The role of polyamines in potato tuber formation

Summary The involvement of free and conjugated polyamines in tuber formation was studied in in vitro cultured node explants ofSolanum tuberosum cv. Superior. Tubers developed from the axillary buds in 100% of the explants cultured in MS medium containing high sucrose levels and supplemented with kinetin (Kin) and chlorocholine chloride (CCC). The addition of growth regulators was not essential for tuber formation, although smaller tubers were formed in the medium devoid of Kin and CCC. Tuber formation was inhibited in about 75% of node explants treated with 0.5 mM difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase. The inhibitory effect of DFMO was almost completely reversed by putrescine addition. Addition of difluoromethylarginine (DFMA), the analogous inhibitor of arginine decarboxylase, had no effect on tuber formation. DFMO, but not DFMA, also inhibited the development of axillary buds into shoots in light-grown node explants. Aminooxyphenylpropionic acid (0.1 to 0.25 mM), an inhibitor of phenylalanine ammonia lyase, caused a sharp reduction in cinnamoyl putrescines, but had no effect on tuber formation. Our results suggest that hydroxycinnamic acids are not causal in tuber formation but may serve as polyamine storage pools. Our findings support the hypothesis that polyamines derived via the ornithine decarboxylase-mediated pathway are necessary for tuber formation in vitro, probably at the early phase of morphogenesis involving active cell division.     

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.     

Sucrose and light effects on in vitro cultures of potato,chokecherry and saskatoon berry during low temperature storage

Cultures of potato (Solanum tuberosum) cv. Atlantic, chokecherry (Prunus virginiana L.) cv. Garrington and saskatoon berry (Amelancher alnifolia Nutt.) cv. Northline grown in vitro for 3 weeks at 24/22 °C, 16-h photoperiod, 150 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) mixed fluorescent/incandescent light were stored for 6, 9 and 12 weeks at 4 °C under 0 (darkness) and 3 μmol m⁻² s⁻¹ PPFD (690 nm red light continuous illumination). Growth regulators free MSMO medium either with or without 30 g l⁻¹ sucrose was used to store the cultures. All cultures retained capacity to re-grow after storage. Tested factors, sucrose, light and the length of the storage period had an impact on shoot quality and re-growth capacity of the cultures. For either light treatment sucrose was essential for the low temperature maintenance of vigorous stock plants of potato, if stored for over 6 weeks. Chokecherry and saskatoon cultures stored well without sucrose; although chokecherry benefited from sucrose in the storage medium when the stock cultures were kept at the low temperature for 12 weeks. Low light significantly improved quality of the stored potato cultures, but had very little effect on both chokecherry and saskatoon berry cultures. The woody plant cultures grew during storage, and the longer the stock plants were stored, the more vigorous cultures they generated. The results indicate that growers can successfully use their existing facilities, small refrigerators and coolers with low light intensity, set at 4 °C, for short term storage of potato, chokecherry and saskatoon berry cultures. The potato cultures, which are known to be sensitive to prolonged low temperature storage, should be frequently monitored and subcultured as required. On the other hand, the woody plant stock cultures do not require any special attention when kept at 4 °C and re-grow the most vigorous shoots if stored for at least 12 weeks. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of gamma irradiation on potato microtuber production in vitro

The effects of low doses of gamma irradiation and potato (Solanum tuberosum L.) cultivar on the production of microtubers in vitro were investigated. Nodal segments from virus free explants of three potato cultivars (cv.) were placed on tuberization inducing medium and irradiated with 4 doses of gamma radiation (2.5, 5, 10, 15 Gy). Cv. Diamant produced the highest number of microtubers followed by Draga and Spunta. Irradiation of the explants with 2.5 Gy of gamma radiation led to a significant increase in the number of microtubers (38% increase over the control). Average weight of microtubers was not significantly influenced by low doses of gamma irradiation. Draga microtubers were the largest followed by Diamant and Spunta. Microtubers resembled mature tubers in shape (Spunta was oval and Draga and Diamant were spherical). Size of microtubers was crucial for sprouting in vivo. It is suggested that only microtubers larger than 5 mm in diameter (250 mg) be used to produce minitubers in vivo. Since 2.5 Gy is a low irradiation dose, it can be used to enhance tuberization in vitro without fear of genetic changes in the used cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transformation of potato using mannopine and cucumopine strains of Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberosum L.). An overnight pretreatment of stem fragments with NAA prior to bacterial infection was necessary to induce root formation, otherwise very few roots were produced. Whatever the potato cultivar used, rhizogenesis induced by NAA pretreament depended on the bacterial strain. In fact, when explants from both potato cultivars were pretreated with 26.5 M NAA, on average 84.4% and 71.9% produced roots after inoculation with the strains 2659 and 2659 GUS respectively. On the contrary, few rhizogenic responses (2.0–17.0%) or no response at all (0.0%) were obtained with the strains 15834 and 8196 GUS whatever NAA concentration used. Tests for confirming stable transformation of plant explants by examining both -glucuronidase activity and the presence of opines showed that 85% of the selected roots were cotransformed. Most of the transformed roots were highly branched and grew rapidly, compared to non-transformed roots with no branching and poor growth. Transgenic plants were readily regenerated with a frequency reaching 80% of total explants tested for both potato cultivars.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - df degree of freedom - F F distribution - GA₃ gibberellic acid - MS Murashige and Skoog basal medium - NAA -naphthaleneacetic acid - P probability - T-DNA transferred DNA     

