Protective Effect of Arachidonic Acid during Viral Infection: Synthesis of New Proteins by in vitroPotato Plants

The mechanisms of the protective, immunostimulating effects of arachidonic acid (AA) were studied, and its efficiency in the induction of defense reactions in the moderately virus-resistant potato cultivar Nevskii (Solanum tuberosumL.) was determined. Virus-free in vitropotato plants treated with AA and inoculated with phytopathogenic viruses were used as a model. The data on the X virus accumulation obtained by the enzyme-linked immunosorbent assay confirmed the immunizing effect of AA; the optimum concentration of the compound was 10^–8M. The antiviral effect of AA was maintained in infected in vitropotato plants for at least two or three weeks. The electrophoretic analysis of leaf proteins revealed a 33-kD polypeptide induced by the potato virus Y. Two weeks after inoculation with virus X, a 40-kD protein was identified in potato plants pretreated with AA. In addition, the relative content of the two groups of proteins consisting of two or three components with mol wts about 50 kD and above70 kD changed both upon viral infection and pretreatment with AA. Only small changes in the isozyme patterns of peroxidase in potato plants were observed during the development of systemic acquired resistance; they were manifested in some treatments in the band intensities. The existence of the alternative pathways of systemic acquired resistance in potato plants specifically activated by viral infection and AA was suggested.     

Influence of arachidonic acid and phytoviruses on the hormonal system of potato plants grown in vitro

Changes in the phytohormonal system during the development of antivirus resistance was investigated. This resistance was induced by arachidonic acid (AA) in potato plants (Solanum tuberosum L.) grown in vitro. The plants in tubes were treated with AA 7 days before infection with tobacco mosaic virus or potato X virus. These plants were used for ABA, IAA, and cytokinin quantification. As a rule, phytoviruses increased ABA and cytokinin levels and decreased that of IAA in both immunized and unimmunized plants. AA significantly decreased the phytovirus content in potato plants at least during 3 weeks. In general, AA immunization acted as a limiting factor for increasing the ABA and cytokinin concentrations during virus pathogenesis in experimental plants. However, no regularity in the changes of the IAA concentration was noted. The data obtained showed that AA-induced changes in the hormonal system could be an important factor in the mechanism of plant resistance against phytopatogenic viruses.     

Accumulation of potato virus Y is enhanced in Solatium brevidens also infected with tobacco mosaic virus or potato spindle tuber viroid

The accumulation of potato virus Y?(PVY?) and potato leaf roll virus (PLRV) was studied in plants of Solanum brevidens co-infected with each of six viruses or a viroid. Virus could not be detected by ELISA in plants of S. brevidens infected solely with PVY. However, accumulation of PVY was increased c. 1000-fold in plants doubly infected with tobacco mosaic virus or potato spindle tuber viroid (PSTVd). PVY titres in doubly infected plants of S. brevidens were between 1% and 0.1% of those found in the PVY-susceptible interspecific Solanum hybrid DTO-33. Double infections of 5. brevidens by PVY and alfalfa mosaic virus or potato viruses M, S, T or X did not significantly enhance PVY accumulation. Accumulation of PLRV was not enhanced in plants co-infected with any of the six viruses or PSTVd.     

Somatic Hybrids Between Potato and S. berthaultii Show Partial Resistance to Soil‐Borne Fungi and Potato Virus Y

Three tetraploid somatic hybrid lines produced by protoplast fusion between a dihaploid potato, Solanum tuberosum, cultivar BF15 and the wild potato species Solanum berthaultii were evaluated here for their response to different soil‐borne pathogens, that is Fusarium solani, Pythium aphanidermatum and Rhizoctonia solani as well as to infection by potato virus Y (PVY). Both hybrid and BF15 plants grown in vitro were inoculated with the tested pathogen strains, that is R. solani, P. aphanidermatum, or F. solani. The growth level and disease severity index of these plants were compared to the susceptible commercial cultivar Spunta. A better growth of inoculated hybrid plants and restricted disease symptoms were observed in comparison with the commercial plants. Under glasshouse conditions and after inoculation with R. solani and P. aphanidermatum, improved resistance of the hybrid plants to these pathogens was confirmed. Indeed, these plants showed no significant damage following inoculation and a better development in R. solani‐infected plants. The susceptibility of the hybrid tubers to R. solani, P. aphanidermatum, and to F. solani infection was also determined. A significant reduction of tissue colonisation was observed in all the hybrid lines compared to the cultivated cultivars. The STBc and STBd hybrids also showed improved resistance to the PVY ordinary strain (PVY^o) under glasshouse conditions.     

