Patatin Gene Research Articles

Expressing the Saccharomyces cerevisiae SUC2 gene encoding invertase apoplastic localization in the transgenic potato (Solanum tuberosum L.) does not contribute to microtubers production of identical physiological age

Study was aimed at assessing the role of apoplastic invertase in synchronization of the microtuber formation in potato plants in vitro. A non-transgenic potato (Solanum tuberosum L.) plants (hereinafter nonTPs) and a line of potato plants, which expressed the SUC2 gene of Saccharomyces cerevisiae encoding invertase apoplastic localization under the control of the B33 promoter of the patatin gene (hereinafter TPs), was used to achieve this purpose. Cell divisions in the axillary meristems of single-node cuttings were synchronized by exposing to 7°C for 24 h in a MS medium (without agar). After the low-temperature, the synchronization (LTS) of proportion of simultaneously dividing cells in meristems of nonTPs was four-fold greater than without chilling. The LTS did not change the number of dividing cells in the axillary meristems of TPs. The LTS contributed to an increase in the mass and size of microtubers in both potato lines. All microtubers of nonTPs were of physiological maturity. At the same time, microtubers of TPs were physiologically immature, with high glucose content. During the tuber formation, the activity of acidic invertases in microtubers of TPs was higher than that of nonTPs. Microtubers of TPs were larger than the nonTPs, more hydrated with low starch amount. Propably glucose accumulated in the microtubers of the TPs may act as a signal to trigger cell division. It indicates the regulatory function of apoplastic invertase and sugars. The high activity of apoplastic invertase plays a negative role in the production of potato microtubers of identical physiological age.

Drought-Induced Regulatory Cascades and Their Effects on the Nutritional Quality of Developing Potato Tubers.

Competition for scarce water resources and the continued effects of global warming exacerbate current constraints on potato crop production. While plants’ response to drought in above-ground tissues has been well documented, the regulatory cascades and subsequent nutritive changes in developing tubers have been largely unexplored. Using the commercial Canadian cultivar “Vigor”, plants were subjected to a gradual drought treatment under high tunnels causing a 4 °C increase in the canopy temperature. Tubers were sampled for RNAseq and metabolite analysis. Approximately 2600 genes and 3898 transcripts were differentially expressed by at least 4-fold in drought-stressed potato tubers, with 75% and 69% being down-regulated, respectively. A further 229 small RNAs were implicated in gene regulation during drought. Expression of several small RNA clusters negatively correlated with expression of their six target patatin genes, suggesting involvement in the regulation of storage proteins during drought. The comparison of protein homologues between Solanum tuberosum L. and Arabidopsis thaliana L. indicated that down-regulated genes were associated with phenylpropanoid and carotenoid biosynthesis. As is indicative of reduced flow through the phenylpropanoid pathway, phenylalanine accumulated in drought-stressed tubers. This suggests that there may be nutritive implications to drought stress occurring during the potato tuber bulking phase in sensitive cultivars.

Structural and Thermodynamic Characteristics of Potato Starches Depending on the Plant Genotype and Conditions of Their Cultivation

The creation of new highly productive forms of potato by genetic engineering methods raises the question about the quality of transgenic tubers, which first of all relates to the properties of important storage compound such as starch. The article presents the results of studies aiming to the analysis of morphology, structure, and thermodynamic parameters of starch extracted from the potato tubers expressing the tms1 auxin biosynthesis gene from Agrobacterium tumefaciens and characterized by increased productivity in vitro. The transformed and control plants were cultivated in vitro on artificial sterile medium, as well as in vivo in the soil. The methods of scanning electron microscopy, wide-angle X-ray scattering, and differential scanning microcalorimetry were used in the work. It was established that the transformation of plants with the construction with the tms1 gene under the control of the patatin gene promoter causes significant changes in a number of thermodynamic parameters of starch, first of all an increase in the thickness of crystalline lamella and melting temperature, which apparently reflects the increase in the structural ordering of the main starch fraction (approximately 90%) in the tubers of these transformants. Along with this, the effect of accumulating fractions with a less ordered structure in the starch composition, correlating with the ectopic expression of the tms1 auxin biosynthesis gene, was established. At the same time, the B type of the polymorphic structure of starch remains unchanged. However, the detected changes mainly affected starch of the plants, cultivated in vitro. The starch of transgenic plants cultivated in the soil differs little in its basic characteristics from the starch of nontransgenic control plants.

