Agrobacterium-mediated transformation of Solanum tuberosum L. cv. ‘Russet Burbank’

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. Russet Burbank. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.     

Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. ‘Bintje’)

Summary A wide range of phenotypic variation occurred among protoplast — derived plants of tetraploid potato cultivar Bintje. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.     

Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression

Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.     

Genetic instability in protoclones of potato (Solanum tuberosum L. cv. ‘Bintje’): new types of variation after vegetative propagation

Summary The transmission of variation from protoplast-derived plants of tetraploid potato cultivar Bintje to tuber progeny was examined. The morphological alterations of a majority of the variant protoclones were transmitted to corresponding tuber progeny. Some of the normal and variant protoclones gave new phenotypes, or segregated into parental and new phenotypes after vegetative propagation. The ploidy levels of almost all these clones remained unchanged after propagation. It was concluded that the occurrence of variation after vegetative propagation was due to somatic segregation of chimeras resulting from gene mutations or chromosome structural rearrangements in only part of the regenerated plant. The origin of variation is discussed in the light of these results.     

Characterisation of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.)

The cDNA clones of two potato -tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar -tubulin genes, designated TUBST1 and TUBST2. The expression pattern of -tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding -tubulin probes revealed that there are multiple -tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3 non-coding sequences. Phylogenetic analysis of plant -tubulin genes is described.     

Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée

Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.     

茄子(Solanum melongena L. var. grossum)花药培养的研究

通过花药培养进行单倍体育种的研究,目前在我国已取得了很大进展。为了加速蔬菜新品种的选育和现有品种的纯化,我们对茄子花药离体培养进行了一些研究,结果如下:材料与方法1.培养基培养花药及诱导愈伤组织分化,我们采用两种基本培养基,一是 MS 培养基,另一种是 NT 培养基的无机盐加 H 培养基的活性物质。转移小苗时用 H 培养基,并根据需要附加萘乙酸(NAA)、吲(口朶)乙酸(IAA)、2,4-D、生根剂(Racine)、激动素等物质,蔗糖为2—3％,琼脂0.8％,pH 调至5.8,最后热压(15磅,120℃)消毒20分钟。     

马铃薯(Solanum tuberosum L.)幼茎(芽)愈伤组织的诱导和植株再生(简报)

马铃薯的茎尖、花粉和花药培养已有大量报道。由于马铃薯作为组织培养材料难于掌握,因此从它的块茎、茎和叶等主要器官组织诱导愈伤组织并使之分化成再生植株的报道为数不多。为从茎尖培养以外的途径解决生产上的增加繁殖系数,减少籽种消耗和探索马铃薯幼茎再生植株的可能性及其培养条件,特别是激素的影响,我们作了这一工作。供试马铃薯品种为渭源67-5-3。材料在室内发芽,芽长到2厘米时,取下洗净。在无菌条件下,按常规方法灭菌,将     

马铃薯（Solanum tuberosum L.）茎尖的超低温保存及其遗传变异的初步观察

研究马铃薯茎尖超低温保存技术的结果表明,4℃低温下锻炼6d,在添加二甲基亚砜（DMSO）和乙酰胺的培养基中预培养5d,60％PVS2于室温下装载30min,0℃下PVS2脱水40min时,茎尖成活率最高（71．6％）,再生植株生长分化正常。进一步对再生植株进行AFLP分析,6对引物组合共扩增出385条带,超低温保存前后的材料之间未见到明显差的异带,但用MSAP技术分析超低温保存前后植株甲基化的结果显示：超低温保存后的材料均有不同程度的甲基化。在扩增的624条带中,处理与否之间完全一致的带型为584条;有变化的带型为40条,处理2（茎尖经过完整的超低温保存过程,区别于处理1,增加了冷冻、解冻和洗涤后恢复培养）有13个位点的甲基化增加,21个位点去甲基化。     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.     

Somatic hybrids of Solanum tuberosum – application to genetics and breeding

Cultivated potato (Solanum tuberosum L.) is one of the first agricultural crops successfully cultured in vitro and used for obtaining of somatic hybrids. The review presents the current state of knowledge of somatic hybridisation involving this and other species from the genus of Solanum. Methods of somatic hybridisation, in particular factors that must be considered during designing the experiments are presented and discussed. The main attention however is focused on processes that are responsible for somatic hybrid formation. Complex interactions between genomes and plasmones lead to formation of symmetric, asymmetric and cytoplasmic recombinants. The concept of alloplasmic incompatibility is presented and discussed in relation to Solanum hybrids. Selected examples of potato somatic hybrids with agronomically important traits derived from wild species are presented in the table and discussed.     