Boosting the Immune System of Solanaceous Plants by Lysophosphatidylcholine

An in vitro treatment of solanaceous plants, viz. potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.), with lysophosphatidylcholine promoted their resistance to phytopathogens, such as Phytophthora infestans (Mont.) de Bary and potato virus Y. The systemic and long-term lysophospholipid-induced response was independent of the phytopathogen type. It was concluded that lysophosphatidylcholine, a product of phosphatidylcholine hydrolysis with phospholipase A₂, plays an important role in the regulation of plant immune system.     

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.     

Virus elimination and pathogen-free plantlets regeneration in Cucurbita pepo L.

An efficient in vitro protocol was established for developing pathogen-free plantlets in Cucurbita pepo through meristem culture. Meristems of about 0.3–0.5 mm in size were isolated from shoot tips of 25–30 day old in vitro grown plants. For primary establishment of isolated apical meristem, MS liquid medium supplemented with 2.0 mgl KIN and 0.5 mg/l GA₃ was found to be most effective in both cultivars. MS semisolid medium containing 2.0 mg/l BAP were found to be most effective for shoot development from primarily established meristem in both cultivars. A good number of shoots were not concomitant with good rooting. The best root induction was found in media having 1.0 mg/l IBA in cv. Bulum. It was found that cv. Bulum was better than cv. Rumbo in all stages of meristem culture. The presence of virus in plantlets was achieved by DAS-ELISA test, where 68–81% plantlets have been proved to be virus free among the studied viruses. Healthy growth and vigour was observed in meristem derived plants over their source plants after cultivation under natural conditions.     

Nutzung von In-vitro-Pflanzen der Kartoffel zur Prüfung der antiphytoviralen Wirkung von 2,4-Dioxohexahydro-1,3,5-triazin (DHT) in Kombination mit N-Cyanoguanidin

Suitability of in vitro potato plantlets for testing of antiphytoviral effect of a combination of 2,4-dioxo-hexahydro-1,3,5-triazine (DHT) and N-cyano guanidine The suitability of a semiquantitative determination of virus content for the detection of antiphytoviral effects of chemicals was demonstrated by ELISA directly applying at in vitro potato cultures systemically infected and chemotherapeutically treated. The combination of 2, 4-dioxohexahydro-1,3,5-triazine (DHT) and N-cyanoguanidine both applied at a concentration of 0.03% to the culture medium resulted in a high significant reduction of the relative concentration of the potato virus S in the, potato genotype ‘M-812820’ of 44–73 %. A phytotoxic influence of the substances was not observed. The combination of the two antiphytoviral substances had no effect against potato virus Y in explants of the variety ‘Ackersegen’. Differences in the susc, eptibility of various genotypes against biologically active substances were indicated.     

Continous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum dem/ssum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculatedin vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis denovo of PSTV was substantiated by the incorporation of [³H]uridine and of [³²P]orthophosphate into viroid RNA.     

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.     

