Petunia Hybrida Plants Research Articles

Molecular characterization and pathogenicity of a novel monopartite geminivirus infecting tobacco in China

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana, N. tabacum, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development.

Companion plants for conservative management of Tupiocoris cucurbitaceus (Spinola 1852) (Heteroptera: Miridae: Dicyphini) on greenhouse tomato crops

In recent decades, greenhouse crops relevance increased due to the high demand for products outside their growing season, with tomato standing out as one of the most cultivated crops. In these production systems, insects and mites find optimal conditions for their development, achieving high populations that affect crops. Farmers usually control these populations using chemical insecticides, which affect the health of workers and consumers and have negative effects on the environment. Tomato crops suffer damage by lepidopterans and hemipterans, among which those known as whiteflies (Hemiptera: Aleyrodidae) perform regular attacks, causing yield and quality losses in the final product. Currently, the use of zoophytophagous predators of the Miridae family, Dicyphini tribe, as an alternative for their biological control, has intensified studies on Tupiocoris cucurbitaceus (Spinola, 1852), a predator of several species of aphids and whiteflies found in Uruguay. A strategy for the conservative management of those species was designed, using companion plants grown together with the crop. By the time the tomato plants were transplanted, Calendula officinalis, Smallanthus connatus, Tithonia rotundifolia, Nicotiana tabacum, Physalis peruviana and Petunia hybrida plants were established as companion species. These plants were monitored weekly together with the tomato plants, and a greater presence of T. cucurbitaceus in the crops with companion plants was found. The results suggest that the incorporated plants were useful for the preservation and retention of predaceous mirids in productive conditions.

Constitutive overexpression of Nicotiana GA 2 ox leads to compact phenotypes and delayed flowering in Kalanchoë blossfeldiana and Petunia hybrida

This work describes compact phenotypes of Kalanchoe blossfeldiana and Petunia hybrida plants harboring a constitutively overexpressed gibberellin 2-oxidase (GA 2 ox) transgene. A GA 2 ox gene from Nicotiana tabacum under the control of the Ca35S promoter was introduced into the pCAMBIA1303 plasmid. The cloning vector was introduced into leaf explants of Kalanchoe and Petunia via Agrobacterium-mediated transformation. Putative transformants were analysed for the presence, integration and expression of the transgene using polymerase chain reaction (PCR), reverse-transcription (RT)-PCR, and Southern blot analysis, respectively. Phenotypic evaluations revealed that the mean lengths of the Kalanchoe transgenic lines were two-fold shorter than those of wild-type control plants, although the mean numbers of nodes were similar. Moreover, the mean lengths of inflorescence stems of the Kalanchoe transgenic lines were almost three-fold shorter than those of the wild-type control plants. Similarly, the mean lengths of Petunia transgenic lines were four-fold shorter than those of the wild-type plants, except for a single line, while the mean numbers of nodes were either similar or higher in the transgenic lines than in the wild-type control plants. In transgenic lines of both Kalanchoe and Petunia, delayed flowering was observed with a mean of 24 days for Kalanchoe and a range of three to 12 days for Petunia. Although the flower morphology of the transgenic lines did not exhibit any differences from their respective wild-type control plants, transgenic lines of both species exhibited darker green pigmented leaves containing an approximately two-fold increase in chlorophyll contents over the wild-type control plants.

Selection of transgenic Petunia plants using the green fluorescent protein (GFP)

Selectable marker genes are needed for efficient transformation of plants. The present study focused on testing the applicability of green fluorescent protein (GFP) for selecting transgenic Petunia hybrida plants without applying antibiotics or herbicides. Based on a transient gene expression assay, the efficiency of two gfp genes, mGFP-4 and smRS-GFP, was compared. Two days after infiltration of Agrobacterium tumefaciens, GFP expression was recorded in leaf epidermal cells. The intensity of smRS-GFP fluorescence was higher than that of mGFP-4 and easier to distinguish from other unspecific fluorescent signals in Petunia. Transformations using the pMen65smRS-GFP vector, which contained the neomycin phosphotransferase II (nptII) gene, resulted in callus and shoots that visually and clearly expressed detectable GFP levels; in addition, this vector made it possible to exclusively select transformed plants using GFP. The transformation efficiencies achieved by using GFP selection versus combined kanamycin and GFP selection (nptII+GFP) were compared in four Petunia genotypes with a transformation experiment with four replications. In three out of four Petunia cultivars a higher transformation frequency was achieved by using nptII+GFP selection. Southern blot hybridisation revealed single and multiple integrations of smRS-GFP in Petunia. Single copy plants showed intensive expression in all parts of the plants, whereas a higher copy number led to only weak or partial expression of smRS-GFP allowing the visual selection of single copy events. Thus, it is possible to select transgenic Petunia plants based on their GFP expressions without applying antibiotics or herbicides.

