RNA synthesis in the early stage of aerobic incubation of potato tuber discs

RNA synthesis of potato tuber discs during the early periodof their aerobic incubation was investigated by feeding thediscs with ³H-uridine. The rate of total RNA synthesis increasedin two steps during the incubation. The increase during thefirst 2 to 3 hr was small, but that after 3 hr was large. Thelabeled RNAs were separated into poly(A) containing RNA [poly(A)(+) RNA] and poly(A) lacking RNA [poly(A) (–) RNA] bythe use of a poly(U)-Sepharose column. Poly(A) (+) RNA was synthesizedeven in the freshly prepared discs which incorporated little¹⁴C-leucine into a protein fraction, and the synthetic rateof poly(A) (+) RNA increased by about 50% during the first 3hr incubation period, then gradually decreased thereafter. Synthesisof poly(A) (–) RNA continued to increase up to 7 hr afterslicing. When the discs were pulse labeled, the proportion ofradioactivity in poly(A) (+) RNA to that in the total RNA wasmaintained at about 50% until about 3 hr after slicing, butit abruptly decreased between 3 and 5 hr to about 35% whichwas maintained up to 9 hr after slicing. (Received October 12, 1977; )     

Effect of ethylene on DNA synthesis in potato tuber discs

The effect of ethylene on DNA synthesis in potato tuber discsinduced by cutting was examined. Continuous presence of ethylenein the ambient atmosphere of the slices lowered the rate ofinduced DNA synthesis by about 50%, but did not alter the timecourse pattern of development of DNA synthesis. RNA and proteinsyntheses were not affected. The inhibitory effect on DNA synthesiswas observed at as low as 0.01 µl/liter and was due tothe specific action of ethylene, not to a non-specific actionof gaseous hydrocarbons. Ethylene also decreased the numberof cells which could synthesize DNA. The results of ethylenetreatment of various durations at various times after cuttingindicate that a process prerequisite for DNA synthesis and susceptibleto ethylene action starts at about 6 hr after cutting and continuesfor only a limited period. (Received July 5, 1976; )     

Ultrasound-mediated stable transformation of potato tuber discs

Summary Plasmid DNA (pBARGUS), containing the selectable bar gene for resistance to the herbicide Basta, was delivered into potato tuber discs via ultrasonication. Transformed plants were identified by their ability to grow on a medium containing 1mg phosphinothricin/l. Southern hybridization and plant resistance to the application of Basta indicated that a functional bar gene had integrated into potato chromosomal DNA.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Biochemical studies on mitochondria formed during aging of sliced potato tuber tissue

Respiratory activities in mitochondria of potato tuber tissueincreased about 3-fold after aging sliced tissue for 1 day.Increased activity was insensitive to cyanide. The number ofmitochondrial particles also increased during aging. It seemslikely that newly formed mitochondria are heavier than pre-existingones and that respiration in the former is insensitive to cyanide. (Received January 26, 1970; )     

Adenine nucleotide contents in callus-forming potato tuber discs with different respiratory characteristics

ATP content (per g fresh weight) and energy charge were higher during incubation at 8°C than at 28°C in the early stages of callus induction of potato tuber discs ( Solanum luberosum L. cv. Bintje). After a transfer from 28°C to 8°C, ATP content increased while a sharp decline in ATP content was observed after a transfer from 8°C to 28°C. ADP and AMP pools did not increase correspondingly. When the callus discs had entered the logarithmic growth phase, the energy charge was maintained within relatively narrow limits (0.77–0.80) at all culture temperatures.     

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.     

Influence of ethanol on alternative oxidase in mitochondria from callus-forming potato tuber discs

Ethanol, when added to the incubation medium of callus-forming potato tuber discs, inhibits callus growth and causes an increase of the mitochondrial antimycin-A resistant respiration, expressed as a percentage of state III-respiration. This increase in resistance to antimycin-A is the result of a poor development of the cytochrome pathway in tissue discs treated with ethanol. The development of the antimycin-A resistant alternative oxidase sensitive to chelator is about the same for treated and untreated discs. The respiratory control (RC) ratio of the mitochondrial respiration increases after addition of a chelator, which inhibits the alternative pathway. The RC ratio of the uninhibited mitochondrial respiration appears to be inversely related to the capacity of the alternative pathway, when mitochondrial preparations with different capacities to transfer electrons via the alternative path are compared. From the experimentally observed relation between RC-ratio and alternative oxidase capacity, it was concluded that at least half of the capacity of the alternative path is used in uninhibited state IV respiration.     

