Echinacea, better known as purple coneflower, has received a global attention because of its increasing medicinal value. There is enormous potential for the discovery of new medicinal compounds in this species and an immediate need for techniques to facilitate the production of high quality, chemically consistent plant material for drug development and clinical trials. In vitro tissue culture of Echinacea can play a vital role in the development of novel germplasm, rapid multiplication, and genetic modifications for an enhanced phytochemical production. Recent establishment of liquid culture techniques, large-scale bioreactors, and Agrobacterium-mediated transformation are changing the production parameters of the Echinacea species. This review provides an overview of the recent developments in in vitro technologies and challenges that remain in the Echinacea biotechnology.

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus, bar, and hpt. Immature embryos and callus derived from immature embryos were targeted for transformation. Subsequently, genes of agronomic and malting importance have been deployed in barley. Particle bombardment appears to be the preferred choice for barley transformation in the majority of the reports, although Agrobacterium-mediated transformation is being used more often. The current review focuses on the challenges encountered in barley transformation such as somaclonal variation, development of transformation systems for commercial cultivars, gene expression, stability and inheritance, and gene flow. Newer markers such as the green fluorescent protein (gfp), firefly luciferase, and phosphomannose isomerase were found to be useful in the selection of transgenic plants. Tissue-specific promoters such as those for B1-hordein and D-hordein genes, and spike-specific promoters, are increasingly used to drive gene expression. The review also describes recent research on gene-tagging through transformation, insertion of disease resistance, and abiotic stress resistance genes, transformation with genes for improved malting quality, nutrient content, feed quality, and the production of feed enzymes and pharmaceutical compounds.

Genetic improvement of coffee, an important commercial crop, through classical breeding is slow and cumbersome. Biotechnology offers alternative strategies for generating new and improved coffee varieties, including those resistances to environmental extremes, pests, and diseases, low in caffeine, and with uniform fruit maturation. Large improvements in somatic embryogenesis, development of haploids, and scale-up of micropropagation have been accomplished in the last 5 yr. The recent identification of expressed sequence tags (EST) that are differentially expressed during the infestation of coffee plants by coffee leaf miners and the isolation and cloning of the promoter for the N-methyltransferase gene associated with caffeine production open up the possibility of producing varieties of coffee with new traits. This review provides a summary of in vitro biological advances and directions as to how they could be applied to improve the production and quality of coffee.

Agrobacterium rhizogenes strain K599 (pRi2659), a causative agent of hairy root disease, effectively induces hairy root formation in a variety of plant species, including numerous soybean (Glycine max) cultivars. Because Agrobacterium-mediated transformation of soybean remains challenging and labor intensive, K599 appeared a suitable progenitor for new agrobacteria strains for plant transformation. In this paper, we report the disarming and sequencing of pRi2659 and the usefulness of the resulting disarmed strain in plant transformation studies of Arabidopsis thaliana, maize (Zea mays), tomato (Lycopersicon esculentum), and soybean (G. max).

A novel Agrobacterium rhizogenes-mediated transformation method using a primary-node explant from Dairyland cultivar 93061 was developed for soybean using the disarmed Agrobacterium strain SHA17. Transformed plants regenerated from explants inoculated with SHA17 were fertile and phenotypically normal. In a comparative experiment, regeneration frequencies were not significantly different between explants inoculated with A. rhizogenes strain SHA17 and Agrobacterium tumefaciens strain AGL1; however, a 3.5-fold increase in transformation efficiency [(number of Southern or TaqMan-positive independent events/total number of explants inoculated)×100] was found for explants cocultured with SHA17 compared to AGL1 (6.6 and 1.64%, respectively). Southern analysis of 48 T₀ plants suggested that 37.5, 23, and 39.6% of the T₀ plants contained 1, 2, and 3 or more T-DNA fragments integrated into the genome, respectively. Additionally, T₁ progeny analysis of 8 independent events resulted in typical Mendelian inheritance of T-DNA genes. Of seven T₀ plants that had two or more T-DNA fragments, six contained multiple loci segregating in T₁ progenies. Further analysis of four lines confirmed the presence of PAT, GUS, and/or DsRED2 proteins in transgenic plants that were encoded on the T-DNA into the T₂ generation.

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90∼120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.

Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(γ-γ-dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 μM BA) compared to auxin (4.5 μM 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 μM 2,4-D and 1.3 and 4.0 μM BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.

This is the first report where shoot regeneration in strawberry cultivar Chandler has been achieved simultaneously through both somatic embryogenesis and shoot bud formation. Direct somatic embryogenesis was observed in leaf discs which were cultured on medium containing MS salts B₅ vitamins 2% glucose 18.16 μM thidiazuron (TDZ) and given both chilling and dark treatment for 2 wk at 4±2°C followed by incubation at 25±2°C under 16-h photoperiod for third wk. After 3 wk, these explants were then subcultured on medium containing MS salts B₅ vitamins 2% glucose and incubated under 16-h photoperiod at 25±2°C for further growth and development. Direct regeneration via de novo shoot bud formation was observed in leaf disks which were given dark treatment and were cultured on medium containing MS salts B₅ vitamins 2% glucose supplemented with 9.08 μM TDZ. There was a synergistic effect of photoperiod, dark, and chilling treatments on somatic embryogenesis, whereas chilling treatment had an inhibitory effect on shoot organogenesis.

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog's medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36±2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D 1 μM BA, the average number of somatic embryos was much less (7.6±0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04±2.10 shoots and shoot length of 2.26±0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12±0.58 roots on half strength MS 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.

Transgenic plants of grapefruit (Citrus paradisi Macf.) cvs. ‘Duncan’, ‘Flame’, ‘Marsh’, and ‘Ruby Red’ were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Two citrus tristeza virus (CTV)-derived candidate resistance genes: ‘392’ (3′ region of the p23 ORF plus 3′ untranslated region—UTR) and ‘p23 hairpin’ (sense-p23 ORF plus UTR plus antisense-p23 ORF) were introduced into grapefruit using Agrobacterium strains EHA105 and EHA101, respectively. Epicotyl explants from 1-mo.-old in vitro etiolated seedlings were incubated in bacterial suspension. Green shoots that formed on explants after 4–5 wk after bacterial incubation were tested for the presence of the GUS gene by histochemical analysis. The percentage of GUS-positive shoots and transformation efficiency was 30.3±3.3% and 3.5% for treatment with EHA101 and 15.3±1.7% and 1.3% for treatment with EHA105. GUS-positive shoots were micrografted onto Carrizo citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) seedling rootstocks, and the presence of transgene sequences in these plants was confirmed by polymerase chain reaction (PCR), Southern blot, and reverse transcription PCR analyses. Resulting transgenic grapefruit plants were challenged with CTV and tobacco mosaic virus using a protoplast challenge assay as an initial screen to determine the effects of the transgenes on virus replication. Although complete RNA-mediated resistance was not achieved, preliminary results showed that 5.2% of the recovered transgenic plants containing the ‘392’ CTV-derived sequence repeatedly exhibited reduced CTV replication in protoplasts. These plants are being further evaluated using the traditional method of virus inoculation followed by enzyme-linked immunosorbent assay.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40 μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1 μM N⁶ benzyladenine and 2.85 μM indole-3-acetic acid was used for the study. At 20 and 40 μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38 ± 7 and 153 ± 27 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.

Murashige and Skoog's (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.

In an effort to develop a sustainable protocol for the micropropagation of a shy suckering elite chrysanthemum cv. Arka Swarna (yellow pompon type), in vitro cultures were established using surface-sterilized nodal microcuttings (1–1.5 cm) from polyhouse-grown plants on MS medium containing 3% sucrose, 0.25% phytagel, and 5 μM benzyl adenine (BA) or kinetin. Microbial contamination in the range of 6–24% was encountered during the first in vitro passage. Apparently clean cultures after one passage on MS basal medium were transferred to medium with BA or kinetin (0, 1, 5, 10, or 20 μM) in culture bottles, and were monitored for eight in vitro passages (1 mo. each) for growth and microbial contamination. Plant growth regulator (PGR)-free medium was the best for sustainable micropropagation over successive in vitro passages yielding a single shoot from cultured microcuttings. Higher cytokinin levels inhibited rooting and induced one or more shorter shoots with close nodes resulting in low propagation rates. All apparently clean stocks revealed covert endophytic bacteria during tissue-indexing using bacteriological media. Three distinct bacterial morphotypes were isolated from such stocks, identified based on 16S rRNA gene sequence analysis as different morphotypes of Curtobacterium citreum. The endophytes tended to show obvious growth on chrysanthemum culture medium with increase in cytokinin levels (5–20 μM), but such growth was not noticed in inoculations on MS medium without plants. Sustainable micropropagation of cv. Arka Swarna for more than 2 yr with the resident endophytic bacteria in covert form was realized on PGR-free MS medium giving a net propagation rate of three to four times over a subculture cycle of 2–3 wk.

