Germination Promotion of Loblolly Pine and Baldcypress Seeds by Stratification and Chemical Treatments

The effects of 4 chemicals on the germination promotion of stratified and unstratified seeds of loblolly pine (Pinus taeda) and baldcypress (Taxodium distichum) were studied. The chemicals used were gibberellic acid, kinetin, potassium nitrate and thiourea, each at 3 different concentrations. Stratification promoted the germination of both seed species. Certain concentrations of gibberellic acid, potassium nitrate and thiourea improved the germination of unstratified loblolly pine and baldcypress seeds while kinetin had no stimulatory effect. All 4 chemicals at specific concentrations promoted the germination of loblolly pine seeds stratified for a short period of time. Considering both speed and completeness of germination, best results were obtained when 21-day stratified seeds were treated with either gibberellic acid (100 mg/1) or kinetin (10 mg/1). In baldcypress, on the other hand, none of these chemicals had any stimulatory effect on the germination of stratified seeds. Germination of both species of seeds was either partially or completely inhibited by the highest concentration of thiourea (30,000 mg/1) used.     

Cold stratification and growth of radicles of loblolly pine (Pinus taeda) embryos

The mechanism of germination enhancement by cold stratification was examined in seeds of loblolly pine ( Pinus taeda L. ), Removal of the seed coat permitted elongation of radicles from unstratified embryos, but both rates of germination and radicle elongation were increased by stratification. Radicles of both stratified and unstratified embryos excised from the megagamethophyte elongated only when in contact with solid incubation media supplemented with sucrose. Stratification of embryos either in the presence or absence of the megagametophyte resulted in similar enhancement of radicle elongation. Elongation rates of radicles were increased after stratification independent of sucrose concentration, and changes in sucrose content in the megagamethophyte during stratification or incubations subsequent to stratification were insufficient to regulate radicle growth. Our results support the hypothesis that the embryos of pine seeds perceive the low temperature stimulus directly and this stimulus results in a growth potential increase in the embryonic axes. We propose that this growth potential increase enables the embryos to overcome the mechanical restraint of the seeds coats and to germinate.     

Changes in Endogenous Growth Kegulators in Loblolly Pine Seeds During the Process of Stratification and Germination

The seeds of loblolly pine (Pirns taeda L.) were cold-stratified for 0, 14, 28, 42, and 56 days. Endogenous growth regulators were extracted from these seeds, and also from the germinating and the 28-day warm stratified seeds. Partially purified extracts were separated on chromatographic paper. The chromatograms were cut into 10 equal segments, and these were tested for biological activity using three different btoassays. The results indicated that the unstratified seeds and those stratified up to a period of 28 days contained very little or no growth promoter (GA-like substances), and a relatively high concentration of an inhibitor (presumably abscisic acid), Following 42-day stratification, the promoter concentration gradually increased while the inhibitor level fell almost to zero. A high level of promoter but no inhibitor was detected in germinating seeds. No auxin-like activity was noted in the unstratified seeds. This activity slowly increased up to a period of 28 days and remained at this level for the subsequent stratification periods. However, the activity greatly increased in the germinating seeds. Very little changes in the levels of growth regulators were noted in warm-stratified seeds as compared to the unstrati-fied controls.     

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.     

Paternal chloroplast inheritance patterns in pine hybrids detected with trnL-trnF intergenic region polymorphism

The inheritance patterns of the chloroplast genomes of shortleaf pine (Pinus echinata Mill.), loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) were investigated through the trnL-trnF intergenic spacer polymorphism analysis. The DNA sequences of this spacer differ among these three closely related Pinus species. A modified 'cold' PCR-SSCP (single-strand conformation polymorphism) analysis of this spacer shows that the artificial hybrids (F1) from the shortleaf pine (seed parent) 2 loblolly pine (pollen parent) cross, exhibit the loblolly pine profile. Additionally, nine putative hybrids between shortleaf pine and loblolly pine, previously identified by the IDH (Isocitrate dehydrogenase) allozyme marker, presented the shortleaf pine profile indicating that shortleaf pine, not loblolly pine, sired all of the putative hybrids. Nondenatured polyacrylamide-gel electrophoresis of the trnL-trnF intergenic spacer demonstrated that the artificial hybrids (F1) from the cross, slash pine (seed parent) 2 shortleaf pine (pollen parent), present the shortleaf pine profile. Those results confirmed that the chloroplast genome is paternally inherited in these three species of the genus Pinus. The significance of the trnL-trnF intergenic region polymorphism and our modified 'cold' SSCP protocol for population genetic studies is discussed.     