Factors affecting shoot initiation from tuber discs of potato (Solanum tuberosum)

Discs of cortical and perimedullary tissue from tubers of potato (Solanum tuberosum L. cv. Superior) formed adventitious shoots when cultured on a medium containing Murashige and Skoog's salts, myo-inositol, 100 mg/l; folic acid, 0.5 mg/l; D-biotin, 0.05 mg/l; gibberellic acid (GA₃), 0.5 mg/l; thiamine ˙ HCl, 0.1 mg/l; glycine, 2.0 mg/l; pyridoxine ˙ HCl, 0.5 mg/l; nicotinic acid, 0.5 mg/l; sucrose, 25 g/l; casein hydrolysate, 1 g/l; Bacto agar, 9.0 g/l; and a cytokinin [N⁶-benzylaminopurine (BAP), N⁶-γ,γ-dimethylallylaminopurine (2iP), or N⁶-furfurylaminopurine (kinetin)]. Discs of pith tissue either failed to survive or produced callus but did not undergo morphogenesis. Cytokinin was essential for explant survival, while BAP at 3.0 mg/l was most efficacious in promoting shoot initiation. Auxin was not essential for shoot initiation or development; however, a low concentration (0.03 mg/l) of α-naphthaleneacetic acid (NAA) stimulated both explant survival and the number of shoots produced per disc. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) did not stimulate shoot initiation. GA₃ was essential for both shoot initiation and subsequent shoot development. The highest incidence of morphogenesis (over 100 shoots in 12 weeks) occurred from tuber discs cultured at 18°C constant and under a photon flux density of 60 μE m^-2s^-1. No difference in regenerative ability was found in explants taken from source tubers stored for 0 to 20 weeks at 4°C. A histological examination indicated that shoots developed from small clusters of meristematic cells which were initiated from within small protuberances on the surface of the tuber disc 3 weeks after transfer.     

The synthesis and properties of polysomal RNA in potato tuber slices during the early stage of aging

The synthesis, molecular size, and coding properties of polysome-associatedpolyadenylated RNA[poly(A)(+)RNA]and non-polyadenylated RNA[poly(A)(–)RNA] were investigated in potato tuber discsduring the early stage of aging. Tissue discs were labeled for6 hr with ³H-uridine in the presence of 5-fluorouracil to suppressrRNA synthesis, and polysomal RNA was isolated from the discs.Poly(A)(+)RNA accounted for 70% of the radioactivity in polysomalRNA and had a molecular size ranging from 6S to 30S with a peakat about 15S, when measured by formamide-polyacrylamide gelelectrophoresis. The rest of the radioactivity was in poly(A)(–)RNAwhich had nearly the same range in molecular size, but had noconspicuous peaks on the gel. The polysomal RNA could programthe synthesis of a wide variety of polypeptides in a cell-freetranslation system of wheat germ. Seventy percent of the translationalcapacity of polysomal RNA was attributed to poly(A)(+)RNA. Theelectrophoretic behaviour of the majority of the products frompoly(A)(+)RNA was similar to that of products from poly(A)(–)RNA,but the former could program the synthesis of five polypeptidesin addition to those translated from the latter. There was atendency for poly(A)(–)RNA to be a more efficient messengerfor large polypeptides. ¹Present address: Department of Agricultural Chemistry, Facultyof Horticulture, Chiba University, Matsudo 271, Japan. (Received November 16, 1979; )     

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.     

Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs

The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28°C, is higher than that of mitochondria from tissue incubated at 8°C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28°C tissue than in mitochondria from 8°C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28°C tissue and 8°C tissue.

The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8°C tissue, but also by lower values of respiration via the cytochrome pathway.

A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37°C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs.