Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the ??cytokinin deficiency syndrome?? were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.     

Vacuolar Invertase Gene Silencing in Potato (Solanum tuberosum L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.     

Impact of invertase overexpression on cell size, starch granule formation and cell wall properties during tuber development in potatoes with modified carbon allocation patterns

Transgenic potato tubers that overexpressed either a cytosolic or an apoplastic invertase in the wild type or AGPase antisense background were used to analyse the effect of invertase activity on cell expansion, starch granule formation and turgor pressure during tuber development. Although the transgenic plants did not develop a visible phenotype in aerial regions the size and number of tubers were significantly modified in the various lines. Transmission electron and light microscopy were performed to monitor starch grain size and number, cell size and cell wall thickness. Water potential, osmotic pressure, and indirectly, turgor pressure were determined during the final stages of tuber development. Glucose levels were high in transgenic tubers that overexpressed a yeast-derived invertase. The number of starch grains per cell was almost identical in all transgenic lines. However, the amount of starch was modified in the transgenics as compared to the wild type. As expected, the size of starch grains was reduced in all lines that expressed an AGPase antisense mRNA. These results indicate that invertase activity and glucose levels do not affect initiation of starch grain formation during the early stages of tuber development, but growth of starch corns in the later stages of tuber maturation.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

Control of potato tuber dormancy and sprouting by expression of sense and antisense genes of pyrophosphatase in potato

Inorganic pyrophosphate (PPi) is an enzyme involved in sugar metabolism in potato tubers. In our previous study, we isolated an inorganic pyrophosphatase (PPase) gene from potato and obtained the transgenic potato plants transformed with the sense and antisense PPase genes respectively. In the present experiment, the physiological indexes, tuber dormancy, and sprouting characteristics of the transgenic potatoes were analyzed and evaluated. The result showed that the PPase activity and the inorganic phosphate content of tubers were lower in the antisense transgenic plant lines but were higher in the sense transgenic plant lines, compared with wild-type tubers. Soluble sugars, such as glucose, fructose and sucrose increased in transgenic plants that had overexpression of the sense PPase gene, but decreased in the antisense transgenic plant lines, compared with wild-type tubers. Tuber sprouting time of the antisense transgenic plants were delayed for 2 and 3 weeks and reached the 100 % sprouting rate only after 14 and 16 weeks storage compared with the wild-type when tubers are stored under 25 and 4 °C, respectively. In contrast, tuber sprouting time of the sense transgenic plants was earlier by approximately 2 weeks than that of wild-type tubers under these storage temperatures.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

The effect of thaumatin gene overexpression on the properties of H+-ATPase from the plasmalemma of potato tuber cells

The introduction of the thaumatin gene into potato plants was accompanied by a decrease in the activity of H⁺-ATPase in the plasmalemma (PL) of tuber cells. When tubers were released from dormancy, the enzyme was activated in the tuber cells of both the original and transgenic plants. Experiments performed in vitro demonstrated that sensitivities to ambiol (AM) and jasmonic acid (JA) of H⁺-ATPase in the PL of tubers from the original plants were lower after the release from a period of deep dormancy. In preparations from the tubers of transgenic plants, the situation was reversed. The differences between the activities of H⁺-ATPase in the PL preparations produced from the original and transgenic tubers that sprouted under the action of AM and JA were detected. Thus, the overexpression of the thaumatin gene in potato plants changed the properties of H⁺-ATPase from PL.     

RNAi-mediated silencing of endogenous <Emphasis Type="Italic">Vlnv</Emphasis> gene confers stable reduction of cold-induced sweetening in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Désirée) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Désirée results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.     

Agrobacterial <Emphasis Type="Italic">rol</Emphasis> genes modify thermodynamic and structural properties of starch in microtubers of transgenic potato

Wild-type (WT) plants of potato (Solanum tuberosum L.) and their transgenic forms carrying agrobacterial genes rolB or rolC under the control of B33 class I patatin promoter were cultured in vitro on MS medium with 2% sucrose in a controlled-climate chamber at 16-h illumination and 22°C. These plants were used as a source of single-node stem cuttings, which were cultured in darkness on the same medium supplemented with 8% sucrose. The tubers formed on them were used for determination of the structure of native starch using the methods of differential scanning microcalorimetry (DSC), X-ray scattering, and scanning electron microscopy. It was found that, in starch from the tubers of rolB-plants, the temperature of crystalline lamella melting was lower and their thickness was less than in WT potato. In tubers of rolC plants, starch differed from starch in WT plants by a higher melting temperature, considerably reduced melting enthalpy, and a greater thickness of crystalline lamellae. Deconvolution of DSC thermogram makes it possible to interpret the melting of starch from the tubers of rolC plants as the melting of two independent crystalline structures with melting temperatures of 65.0 and 69.8°C. Electron microscopic examination confirmed the earlier obtained data indicating that, in the tubers of rolC plants, starch granules are smaller and in the tubers of rolB plants larger than in WT plants. Possible ways of influence of rol transgenes on structural properties of starch in amyloplasts of potato tubers are discussed.     