Seed Coat Dormancy in Two Species of Grevillea(Proteaceae)

The role played by the seed coat in seed dormancy of Grevillealinearifolia(Cav.) Druce and G. wilsonii(A. Cunn.) was testedby a series of manipulations in which the seed coat was dissectedand removed, dissected and returned to the decoated seed, ordissected, removed and given a heat shock, and returned to thedecoated seed. Germination of intact seeds of both species wasalso examined after exposure to heat shock, smoke, or heat shockand smoke combined. Water permeability of the seed coat wasinvestigated by examining imbibition. For intact seeds, virtuallyno germination occurred under any treatment (G. wilsonii), orgermination was increased by exposure to either heat or smoke(G. linearifolia). Removal of the seed coat led to germinationof all decoated seeds for G. linearifolia, or a proportion ofdecoated seeds for G. wilsonii. Inclusion of smoked water inthe incubation medium led to a higher proportion of decoatedseeds germinating for G. wilsonii. Returning the seed coat,either with or without heat shock to the seed coat, did notsignificantly affect germination in either species. Seed coatswere permeable to water in both species. For the two Grevilleaspecies, there were different dormancy mechanisms that werecontrolled by the seed coat (G. linearifolia) or by both theseed coat and embryo (G. wilsonii). Copyright 2000 Annals ofBotany Company Grevillea linearifolia, Grevillea wilsonii, dormancy, seed coat dormancy, seed coat permeability, smoke, heat shock, germination     

Changes in phenolic acids and abscisic acid in sugar pine seed coats during stratification

The phenolic acids and abscisic acid (ABA) of sugar pine ( Pinus lambertiana Dougl.) seeds coats, separated by high-pressure liquid chromatography, were analyzed during 90 days stratification of the seeds. Although levels of seed coat phenolic acids and ABA declined significantly during, stratification, this decrease did not appear to be responsible for the loss of dormancy due to stratification. Lack of improved germination following washing, cracking, or removal of the seed coats, plus additional evidence, did not support a significant role for the seed coat in the dormancy of sugar pine seeds.     

Episodic Growth and Relative Shoot: Root Balance in Loblolly Pine Seedlings

Leaf, root and stem systems of loblolly pine seedlings are characterizedby a seasonal periodicity in growth, during which they alternatein spurts of activity. Despite this periodicity, the allometriccoefficient describing the ratio of the relative growth ratesof leaf to root remains constant for at least the first twoyears of development. In part, constancy results from the inabilityof variation in the allocation of growth increment during briefperiods to change a pre-existing structure accumulated overthe life of the seedling. In addition, alternating periods ofleaf, root and stem growth may represent the action of feedbackmechanisms which operate to maintain an adaptive balance betweenorgan systems. Pinus taeda L., loblolly pine, allometric coefficient, homeostatic control of growth     

Metabolome profiling of stratified seeds provides insight into the regulation of dormancy in Davidia involucrata

Dove tree (Davidia involucrata), a tertiary vestige species, is well-adapted to cool conditions. Dormancy in D. involucrata seed lasts for an extremely long period of time, typically between 3 and 4 years, and this characteristic makes the species an excellent model for studying the mechanisms of seed dormancy. The molecular mechanisms governing germination control in D. involucrata are still unknown. Seed stratification have been reported to enhance germination in recalcitrant seeds. We performed a widely targeted metabolome profiling to identify metabolites and associated pathways in D. involucrata seeds from six different moist sand stratification durations (0–30 months) using the ultra-high-performance liquid chromatography-Q Exactive Orbitrap-Mass spectrometry. There was an increasing germination rate with prolonged stratification durations (12–30 months). Furthermore, we detected 10,008 metabolites in the stratified seeds. We also detected 48 differentially accumulated metabolites (DAMs) between all stratification periods in the seeds, with 10 highly conserved metabolites. Most of the differentially accumulated metabolites between unstratified and stratified seeds were enriched in purine metabolism, pyrimidine metabolism, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis, and arginine biosynthesis pathways. Key phytohormones, abscisic acid, indole-3 acetic acid, and sinapic acid were differentially accumulated in the seeds and are predicted to regulate dormancy in D. involucrata. We have provided extensive metabolic information useful for future works on dove tree germination study.     

Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.)

Summary A culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine.     