Effect of jasmonic acid on in vitro explant growth and microtuberization in potato

The shoot fresh mass, root length and root numbers of two potato (Solanum tuberosum L.) cultivars Favorita and Helanwuhua were increased significantly by the application of 0.2 – 2 mg dm⁻³ jasmonic acid (JA) in the Murashige and Skoog medium. However, the growth of potato explants was inhibited by JA at high concentrations (20 – 50 mg dm⁻³). Chlorophyll content in explant leaves decreased with an increase in the concentration of JA. In leaves treated with 0.2 mg dm⁻³ JA acid peroxidase activity increased, while in the leaves treated with more than 2 mg dm⁻³ JA peroxidase activity decreased. Under the dark, the microtuber numbers, fresh mass and percentage of big microtubers of two potato cultivars were not promoted by the application of 0.2 – 50 mg dm⁻³ JA.     

Genetical analysis of in vitro cell and tissue culture response in potato

The genetics of tissue culture response in potato has been examined by analysing a sample of dihaploids (2n=2x=24) extracted from tetraploid parents (4n=4x=48). The genotypes were screened for rate of nodal multiplication, in vitro tuberisation, regeneration from leaf discs and protoplast plating efficiency. Significant differences were detected between dihaploids for the traits measured and this indicates that tissue culture response in the tetraploid parents must be in the heterozygous condition. Estimates of the broad sense heritabilities were calculated together with the number of genes or effective factors involved in the control of the traits. These estimates indicate that tissue culture response in potato is under relatively simple genetic control and blocks of genes may be located on specific chromosomes. The inheritance of RFLP markers in the segregating dihaploid population was also monitored and the potential of using molecular markers linked to gene(s) controlling tissue culture response is discussed.     

Induction and development of potato tubers in a jar fermentor

Potato (Solanum tuberosum L.) tubers were mass propagated in a jar fermentor using a two-step method consisting of a shoot multiplication step (phase 1) followed by a tuber induction and development step (phase 2). Tuberization was observed within 2 weeks of phase 2 and the number of tubers did not increase after this culture period. In contrast, total tuber weight continuously increased for at least 10 weeks. Although the number of tubers and the total tuber weight clearly decreased under the lower temperature (17°C), this weight decrease was partially prevented by changing the temperature from 17°C to 25°C after 2 weeks of phase 2. This result indicates that tuber development can be controlled independently of induction. Lower temperatures influenced the localization and size distribution of tubers in the jar fermentor.     

Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum)

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA+Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA₃) was the most abundant in all the organs. While GA₁and GA₃ were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.     

Correlation of cell division and cell expansion to potato microtuber growth in vitro

The sink capacity of plant storage organs influences crop economic yield and relates to the number and volume of their cells. To obtain a better understanding of their contributions to the growth of potato microtubers produced in vitro, the number and volume of the cells in the tuber tissues were measured as tubers grew. Two potato cultivars, E-Potato 1 and Mira were employed and the results showed that cortex, perimedulla and pith tissue contributed for about 30, over 65 and up to 3% to the volume of the mature microtuber, respectively. The number of cells and cell volume increased simultaneously as the microtubers grew and the relationships could be described by a power function, Y = aW ^b. However, the rate of cell division was greater than the rate of cell expansion and the former contributed more than the latter to the increase in tuber size. The rate of cell division was greatest in the cortex and least in the pith, but, because the perimedulla forms the largest part of the tuber, cell division in this tissue was particularly important. The regulation of cell division to improve the production of usable microtubers is discussed.     

Influence of the nitrogen level on root growth and morphology of two potato varieties differing in nitrogen acquisition

Two potato (Solanum tuberosum L.) cultivars (Astrid and Bodenkraft) differing in their nitrogen acquisition from the soil (Hunnius, 1981) were used in nutrient solutions to study the effect of increasing concentrations of nitrate (0.05; 0.5; 5.0 mol m^-3) particularly on root growth and morphology. In each variety increasing nitrogen concentrations stimulated shoot growth more than root growth. At all nitrate concentrations, the variety with higher nitrogen acquisition (Astrid) had a significantly larger root system. The larger root system of Astrid compared to Bodenkraft was particularly evident when surface area and total length of the roots, instead of root dry weight were used as parameters. The results stress the importance of root length and surface area for nitrogen acquisition from soils.     