A new plant virus from the high jungle of the Eastern Andes; Solanum apical leaf curling virus (SALCV)

A previously undescribed plant virus, Solanum apical leaf curling virus (SALCV), was found in cultivated potato and indigenous wild solanaceous plants in an area of high jungle near San Ramon, Peru. Symptoms in potato consisting of red, purple or pink discoloration, curling, crinkling and dwarfing of apical leaves develop soon after infection. Symptoms from tuber-borne infection may also include dwarfing and stunting, dormancy may be prolonged and sprouts may be filiform producing small plants with very thin stems. The virus is transmissible by grafting, but was not transmitted through seed, by aphids or leafhoppers tested, nor by mechanical inoculation of sap. Infected Datura tatula and D. stramonium, the most useful indicator hosts, developed yellowing of the small veins of newly formed leaves followed by distortion, dwarfing, and cupping of subsequently formed leaves. Tomato, Solanum nigrum, Nicandra physalodes and Nicotiana benthamiana were also infected experimentally. N. physalodes, Solanum basendopogon, D. tatula and Physalis peruviana were naturally infected in the field. Antiserum produced in rabbits was suitable for ELISA which detected SALCV in a range of graft-inoculated and naturally infected plants. Most virus particles in purified preparations and those trapped on antiserum sensitised grids treated with infective sap were c. 52 times 17 nm and consisted of three quasi-isometric units in a straight chain. This particle morphology although novel, suggests possible affinities with geminiviruses.     

Tuber Formation and Growth of in vitro Cultivated Transgenic Potato Plants Overproducing Phytochrome B

We studied the effect of the ectopic expression of the Arabidopsis PHYB gene, which encodes the phytochrome B (phyB) apoprotein, under the control of cauliflower mosaic virus 35S promoter on the photoperiodic response of tuberization and growth of potato (Solanum tuberosum L., cv. Désirée) transformed lines. Stem cuttings of transformed and control plants were cultured on Murashige and Skoog nutrient medium containing 5 or 8% sucrose in the phytotron chambers at 20°C under conditions of a long day (16 h), a short day (10 h), or in darkness. We showed that the overexpression of the PHYB gene enhanced the inhibitory effect of the long day on tuberization. In addition, tuber initiation in these transformed plants occurred at a higher sucrose concentration. The insertion of the PHYB gene decreased plant and tuber weights and shortened stems and internodes. Thus, we demonstrated the complex result of the PHYB gene insertion: it affected the photoperiodic response of tuberization, the control of tuber initiation by sucrose, and the growth of potato vegetative organs.     

Anin vitro procedure to eradicate potato viruses X,Y, and S from Russet Norkotah and two of its strains

Summary A tissue culture method was developed for the eradication of three important potato viruses, PVX, PVY, and PVS, from the Russet Norkotah variety and two of its strains (TXNS 112 and TXNS 278). The method combined the use of liquid medium, thermotherapy, and chemotherapy. Initially, virus-free plants were inoculated with PVX, PVY, and PVS and, after 10 d, tested quantitatively for virus titer by ELISA to determine the initial virus concentration. The apical tip and the roots were removed from the inoculated plants, and only stem sections from inoculated plants were cultured in liquid medium containing MS inorganic salts, vitamins, and ribavirin (40 μM or 80 μM). After 5 d, half of the plants were subjected to thermotherapy and half were kept at room temperature. At 6 wk, the uppermost node (5–7 mm) was removed and recultured, and plants were tested then and again after 6 wk using ELISA to identify the virus-free plants. Ribavirin alone eradicated the viruses from some plants; however, more virus-free plants were obtained when the treatments included heat. Additionally, thein vitro culture technique using liquid medium promoted rapid lateral shoot elongation and resulted in significantly faster plant production. Also this approach, which required less skilled labor, produced more plants than the meristem culture method for virus eradication. A detailed procedure for elimination of multiple viruses is described. This procedure resulted in production of more than 10% virus-free plants.     

The effects of nor-spermidine and dicyclohexylamine on in vitro potato development and free polyamines content

The in vitro micropropagation of potato (Solanum tuberosum L., var. Spunta) on media containing nor-spermidine (nor-SPD), a natural polaymine (PA) or dicyclohexlamine (DCHA), a spermidine synthesis inhibitor was studied to test their effects on plantlet growth and on the level of free cellular polyamines. The triamine nor-SPD, inhibited spermidine synthesis and substantially reduced root growth. DCHA strongly inhibited potato growth but surprisingly the free spermidine level seemed unaffected. These data suggest that dicyclohexylamine acts on the growth and on the development of plants by mechanisms unrelated to polyamine metabolism. Conversely, nor-spermidine was effective in reducing cellular spermidine content and seems to be a spermidine biosynthesis inhibitor in plants.     

