A Low Temperature Therapy and Meristem-Tip Culture for Eliminating four Viroids from Infected Plants

Viroid-free potato and chrysanthemum plants were obtained from meristem-tips cut from potato spindle tuber viroid-infected potato plants and from chrysanthemum plants infected with chrysanthemum stunt, chrysanthemum chlorotic mottle or cucumber palefruit viroids after 6 months therapy in a growth chamber at 5 °C and 16 hours daily light of 5.000 lx intensity. Chrysanthemum plants survived quite well the conditions of therapy while potato plants grown from stem cuttings survived these conditions much worse and potato plants grown from tubers did not survive these conditions. PSTV-free plants were obtained from meristem-tips cut from sprouts grown from potato tubers infected with severe (s-PSTV) or mild (m-PSTV) strains of potato spindle tuber viroid after 6 months therapy at 6–7 °C in the dark. The tubers survived these conditions quite well. The 3 months therapy period was found too short for any plant material. The efficiency of 6 months therapy in viroid elimination varied for different viroids and different plant material from 18.5 to 80.0 %.     

Photoperiodic control of growth, development and phytohormone balance in Solanum tuberosum

Some lines of Solanum tuberosum ssp. andigena are strictly photoperiodic, forming tubers only in short days and flowers only in long days. We used this advantageous phenomenon to study phytohormone involvement in the development of the plants, mainly that of tuber formation. Plants grown for 2 months under short days (SD) of 14 h darkness, night break (1 h white light in the middle of the 14‐h dark period) and continuous light (LD) were compared. Short day‐grown plants formed tubers, while plants in LD flowered. Night break prevented tuber formation, but caused flowering, although it was weaker than in LD. Plants grown under night break displayed many growth characteristics intermediate between SD and LD. Under LD and night break regimes ABA levels in all organs were about one‐fourth of those under SD. An opposite trend was found for gibberellin content: it was very low in SD‐grown plants and 4‐10 times higher under both other conditions with the exception of roots and stolons in night break. Thus, the ratio of ABA/GA, known to be important for tuber formation, was high in SD and low in night break and LD. The level of free indoleacetic acid (IAA) was high in LD‐ and night break‐grown plants and it was much lower in SD‐grown plants, including tubers. Cytokinin (CK) levels were also high in LD‐ and night break‐grown plants. In SD, leaves had slightly decreased and stems and roots + stolons had more pronouncedly decreased cytokinin levels. The CK/IAA ratio was thus higher in SD in stems and roots + stolons; it was also high in tubers and no difference was found in leaves. These results indicate that the effect of photoperiod on tuber formation and development may be mediated by photoperiod‐induced changes in hormone levels.     

Susceptibility of potato plants grown from tubers irradiated with stimulation doses of gamma irradiation to potato tuber moth, Phthorimaea operculella Zeller (Lep., Gelechiidae)

The feeding behaviour of potato tuber moth, Phthorimaea operculella Zeller (Lep., Gelechiidae), reared on leaves and tubers of potato plants, which were irradiated as seed (tubers) with stimulation doses of gamma irradiation (1, 3, 5 and 10 Gy), was studied. Significant differences in the larvae and pupae developmental time, pupal weight, mortality, fecundity and percentage egg hatch, were observed between insects fed on plants which resulted from the irradiated seeds and those of the control. It appears that leaves of potato plants grown from irradiated seeds particularly those of 3 Gy, became more attractive to the larvae, although the resulting tubers, with the exception of those of 10 Gy, became more resistant to potato tuber moth. Storing the tubers at ambient temperature conditions affected the degree of larval sensitivity. The leaves and tubers of 10 Gy-irradiated seeds became more suitable for insect development, indicating that the later dose may inhibit the production of secondary plant metabolities and chemical compounds.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.     

Ultrastructure of potato tubers formed in microgravity under controlled environmental conditions

Previous spaceflight reports attribute changes in plant ultrastructure to microgravity, but it was thought that the changes might result from growth in uncontrolled environments during spaceflight. To test this possibility, potato explants were examined (a leaf, axillary bud, and small stem segment) grown in the ASTROCULTURETM plant growth unit, which provided a controlled environment. During the 16 d flight of space shuttle Columbia (STS-73), the axillary bud of each explant developed into a mature tuber. Upon return to Earth, tuber slices were examined by transmission electron microscopy. Results showed that the cell ultrastructure of flight-grown tubers could not be distinguished from that of tuber cells grown in the same growth unit on the ground. No differences were observed in cellular features such as protein crystals, plastids with starch grains, mitochondria, rough ER, or plasmodesmata. Cell wall structure, including underlying microtubules, was typical of ground-grown plants. Because cell walls of tubers formed in space were not required to provide support against the force due to gravity, it was hypothesized that these walls might exhibit differences in wall components as compared with walls formed in Earth-grown tubers. Wall components were immunolocalized at the TEM level using monoclonal antibodies JIM 5 and JIM 7, which recognize epitopes of pectins, molecules thought to contribute to wall rigidity and cell adhesion. No difference in presence, abundance or distribution of these pectin epitopes was seen between space- and Earth-grown tubers. This evidence indicates that for the parameters studied, microgravity does not affect the cellular structure of plants grown under controlled environmental conditions.     