Conditions for obtaining an efficient mass propagation procedure to overcome isolated Taxus baccata embryo dormancy were investigated. The protocol herein described was efficient for overcoming the dormancy of T. baccata isolated embryos under in vitro conditions, enabling the conservation and propagation of this species. T. baccata seeds were unable to germinate directly after collection under in vitro conditions. Very good sterility and germination was achieved by soaking seeds in distilled water at a low temperature ( 4°C) at least for 48 h instead of leaching them for 7 d under running water, followed by maintaining isolated embryos on the Murashige and Skoog medium (MS) supplemented with 5 g l⁻¹ activated charcoal. That treatment allowed one to shorten the time of the experiment and gave almost 100% sterility. The best germination was observed in darkness, but to obtain worthy seedlings, it was necessary to place cultures in a 16-h photoperiod after a 2-wk incubation. There was no significant difference in germination between seeds collected from different populations of Southern Poland.

The present study describes the micropropaga-tion of Swertia chirayita, an endangered medicinal herb of the temperate Himalayas, through axillary multiplication from 4-wk-old seedling-derived nodal explants. We obtained 4.5-fold multiplication every 4 wk on Murashige and Skoog (MS) medium supplemented with 4 μM benzyl amino purine (BA) and 1.5 μM 6-(γ,γ-dimethylallylamino) purine (2iP). Rooting was optimized on modified MS medium supplemented with 1 μM naphthalene acetic acid (NAA) and 500 mg l⁻¹ of activated charcoal (AC). A success rate of 94 % was obtained by in vitro hardening in the growth-room and by ex vitro hardening in greenhouse conditions. The present study can serve as a tool for the mass multiplication of elite genotypes of this critically endangered species and can also be utilized for multiplying large numbers of quality planting material for the replantation in their natural habitat. This approach can also help meet the requirements of the growing pharmaceutical industry by ensuring the regular supply of authentic planting material.

The oligogalacturonides derived from trifluoroacetic acid hydrolysis and their monomer galacturonic acid were tested for their effect on development of strawberry explants (Fragaria × ananassa) in vitro. The addition of oligogalacturonides or galacturonic acid in the plant tissue culture medium at concentrations of 0.1 or 1.0 mM increased shoot and leaf number in the presence of added benzyl adenine. The response to oligogalacturonides or galacturonic acid was maintained in the absence of benzyl adenine but was lessened. Conversely, root elongation was increased in the absence of benzyl adenine. A combined analysis of these experiments also indicated the increase in shoot or leaf number. High-performance anion exchange chromatography of the bioassayed oligogalacturonide samples using a pulsed amperometric detector revealed that the sample consisted of galactouronides with a degree of polymerization from 1 to 5 and the proportion of the monomer was very high (94%).

Roots of Primula veris L. contain considerable amounts of triterpene saponins, which are used in medicine as expectorants. P. veris is in many places an endangered plant, and its production in the field is laborious and a low yielding process. Plant tissue culture provides an alternative means for producing secondary metabolites. Shoot apex, callus, suspension, and root cultures of P. veris were developed for saponin production. In these cultures, the content of triterpene saponins, with focus on primula acid I, the most dominant saponin in Primula species, was determined and compared to that in soil-grown plants. The highest content of primula acid I was observed in root cultures, on average 29.5 mg/g dry weight. Some culture lines contained higher amounts of primula acid I (62.6 mg/g dry weight) than the roots of plants grown in soil.

