Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata

Biondi, S., Torrigiani, P., Sansovini, A. and Bagni, N. 1988. Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata. - Physiol. Plant. 72: 471–476.
The effect of 1 mAf dicyclohexylamine (DCHA) on the synthesis of spermidine and spermine was examined in excised cotyledons of radiata pine ( Pinus radiata D. Don) cultured under shoot-forming (with cytokinin) and non-shoot-forming (minus cytokinin) conditions by incubation with [¹⁴C]-putrescine. In control cotyledons incorporation into spermidine showed a peak at day 2 in the presence and at day 5 in the absence of N⁶-benzyldenine (BA). DCHA-treated cotyledons gave the same labeling pattern, both in the presence and absence of benzyladenine, with a much smaller peak at day 2. The incorporation into spermidine and spermine was insignificant at day 5 and later. The total radioactivity in the trichloroacetic acid supernatant indicated that precursor uptake was strongly reduced by the drug. In addition, the percentage label found in the benzene phase and combined in the 3 polyamines was lower in DCHA-treated cotyledons. Thus, treatment with DCHA not only inhibited the conversion from putrescine to spermidine and spermine, but also reduced its conversion to other benzene-extractable compounds. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity, which furnishes the propylamine group to spermidine and spermine synthases (EC 2.5.1.16 and EC 2.5.1.-), was not significantly affected by DCHA and appeared to be independent of the spermidine and spermine synthase reactions, suggesting that spermine synthesis decreased as a result of substrate depletion. The correlation between morphological development and polyamine biosynthesis is discussed.     

Cytokinin-induced switch in development in excised cotyledons of radiata pine cultured in vitro

Cotyledons of Pinus radiata D. Don were cultured under shoot-forming (plus cytokinin) and elongating (minus cytokinin) conditions. Using. autoradiographic and precursor incorporation techniques, the sites and rate of macromolecular synthesis were examined during the first five days in culture. Active incorporation of ³H-thymidine, ³ H-uridine and ³H-leucine occurred. In shoot-forming cotyledons the incorporation became preferentially located in the epidermal and sub-epidermal cell layers in contact with the medium. In elongating cotyledons, in contrast, incorporation was randomly distributed, and the amount of incorporation declined with time. Biochemically, differences in DNA, RNA and total protein synthetic patterns were observed. In elongating cotyledons the rates of RNA and protein synthesis were higher during the first 48 h than in shoot-forming tissues, after which the synthetic rates were similar. Two peaks of newly formed DNA were observed in both tissues. These findings indicate that the cytokinin-induced changes in developmental pathways began within 24 h in culture.     

Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.     

Comparative studies on pyrimidine metabolism in excised cotyledons of Pinus radiata during shoot formation in vitro

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.     

Dicyclohexylamine uptake and effects on polyamine content in cultured cotyledons of radiata pine

Excised cotyledons of radiata pine ( Pinus radiata D. Don) were cultured in the presence or absence of benzyladenine and two concentrations of dicyclohexylamine, a potent inhibitor of spermidine synthesis in animals and bacteria. Cellular levels of the drug and of putrescine, spermidine and spermine were determined by dansylation followed by thin layer chromatography. The rate of uptake of the drug was rapid during the first 6 h of incubation and then diminished; nevertheless, its cellular content increased with time in culture and uptake was greater if more was present in the medium. Cotyledons cultured on benzyladenine-containing media accumulated more dicyclohexylamine than on benzyladenine-free media and this resulted in toxic side-effects. The drug had no significant effect on the elongation growth occurring in the absence of the hormone. In cotyledons treated with the drug, spermidine levels fell to zero after a 5- to 10-day exposure. Putrescine increased transiently at 24 h and then declined significantly. Spermine levels also declined to 11% of control values by day 5 and remained low throughout. Except for a marked decrease in all three polyamines during the first 24 h of culture, all control cotyledons maintained significantly higher polyamine levels than dicyclohexylamine-treated ones.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Metabolism of 14C-aspartate during shoot bud formation in cultured cotyledon explants of radiata pine