MYB class transcription factors bind to the tuber-specific and sucrose-response element of a class-I patatin promoter

Class-I patatin genes, which encode the potato storage protein patatin, are tuber specific in their expression, and several promoter elements directing the tissue-specific expression of the patatin genes have been known for long time. However, the identity of the relevant transcription factors is still unknown. In this study, we show that K box, one of the protein-binding sites in a class-I patatin gene promoter, is a novel cis-regulatory element required for the tuber-specific expression of the gene. Subsequently, we isolated K-box-binding factors by yeast one-hybrid screening. Analysis of the factors revealed that they are MYB class transcription factors, which we named TSF1–4 (tuber-specific and sucrose-inducible element-binding factors 1–4). TSFs possess transcriptional activity to various degrees, and overexpression of TSF2, which possesses the highest transcriptional activity, induced the ectopic expression of patatin genes in potato leaves. Our results strongly suggest that TSFs could play regulatory function in the expression of patatin genes in planta.

Auxin synthesis gene tms1 driven by tuber-specific promoter alters hormonal status of transgenic potato plants and their responses to exogenous phytohormones

Ectopic auxin overproduction in transgenic potato leads to enhanced productivity accompanied with concerted and occasional changes in hormonal status, and causing altered response of transformants to exogenous auxin or cytokinin. Previously, we generated potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 driven by tuber-specific patatin gene promoter (B33-promoter). Here, we studied the endogenous hormonal status and the response to exogenous phytohormones in tms1 transformants cultured in vitro. Adding indole-3-acetic acid (IAA) or kinetin to culture medium affected differently tuberization of tms1-transformed and control plants, depending also on sucrose content in the medium. Exogenous phytohormones ceased to stimulate the tuber initiation in transformants at high (5-8%) sucrose concentration, while in control plants the stimulation was observed in all experimental settings. Furthermore, exogenous auxin partly inhibited the tuber initiation, and exogenous cytokinin reduced the average tuber weight in most transformants at high sucrose content. The elevated auxin level in tubers of the transformants was accompanied with a decrease in content of cytokinin bases and their ribosides in tubers and most shoots. No concerted changes in contents of abscisic, jasmonic, salicylic acids and gibberellins in tubers were detected. The data on hormonal status indicated that the enhanced productivity of tms1 transformants was due to auxin and not mediated by other phytohormones. In addition, exogenous cytokinin was shown to upregulate the expression of genes encoding orthologs of auxin receptors. Overall, the results showed that tms1 expression and local increase in IAA level in transformants affect both the balance of endogenous cytokinins and the dynamics of tuberization in response to exogenous hormones (auxin, cytokinin), the latter reaction depending also on the carbohydrate supply. We introduce a basic model for the hormonal network controlling tuberization.

Negative association between chickpea response to competition and crop yield: Phenotypic and genetic analysis

Donald’s ideotype and empirical evidence in cereal and oilseed crops indicate high yield is associated with less competitive plants. In this study we grew 20 chickpea lines in six environments to investigate the association between yield and intra-specific competitive ability and its genetic underpinnings using Fst genome scan based on whole genome resequencing data. We measured yield and its components and calculated response to competition (RC) as the ratio between the trait in outer rows (relaxed competition) and the trait in inner rows (higher competition). Crop yield correlated negatively with RC for yield, biomass, harvest index, seed number, and pod number. Fst genome scan revealed 14 genomic regions under selection for response to competition of yield, seed number or biomass, and 6 genomic regions under selection for yield in inner or outer canopy rows. Candidate genes in these regions include members of the nitrate-transporter 1 family, patatin and hormone-related genes. The top genomic regions found to be under selection for yield in inner rows and outer rows did not coincide. This genetic architecture provides a mechanistic basis for the observation that phenotypes that are adequate for relaxed competition often perform poorly in dense stands.

Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro.

Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.

Class I patatin genes from potato (Solanum tuberosum L.) cultivars: molecular cloning, sequence comparison, prediction of diverse cis-regulatory motifs, and assessment of the promoter activities under field and in vitro conditions

Patatin, the most abundant glycoprotein (∼40 kDa) in potato (Solanum tuberosum L.) tuber is encoded by a large multigene family comprising of two classes. Class I patatin gene promoters are attractive molecular tools for studying multiple facets of transcriptional regulation. Only a few members of this class are relatively well understood at the molecular level. Here, we report the isolation of two new partial class I patatin genes with corresponding 5′-flanking regions, designated as StPN02 and StPK01, from two commercially important Indian potato cultivars, ‘Kufri Jyoti’ and ‘Kufri Chipsona-1’, respectively. Their 5′-flanking regions were found to vary significantly because of insertions/deletions in addition to point mutations. A major 487-bp insertion in the 5′-flanking region of StPN02 was a distinctive from other family members. Many of the diverse cis-regulatory sequence motifs predicted in this study have not been previously documented. The 1,215 and 2,225 bp 5′-flanking regions of StPN03 (derived from StPN02) and StPK01, respectively, were used in generating patatin promoter–GUS gene fusions for assessment of promoter activities in potato. Under field conditions, both StPK01 and StPN03 promoters showed tuber-specificity. The former was significantly stronger than the latter as examined by histochemical and fluorometric β-glucuronidase assays. Under in vitro conditions, these promoters were highly inducible by sucrose in stems and leaves. Apart from the applied aspects of this research, this study may help in unraveling the molecular mechanisms involved in the regulation of patatin gene expression.

Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

Expression of a class I patatin cDNA clone, SK24-1, in Escherichia coli revealed that the cDNA clone possessed lipid acyl hydrolase (LAH) activity. Transformed potato plants were obtained via Agrobacterium-mediated transformation using the chimeric constructs containing the sense and antisense cDNA under the control cauliflower mosaic virus 35S (CaMV 35S) promoter. In some sense transformed plants, both sense patatin RNA and LAH activity were increased and further resulted in a significant increase of percentage of plantlets that formed microtubers and numbers of microtubers per plantlet in vitro. All antisense plants displayed a reduction in LAH activity. Both sense and antisense RNA could be detected in antisense plants, but transcripts of antisense RNA resulted in a reduction of endogenous sense RNA. Moreover, expression of antisense cDNA in some antisense transformed plants led to a significant decrease in the number of microtubers formed. These results suggest that SK24-1 was involved in regulating microtuber formation. Key words: Patatin, potato, Escherichia coli, sense RNA, antisense RNA

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins

BackgroundPatatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.ResultsBased upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts) of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L.) was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi) vector. The CaMV 35S promoter-driven patatin hpRNAi vector was transformed into the potato cultivar Desiree by Agrobacterium-mediated transformation. Ten transgenic potato lines bearing patatin hpRNA were generated. The effects of RNA interference were characterized at both the protein and mRNA levels using 1D and 2D SDS/PAGE and quantitative real-time RT-PCR analysis. Dependent upon the patatin hpRNAi line, patatins decreased by approximately 99% at both the protein and mRNA levels. However, the phenotype (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc.) of hpRNAi lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. During glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins.ConclusionPatatin-specific hpRNAi effectively suppressed the expression of a majority of patatin variants in potato tubers via the specific degradation of individual mRNAs of the patatin multi-gene family. More importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins.

