins, plant expression systems have the potential to offer lucrative benefits from the development of edible vaccines and prophylactics, as these would not require "cold chain" storage and transportation, and could be administered in mass volumes with minimal processing. Several biotechnology companies currently have developed and adopted plant-based platforms for commercial production of recombinant protein therapeutics. In this manuscript, we outline the challenges in the process of livestock immunization as well as the current plant biotechnology developments aimed to address these challenges.

Background: Recently, we identified neutralizing autoantibodies against progranulin (PGRN) in a wide spectrum of rheumatic diseases including cases with enteropathic spondylarthritis. PGRN is a secreted protein with strong anti-inflammatory effects, believed to be mediated by the direct inhibition of TNF receptors 1&2. Given the central role of TNF-α as proinflammatory cytokine, a neutralizing antibody directed against its physiologic antagonist PGRN might entertain a proinflammatory environment., Objective: The aim of the present study was to investigate a possible occurrence of PGRN-antibodies (PGRN-Abs) in inflammatory bowel disease (IBD), and to investigate a possible pathogenic effect., Materials and Methods: Sera samples of 141 patients with Crohn's disease (CD) and of 71 patients with ulcerative colitis (UC) were tested for PGRN-Abs by ELISA. PGRN plasma levels were detected by ELISA. Proinflammatory effects of progranulin-antibodies were analyzed by TNF-α-mediated cytotoxicity assays using HT29 cells and by examination of possible effects of PGRN and of PGRN-antibodies on TNF-α-induced downmodulation of FOXP3 expression in CD4(+)CD25(hi) Tregs., Results: PGRN-Abs were found in sera of 23/141 (16.31%) patients with CD, and 15/71 (21.13%) patients with UC. PGRN-Abs were more frequent than anti-neutrophil cytoplasmic autoantibodies (ANCAs) in UC, but less frequent than anti-Saccharomyces cerevisiae antibodies (ASCAs) in CD. PGRN-Abs belonged mostly to IgG1 (71.1%) and IgA (26.3%). They occurred in relevant titres and had significant neutralizing effects on PGRN plasma levels. Cytotoxicity assays comparing PGRN-antibody-positive sera with negative sera from matched patients with IBD showed a proinflammatory effect of PGRN-Abs on HT29 cells. Moreover, PGRN-antibodies led to an increase of TNF-α-induced downmodulation of FOXP3 in CD4(+)CD25(hi) Tregs., Conclusion: The results suggest that PGRN-Abs occur frequently in CD and UC, and have a proinflammatory effect.

Objective: Cardiac reoperations have been associated with increased morbidity and mortality compared with first-time surgery. We analyzed our experience with reoperative aortic valve replacement (redo-AVR) and compared these results with those from patients who had undergone transapical aortic valve implantation (TA-AVI) as a second heart operation., Methods: In the present retrospective observational comparative study, we analyzed the outcome of 136 patients with previous cardiac surgery who had undergone conventional redo-AVR (n = 59; since 2006) or TA-AVI (n = 77; since 2008) with respect to the 30-day outcomes (Valve Academic Research Consortium criteria), 1- and 3-year survival, and the risk factors for both approaches after previous heart surgery., Results: Neither group differed significantly in their risk profile, leading to similar Society of Thoracic Surgeon score and EuroSCORE. The 30-day mortality was 3.39% (n = 2) in the redo-AVR group and 7.8% (n = 6) in the redo TA-AVI group (P = .465). The overall combined safety endpoint at 30 days was significantly lower for the TA-AVI patients (18.1% vs 33.9% in redo-AVR; P = .036). The unadjusted and adjusted 1-year survival showed no difference between the 2 groups. The unadjusted 3-year survival revealed a 2.1-fold greater mortality risk after TA-AVI (P = .055). Adjustment by multivariate Cox regression analysis (hazard ratio, 1.427; 95% confidence interval, 0.635-3.209; P = .389) and propensity score (hazard ratio, 1.571; 95% confidence interval, 0.575-4.291; P = .378) led to a >50% risk reduction, resulting in similar 3-year survival in the 2 groups., Conclusions: Redo-AVR can be performed with acceptable results in high-risk patients and still serves as the reference standard. Reoperative valve surgery by TA-AVI is feasible and results in comparable short- and mid-term survival., (Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Cereal seeds are versatile platforms for the production of recombinant proteins because they provide a stable environment for protein accumulation. Endogenous seed storage proteins, however, include several prolamin-type polypeptides that aggregate and crosslink via intermolecular disulfide bridges, which could potentially interact with multimeric recombinant proteins such as antibodies, which assemble in the same manner. We investigated this possibility by sequentially extracting a human antibody expressed in maize endosperm, followed by precipitation in vitro with zein. We provide evidence that a significant proportion of the antibody pool interacts with zein and therefore cannot be extracted using non-reducing buffers. Immunolocalization experiments demonstrated that antibodies targeted for secretion were instead retained within zein bodies because of such covalent interactions. Our findings suggest that the production of soluble recombinant antibodies in maize could be enhanced by eliminating or minimizing interactions with endogenous storage proteins., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