Aspartate metabolism was investigated in excised cotyledons of radiata pine ( Pinus radiata D. Don). These cotyledons were cultured under shoot-forming (plus N⁶-benzyladenine, SF), non-shoot-forming (minus N⁶-benzyladenine, NSF) and unresponsive (plus N⁶-benzyladenine, OLD) conditions, then incubated with [¹⁴C]-aspartate for 3-h pulse treatments followed by 3-h chase treatments with cold aspartate. The majority of label was recovered in the CO₂, amino acid, organic acid and pellet fractions. Uptake was greatest in all tissue types early in culture. Most (over 80%) of the [¹⁴C]-aspartate taken up by the tissues was converted to CO₂ at day 0 in SF and NSF tissues, CO₂ accounted for less than 50% of the total radioactivity in other tissues. Greater incorporation into fractions was observed in SF tissues during promeristemoid formation, while in NSF tissues the greatest incorporation was observed during a period of rapid elongation. Generally, less incorporation was observed in OLD cotyledons than in SF and NSF cotyledons. Analysis of the amino acid fraction showed that labelled aspartate was converted to other amino acids, mainly glutamate, glutamine, asparagine and 4-aminobutyric acid.     

The role of ethylene and carbon dioxide in differentiation of shoot buds in excised cotyledons of Pinus radiata in vitro

The production of ethylene and carbon dioxide by Pinus radiata D. Don cotyledons cultured on shoot-forming medium in sealed Erlenmeyer flasks was studied. To establish the role of these gases on morphogenesis three experimental approaches were used: (1) capping the flasks with serum caps on various days, and removing the serum caps at different stages of morphogenesis, (2) absorbing the gases by setting up traps singly and in combination during the various stages of differentiation and (3) incubating the cultures under continuous flow of constant gas mixtures. The results indicate that both ethylene and carbon dioxide, which build up during the first 10 to 15 days of culture, promote morphogenesis. Excessive accumulation after the initiation of buds causes some degree of dedifferentiation. When both the gases were eliminated from the flasks, bud formation and growth were inhibited. In the absence of oxygen, ethylene and carbon dioxide fail to influence growth and morphogenesis.     

Plantlet regeneration from subculturable nodular callus of Pinus radiata

An efficient planlet regeneration system via nodular callus formation is described for Pinus radiata. Subculturable nodular callus was induced at its highest frequency (93%) on embryonic explants excised from seeds at an early stage of germination (radicle length 2–5 mm). The optimal medium for nodular callus tissue proliferation was LP basal medium that was modified by reducing the concentration of potassium nitrate to 500 mg l^–1 and supplemented with 22.2 M 6-benzyladenine (BAP) and 2.85 M indole-3-butyric acid (IBA). Bud differentiation from the nodules was achieved by reducing BAP and sucrose concentrations in the culture medium. The maximum frequency of adventitious bud formation occurred on LP basal medium containing 2% sucrose and 0.44 M BAP on which about 61% of the transferred nodules formed buds. During the next 6 weeks of culture on the same cytokinin-free medium multiple shoots elongated from the buds. These shoots were excised and transferred to root initiation medium (RIM2.1), consisting of full-stregth SH macro- and micro-salts, 1000 mg l^–1 myo-inositol, 0.4 mg l^–1 thiamine-HCl, 2% sucrose and a combination of naphthaleneacetic acid (NAA), IBA and BAP at concentrations of 2.69, 4.93 and 0.11 M, respectively. After 5–15 days, root meristems were initiated on the stem bases. The highest rooting frequency was achieved when shoots were treated for 10 days on RIM2.1 medium, before being transferred to half-strength Schenk and Hildebrandt medium with 1% sucrose and without growth regulators for root growth.     

Nitrogen metabolism in cultured cotyledon explants of Pinus radiata during de novo organogenesis

Nitrogen metabolism was investigated under shoot-forming (SF) and non-shoot-forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [¹⁴C]-l,2-acetate into various metabolites. Early in culture, the lipid fraction contained the most ¹⁴C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21-day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg^-1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21-day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non-organ-forming tissue senescence-like metabolism was exhibited later in culture.     

Histological and scanning electron microscopic observations on plant regeneration in mungbean cotyledon (Vigna radiata (L.) Wilczek) cultured in vitro

Explanted cotyledons of mungbean Vigna radiata (L). Wilczek, variety Pag-asa-1, regenerated shoots directly from the basal adaxial side of the petiolar residue on MS medium supplemented with 8.9 M 6-benzyladenine. A simplified and rapid procedure for glycol methacrylate sectioning for histological observations was used to observe shoot initiation. At the time of culture, comparatively smaller and differentially stained epidermal cells were present on the basal adaxial region of the petiolar residue. A meristematic cell mass that developed at 48 h after culture appeared to be of epidermal and subepidermal cell origin. Scanning electron microscopy revealed shoot primordia and approximately 2 nodules at the base of the petiole as early as 48 h after culture. All of these structures developed into shoots during incubation.Abbreviations FAA formalin 5%–70% ethanol, 90%-acetic acid 5% - GMA glycol methacrylate - BA 6-benzyladenine     

