Comparison of tissue culture and whole plant responses to salinity in potato

Salt sensitivities of six potato cultivars using six levels of sodium chloride (0.0 to 0.25M) were studied in a greenhouse. Responses of these cultivars were also determined in tissue culture by studying rooting of stem segments, increase in length of cultured roots and inhibition of growth of cell suspension cultures using similar salt concentrations. Responses of cultured stem segments and cell suspensions differed from those expressed by whole plants. A close similarity was observed between the salt stress response of whole plants and of cultured roots. The latter technique may provide a preliminary screening method for assessing salt tolerance in potato genotypes.     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

An efficient genotype-independent method for regeneration of potato plants from leaf tissue

The regeneration of plants from leaf explants of a number of potato cultivars using a number of published one-, two- and three-step methods was assessed. A method using a pretreatment with high levels of auxin and cytokinin coupled with silver thiosulphate in the regeneration medium proved the most rapid and efficient for the eight cultivars examined.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid - STS silver thiosulphate     

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.     

Growth rate, water relations and ion accumulation of soybean callus lines differing in salinity tolerance under salinity stress and its subsequent relief

A selected Glycine max (L.) salt-tolerant calluscell line (R100) was significantly more tolerant to salt than a salt-sensitiveline (S100) during exposure to salt stress. Growth (Fresh and Dry weights) ofthe R100 cell line declined significantly at NaCl concentrations greater than 75mM, while growth of the S100 cell line was already impaired at 25mM NaCl. Levels of Na⁺ and Cl^– inthe callus were elevated as the salt concentration increased, whileK⁺, Ca²⁺ and Mg²⁺ levels weremarkedly reduced. The lower s reduction and Na⁺accumulation found in the S100 callus corresponded with the higher callusdehydration during salinity. Calli grown on Miller's basal medium weresupplied with 100 mM NaCl for 12 days and then supplied with mediumwithout NaCl to relieve salinity stress. The Na⁺ andCl^– content decreased in both R100 and S100 cell lines duringthe first 24 h and reached normal levels four days after transferto the normal medium. This lower concentration was maintained until the end ofthe experiment. Concurrently, the K⁺ content andK⁺/Na⁺ ratio increased sharply and reached theirhighest levels within 24 h in both salt-sensitive and salt-tolerantcell lines. These data suggest that the inhibitory effects of salinization ongrowth and accumulation of potentially toxic ions (Na⁺,Cl^–) can be readily reversed when salinity is relieved.     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

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.     

Starch synthesis in potato (Solanum tuberosum) tubers: Activity of selected enzymes in dependence of potassium content in storage tissue

Starch synthesis in potato tubers grown at varied K nutrition 0.1 (K₁), 0.25 (K₂) and 1.0 mmol K L^- nutrient solution (K₃) was investigated with particular regard to the activity of selected enzymes (sucrose synthase, UDP-D-glucose pyrophosphatase, starch phosphorylase, amylases) in dependence on tuber K content. Allocation of K to the tubers was nearly the same in all treatments. The activity of enzymes related to tuber K content did not differ significantly. Starch and K content of tubers increased with progressing age, whereas a decrease was observed in growth rate, starch synthesis per day and K uptake per day. Positive correlations between the rates of K uptake, starch production and growth indicate that the dynamic phase of K supply to the tubers is of greater importance for starch synthesizing processes than the influence of total K content.     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

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.     

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.     

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.     

Lipolytic and peroxidative enzyme expressions in cultured potato tuber cells

Potato cells (cv. Norchip) were cultured from tuber parenchymal tissue and subcultured to dissociate and habituate the despecialized cells. After several subculturings on a minimal nutrient media, this line of cells demonstrated repeatable physical growth profiles for dry weight (DW), fresh weight (FW) and protein. Two enzymes of plant lipid metabolism were investigated, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX), which respectively liberate and peroxidize fatty acids from lipid in cellular membranes. LAH, measured as p-nitrophenyl palmitate hydrolase, was present in this line of cells in easily detectable amounts (317 units g^-1 DW) albeit much lower than that found in mother tuber (9878 units g^-1 DW). The presence of LAH in this line is significant because LAH isozymes are often described as storage proteins, yet activity per gram fresh weight in these unorganized cells is reasonably constant until culture growth exits the linear phase. However, LOX, the most active free fatty acid metabolizing enzyme in potato tubers (89,800 units g^-1 DW), was not detectable in this line of callus or suspension cultured cells. The absence of LOX activity in this line of cells was verified by a number of assay approaches and was confirmed by activity staining of extracted enzymes separated in polyacrylamide gels. The absence of LOX in these cultured cells is especially important in determining the functions of this lipid peroxidation system and how it may be genetically regulated.Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.A laboratory cooperatively operated by the Midwest Area, Agricultural Research Service, U.S. Department of Agriculture, The Minnesota Agricultural Experiment Station, the North Dakota Agrcultural Experiment Station, and the Red River Valley Potato Grower's Association.     