Botrytis cinerea-resistant marker-free Petunia hybrida produced using the MAT vector system

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacteriumtumefaciens strain EHA105. Infected leaf explants of P.hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida.

Production and selection of marker-free transgenic plants of Petunia hybrida using site-specific recombination

MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately 4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1–3 for ipt gene and 1–2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium.

Molecular characterization and infectivity of Papaya leaf curl China virus infecting tomato in China.

Papaya leaf curl China virus (PaLCuCNV) was previously reported as a distinct begomovirus infecting papaya in southern China. Based on molecular diagnostic survey, 13 PaLCuCNV isolates were obtained from tomato plants showing leaf curl symptoms in Henan and Guangxi Provinces of China. Complete nucleotide sequences of 5 representative isolates (AJ558116, AJ558117, AJ704604, FN256260, and FN297834) were determined to be 2738-2751 nucleotides, which share 91.7%-97.9% sequence identities with PaLCuCNV isolate G2 (AJ558123). DNA-beta was not found to be associated with PaLCuCNV isolates. To investigate the infectivity of PaLCuCNV, an infectious clone of PaLCuCNV-[CN:HeNZM1] was constructed and agro-inoculated into Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants, which induced severe leaf curling and crinkling symptoms in these plants. Southern blot analysis and polymerase chain reaction (PCR) indicated a systemic infection of test plants by the agro-infectious clone.

Efficient virus‐induced gene silencing in plants using a modified geminivirus DNA1 component

Virus-induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite-like and single-stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication-associated protein open reading frame and the A-rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co-agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co-agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene-silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.

Pathogenicity of a naturally occurring recombinant DNA satellite associated with tomato yellow leaf curl China virus

Recombinant DNA beta molecules (RecDNA-Abeta) comprising parts of DNA A and DNA beta associated with tomato yellow leaf curl China virus (TYLCCNV) have been identified in naturally infected tobacco plants. Several examples of the recombinant DNA have been cloned and characterized by sequence analysis. All are approximately half the size of TYLCCNV genomic DNA, and all contain the betaC1 gene and the A-rich region from TYLCCNV DNA beta as well as intergenic region sequences and the 5' terminus of the AC1 gene from TYLCCNV DNA A. RecDNA-Abeta was detected by PCR in five of 25 TYLCCNV isolates. Co-inoculation of TYLCCNV DNA A and RecDNA-Abeta induced symptoms indistinguishable from those induced by TYLCCNV DNA A and DNA beta in Nicotiana benthamiana, Nicotiana glutinosa, Solanum lycopersicum and Petunia hybrida plants, and Southern blot hybridization results showed that RecDNA-Abeta could replicate stably in N. benthamiana plants.

Size reversion of a truncated DNAβ associated with Tobacco curly shoot virus

Our previous results demonstrated that DNAβ associated with Tobacco curly shoot virus (TbCSV) is not necessary for infection but intensifies symptoms in some hosts. To better understand the function of DNAβ in virus infection, a βC1 deleted infectious clone of the TbCSV DNAβ was constructed. Agroinoculation showed that the truncated DNAβ (DNAΔC1β) was trans-replicated by TbCSV in tobacco and Petunia hybrida plants. However, PCR and Southern blot analysis demonstrated that the truncated DNAβ reverted to near wild type component size in some Nicotiana benthamiana, N. glutinosa, N. tabacum Samsun and P. hybrida plants co-inoculated with TbCSV and DNAΔC1β. Sequence analysis of four DNAΔC1β derivatives revealed that the wild type size DNAβ molecules were recombinants between TbCSV DNAβ and the pBinPLUS vector in which dimeric constructs were produced for inoculation. The significance of these findings is discussed with respect to the constraints imposed on begomovirus genome size.

HlMyb3, a Putative Regulatory Factor in Hop (Humulus lupulus L.), Shows Diverse Biological Effects in Heterologous Transgenotes

