Transformation Of Potato Research Articles

Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T‐DNA loci in potato

SummaryIn this study the T‐DNA composition of four antisense potato transformants showing complete or very strong inhibition of granule‐bound starch synthase (GBSS) activity was analysed in detail. By Southern blot hybridizations, it was determined that all four transformants contained T‐DNAs on multiple linkage groups and that most linkage groups contained multiple T‐DNA copies, often in combination with non‐T‐DNA vector sequences. Subsequently, fluorescence in situ hybridization was performed on extended DNA fibres (‘fibre‐FISH’) of three progeny plants each containing a single linkage group with a complex T‐DNA organization. By using two differently labelled probes, one consisting of T‐DNA sequences and the other of vector DNA sequences, it was possible to visualize the composition of complex loci. DNA sequences of 5–6 kb were well distinguishable. With this technique it is possible to determine T‐DNA copy number, and arrangement of T‐DNA and vector DNA sequences in a locus, more accurately than by Southern blot analysis alone. Therefore, fibre‐FISH is a valuable supplementary tool to study T‐DNA integrations in detail.

Composition of endogenous alleles can influence the level of antisense inhibition of granule-bound starch synthase gene expression in tetraploid potato plants.

The T-DNA composition was analysed of twelve potato genotypes obtained after transforming a tetraploid cultivar with an antisense granule-bound starch synthase (GBSSI) gene. In five transformants (labelled TB50 nos.) the antisense GBSSI gene was driven by the CaMV 35S promoter, while in the remaining seven (labelled TBK50 nos.) the GBSSI promoter was used. In these twelve transformants the antisense effect on amylose production in potato tuber starch ranged from complete suppression to no discernible inhibition, and the number of T-DNA insertions ranged from one to at least fifteen. The antisense effect of individual T-DNA loci in progeny of these transformants was studied. Progeny containing a single T-DNA showed no inhibition of GBSSI activity. Only multiple, linked T-DNA insertions resulted in substantial antisense inhibition. T-DNA fragments present in duplex in selfed progeny resulted in a larger antisense effect than that in the parent (which contained the T-DNA insertions in simplex). Furthermore, the antisense effects of some T-DNA-containing linkage groups were influenced by the composition of endogenous GBSSI alleles. For practical breeding this implies that (1) the efficiency of obtaining primary potato transformants showing complete inhibition of GBSSI gene expression by antisense RNA is genotype-dependent, and (2) many transformants have to be produced per genotype to be able to select plants with maximum suppression of GBSSI and a minimum number of T-DNA loci.

Pectate lyase in transgenic potatoes confers pre-activation of defence againstErwinia carotovora

Erwinia carotovorabacteria cause blackleg and soft-rot of potatoes by enzymic maceration of host tissue. Pectate lyases (PLs), the mainE. carotovoraenzymes, depolymerize pectic substances of plant cell walls. The degradation products, unsaturated oligogalacturonates, have been proposed to induce plant defence reactions. The expression of the isoenzyme PL3in plantaand its effect on the soft-rot resistance of transgenic potatoes was investigated. The DNA sequence encoding the mature enzyme was fused to the promoter of the potato patatin B33 gene and to the 35S promoter of the cauliflower mosaic virus. The expression cassettes were inserted into the binary vector pBin19. The plasmids pB33-PL3 and p35S-PL3 were used forAgrobacterium-mediated transformation of potato, cv. Dèsirèe. Enzyme production in plant lines transformed with plasmid pB33-PL3 was confined to tuber tissue, yielding a maximum of 340 mU ml−1extract. Plants harbouring the 35S-PL3 chimeric gene exhibited constitutive expression of PL in various tissues, with activities up to 280 mU ml−1in leaf extracts and 50 mU ml−1in tuber tissue. After wounding the tuber tissue, the liberated PL3 caused a decrease of cell viability. Compared with non-transformed plants, the transgenic lines which synthesized PL3 were more resistant to tissue maceration byE. carotovoraor its enzymes. Wounding induced the transcription of the plant defence related gene encoding phenylalanine ammonia-lyase to a high level in tubers of a PL3-expressing transgenic line. The possible implications of PLs and their enzymatic products for protection against invading microbes are discussed.