Genetically engineered resistance to potato virus X in four commercial potato cultivars

The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP benzyl-aminopurine - BCIP 5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt - CaMV cauliflower mosaic virus - CP capsid protein - GA₃ gibberellic acid - Kbp kilobase pair - NAA naphthalene acetic acid - NBT nitroblue tetrazolium chloride - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PMSF phenyl methyl sulfonyl fluoride - PVX potato virus X - PVY potato virus Y     

High frequency of tetraploidy in Agrobacterium-mediated transformants regenerated from tuber discs of diploid potato lines

Summary Variations in the ploidy level of 69 transgenic potato (Solanum tuberosum L.) plants regenerated from the tuber discs of 17 diploid lines were studied: 24 plants (35%) were diploid, the other 45 plants (65%) were tetraploid. Seventy-eight control regenerants obtained without Agrobacterium inoculation showed a relatively low tendency to tetraploidization (35%). The results obtained suggested that chromosome doubling occurred frequently in diploid potato lines during the tissue culture process for regeneration. Putative somaclonal changes in in vitro-formed tuber proteins were detected in three out of six transformants by electrophoretic analysis.     

Reproductive ecology and genetic variability in natural populations of the wild potato,Solanum kurtzianum

The cultivated potato (Solanum tuberosum ssp. tuberosum) has more than 200 related wild species distributed along the Andes, adapted to a wide range of geographical and ecological areas. Since the last century, several collection expeditions were carried out to incorporate genetic variability into the potato germplasm around the world. However, little is known about the reproductive ecology and genetic population structure of natural potato population from field studies. The aim of this work is to study, in the field, the genetic variability and reproductive strategies of populations of one of the most widely distributed potato species in Argentina, Solanum kurtzianum, growing in Mendoza province. AFLP markers showed that the genetic variability is mainly present among plants within populations, indicating that in the sampled populations, sexual reproduction is more relevant than clonal multiplication (by tubers). Additional evidence was obtained evaluating the genetic diversity in populations with a distribution in patches, where several genotypes were always detected. From a field study performed in the Villavicencio Natural Reserve, we found that the average number of plump seeds per fruit was 94.3, identified and calculated the foraging distance of four insect pollinators, and demonstrated the seed dispersal by storm water channels. We argue that the breeding system, the two modes of reproduction and the ecological interaction described here may have a prominent role in determining the genetic structure of S. kurtzianum populations, and discuss the importance of field studies on population genetics, reproductive biology and ecology to design collections and conservation strategies.     

Relationship between inoculum density and vector phenology on the incidence of potato virus Y in tobacco

The epidemiology of potato virus Y (PVY) in the tobacco crop, Nicotiana tabacum, was examined in the context of the seasonal abundance of aphid vectors, rate of disease progress, and disease gradient from a known virus source. The spring potato crop, Solanum tuberosum, was suspected of being the main source of inoculum; therefore, varying numbers of infected potato plants were used as the inoculum source in different test plots. A 3-wk lag phase was present in all disease progress curves prior to an exponential increase in disease incidence. The relatively low numbers of aphid vectors, primarily transient species, alighting on the crop during the lag phase were responsible for the primary spread of PVY from potato to tobacco. The arrival of large numbers of colonising aphid vectors, Myzus persicae, presumably from the harvested potatoes, coincided with the exponential increase in PVY incidence in tobacco. The initial number of potato plants infected with PVY was positively correlated with the final disease incidence, rate of disease progress, and the magnitude of radial dispersion of PVY into the tobacco. Aphid vector pressure was not a significant variable in the differences in spatial and temporal characteristics of PVY epidemics among test plots.