A hop-specific cDNA library from glandular tissue-enriched hop cones was screened for Myb transcription factors. cDNA encoding for R2R3 Myb, designated HlMyb3, was cloned and characterized. According to the amino acid (aa) sequence, HlMyb3 shows the highest homology to GhMyb5 from cotton and is unrelated to the previously characterized HlMyb1 from the hop. Southern blot analyses indicated that HlMyb3 is a unique gene, which was detected in various Humulus lupulus cultivars, but not in Humulus japonicus. Reverse transcription and real-time PCR revealed the highest levels of HlMyb3 mRNA in hop cones at a late stage of maturation and in colored petiole epidermis, while the lowest levels were observed in hop flowers. Two alternative open reading frames starting in the N-terminal domain of HlMyb3, encoding for proteins having 269 and 265 amino acids with apparent molecular masses of 30.3 and 29.9 kDa, respectively, were analyzed as transgenes that were overexpressed in Arabidopsis thaliana, Nicotiana benthamiana, and Petunia hybrida plants. Transformation with the longer 269 aa variant designated l-HlMyb3 led to a flowering delay and to a strong inhibition of seed germination in A. thaliana. Nearly complete flower sterility, dwarfing, and leaf curling of P. hybrida and N. benthamiana l-HlMyb3 transgenotes were noted. On the contrary, the shorter 265-aa-encoding s-HlMyb3 transgene led in A. thaliana to the stimulation of initial seed germination, to fast initiation of the lateral roots, and to quite specific branching phenotypes with many long lateral stems formed at angles near 90 degrees . Limited plant sterility but growth stimulation and rather branched phenotypes were evident for s-HlMyb3 transgenotes of P. hybrida and N. benthamiana. It was found that both HlMyb3 transgenes interfere in the accumulation and composition of flavonol glycosides and phenolic acids in transformed plants. These effects on heterologous transgenotes suggest that the HlMyb3 gene may influence hop morphogenesis, as well as metabolome composition during lupulin gland maturation.

Delayed flower senescence of Petunia hybrida plants transformed with antisense broccoli ACC synthase and ACC oxidase genes

Petunia ( Petunia × hybrida Hort. Vilm.-Andr.) plants were transformed by Agrobacterium with antisense BoACS1 (broccoli ACC synthase) and antisense BoACO1 (broccoli ACC oxidase) coding sequences of enzymes involved in biosynthesis of ethylene in broccoli plants. The integration of these genes with an antisense orientation was verified by PCR analyses of kanamycin-resistant regenerants. The expression of transgenes and endogenous genes was further confirmed by RT-PCR analysis. Production of ethylene in shoot tissues was reduced among most transgenic plants. Flowers of transformants, especially excised flowers, generally remained fresh longer than those of untransformed controls. The delayed flower senescence was more pronounced with the antisense BoACO1 than the antisense BoACS1. Transgenic tissues were, nevertheless, still responsive to ethylene. We conclude that the antisense BoACO1 gene from Brassica oleracea is able to reduce ethylene biosynthesis and delay flower senescence of Petunia hybrida more efficiently than the antisense BoACS1 gene.

Tomato leaf curl Guangxi virus is a distinct monopartite begomovirus species

Three begomovirus isolates were obtained from tomato plants showing leaf curl symptoms in Guangxi province of China. Typical begomovirus DNA components representing the three isolates (GX-1, GX-2 and GX-3) were cloned and their full-length sequences were determined to be 2752 nucleotides. Nucleotide identities among the three viral sequences were 98.9–99.7%, but all shared <86.7% nucleotide sequence identity with other reported begomoviruses. The sequence data indicated that GX-1, GX-2 and GX-3 are isolates of a distinct begomovirus species for which the name Tomato leaf curl Guangxi virus (ToLCGXV) is proposed. Further analysis indicated that ToLCGXV probably originated through recombination among viruses related to Ageratum yellow vein virus, Tomato leaf curl China virus and Euphorbia leaf curl virus. PCR and Southern blot analyses demonstrated that isolates GX-1 and GX-2 were associated with DNAβ components, but not isolate GX-3. Sequence comparisons revealed that GX-1 and GX-2 DNAβ components shared the highest sequence identity (86.2%) with that of Tomato yellow leaf curl China virus (TYLCCNV). An infectious construct of ToLCGXV isolate GX-1 (ToLCGXV-GX) was produced and determined to be highly infectious in Nicotiana benthamiana, N. glutinosa, tobacco cvs. Samsun and Xanthi, tomato and Petunia hybrida plants inducing leaf curl and stunting symptoms. Co-inoculation of tomato plants with ToLCGXV-GX and TYLCCNV DNAβ resulted in disease symptoms similar to that caused by ToLCGXV-GX alone or that observed in infected field tomato plants.

Sequence Analysis of a “True” Chalcone Synthase (chs_H1) Oligofamily from hop (Humulus lupulus L.) and PAP1 Activation of chs_H1 in Heterologous Systems

Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for "true" chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the "native" chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome.

Stachytarpheta leaf curl virus is a novel monopartite begomovirus species

Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAbeta or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAbeta molecules in plants.