Organ-specificity and inducibility of patatin class I promoter from potato in transgenic arabidopsis plants

Patatin class I promoter (B33 promoter) is a tissue-specific potato (Solanum tuberosum L.) promoter expressing the patatin gene mainly in tubers. However, it can be induced in other organs by sucrose or light. We compared the activity of this promoter fused with the reporter gene during heterological expression in B33::GUS transgenic arabidopsis (Arabidopsis thaliana L.) plants and homological expression of the same DNA construct in potato. Promoter activity was estimated from quantification of β-glucuronidase (GUS) activity. It was shown that, during heterological expression in arabidopsis seedlings, B33 promoter manifested a tissue-specificity and inducibility, although in a different manner than during homological expression in potato. In noninduced arabidopsis seedlings, B33 promoter was most active in the roots, whereas, after induction with sucrose treatment, it became most active in cotyledons. 10 mM sucrose was sufficient for a manifold activation of B33 promoter in intact seedlings. The degree of B33 promoter induction by sucrose in arabidopsis seedlings was strictly organ-specific and increased in the following sequence: root < hypocotyl < cotyledons. 150–200 mM sucrose enhanced B33 promoter activity in cotyledons by 200 to 300 times, i.e., much stronger than in potato organs. Glucose and fructose were less efficient than sucrose. Phytohormones affecting tuber formation in potato (gibberellins, auxins, and cytokinins) did not affect significantly B33 promoter activity in arabidopsis. A lag period of approximately 6 h preceded sucrose-induced B33 promoter activation. This indicates that the patatin promoter is not the primary target for the sucrose signal. The quantitative examination of heterological expression of patatin class I promoter further clarifies its basic functional characteristics and permits a better prognosis of its behavior after transferring into other plant species.

Structural Diversity and Differential Transcription of the Patatin Multicopy Gene Family During Potato Tuber Development

The patatin multicopy gene family encodes the major storage protein in potato tubers and is organized as a single cluster in the potato genome. We sequenced a 154-kb bacterial artificial chromosome (BAC) clone containing a portion of the patatin gene cluster. Two putatively functional patatin genes were found in this BAC. These two genes are embedded within arrays of patatin pseudogenes. Using a chromatin immunoprecipitation method we demonstrate that the dramatic increase of patatin gene expression during the transition from stolons to tubers coincides with an increase of histone H4 lysine acetylation. We used 3' rapid amplification of cDNA ends to profile expression of different patatin genes during tuber development. The profiling results revealed differential expression patterns of specific patatin gene groups throughout six different stages of tuber development. One group of patatin gene transcripts, designated patatin gene group A, was found to be the most abundant group during all stages of tuber development. Other patatin gene groups, with a 48-bp insertion in the 3'-untranslated region, are not expressed in stolons but display a gradual increase in expression level following the onset of tuberization. These results demonstrate that the patatin genes exhibit alterations in chromatin state and differential transcriptional regulation during the developmental transition from stolons into tubers, in which there is an increased demand for protein storage.

Transformation of potato using an antisense class I patatin gene and its effect on microtuber formation

AbstractAn antisense class I patatin gene under control of the CaMV 35S promoter was introduced into potato (Solanum tuberosum) cultivar E-potato 3 using the Agrobacterium tumefaciens system. PCR amplification and PCR–Southern blot analysis indicated that the antisense class I patatin gene had been integrated into the potato genome. Northern hybridization analysis showed that the antisense gene transcribed normally in the transgenic potato plants and resulted in a reduction of endogenous class I patatin mRNA. Total soluble protein content and lipid acyl hydrolase activity of microtubers, derived from transformed plants, decreased by a maximum of 36.4% and 31.4%, respectively, compared with control plants. The expression of this antisense gene also resulted in reductions of the plantlets forming tubers, tubers per plantlet and the effective tubers (≥50 mg) of the transformed plants.

Expression and Characterization of Hepatitis B Surface Antigen in Transgenic Potato Plants

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression.

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber-specific and sucrose-inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B-box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA-binding protein designated Storekeeper (STK) that specifically recognises the B-box motif in vitro. Gel shift experiments with an STK-specific antibody suggest that STK is the B-box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B-box motif.

Cloning and characterization of a latex allergen (Hev b 7): homology to patatin, a plant PLA2.