The pleiotropic effects induced by therolC gene in transgenic plants are caused by expression restricted to protophloem and companion cells

Expression of therolC gene fromAgrobacterium rhizogenes causes morphological and developmental alterations in transgenic plants. The histological alterations underlying the macroscopic changes and the cellular localization of the site of expression of therolC gene have shown that: (i) the expression of therolC gene is developmentally regulated, (ii) in vegetative transgenic plants, the expression of therolC gene under the control of its own promoter is restricted to companion and protophloem cells, (iii) the site of action of the product(s) of the activity of the rolC enzyme is distinct from its site of expression, (iv) precise localization of the rolC peptide has been achieved by immunocytochemistry but not by the histochemical GUS assay. These results imply that the sites of action and expression of therolC gene in trangenic plants are physically separated. Thus the product(s) of the activity of the rolC enzyme must be a factor capable of being transported. Current models forrolC gene action are discussed taking into account the reported results.     

The influence of the rolC gene on isoflavonoid production in callus cultures of Maackia amurensis

The polyphenolic complex of Maackia amurensis, as well as a complex of isoflavonoids from M. amurensis callus cultures, display strong hepatoprotective effects in experimental animal and human studies. To increase the yield of polyphenols in cultures of M. amurensis, calli were transformed with the rolC gene as well as with an empty vector that was used as a control. HPLC analysis revealed that the transgenic cultures produced the same complex of isoflavonoids. The complex consisted of 20 compounds, including isoflavones and their glucosides as well as pterocarpans and their glucosides. The cultures transformed with either the empty vector or the rolC gene construct produced on average 1.22 % dry weight (DW) and 1.39 % DW of isoflavonoids, respectively. Isoflavonoid production in the transformed callus lines carrying the empty vector and the rolC gene construct reached 106 and 146 mg/L, respectively. Moreover, the rolC gene construct promoted cell growth and overall cell productivity. The transgenic callus lines expressing the rolC gene exhibited higher levels of the following six isoflavonoids: daidzein, calycosin, formononetin, 4′-Ο-β-glucopyranosyldaidzin, maackiain and 6′-O-malonyl-3-O-β-D-glucopyranosylmaackiain. However, lower levels of genistin were observed in rolC calli than in those carrying the empty vector.     

Effect of indole-3-acetic acid and kinetin on tuberisation parameters of different cultivars and transgenic lines of potato in vitro

The effect of indole-3-acetic acid or kinetin on the weight and numberof microtubers formed was studied on single node cuttings of sevendifferent potato (Solanum tuberosum L.) cultivars as well astransgenic lines harbouring rolB or rolC genes undercontrol of the patatin class I (B33) promoter. Plants were cultivatedin vitro in the dark on solidified MS medium containing 1 to8% sucrose with or without phytohormones. Most of thenontransformed potato cultivars and transgenic lines responded tohormone application by an increase in tuber yield. Auxin and cytokininacted differently: IAA increased predominantly the tuber size whilekinetin increased the number of tubers. RolC transformantsdisplayed an altered response to sucrose and especially to auxin. Thedegree of phytohormone effect on tuberisation parameters depended onsucrose content of the medium and potato genotype.     

Growth pattern, tuber formation and hormonal balance in in vitro potato plants carrying ipt gene

Nodal cuttings of in vitro grown potato plants (Solanum tuberosum, cv. Miranda) were transformed by a vector plasmid carrying ipt gene of Agrobacterium tumefaciens. From the initial teratoma stage 5 clones of transgenic plants (1, 2, 11, 13 and 15) were obtained, which displayed in varying degree shortening of the internodes, decrease of the leaf size, decrease of apical dominance and poor rooting. In addition, two of the clones (11 and 13) showed increased stolon and tuber formation. In all these clones the endogenous level of free cytokinins (CKs) was increased: from 40% in clone 11 to almost 300% in clone 1. Also free indole-3-acetic acid (IAA) level was increased, but to a lower degree; the maximal increase was 160% (clone 13). Applied kinetin or IAA (1 mg·l^-1) strongly suppressed root and tuber formation in clones 11 and 13, although they did not affect or even stimulated these processes in control plants. For control plants the minimal medium sucrose concentration necessary for tuber initiation was 6% whereas in clone 11 plants 2% was sufficient. Different distribution of endogenous CKs and IAA was observed in clone 11 and control plants. The highest CK content was found in transgenic plants in stems and in controls in leaves. In clone 11 plants abscisic acid (ABA) level was significantly increased in comparison to the control throughout the cultivation period. Ethylene formation was strongly increased the first week after the subcultivation and later on the difference between transgenic and control plants rapidly diminished. Reactions of clone 11 plants to red (RL) and blue light (BL) were similar to reactions of control plants. In RL clone 11 plants were tall and thin with stunted leaves; in BL they had a teratoma-like appearance and formed a very high number of tubers. The role of hormones in these changes in growth and tuber formation is discussed.     

Stability of the rolC gene and its expression in 15-year-old cell cultures of Panax ginseng

After 15 years of cultivation of Panax ginseng, transgenic cell cultures were shown to express rolC gene at a high level. We determined that the rolC gene underwent mutagenesis. In particular, from one to four nucleotides were changed for the whole gene sequence (540 bp). These substitutions were synonymous in half of the cases or resulted in the modification of single amino acids in the rolC-encoded protein. With the example of β-1,3-glucanases we showed that long cultivation and the observed changes in nucleotide sequences of the transgene did not inhibit the activating effect of rolC on enzymatic activity of β-1,3-glucanases.