The Influence of Volatile Substances from Incubated Litter of Pinus radiata on Seed Germination

Vapour from incubated Pinus radiata D. Don litter caused a depressionin the germination of Lolium perenne L. seeds and responsesvarying from stimulation to depression in germination of Trifoliumrepens L. seeds. These effects could be attributed to ethyleneand carbon dioxide. Experiments using ethylene and carbon dioxidesupported this conclusion. Pinus radiata, litter, Lolium perenne, Trifolium repens, seed germination, ethylene, carbon dioxide     

红松种子休眠与种皮的关系

本文探讨红松(Pinus koraiensis)种子休眠与其种皮之间的关系。夹破中种皮后,种子萌发率很低。在离体胚培养基中外加 ABA 及经 ABA 溶液浸泡种子的萌发实验表明,ABA也不是导致休眠的关键因素。试验确认红松种子存在透气障碍,即中、内种皮对氧气的进入都有阻碍作用。经低温砂藏后,种皮的阻碍作用明显减小。种皮的透气性障碍可能是诱导休限的主导因素。     

Purification and properties of Pinus taeda arginase from germinated seedlings

In germinated loblolly pine (Pinus taeda L.) seeds arginine accumulates in the seedling during its growth immediately following germination. The enzyme arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is responsible for hydrolyzing this arginine into ornithine and urea. Loblolly pine arginase was purified to homogeneity from seedling cotyledons by chromatographic separation on DE-52 cellulose, Matrex Green and arginine-linked Sepharose 4B. The enzyme was purified 148-fold and a single polypeptide band was identified as arginase. The molecular mass was determined to be 140 kDa by FPLC, while the subunit size was shown to be 37 kDa by SDS-PAGE, predicting a homotetramer holoprotein. Removal of manganese from the enzyme abolishes catalytic activity, which can be restored by incubating the protein with Mn²⁺. Antibodies, raised against the arginase subunit, are able to immunotitrate arginase activity and are monospecific for arginase on immunoblots.     

Purification and Characterization of Catalase from Loblolly Pine (Pinus taeda L.) Megagametophytes

Catalase (EC 1.11.1.6) was purified to near homogeneity from isolated megagametophytes of germinated loblolly pine (Pinus taeda L.) seeds, and monospecific antibodies were elicited in rabbits. Following a procedure that involved acetone extraction, (NH4)2SO4 fractionation, and four chromatographic steps (i.e. DE-52 cellulose, Superdex-200, hydroxylapatite, and phenyl-Sepharose CL-4B), catalase was purified about 140-fold to a final specific activity of 2215 mmol min-1 mg-1 of protein. Cotton isocitrate lyase antibodies were used, and protein immunoblots revealed that the resolution on hydroxylapatite and phenyl-Sepharose allowed for the complete separation of catalase from contaminating isocitrate lyase. The molecular masses of the native enzyme and its subunit are 235 and 59 kD, respectively, indicating that the pine holoenzyme is a homotetramer. Loblolly pine catalase exists as multiple isoforms. When megagametophytes taken 7 d after imbibition at 30[deg]C were extracted, subjected to nondenaturing isoelectric focusing, and stained for catalase activity, at least four catalase isoforms were observed, including one dominant form with an isoelectric point of 6.87. Purified pine catalase is not a glycoprotein and has a ratio of absorbance at 208 nm to absorbance at 405 nm of 1.5. When probed with loblolly pine catalase antibodies, protein blots of cell-free extracts from megagametophytes of mature, stratified, and germinated loblolly pine seeds, the megagametophyte glyoxysomal fraction, and purified loblolly pine catalase all revealed one immunoreactive 59-kD polypeptide. This indicates that no detectable change in the enzyme's monomeric molecular mass occurs during seed stratification and germination, early seedling growth, and purification.     

Changes in Ultrastructure and Abscisic Acid Level, and Response to Applied Gibberellins inTaxus maireiSeeds Treated With Warm and Cold Stratification

