Effect of ambiol on stem growth in regenerants of source and transgenic potato plants

The effects of ambiol, a new growth regulator, on stem growth and morphological features of stem development have been compared in regenerants of potato (Solanum tuberosum L., var. Desire) plants transgenic for a defensin gene and in original potato plants. The original and transgenic plants exhibited differences in shoot development, which were observed both in control settings (no ambiol) and in the presence of various ambiol concentrations. In addition to normal plants of both forms, plant regenerants with morphological deviations were present in ambiol-treated groups. It is suggested that the abnormal shoot development observed in original and transgenic potato plants treated with ambiol is associated with (a) hormonal changes caused by expression of the defensin gene in the transgenic plants and (b) effects of ambiol on the hormonal balance of the plants.     

Morphogenetic Changes in Ambiol-Treated Regenerants of Parent and Transgenic Potato Plants

The effects of ambiol, a new growth regulator, on the formation of leaves and roots in parent and defensin-gene-transformed regenerants of potato Solanum tuberosum L. (cultivarDesire) were studied. Various concentrations of ambiol induced differences in morphogenetic parameters between parent and transgenic plants. In some cases, ambiol caused the formation of shoots without leaves or with rudimentary leaves. The data suggest that features of root and leaf formation in parent and transgenic regenerants induced by ambiol are due to changes in hormone balance in transgenic plants caused by expression of the defensin gene and the effect of ambiol on the plant hormonal balance.     

Morphogenetic changes in ambiol-treated regenerates of parent and transgenic potato plants

The effects of ambiol, a new growth regulator, on the formation of leaves and roots in parent and defensin gene-transformed regenerants of potato Solanum tuberosum L. (cultivar Desire). Various concentrations of ambiol induced differences in morphogenetic parameters between parent and transgenic plants. In some cases, ambiol caused the formation of shoots without leaves or with rudimentary leaves. The data suggest that features of root and leaves formation in parent and transgenic regenerants induced by ambiol are due to changes in hormone balance in transgenic plants caused by expression of the defensin gene and the effect of ambiol on the plant hormonal balance.     

Effect of ambiol on the ultrastructure of mitochondria in the apical cells of tubers of original and transgenic potato plants

The ultrastructure of the mitochondrial apparatus of apical tuber cells of original and transgenic (defensin gene-transfected) potato have been compared in normal and ambiol-treated plants, using morphometric approaches. No qualitative or quantitative differences were found between the mitochondria of original and transgenic plants under normal conditions (control). Treatment with ambiol produced only quantitative differences (in the number of mitochondria and their volume) between the cells of original and transgenic plants. This observation has been attributed to (1) changes in the physiology and biochemistry of transgenic plants, induced by the expression of the gene of defensin (hormonal balance, functional activity of the plasmalemmata, etc.), and (2) direct effects of ambiol.     

Effect of Ambiol on the Ultrastructure of Mitochondria in the Apical Cells of Tubers of Original and Transgenic Potato Plants

The ultrastructure of the mitochondrial apparatus of apical tuber cells of original and transgenic (defensin gene-transfected) potatoes have been compared in normal and ambiol-treated plants, using morphometric approaches. No qualitative or quantitative differences were found between the mitochondria of original and transgenic plants under normal conditions (control). Treatment with ambiol produced only quantitative differences (in the number of mitochondria and their volume) between the cells of original and transgenic plants. This observation has been attributed to (1) changes in the physiology and biochemistry of transgenic plants, induced by the expression of the gene of defensin (hormonal balance, functional activity of the plasmalemmata, etc.), and (2) direct effects of ambiol.     

Ultramorphometric study of the plastids in apical tuber cells of original and transgenic potato plants treated with ambiol

Ultramorphometric characteristics of plastids in cells of apical tuber meristems of original and defensin gene-transfected potato (Solanum tuberosum L.) plants, either maintained under normal conditions or subjected to treatment with the antioxidant ambiol, were compared. Under normal conditions, the tuber cells of the original and transgenic potato plants differed in neither the number nor size of the plastids. Only certain quantitative distinctions in the development of individual ultrastructural characteristics of plastids were detected. Treatment with ambiol enhanced the differentiation of the internal membrane system of plastids in the cells of original and transgenic plants, especially the tubular membrane systems. Certain differences in the responses to ambiol of cell plastids of original and transgenic plants were related to plastid sizes and development of individual intraplastid structures. The results comply with earlier data on varying responses of mitochondria of original and transgenic plants to ambiol treatment.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 330–339.Original Russian Text Copyright © 2005 by Platonova, Evsyunina, Belikov, Korableva.     