SILENCING THE EXPRESSION OF AN AGROBACTERIUM VITIS ONCOGENE IN A MODEL PLANT

The purpose of this study was to demonstrate the feasibility of silencing the expression of an essential oncogene from Agrobacterium vitis. Plants that silence Agrobacterium oncogenes are expected to resist the formation of tumors, and therefore better tolerate crown gall disease. Transgenic Petunia hybrida plants that expressed the coding sequence of the Agrobacterium vitis Tm4 TA-iaaM gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter produced a readily recognizable phenotype that was characteristic of elevated auxin levels. A transgenic line that stably expressed the 35S-iaaM transgene was double-transformed with a DNA construct that contained two convergently repeated copies of a partial iaaM sequence. A subset of the double-transformed plants appeared phenotypically wild-type even though they maintained an apparently intact 35S-iaaM transgene in their genomes. These plants had reduced iaaM RNA levels relative to the progenitor 35S-iaaM line and to those double-tranformants that retained the elevated-auxin-levels phenotype. These data suggest that the down-regulation of the 35S-iaaM transgene was mediated through a gene silencing mechanism.

Extended flower longevity of Petunia hybrida plants transformed with boers, a mutated ERS gene of Brassica oleracea

Petunia x hybridaHort.Vilm.-Andr. was transformed with boers, a mutatedallele of BOERS, an ethylene receptor sensor gene ofBrassicaoleracea.boers was obtained by removing anEcoRI cutting site with a silent mutation at Gly-521 andintroducing a point mutation at Ile-62, replacing isoleucine withphenylalanine. Transformation was Agrobacterium tumefaciens mediated.Hygromycin resistant regenerants were tentatively confirmed as transformants byPCR's for HPH and boers and moredefinitively by Southern hybridization of genomic DNA with pBOERS4421. Flowersof transgenic plants retained turgidity and pigmentation considerably longerthan those of untransformed controls, whether left undisturbed on plants orexcised and placed in water. Furthermore, flowers were unaffected by exposureto exogenous ethylene. Excised shoots of transgenic plants released considerablymore ethylene than those of untransformed plants. Transformed plants alsoproduced apparently larger flowers. Unexpectedly higher mortality was observed,suggesting that the ethylene insensitive petunia plants were also lower indisease resistance.

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.

Post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector.

A vector that produces DNA replicons (multicopy plant episomes) was constructed using elements of the geminivirus tobacco yellow dwarf virus (TYDV). All plant cells contain an integrated chromosomal T-DNA copy of the TYDV elements that provides a template for the production of episomes in the cell nucleus. Transgenic Petunia hybrida plants containing a CaMV 35S promoter-driven chalcone synthase A (ChsA) gene cloned into the episomal vector produced flowers with a white-spotted phenotype at high frequency. The spots were found at random locations in the petals and occurred in corresponding positions in both the upper and lower epidermis, indicating that the spots were non-clonal. The spotted phenotype was somatically stable and was inherited through meiosis. In white-spotted flower tissue, steady-state ChsA mRNA levels were downregulated but rates of RNA transcription were unaffected, suggesting that the phenotype resulted from post-transcriptional gene silencing of the endogenous and episomal ChsA genes. Increases in both the frequency and extent of gene silencing in flowers correlated with increases in episome copy number in mature flowers, flower buds and young and fully expanded leaves. Relatively small increases in episome copy number (less than threefold) appeared sufficient to trigger the gene-silenced phenotype.

Stress inactivation of foreign genes in transgenic plants

The inactivation of endogenous plant genes during periods of stress or environmental changes has already been demonstrated. Although the expanding use of transgenic plants in scientific research has given hints for transgene inactivation caused by transinactivation, the first inactivation of transgene-encoded proteins caused by environmental changes was observed not before 1990. Transgenic Petunia hybrida plants, carrying the maize A1 cDNA under the control of the CaMV35S RNA promoter causing a salmon red flower phenotype, were released to the field. Environmental factors, possibly including heat, lead to a stable loss of flower pigmentation, accompanied by the methylation of the viral promoter. In the same year, the inactivation of the phosphinothricin resistance gene in single-cell cultures of a transgenic Medicago sativa line was observed in more than 90% of the cells after a 10-day heat treatment. In transgenic tobacco, different transgenes could be inactivated by a heat treatment (37°C), which did not lead to changes in the growth of the plant. These inactivations seem to be not correlated with methylation. The heat-induced inactivation of herbicide resistance genes led to sensitivity to the herbicide during the heat treatment. The transgene activity was regained after the temperature decreased to normal cultivation conditions. To date, heat-induced transgene inactivation has not been reported in the field, but it may give rise to problems if it does occur. Therefore, strategies to analyse the causes of heat-induced transgene inactivation and to circumvent the problem have to be developed.