Infection of potato tubers with soft rot bacteria

Stolons attached to developing potato tubers were inoculated with the soft rot bacterium Erwinia carotovora var. atroseptica. Almost all the stolons rotted, but soft rots developed in less than 10% of new tubers; the bacterium was isolated later from these tubers. No rots developed in the other tubers but the bacterium was later isolated from about half of them. It could not be isolated from tubers attached to inoculated stolons where the rot on them did not extend to the tuber or from tubers attached to stolons that were not inoculated though many of these rotted. The bacterium was reisolated from almost all arrested lesions in tubers inoculated 8 month earlier with E. carotovora var. atroseptica. Blackleg did not develop from plants grown fom these tubers under various soil conditions. It did develop in a large proportion of plants from tubers inoculated shortly before planting and grown in cool, wet soil. Less than 1% blackleg developed in plants grown from tubers from plants with blackleg or from plants immediately adjacent. The presence of pectolytic bacteria and E. caratovora var. atroseptica in seed and new tubers was investigated during June, July and August. Although E. caratovora var. atroseptica was obtained from c. 40% tubers, only c. 0·3% of c. 8400 plants developed blackleg. The bacterium was isolated from only three of 160 new tubers sampled during the summer.     

Early generation selection for agronomic characters in a potato breeding programme

Summary A random sample of seedlings representing high, medium and poor vigour was studied for tuber colour, tuber shape, eye depth, tuber cracking, tuber yield per plant, average tuber weight and number of tubers per plant in four successive generations (F₁, F₁, F₁C₂, and F₁C₃). Based on the performance of vigour groups in various generations and inter-generation correlation coefficients, we propose a procedure for the elimination of unproductive genotypes early in the breeding programme. The data indicates that seedlings of poor vigour can be discarded at the seedling stage prior to transplantation in the field. The rejection of clones on the basis of tuber colour, tuber shape, eye depth and tuber cracking can also be initiated at the seedling stage. For tuber yield and average tuber weight a negative selection (rejection of poor phenotypes) is suggested from the first clonal generation and for number of tubers, from second clonal generation, until statistically sound replicated trials can be conducted for carrying out positive selection.     

Development of black dot disease (Colletotrichum coccodes (Wallr.) Hughes) and its effects on the growth and yield of potato plants

Seed tubers of cvs Désirée and Pentland Crown with different severities of black dot were planted in 1988 and 1989 at Rothamsted in fields in 4– or 7-course rotations, respectively. Tubers treated with prochloraz (1988) or imazalil (1989) were planted in some plots, and in others Colletotrichum coccodes inoculum was added to the soil at planting. In further experiments at Mepal, Cambridgeshire in 1989 and 1990 and at Rothamsted in 1990 on sites where potatoes had not been grown for more than 15 years, large amounts of inoculum were added to the soil around disease-free seed tubers of two (1989) or three (1990) cultivars at planting. In all experiments plants were sampled during the season and the effects of treatments on disease development, growth and yield were recorded. Disease on roots, stem bases and tubers was found early in the season and was more severe on Désirée than on Pentland Crown plants from fields in 4– or 7-course rotations. Severity increased throughout the season and with increasing amounts of disease on the seed tubers, especially with Desiree. Disease was also found on plants from disease-free tubers and was more severe in 1988 than 1989. At harvest, black dot on tubers was significantly more severe from severely affected than from disease-free seed, and was most severe where inoculum, especially large amounts, had been added at planting. Fungicide treatment decreased disease early in the season but had no effect on tuber infection at harvest. In 1989 the weight loss of seed tubers during sprouting increased with increasing amounts of black dot, but the disease had little effect on plant size through the season. At harvest the yield of ware tubers (>50 mm) decreased with severe disease but total tuber yields were not significantly affected. However, at harvest in 1988 severely affected seed yielded significantly less than healthy seed. Plants grown from mini-tubers were free from disease on sites where potatoes had not been grown for at least 15 years. Inoculum applied at planting caused severe disease on all cultivars in both years, whereas disease was slight on uninoculated plants. Inoculated plants senesced early at Mepal in 1990, but there were no significant differences in total tuber yield in any experiment. However, yields of ware tubers (>50 mm) were sometimes decreased and the total tuber number per plant increased.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: III. RELATIONSHIP BETWEEN GROWTH RATE OF INDIVIDUAL TUBERS, TUBER WEIGHT AND STOLON GROWTH PRIOR TO TUBER INITIATION