From plant biotechnology to bio-based products - this Special Issue of Biotechnology Journal is dedicated to plant biotechnology and is edited by Prof. Eva Stöger (University of Natural Resources and Life Sciences, Vienna, Austria). The Special Issue covers a wide range of topics in plant biotechnology, including metabolic engineering of biosynthesis pathways in plants; taking advantage of the scalability of the plant system for the production of innovative materials; as well as the regulatory challenges and society acceptance of plant biotechnology., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N-Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N-glycans and that the ELP fusions had no significant effect on N-glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.

A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Protein-based microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.

Aim: Recent results from animal experiments have shown that radix astragali (RA), a traditional Chinese herbal tonic, alleviates muscle atrophy under simulated weightlessness conditions, rendering RA a candidate for human use as a countermeasure against muscular atrophy. Possible cardiovascular side effects have not yet been investigated. We analyzed the effects of RA on the orthostatic stability of healthy men., Methods: There were 10 test subjects who were assigned to a double-blinded, randomized crossover design using RA or placebo (PL) for 14 d each, respectively. Test runs were separated by a 14-d 'washout' interval. At the beginning and the end of every 14-d test run, graded orthostatic stress (GOS) consisting of head-up tilt (HUT) combined with lower body negative pressure (LBNP) was used to achieve a presyncopal endpoint. Orthostatic effects on cardiac and vascular function were continuously monitored., Results: There were no significant differences between the RA vs. PL groups: mean arterial blood pressure dropped by 13 vs. 17%, pulse pressure 46 vs. 35%, heart rate increased 108 vs. 117%, and stroke volume index decreased 54 vs. 49% from supine control to presyncope. Neither did RA influence standing time compared to PL (18 +/- 7 vs. 17 +/- 6 min), nor did progression from the first to the fourth trial (15 +/- 6 to 18 +/- 7 min)., Conclusion: RA does not influence resting cardiovascular variables and orthostatic capacity in humans. It can be expected that human studies of RA's musculo-skeletal countermeasure potential will not be compromised by any cardiovascular side effects at the dosage employed in this study.

The present paper addresses the question of thermotolerance of in planta synthesized heterologous enzymes using phytase as a model. Two individual transgenic wheat materials expressing an Aspergillus fumigatus phytase with a low denaturation temperature (62.5 degrees C) but a high refolding capacity, and a rationally designed consensus phytase engineered to a high denaturation temperature (89.3 degrees C), were evaluated. High levels of endosperm specific expression were ensured by the wheat high molecular weight glutenin 1DX5 promoter. Immunodetection at the light and electron microscopical level shows unequivocally that the heterologous phytase is deposited in the vacuole, albeit that the transformation constructs were designed for secretion to the apoplast. Evaluation of heat stability properties and kinetic properties unraveled that, under these deposition conditions, heat stability based on high unfolding temperature is superior to high refolding capacity and represents a realistic strategy for improving phosphate and mineral bioavailability in cereal-based feed and food.