The effects of IAA and tetcyclacis on tuberization in potato (Solanum tuberosum L.) shoot cultures in vitro

Potato (Solanum tuberosum cv. Désirée) shoots grown in vitro in continuous darkness or in long days (LDs), were used to investigate indole-3-acetic acid (IAA) effects on stolon initiation and tuber formation, combining IAA with increased or decreased gibberellin levels. An increased gibberellin (GA) level was achieved by the applying 1 μM GA₃, while decreased gibberellin level was presumably realized by the adding 3 μM tetcyclacis (Tc). About 15% of potato shoots developed stolons both in LDs and in darkness. Stolon initiation was stimulated by GA₃ in darkness and by Tc in LDs. Tuber formation was strongly inhibited in LDs and by GA₃ both in light and darkness, but stimulated in darkness at low GA level. Exceptionally, tuber formation occurred in LDs at the highest Tc concentrations, in about 25% of explants. Indole-3-acetic acid alone stimulated stolon formation in LDs, both in the presence or absence of GA₃. IAA alone also stimulated tuber formation in dark-grown shoots, but could not overcome the inhibitory effect of LDs. Indications that, depending on their concentration ratio, IAA may interact with GA₃ in different tuberization phases, have been discussed. Radomir Konjević—Deceased in July 2006.     

Parasite fauna of the burbot Lota lota L. in body of water of the Kol'skiĭ peninsula

The results of a parasitological study of the burbot Lota Iota L. inhabiting the Kola region are presented. 51 species of parasite were found on burbot in 16 waterbodies belonging to the White Sea and Barents Sea basins (Muxosporea - 7, Suctoria - 1, Peritricha - 6, Monogenea - 1, Cestoda - 6, Trematoda - 13, Nematoda - 6, Acanthocephala - 5, Hirudinea - 3, Bivalvia - 1 and Crustacea - 2 species). Data on the infestation of burbot by different parasite species and their prevalence in investigated waterbodies were obtained.     

Does light spectral quality affect survival and regeneration of potato (Solanum tuberosum L.) shoot tips after cryopreservation?

Cryopreservation, the storage of germplasm at ultra-low temperature is the most reliable tool for long-term preservation of plant genetic resources. Cryopreservation techniques are widely applied but the effect of light spectra on plant recovery after cryopreservation is largely unknown. Therefore, we investigated the effect of different light spectral qualities on survival and regeneration of shoot tips of potato (Solanum tuberosum L.) cultivars Agrie Dzeltenie, Maret, Bintje, Désirée and Anti cryopreserved by the DMSO-droplet method. Prior to cryopreservation, the plants were stored under cool white fluorescent light (CW). Post-cryopreservation, the plants were allowed to regenerate under six different light qualities: CW, warm white light (HQI), blue LEDs (B), red LEDs (R), red with 10 % of blue (RB) and RBF - red with 10 % of blue with addition of 20 % of far-red LEDs. The light spectral quality had a significant effect on the survival and regeneration of potato shoot tips after cryopreservation. The combination of red light with 10 % of blue (RB) doubled the regeneration percentage of all cultivars, whereas red light (R) was not suitable for regeneration after cryopreservation. Specifically, the regeneration percentages were increased in RB compared to CW from 25.5 to 52.6 % for ‘Agrie Dzeltenie’, 25.0–43.6 % for ‘Maret’, 8.1–26.1 % for ‘Bintje’, 0.0–17.1 % for ‘Anti’ and 18.2–36.6 % for ‘Désirée‘. Therefore, the modification of light spectra during the recovery phase is a promising tool for increasing the regeneration of potato shoot tips after cryopreservation.     

The expression of petunia flavonoid 3′ and 3′5′ hydroxylase genes in potatoes (Solanum tuberosum cv. Jopung)

Flavonoid 3′ (F3′OH) and 3′5′ hydroxylase (F3′5′OH) play a major role in the synthesis of flavonoids. They are involved in the flavonoid modification and the B-ring hydroxylation produces quercetin and myricetin, respectively. We introduced the petunia F3′OH and F3′5′OH genes in potato and expression of these enzyme was confirmed by Southern and Northern blot analyses in these transgenic plants. In the flavonoid, staining experiment, all transgenic plants with petunia F3′OH and F3′5′OH genes were successfully changed with their green color to orange, confirming that quercetin was synthesized in those plants. Especially, the F3′5′OH transgenic potatoes showed the strongest orange color, and it was revealed by capillary electrophoresis that they produce quercetin one and a half times as much as the untransformed potatoes.     

Modelling the recovery of hypertrophic L. Glumsø (Denmark)

Diversion of sewage from L. Glumsø reduced phosphorus loading from 6.0 g P.m^–2.yr^–1 to 1.6 g P.m^–2.yr^–1. Chlorophyll levels during summer were reduced from 6–800 mg Chl.m^–3 to about 200 mg Chl.m^–3 mainly by extended periods with phosphorus limitation. Internal phosphorus loading was significant in the first 2 years after nutrient reduction. Predictions of the recovery were made by both simple, empirical models and with complex, dynamic model versions. The actual responses of L. Glumsø were compared with both previously published predictions and predictions made with improved model versions. Objective functions of 0.18 and global correlation coefficients of 0.89 could be achieved.     

The significance of alpha-amylase under anoxia stress in tolerant rhizomes (Acorus calamus L.) and non-tolerant tubers (Solanum tuberosum l., var. Désirée)

Rhizomes of Acorus calamus L. were able to maintain a functional alpha-amylase under anoxia, whereas a steep decrease in the enzyme protein content and activity took place in potato tubers. The stress-induced control in tubers occurred on the translational level. It is suggested that this decrease is one of the key factors with regard to anoxia intolerance.