A synergism between oxalic acid and polygalacturonases in the depolymerization of potato tuber tissue

Rhizoctonia bataticola produced oxalic acid in vitro and in vivo during pathogenesis of patato tuber. Polygalacturonase (PG) was also detected in culture filtrates of the rot-causing organism. Levels of maceration and cell death in tuber tissue were higher when a mixture of oxalic acid and PG was used than when either oxalic acid or PG were used alone.     

Somaclonal variation in tuber disc-derived populations of potato

Summary Approximately 1,600 potato (Solanum tuberosum L.) plants of the cultivar Superior were regeneratedin vitro from meristems adventitiously initiated on tuber disc expiants. Direct regeneration from tuber disc cells, by passing a callus intermediary, is efficient and results in low frequencies of plants with gross phenotypic aberrations. The somaclonal plant population was statistically characterized in field plots over five asexual generations and in three diverse locations. When compared in advanced generations to a large population of control plants propagated from stem cuttings, the means of the somaclonal population were significantly different, often shifted in the desirable direction, for 16 of 22 horticulturally important traits. Somaclonal population variances statistically exceeded those of the controls for 13 of the 22 traits. Regressions between consecutive tuber generations and between locations or replications (blocks) within a generation were significant in the somaclonal population for all traits analyzed. In a few instances, significant control population regressions occurred that are interpreted to be the result of non-random, non-genetic factors primarily affecting control plants of low vigor. Selected somaclones exhibiting desirable alterations for yield, tuber number and shape, and vigor were stable over more than two consecutive asexual generations.Research supported by a grant from NPI, 417 Wakara Way, Salt Lake City, UT 84108     

Nitrate reductase and glutamine synthetase activities in relation to growth and nitrogen assimilation in red oak and red ash seedlings: effects of N-forms,N concentration and light intensity

The effects of growing seedlings of red oak (Quercus rubra) and red ash (Fraxinus pennsylvanica) with Hoagland solutions containing five N-regimes, differing in the N-forms (NH₄, NO₃) and concentrations (High and Low), in relation to light intensity were investigated by the utilization of enzymatic markers of the N assimilation pathway, nitrate reductase (NR) and glutamine synthetase (GS). Red oak and red ash showed different patterns of N-assimilation. Red oak seedlings assimilated NO₃ in low amounts in their roots and leaves, whereas red ash seedlings assimilated high amounts of NO₃, mostly in the leaves. A significant amount of constitutive NR activity was found in red oak seedlings supplied with NH₄ N-regime. This could be characteristic of a species adapted to soils that are poor in nitrogen. Root GS activity was lower in red oak seedlings than in red ash seedlings, indicating that the rate of NH₄ assimilation differed in these two hardwood species. Low irradiance reduced growth of both hardwood species, but greatly affected the specific leaf area of red ash and reduced NO₃ assimilation (when data are expressed per leaf area). Both species reacted similarly to N-regimes in terms of relative growth rate.     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.     

Isolation and culture of protoplasts from callus tissue of Hibiscus syriacus L.

Protoplasts were isolated from callus tissue of Hibiscus syriacus L. using a solution of 3% Onozuka cellulase, 1% Onozuka macerozyme, and 0.5% hemicellulase. Highest yields of viable protoplasts were obtained from friable, white or yellow callus 8–9 days after subculture on Murashige & Skoog medium with 0.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. Protoplasts cultured in thin liquid layers of this medium with mannitol continued dividing for longer than those cultured in droplets or in an agar medium. Cultures were maintained until protoplasts had divided to form groups of more than ten cells. Cell groups developed into callus and continued to grow on an agar medium, but failed to differentiate on a regeneration medium with 2 mg l^-1 naphthalene acetic acid and 1 mg l^-1 benzylaminopurine.     

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.