岷江上游本地种油松和外来种辐射松造林对土壤磷的影响

通过测定岷江上游16年生本地种油松和外来种辐射松人工林下不同土壤层次中各形态磷素含量以及磷酸酶活力,阐述两种林分对土壤磷素含量及其分布的影响.结果表明,在各个土层中,两种林分下的土壤含水量、pH值、有机碳的含量以及微生物生物量磷均无显著差异.在0～20cm土层中,油松林与辐射松林土壤全磷、Al-P、Fe-P、Ca-P含量以及磷酸酶活力均无显著差异,而油松林土壤有效磷和有机磷显著高于辐射松林;在20～40cm土壤中,油松林土壤全磷、有效磷、有机磷、Al-P、Fe-P含量与辐射松林差异不显著,油松林土壤Ca-P含量、酸性磷酸酶和中性磷酸酶活力显著高于辐射松林;在40～60cm土壤中,油松林土壤除中性磷酸酶活力与辐射松林的差异不显著外,其余各形态磷素含量和酸性磷酸酶活力变化与20～40cm土壤中的一致.此外,随土壤深度的增加,两种人工林土壤各形态磷素含量、磷酸酶活力都呈降低的趋势.单从土壤磷的状况看,油松林土壤中磷素含量高于辐射松林.     

Differential response of the two cotyledons of Vigna radiata in vitro

Summary Cotyledons of mature seeds of Vigna radiata were found to be variable in their response to N 6-benzytadenine, kinet in and zeatin. The two cotyledons of a seed were designated as CE and C; where CE referred to the cotyledon that remained closely attached to the embryonal axis, and the other more loosely attached cotyledon was referred to as C. Shoots formed from the proximal end of both explants in all nine cultivars studied. Shoot regeneration was faster and regeneration efficiency was higher in CE explants than in C explants in these cultivars. BA was found to be the most suitable cytokinin for both multiple shoot induction and regeneration.Abbreviations BA N6- benzyladenine - Cv cultivar - KIN kinetin - NAA naphthalene acetic acid - RE regeneration efficiency - ZEA zeatin     

In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons

Summary The anatomy and morphology of bud regeneration were investigated in melon (Cucumis melo L.) cv. Galia, which regenerates in vitro only by direct organogenesis from the cotyledon explant. Explants were cut from the cotyledon proximal to the apex from 3-d-old in vitro seedlings. After 3 d on Murashige and Skoog medium with N⁶-benzyladenine, cell division can be observed in the epidermal layer on the adaxial side in the center of the explant, near the most proximal (wounded) cut edge. Over the next week, the area of the meristem increases laterally. Additional cell layers are added to the meristematic area by cell division in the epidermis. In places the epidermis remains active in cell division. Alongside those active areas there are zones where the epidermis has become inactive, although the subepidermal layers continue to divide. In transverse section, the explant now has small protuberances on the adaxial surface. After 10 d on cytokinin-containing medium, the first signs of development are visible on the adaxial surface adjacent to the proximal cut edge. The protuberances observed after 10 d are neither primordia nor buds, although some meristematic bulges are observed. The first regenerated shoot buds are observed histologically after 15 d, by which time the surface has many protuberances and some small leaves. The first shoot is found by histology after 22 d. By this time the surface is covered with protrusions and leaves, mostly without accompanying buds. The leaves may be produced from the protrusions initially visible after 10 d.     

Reliable plantlet formation from embryos and seedling shoot tips of radiata pine

A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips of Pinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/l benzylaminopurine (BAP). Except for some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non-sterile conditions in a humid environment with occasional misting. An intervening 5-day treatment of shoots in an agar medium containing 2.0 mg/l indolebutyric acid (IBA) and 0.5 mg/l napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mix. An enhanced level of CO₂ during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.     