Stability of a Transgene in Potato Depends on Endogenous Plant Tissue Factors

Transformation of potato cultivar (Desiree) has been achieved using different types of explant tissue (minituber, stem and leaf discs) via infection with an Agrobacterium tumefaciens harboring a kanamycin resistance gene. The product of it, an aminoglycoside phosphotransferase, has been determined in the protein extracts of the transformants in addition to the common rooting tests. Certain influences of the type of the explants upon the transformation efficiency and the morphology of the regenerated transgenic plantlets have been noticed. A selection and regeneration protocol for the potato transformants is described. A periodical study of the foreign gene stability and reversibility tests with demethylating agent of the relevant lines have been performed. A possible involvement of DNA methylation processes in the variable transgene expression in potato plantlets is discussed.

Synthesis of active oryzacystatin I in transgenic potato plants

Transformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.

POTENTIAL OF PROTEINASE INHIBITORS FOR THE PRODUCTION OF TRANSGENIC PLANTS TOLERANT TO INSECT PESTS. I. FUNCTIONAL ASPECTS

In the past few years, transformation of plant genomes with proteinase inhibitor (PI) genes has been proposed as an effective way to produce insect-tolerant plants. For such a control approach, however, biochemical studies are necessary to assess the effect of PIs on not only insect digestive proteinases (target enzymes) but also plant endogenous proteinases (nontarget enzymes). As an example, transformation of potato (Solanum tuberosum L.) with oryzacystatin (OC) genes, two cysteine PIs, was considered for controlling Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The use of electrophoretic approaches and standard assays showed that CPB uses at least 14 cysteine proteinases for protein digestion throughout its development. Proteinases of the same class were also detected in sprouting potato tuber extracts, suggesting a potential interference of cPIs in transgenic plants. While OCs inhibit a significant fraction of CPB digestive proteinases, no inactivation of potato proteinases was detected. This apparent absence of direct interference suggests the real potential of OCs for producing CPB-tolerant transgenic potato plants.

Genetic transformation of sweet potato by particle bombardment

Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.

Early events in transformation of potato by Agrobacterium tumefaciens

We have used an intron containing β-glucuronidase gene to monitor transformation events after Agrobacterium tumefaciens cocultivation with leaf explants of potato. We find that the number of transformation events greatly exceeds the number of transformed plants which can be regenerated, and that some explants have many transformed foci while a significant number have none. Cultivar differences in gene transfer are apparent, and frequencies of gene transfer can be improved with acetosyringone and silver thiosulphate. We also show that, with the transformation vector we used, some Agrobacterium tumefaciens virulence plasmids are more efficient than others.

PEG-mediated transformation of leaf protoplasts of Solanum tuberosum L. cultivars

As an alternative to Agrobacterium-mediated gene transfer, direct transformation of potato (Solanum tuberosum L.) cultivars was achieved by using the Mg2+/PEG protocol (Negrutiu et al. 1987) to stimulate DNA uptake into leaf protoplasts. The frequency of kanamycin-resistant clones varied between 1–12% from experiment to experiment independently of the genotype used. A deleterious effect of heat shock treatment (45°C for 5 min.) has been found on colony formation during optimization of the transformation procedure. The Mg2+ ion concentration (15–35 mM), the denaturation (100°C, 10 min. right before the treatment) and the form of the plasmid DNA (linear or circular) had no significant effect on the efficacy of the transformation. Application of denatured calf thymus DNA as carrier molecules (at concentration of 50 μg ml-1, however, resulted in a significant decrease in the number of the resistant cell colonies). Sixty to 80 percent of the kanamycin-selected callus tissues regenerated shoots. The presence and expression of the introduced neomycin phosphotransferase neo gene in regenerants with normal morphology and tetraploid chromosome number was proved by biological tests based on rooting and callus formation in the presence of the antibiotic and by NPT enzyme activity assay. Southern analysis revealed the integration of the introduced DNA molecules carrying the neo marker gene, into the genome of potato.

Potato transformation by T-DNA Cytokinin synthesis gene

Potato plants were transformed by the A.tumefaciens vector which integrates into the plant genome two foreign genes only: pTi C58 T-DNA gene 4 (ipt) responsible for elevated synthesis of cytokinins and kanamycin resistance gene. Three teratoma clones studied showed approximately 3 times, 6 times and 9 times increased levels of zeatin riboside and zeatin in comparison with untransformed controls and slight increase of the IAA level. Shoots formed roots in agar cultures sporadically, if the increase of zeatin riboside and zeatin levels was not higher than 6 times the control level. The chlorophyll content and net photosynthetic rate decreased with increasing levels of zeatin and zeatin riboside.

The use of in vitro-grown microtuber discs inAgrobacterium-mediated transformation of Russet Burbank and Lemhi Russet potatoes

We have usedin vitro-grown microtuber discs in the transformation of Russet Burbank and Lemhi Russet potato (Solanum tuberosum L.) cultivars byAgrobacterium-mediated gene transfer. Transformed plants were selected by their resistance to kanamycin and identified by β-glucuronidase activity. Northern blot analysis confirmed the presence of the corresponding messenger RNA. The ability to transform these two cultivars promises significant improvements to agronomically important varieties.

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.

Genetic transformation of potato ( Solanum tuberosum): An efficient method to obtain transgenic plants

A quick procedure for efficient transformation of potato (cv. Désirée) has been devised. Leaf disc have been inoculated with an Agrobacterium tumefaciens harbouring a Ti plasmid derived binary vector. Transformed shoots carrying the neomycin phosphotransferase gene were regenerated using a feeder layer technique after 4 weeks on selective medium containing kanamycin. Transgenic plants obtained in large numbers appeared phenotypically normal and expressed the NPT II gene. The present work points out that culture conditions are fundamental to obtain the highest transformation efficiency.

Structural diversity of the patatin gene family in potato cv. Desiree.

We have used a combined genetical and molecular approach to study the structural diversity of the patatin gene family in tetraploid Solanum tuberosum L. cv. Desiree (2n = 4x = 48). Nine dihaploid derivatives (2n = 2x = 24) of cv. Desiree were isolated by gynogenesis through prickle pollination with S. phureja Juz et Buk. Patatin DNA sequences in Desiree and in the dihaploids were examined by probing Southern blots of restriction endonucleases HindIII and XbaI digested DNA with patatin cDNA region-specific gene probes and by more detailed examination (restriction endonuclease mapping and partial DNA sequencing) of 10 patatin genomic clones in bacteriophage lambda replacement vector EMBL4. This provided positive identification for most individual patatin gene family members and some estimate of their organisation and diversity. Most of the 64-72 patatin DNA copies showed little allelic variation based on HindIII and XbaI restriction fragment length polymorphism (RFLP) mapping and did not appear to be very tightly clustered. Four of the 6-8 class I patatin genes (without a characteristic 22 bp insert in their untranslated leader DNA), showed apparent allelic homogeneity, whilst the remaining class I genes comigrated with a single class II patatin gene RFLP subclass. Of the isolated clones, 4 contained apparent pseudogenes lacking 5' control sequences and exon-1 DNA while another clone contained a patatin gene truncated at the 5' region due to the cloning event. The remaining 5 all contained class II genes (with the 22 bp insert) and these showed varying degrees of sequence homology for 400 bp of conserved 5' coding and non-coding DNA (from 77%-95%). In one case the extent of homology differed, with complete sequence divergence upstream of position -80 from the start of transcription. The structural diversity of the patatin gene family is discussed in relation to expression of individual patatin genes and the use of cv. Desiree as a host for potato transformation experiments.

Precise Incubation Period for the Agrobacterium- mediated Transformation Efficiency in Potato (Solanum tuberosum L.) cvs. Cardinal and Atlas

Precise incubation period facilitates significant response to Agrobacterium- mediated genetic transformation of potato (Solanum tuberosum L.) cvs. Atlas and Cardinal. The protocol yielded an average transformation rate of 58.4% for internodal explants compared to 38% for leaf explants in Atlas while 47.6% for intenodal explants as against 7.6% for leaf explants in Cardinal. Highest survival rate and transient GUS activity were shown by Atlas and Cardinal at 45 min of incubation with Agrobacterium. The plants were analyzed histochemically for GUS activity in their leaves and internodes. All these analysis indicated that each independently selected and regenerated plants of Atlas and Cardinal were GUS positive and transient. Key words: Transformation, Incubation period, Potato cultivars D.O.I. 10.3329/ptcb.v19i2.5440 Plant Tissue Cult. & Biotech. 19(2): 227-235, 2009 (December)