The high molecular weight (HMW) glutenin-encoding genes in wheat are developmentally activated in the endosperm at about 8 days after anthesis. We have investigated the physical changes that occur in these genes in two transgenic lines containing about 20 and 50 copies each of the HMW glutenin genes together with their promoters. Using fluorescence in-situ hybridisation (FISH) and confocal imaging, we demonstrate that, in non-expressing tissue, each transgene locus consists of one or two highly condensed sites, which decondense into many foci upon activation of transcription in endosperm nuclei. Initiation of transcription can precede decondensation but not vice versa. We show that, in one of the lines, cytoplasmic transcript levels are high after onset of transcription but disappear by 14 days after anthesis, whereas small interfering RNAs, which indicate post-transcriptional gene silencing (PTGS), are detected at this stage. However, the transcript levels remain high at the transcription sites, most of the transgene copies are transcriptionally active and transcriptional activity in the nucleus ceases only with cell death at the end of endosperm development.

The FK506-binding proteins (FKBPs) belong to the peptidyl prolyl cis-trans isomerase (PPIase) family, and catalyse the rotation of the peptide bond preceding a proline. They are conserved in organisms from bacteria to man. In order to understand the function of plant FKBP isoforms, we have produced transgenic wheat plants overexpressing each of the two wheat FKBPs: wFKBP73 (which is expressed in young vegetative and reproductive tissues under normal growth conditions) and wFKBP77 (which is induced by heat stress). Transgenic lines overexpressing wFKBP77 at 25 degrees C showed major morphological abnormalities, specifically relating to height, leaf shape, spike morphology and sterility. In these plants, the levels of hsp90 mRNA were over two fold higher than in controls, indicating a common regulatory pathway shared between wFKBP77 and Hsp90. Transgenic lines overexpressing wFKBP73 showed normal vegetative morphology, but the grain weight and composition was altered, corresponding to changes in amylase activity during seed development.

We used particle bombardment to produce transgenic wheat and rice plants expressing recombinant soybean ferritin, a protein that can store large amounts of iron. The cDNA sequence was isolated from soybean by RT-PCR and expressed using the constitutive maize ubiquitin-1 promoter. The presence of ferritin mRNA and protein was confirmed in the vegetative tissues and seeds of transgenic wheat and rice plants by northern and western blot analysis, respectively. The levels of ferritin mRNA were similar in the vegetative tissues of both species, but ferritin protein levels were higher in rice. Both ferritin mRNA and protein levels were lower in wheat and rice seeds. ICAP spectrometry showed that iron levels increased only in vegetative tissues of transgenic plants, and not in the seeds. These data indicate that recombinant ferritin expression under the control of the maize ubiquitin promoter significantly increases iron levels in vegetative tissues, but that the levels of recombinant ferritin in seeds are not sufficient to increase iron levels significantly over those in the seeds of non-transgenic plants.

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

We investigated the suitability of transformed rice cell lines as a system for the production of therapeutic recombinant antibodies. Expression constructs encoding a single-chain Fv fragment (scFvT84.66, specific for CEA, the carcinoembryonic antigen present on many human tumours) were introduced into rice tissue by particle bombardment. We compared antibody production levels when antibodies were either secreted to the apoplast or retained in the endoplasmic reticulum (ER) using a KDEL retention signal. Production levels were up to 14 times higher when antibodies were retained in the ER. Additionally, we compared construct sencoding different leader peptides (plant codon optimised murine immunoglobulin heavy and light chain leader peptides from mAb24) and carrying alternative 5' untranslated regions (the petunia chalcone synthase gene 5' UTR and the tobacco mosaic virus omega sequence). We observed no significant differences in antibody production levels among cell lines transformed with these constructs. The highest level of antibody production we measured was 3.8 micrograms g-1 callus (fresh weight). Immunological analysis of transgenic rice callus confirmed the presence of functional scFvT84.66. We discuss the potential merits of cell culture for the production of recombinant antibodies and other valuable macromolecules.

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8-9 weeks (56-66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4-6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 μg of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4-5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56-66 days, making it possible to obtain R3 homozygous plants in less than a year.

Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10(4) pollen grains were GUS(+). Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS(+) pollen, 1-5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 mum), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.