Stimulation of in vitro root and shoot growth of potato by increasing sucrose concentration in the presence of fluridone,an inhibitor of abscisic acid synthesis

Fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, strongly stimulated rooting of nodal stem segments of potato (Solanum tuberosum L.) cultivar Arran Banner cultured in darkness on tuberisation medium. Inclusion of 10^-6 M ABA in the culture medium prevented this rooting response, indicating that root proliferation in the presence of fluridone could be due to inhibition of ABA synthesis. The rooting response to fluridone (increased total root number and root fresh weight) was obtained only at high sucrose concentrations (0.175 and 0.234 M) and was demonstrated with two potato cultivars and two culture media; one which favoured tuberisation and one which did not. Shoot numbers were also increased, but to a lesser extent than root numbers, and total fresh weight of plant material per culture was greatly increased by inclusion of both fluridone (10^-6 or 10^-5 M) and 0.234 M sucrose in the culture medium. The role of sucrose was not simply osmotic because when the osmolarity of fluridone medium was increased using mixtures of mannitol and sucrose, no root proliferation occurred unless sucrose predominated in the mixture.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Growing of potato microplants in the presence of alginate-silverthiosulfate capsules reduces ethylene-induced culture abnormalities during minimal growth conservation in vitro

Alginate capsules containing anionic complex silverthiosulfate (STS) [Ag(S₂O₃)₂ ^3-] were placed in the culture tubes over minimal growth media for studying whether STS could be used at higher concentrations to sustain ethylene-inhibiting effect during conservation of microplants in six potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of STS (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mM) were incorporated into the alginate capsules, and 12 alginate-STS capsules were tested in semisolid (7 g l^–1 agar) minimal growth medium containing 20 g l^–1 mannitol and 40 g l^–1 sucrose. This indirect supplementation of STS through alginate capsules rendered reduced total availability of STS in the minimal growth medium as compared to when it was directly supplemented in the medium at a given concentration. Growing of microplants in the presence of alginate-STS capsules improved the microplant growth and reduced the culture abnormalities over a period of 16 months under minimal growth conditions. Most significant improvement in microplant growth was in terms of green leaf production and leaf senescence. Vitrification, flaccidity and other growth abnormalities, viz., leaf loss, abnormal stem swelling and necrosis were not observed when the microplants were conserved in the presence of alginate-STS capsules. To foster optimum microplant growth and reduce culture abnormalities, potato microplants could favourably be maintained in the presence of 0.5–1.0 mM alginate-STS capsules during minimal growth conservation. Higher concentrations of alginate-STS capsules (>1.0 mM) were in general detrimental to potato microplant growth and survival during prolonged storage in vitro. Release kinetics of STS from the alginate-STS capsules, its distribution in the medium and accumulation of silver in potato microplants were studied using ^110mAg. The release rate of STS from the capsules was found to be directly proportional to the concentrations of alginate-STS capsules. A distinct concentration gradient of ^110mAg in the medium with increasing depth from top to bottom, and its accumulation in the potato microplants may be attributed to the improved anti-ethylene action of STS at higher concentrations through alginate capsules.     

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 μmol·dcm⁻³ of CdCl₂ solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm⁻³ concentration of CdCl₂ rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of K_I for cadmium and K_M for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.     

Insect-resistant plants with improved horticultural traits from interspecific potato hybrids grown in vitro

Summary Plants were regenerated from petiole calli of interspecific hybrids of Solanum tuberosum x S. berthaultii, an insect-resistant wild species. Callus culture was used to generate genetic changes to overcome the restricted recombination between the two genomes. Two plants out of 58 (3.5%) from calli of hybrid J114-1 showed stable and heritable differences from the hybrid over two cycles of evaluations in the field. Replicated trials were conducted in 1987 and 1988, using two populations of plants propagated by nodal cuttings from the original regenerates maintained in vitro. One regenerate showed insect resistance and increased marketable yield (approximately two fold) in the field. The other had higher levels of phenolic exudate in one of the two types of foliar trichomes associated with the insect resistance mechanism. Some desirable changes were discernible only in sexual progeny of regenerates, not in the regenerates themselves. In a backcross to S. tuberosum, 7 of 14 (50%) regenerates from hybrid F743-4 showed more progeny (up to 15-fold) with improved trichome traits and horticultural characteristics than the original hybrid. The variations were not associated with changes in ploidy. Fifteen plants obtained from these crosses are currently being incorporated into breeding lines. These results suggest that a period of callus culture followed by plant regeneration may aid in the introgression of desirable traits from wild species into crop plants.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Effect of maltose on the response of potato anthers in culture

Anthers of the Solanum tuberosum genotype H3703 were cultured on medium containing equimolar concentrations of sucrose or maltose. It was found that significantly more pollen embryos became plants after culture on maltose and hence the yield of plants per 100 anthers cultured increased significantly. Mechanisms by which carbohydrate source may influence response to anther culture are discussed.