We previously identified a 46-kD protein allergen in latex as having amino acid sequence homology to the patatin gene family. The objective of this study was to characterize this protein by molecular techniques. RNA was isolated from the latex or leaf material from Hevea brasiliensis and from potato tubers. Specific polymerase chain reaction (PCR) primers were designed from the amino acid sequence and reverse transcriptase (RT)-PCR amplified a specific product from latex RNA that was subsequently cloned and sequenced. This product was 1493 bp in length with an 1167 bp open reading frame. The deduced amino acid sequence encodes for a 389 aa protein, pI 4.82 with 43% homology to tobacco patatin. Northern analysis of potato, Hevea leaf, and latex RNA demonstrated the message to be most abundant in latex, weakly present in Hevea leaf, but no hybridization occurred with potato RNA. Patatin has lipid acyl-transferase and PLA2-like activity, suggesting it plays a role as a defence-related protein. Other defence-related proteins in latex such as hevein, glucanase, and hevamine are also allergens. Increased production of defence-related proteins as a result of increased tapping of the rubber trees to meet the demand for latex may explain the increased allergenicity of latex.

Potato and Tomato Forever Young Genes Contain class-I Patatin promoter-like Sequences

Class-I patatin genes from potato, encoding the major storage protein in tubers, contain promoter repeat sequences, which are critical for developmental and metabolic regulation. Previously, RFLP mapping indicated the patatin genes of potato and tomato reside in orthologous loci near the end of the long arm of chromosome VIII. Interestingly, a DNA sequence homologous to the promoter of the potato class-I patatin gene was mapped to the middle of chromosome III in the tomato and potato genomes [Ganal et al., Mol. Gen. Genet. 225, 501-509 (1991)]. We isolated and characterized genomic clones containing the class-I Patatin Promoter-Like sequences (PPL) from tomato, S. bulbocastanum, and S. tuberosum. Sequence analyses reveal that these PPL sequences are highly conserved, as compared to the repeat sequence of the patatin promoter, particularly with respect to the critical regulatory ciselements. These PPL sequences are located upstream of the translational initiation site of Forever Young genes, which encode proteins belonging to a large family of oxidoreductases with conserved structural motifs. The Forever Young genes from tomato and S. bulbocastanuin both contain eight exons and seven introns. Six of the introns are uniquely large, in the range of 418—2,092 bp. Sequence conservation suggests that the class-I patatin repeat and the PPL from Forever Young genes may have a direct phylogenetic relationship and may regulate expression of their respective genes through common cis- and trans-factors.

Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

Growth habit and sugar accumulation in sugarbeet (Beta vulgaris L.) transformed with a cytokinin biosynthesis gene

Expression of a bacterial cytokinin biosynthesis gene fused to a patatin gene promoter was studied in sugarbeet (Beta vulgaris L.). Two independent transformants, Pat-ipt 1 and 2, exhibited a number of distinguishable morphological alterations commonly induced by cytokinins, i.e. less root growth, reduced leaf surface area, and increased axillary shoot development. Concentrations of the cytokinins zeatin and zeatin riboside were increased by twofold in taproots and 7- to 18-fold in leaves. Leaf sucrose and glucose concentrations were not significantly different from those in control plants except in Pat-ipt 2 where glucose levels were elevated ninefold. Since normal taproot development was severely inhibited, sucrose concentrations in the taproots were significantly reduced.

Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet ( Beta vulgaris L.) by Agrobacterium or particle bombardment

Two different methods for sugarbeet (Beta vulgaris L.) transformation were developed, one using Agrobacterium with excised cotyledons, the other, particle bombardment of embryogenic hypocotyl callus. Transformation efficiencies averaged 0.7% for the Agrobacterium method (number of transgenic plants obtained per treated cotyledon) and about 8% for the bombardment method (number of transgenic plants obtained per plate of embryogenic callus treated). Transgenic sugarbeet plants were produced carrying genes encoding either pathogen-defense-related proteins or the reporter enzyme β-glucuronidase (GUS) under transcriptional control of stress- or wound-inducible promoters. In addition, two plants were regenerated carrying a gene associated with enhanced insect resistance, the cytokinin biosynthesis gene, fused to a patatin gene promoter from potato. Expression of the GUS gene (gusA) under the control of the tobacco osmotin promoter was wound inducible with detectable activity at 8 h and maximal activity at 72 h post-wounding.