Light-cytokinin interaction in shoot formation in cultured cotyledon explants of radiata pine

Previous studies utilizing cotyledon explants of radiata pine ( Pinus radiata D. Don) revealed that cytokinin was required for shoot formation. This was confirmed and extended in the present study, which also showed that light was required. As little as three days of exposure to 16 h photoperiod light at a photon fluence rate of ca 80 μmol m⁻² s⁻¹ was sufficient to give some meristematic tissue formation. Longer exposure to light increased this formation. While cytokinin must be present during the first three days in culture for shoot formation, the exposure of the cotyledons to light could be delayed at least until day 10, but after 21 days in darkness, transfer to light did not permit shoot formation. Anatomical examination of the cotyledons confirmed the morphogenetic interactions of light and cytokinin in shoot formation. The data suggest that cytokinin is directly involved in the induction of shoot initiation and both light and cytokinin are required for the development of meristematic tissue and subsequent shoot formation.     

Evaluation of AFLP for genetic mapping in Pinus radiata D. Don

Efficient construction of reasonable density genetic linkage maps is an essential component of QTL detection programmes. The AFLP technique has been used to produce genetic linkage maps in a range of species. We have developed protocols to generate reproducible AFLP profiles in Pinus radiata and have evaluated the inheritance and informativeness of AFLP markers in this important timber species. The large genome size of P. radiata necessitated increased levels of selection at both the pre-amplification and selective amplification steps of the AFLP protocol to generate reproducible AFLP profiles. Once optimised ca. 41.3 scorable AFLP bands were resolvable through denaturing gels, of which 48.4% were polymorphic in a screen of eight unrelated trees. This level of polymorphism is ca. three times higher than with RAPD markers. The total number of bands and the number of polymorphismic bands per PCR were ca. halved when AFLPs were electrophoresed on non-denaturing gels and stained with ethidium bromide. Using the protocols developed, AFLP is an efficient method for generating the DNA markers required for genetic linkage map construction in P. radiata. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

The response of photosynthetic model parameters to temperature and nitrogen concentration in Pinus radiata D. Don

Responses of photosynthesis (A) to intercellular CO₂ concentration (c_i) in 2-year-old Pinus radiata D. Don seedlings were measured at a range of temperatures in order to parametrize a biophysical model of leaf photosynthesis. Increasing leaf temperature from 8 to 30°C caused a 4-fold increase in V_cmax, the maximum rate of carboxylation (10.7–43.3 μol m^?2 s^?1 and a 3-fold increase in J_max, the maximum electron transport rate (20.5–60.2 μmol m ^?2 s^?1). The temperature optimum for J_max was lower than that for V_cmax, causing a decline in the ratio J_max:V_cmax from 2.0 to 1.4 as leaf temperature increased from 8 to 30°C. To determine the response of photosynthesis to leaf nitrogen concentration, additional measurements were made on seedlings grown under four nitrogen treatments. Foliar N concentrations varied between 0.36 and 1.27 mol kg^?1, and there were linear relationships between N concentration and both V_cmax and J_max. Measurements made throughout the crown of a plantation forest tree, where foliar N concentrations varied from 0.83 mol kg^?1 near the base to 1.54 mol kg^?1 near the leader, yielded similar relationships. These results will be useful in scaling carbon assimilation models from leaves to canopies.     

Application of QuickBird and aerial imagery to detect Pinus radiata in remnant vegetation

The invasion of Pinus radiata from long‐term established plantations is contributing to the degradation of fragmented and isolated remnants of native vegetation. Within the south‐east of South Australia, the 20 vegetation communities that occur within 500 m of a plantation edge are at risk, including nine state threatened communities. To plan effective mitigation strategies, the current extent and distribution of P. radiata needs to be ascertained. High spatial resolution, multispectral QuickBird imagery and aerial photography were used to classify P. radiata within eucalypt and acacia woodlands, melaleuca shrubland, modified pasture and an Eucalyptus globulus plantation. Unsupervised classification of aerial photography gave the best result showing reasonable conformity with the observed distribution of P. radiata at the site scale. However, the 9.4 ± 13.5 (SD) cover classified in the quadrats sampled for the accuracy assessment exceeded the 1.4 ± 2.4 (SD) P. radiata cover determined from an independent dataset. Only 30.1 ± 37.9% (SD) of trees within the quadrats and 9.40 ± 13.49% (SD) of their foliage cover were classified. Trees detected by partial classification of canopy were positively correlated with both tree height and canopy diameter. Overall, the low detection rates were attributed to insufficient spectral resolution. Using higher resolution imagery, together with an object‐based image analysis or combination of multispectral and airborne digital image classification, restricted to large emergent adult trees using LiDAR analysis, is likely to improve adult P. radiata detection accuracy.