The objective was to set up a pilot scale process for robusta (Coffea canephora) industrial propagation by somatic embryogenesis in liquid media. A batch production of pre-germinated embryos was initiated once every 2 mo. in 2003 and 2004, then every mo. in 2005. Each run batch requires 4 to 6 mo. to produce the pre-germinated somatic embryos and consists of three phases: (1) the development of torpedo stage embryos in Erlenmeyer flasks, (2) pre-germination in temporary immersion bioreactors to allow maturation from the torpedo stage to the cotyledonary stage, (3) maintaining the embryos under storage conditions before their shipment to coffee producing countries. Starting from 1 kg of embryogenic calluses, a total of 4.4 million pre-germinated embryos for 17 clones were produced over 3 yr. This embryo number was enough to potentially regenerate 2 million plants, as the global embryo-to-plantlet conversion rate was estimated to 46% after acclimatization and complete germination in the greenhouse. At the end of April 2006, 600,000 somatic seedlings were transferred into plastic bags in nurseries or were already planted in the fields, mainly in Thailand. The current capacity allows the production of 2.5 million embryos per year, equivalent to a potential of about 1.0 million plantlets. The technical package has recently been transferred to National Institutes in Mexico, Thailand, and Vietnam.

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.

Toll-like receptors (TLRs) constitute an essential family of pattern recognition molecules that, through the direct recognition of conserved microbial components, initiate inflammatory responses ^after infection. Phylogenetic evidence suggests that vertebrate TLRs are under strong purifying selection for the maintenance of function. Our lab is focused on a related group of vertebrate TLRs that comprise the TLR2 subfamily. The most closely related members, TLRs 1 and 6, appear to have arisen from a recent gene duplication event and have acquired differential microbial recognition specificity. We have characterized two single nucleotide polymorphisms (SNPs), P315L, and I602S, in human TLR1 that effect receptor function through different mechanisms. The 315L variant is associated with deficient recognition of microbial products whereas the 602S variant is associated with aberrant trafficking of the receptor to the cell surface. It is surprising to note that the 602S allele is associated with a decreased incidence of leprosy suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion. TLR1 I602S exhibits strikingly different allele frequencies among different races suggesting that if strong purifying selection took place, it was restricted by either additional genetic or environmental factors that were geographically constrained.

High expression of transgenes is desired for molecular farming. However, transgenes are often subjected to gene silencing pathways in plant cells. Gene silencing may be triggered by the production of aberrant (hairpin) RNA molecule from a complex integration locus in plant genome or by the overexpression of transgene. Excessively transcribed RNA are subject to gene silencing even if they are produced from a single-copy locus. Therefore, we sought to determine how highly a transgene can be expressed before its transcript is subjected to gene silencing. In a previous study, we demonstrated that precise single-copy locus generated by Cre/lox-mediated site-specific integration (SSI) in rice is stably expressed at predictable levels through subsequent generations, and that its expression invariably doubled in homozygous progenies. To further explore the stability of the SSI locus and determine the expression-threshold of rice genomic sites, we generated transformation vectors containing 1–3 copies of 35S-GUS and 35S-GFP transgenes. These vectors were used to develop SSI locus containing 1–3 copies of each transgene in two different varieties of rice, Nipponbare and Taipei 309. SSI lines will be analyzed by PCR and Southern blotting to ascertain the presence of precise integration structures consisting of 1–3 copies of each trans-gene. The precise SSI lines will be subjected to quantitative GUS and GFP assay to determine if gene expression indeed increased with the increase in gene dosage. Molecular and expression data of SSI lines will be presented.

Tobacco plants (Nicotiana tabacum L.) were transformed with a construct based on pCAMBIA 2301 containing a “hairpin” inverted repeat of 598 nucleotides derived from the Potato Virus Y (PVY) replicase (NIb gene) of the N strain (Robaglia et al., J. Gen. Virol. 70, 935, 1989). Such constructs confer virus resistance by a post translational gene silencing mechanism. Homozygous (T3) plants were challenged with a range of PVY strains and resistance was measured by symptom expression, ELISA titer, and back inoculation of controls with extracts from resistant plants. The nucleotide homology of PVY strains to the transgene was: WP (99.5%) PVY-NTN (96.3), PVY-H (95.6%), PVY-O (88.9%), strain 52 (88.3%), and local field isolates from tomato (86.8%), and pepper (86.3%). A transgenic tobacco line was immune to the five PVY strains with which the transgene had the greatest homology (WP, NTN, H, O, 52). In