Ultramorphometric study of the plastids in apical tuber cells of original and transgenic potato plants treated with ambiol

Ultramorphometric characteristics of plastids in cells of apical tuber meristems of original and defensin gene-transfected potato (Solanum tuberosum L.) plants, either maintained under normal conditions or subjected to treatment with the antioxidant ambiol, were compared. Under normal conditions, the tuber cells of the original and transgenic potato plants differed in neither the number nor size of the plastids. Only certain quantitative distinctions in the development of individual ultrastructural characteristics of plastids were detected. Treatment with ambiol enhanced the differentiation of the internal membrane system of plastids in the cells of original and transgenic plants, especially the tubular membrane systems. Certain differences in the responses to ambiol of cell plastids of original and transgenic plants were related to plastid sizes and development of individual intraplastid structures. The results comply with earlier data on varying responses of mitochondria of original and transgenic plants to ambiol treatment.     

Transformed Potato Plants Carrying the Gene of the Antifungal Peptide of Amaranthus caudatus

Potato plants (Solanum tuberosumL., var. Desire) were transformed with a pH22Kneo vector harboring the gene ac2, which encodes the fungicidal peptide (defensin) from the seed of amaranth (Amaranthus caudatusL.). The transformation involved cocultivation on a solid MS medium of potato stem explants (excised from aseptically grown plants) andAgrobacterium tumefaciens. The factors affecting in vitroregeneration of the explants and the transformation efficiency were optimized. Regenerated potato plants carrying the amaranth defensin gene were selected by two traits, growth and ability to form roots on a kanamycin-supplemented MS medium. The transgenic state was confirmed by PCR analysis of ac2in tissues of the kanamycin-resistant plants. The transgenic organisms thus obtained differed from the original plants in their patterns of Ambiol-induced growth and proton translocation across the plasma membrane of the tuber cells.     

Isolation of genetically modified potato plant containing the gene of defensive peptide from Amaranthus]

The plants of potato (Solanum tuberosum L., var. Desire) have been transformed with a pH22Kneo vector carrying the gene ac2, encoding the fungicidal peptide (defensin) from the seed of amaranth (Amaranthus caudatus L.). The transformation involved co-cultivation of potato stem explants (excised from aseptically grown plants) and Agrobacterium tumefaciens on solid MS medium. Factors affecting in vitro regeneration of the explants and the transformation efficiency were optimized. Regenerated potato plants harboring the amaranth defensin gene were selected by two traits, growth and ability to form roots on kanamycin-supplemented MS medium. The transgenic state was confirmed PCR analysis of ac2 in tissues of the kanamycin-resistant plants. The transgenic organisms thus obtained differed from the original ambiol-treated plants in growth patterns and proton translocation across the plasma membrane of the tuber cells.     

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.     

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.     

Establishment of multiple shoot clumps from maize (Zea mays L.) and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene als (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene als has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R₁ plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.     

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum tuberosum L. cv Desiree), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35S promoter, and created transgenic potato plants through Agrobacterium tumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state level of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions. Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.     

Establishment of multiple shoot clumps from maize (Zea mays L.) and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene als (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene als has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R1 plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.     

Extent of ipt gene expression and resulting amount of cytokinins affect activities of carboxylation enzymes in transgenic plants

Three types of transgenic plants of Solanum tuberosum cvs. Kamyk and Oreb, and Nicotiana tabacum cvs. Maryland Mammoth and Trapezond were selected according to intensity of introduced ipt gene expression and resulting amount of synthesised cytokinins (CKs). In comparison with controls, original transgenic regenerants grown in vitro showed a massive increase of CK contents, in tobacco by 379 % and in potato by 159 % (MAS). Potato grown in soil from tubers of transgenic plants demonstrated a moderate increase (44 %) of CK contents (MOD). Transgenic tobacco grown from seeds in vitro did not show any significant change in CK contents (NOT). Initial (RuBPC_i and RuBPO_i) and total (RuBPC_t) activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and the activity of phosphoenolpyruvate carboxylase (PEPC) were not significantly affected by the transformation in the NOT plants. In the MOD plants, the RuBPCO activities were stimulated by up to 34 % whereas the PEPC activity was decreased by 17 %. On the other hand, all the measured enzyme activities were 32 – 91 % lower in the MAS. Leaf area, fresh and dry masses, and chlorophyll and soluble protein contents also went down with increasing CK amounts in the transformants. Dependence of RuBPC_i/RuBPO_i and RuBPC_t/PEPC ratios on the relative CK amounts in transgenic plants revealed that the individual enzyme activities were not affected uniformly. Endogenous CK contents in the MAS thus apparently exceeded an optimum needed for positive effects on many physiological traits and became a stress factor for such plants.