Potato plants (Solanum tuberosum L.) were grown in water culture.About 14 d after tuber initiation no significant differenceswere found between apical and basal tuber parts in ¹⁴C-uptakeand partitioning into various fractions from ¹⁴C-labelled photosynthate.Thus, the fresh weight of these tubers could be used as a parameterfor the sink size. The ¹⁴C-content per tuber (sink strength)20 h after ¹⁴CO₂-supply to the foliage was significantly correlatedwith the tuber fresh weight. No correlation was found betweenthe ¹⁴C-concentration of the tuber (sink activity; ct. min^–g^– fr. wt.) and tuber fresh weight. Consequently, tuberfresh weight (sink size) per se must have been a factor whichinfluenced sink strength. Stolon parameters characterizing theirgrowth prior to tuber initiation (e.g. stolon volume) and theircapacity for photosynthate transport (diameter, length) weremeasured at the time of tuber initiation. Significant correlationswere found between these stolon parameters and subsequent growthof individual tubers. Anatomical studies on the proportion ofvarious tissues in the cross sectional area of stolons supportthe idea of a negative relation between growth of individualtubers and transport resistance in the phloem of the stolons.It is concluded that in the initial phase of tuber growth, mainlyfactors outside of the tuber determine its growth rate. In laterstages of tuber growth, when the sink strength increases, thecompeting strength of individual tubers for photosynthate isdominated mainly by factors within the tuber itself, such astheir sink size and sink activity. Key words: Potato tuber, sink size, tuber initiation, transport resistance     

Reproduction in two submersed macrophytes declines progressively at low pH

• 1 Greenhouse experiments tested the effect of pH 5 v pH 7.5 on reproductive success for the freshwater macrophytes Najas flexilis, an annual, and Vallisneria americana, which perennates as a tuber.
• 2 Seed production by the small Najas plants grown at pH 5 averaged 0.25 seeds/plant, in contrast to 95.5 seeds/plant at pH 7.5. At low pH, Vallisneria grown from seeds produced no flowers and too few tubers to replace themselves, so that sexual reproduction failed nearly completely for both species.
• 3 Vallisneria grown from tubers produced 97% less total tuber mass at pH 5 (0.4 v 14.9 g), the compounded result of producing, on average, 89% fewer and 82% smaller tubers. The smaller tubers developed at pH 5 were less likely to overwinter in the field, and those surviving tubers subsequently grew into smaller plants.
• 4 These findings generated the hypothesis of a closing spiral: growth at low pH (and relatively low CO₂ concentrations) results in small plants that produce a smaller number of progressively smaller tubers each autumn, which in turn develop into progressively smaller plants each summer. This hypothesis was supported by field transplant experiments in two acidic Adirondack Mountain (NY) lakes.

     

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.     

Lactate metabolism in potato tubers deficient in lactate dehydrogenase activity

The aim of this work was to investigate the effect of decreased activity of lactate dehydrogenase (EC 1.1.1.27; LDH) on lactate metabolism in potato tubers. By expressing a cDNA‐encoding potato tuber LDH in the antisense orientation, we generated transgenic potato plants with a preferential decrease in two of the five isozymes of LDH. Surprisingly, transgenic tubers grown under normoxic conditions did not contain less lactate, but rather instead contained approximately two‐fold more lactate than control tubers. This result is explicable if the decreased isozymes of LDH are responsible for the oxidation of lactate to pyruvate in vivo. This was confirmed by measurements of the rate of metabolism of lactate supplied to tuber discs: the rate in transgenic tubers was approximately half that of control tubers. The decrease in LDH activity had no measurable effect on the accumulation of lactate in cold‐stored tubers under anoxia, nor during the subsequent utilization of this lactate upon return to normoxia. In both control and transgenic tubers, the accumulation of lactate during anoxia was not accompanied by an induction of LDH activity or a change in isozyme distribution. In contrast, the metabolism of lactate after a period of anoxia was accompanied by a two‐fold increase in LDH activity and the induction of two isozymes that were distinct from those which had been decreased in the transgenic plants.     

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.     

Response of potato grown under non-inductive condition paclobutrazol: shoot growth, chlorophyll content, net photosynthesis, assimilate partitioning, tuber yield, quality, and dormancy

The effect of foliar and soil applied paclobutrazol on potato were examined under non-inductive condition in a greenhouse. Single stemmed plants of the cultivar BP1 were grown at 35(±2)/20(±2) °C day/night temperatures, relative humidity of 58%, and a 16 h photoperiod. Twenty-eight days after transplanting paclobutrazol was applied as a foliar spray or soil drench at rates of 0, 45.0, 67.5, and 90.0 mg active ingredient paclobutrazol per plant. Regardless of the method of application paclobutrazol increased chlorophyll a and b contents of the leaf tissue, delayed physiological maturity, and increased tuber fresh mass, dry matter content, specific gravity, dormancy period of the tubers. Paclobutrazol reduced the number of tubers per plant. A significant interaction between rates and methods of paclobutrazol application were observed with respect to plant height and tuber crude protein content. Foliar application gave a higher rate of net photosynthesis than the soil drench. Paclobutrazol significantly reduced total leaf area and increased assimilate partitioning to the tubers. The study clearly showed that paclobutrazol is effective to suppress excessive vegetative growth, favor assimilation to the tubers, increase tuber yield, improve tuber quality and extend tuber dormancy of potato grown in high temperatures and long photoperiods.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.