Seeds ofTaxus maireiare known for their deep dormancy whichcan only be broken by a procedure involving warm stratificationfollowed by cold stratification. Treatments with alternatingtemperatures of 25/15 or 23/11 °C (12 h light) for 6 monthsfollowed by 5 °C for 3 months were successful in overcomingseed dormancy. After 6 months of warm stratification, cytologicalchanges observed included: enlargement of the embryo; a decreasein the number of lipid bodies; appearance of ER; and increasesin mitochondria, plastids, dictyosomes, vacuoles and microbodiesin the shoot apical meristem. Cold stratification followingthe warm treatment induced cell division, and one or two distinctnucleoli in the shoot apical meristem cells were observed. Bothwarm and cold stratification reduced endogenous ABA concentrationsfrom the original 8888 pg per freshly harvested seed to 392and 536 pg, respectively. Treatment with exogenous gibberellinsafter seeds had been warm-stratified showed that GA₄and GA₇wereeffective at promoting seed germination, but GA₃was not. Theseresults suggest that the strong seed dormancy ofT. maireicouldbe caused by a high ABA content and underdevelopment of theembryos in freshly shed seeds. We conclude that warm stratificationwith alternating temperatures increases the growth of embryosby cell expansion and enlargement and decreases ABA content,but seeds still remain ungerminated. Cold stratification mayinduce the response to GAs and initiate cell division resultingin release from physiological dormancy and subsequent germinationofT. maireiseeds.Copyright 1998 Annals of Botany Company Taxus mairei; ultrastructure; abscisic acid; gibberellin; seed dormancy; stratification; germination.     

Breaking of Seed Dormancy by Nitrate as a Gap Detection Mechanism

Germination of Planlago lanceolata seeds buried in a chalk grasslandwas higher in bare soil than in vegetated soil, and measurementof soil nitrate concentrations showed that they were high enoughto account for this stimulation. When seeds of P. lanceolatawere sown in pots of soil with or without plants, and wateredwith nutrient solution containing either no nitrate, or 14 mMnitrate (sufficiently high that not all nitrate was absorbedby the plants), the presence of plants inhibited germinationonly when the nutrient solution contained no nitrate. It wasconcluded that breaking of seed dormancy by nitrate can functionas a gap detection mechanism if nitrate concentrations in baresoil are high enough to break seed dormancy, but are too lowto break dormancy when vegetation is present Plantago lanceolata, seed, dormancy, germination, nitrate, gap detection     

Seed Dormancy in Acer: The Role of Testa-imposed and Embryo Dormancy in Acer velutinum

The embryo dormancy shown in freshly harvested samples of Acervelutinum seeds is weakly established and very short-lived.Loss of this embryo dormancy occurred during post-harvest fruitstorage at either 5 or 17 C. In contrast, the dormancy of intactfruits and seeds was overcome only during storage at the lowertemperature. Removal of the cotyledons from embryos of freshlyharvested fruits allowed more rapid germination of the embryonicaxes, indicating that the cotyledons exert an inhibitory effect,although the axes still retained a measure of innate dormancy.The inhibitory effect of the cotyledons became less marked withincreasing duration of fruit storage, this loss of inhibitoryeffect occurring at both storage temperatures. Applied ABA stronglysuppressed germinative capacity in intact embryos and isolatedembryonic axes from freshly harvested fruits, but when ABA wasapplied to embryos of fruits that had been stored for variousperiods at 5 or 17 C, the inhibitory effect was first weakenedand then lost with increased storage. Although dormancy in the seeds of A. velutinum may be describedas intermediate between testa-imposed dormancy and true dormancy,it is perhaps more properly included in the former category. Acer velutinum Boiss. var. vanvolxemii, abscisic acid, embryo dormancy, germination, seed storage, testa-imposed dormancy, tissue sensitivity     

Rapid effects of red light on the isopentenyladenosine content in scots pine seeds

Red light (R) stimulates germination in Scots pine seed (Pinus sylvestris L.). The response is far red (FR) reversible. The dynamics of cytokinin changes following light treatment was investigated. Extracts were purified by immunoaffinity and high performance liquid chromatography. N⁶-(Δ²-Isopentenyl) adenosine (iPA) and trans-zeatin riboside (ZR) were quantified by both UV-absorbance of high performance liquid chromatography peaks and by enzyme-linked immunosorbent assay. Identification of iPA was accomplished by gas chromatography-mass spectrometry. Levels of cytokinins were low in seeds imbibed in the dark. Exposure of seeds imbibed in the dark for 5 hours to R for 15 minutes induced a strong, immediate but transitory increase in iPA content. This increase was not observed when the R treatment was followed by 10 minutes of FR or by storage in darkness before extraction. No ZR was detected during the first 8 hours of imbibition in any treatment. Addition of iPA via acetone enhanced seed germination in the dark. The results suggest that iPA may be involved in the R-mediated release of dormancy of Scots pine seed.     

Rate of Change in Dormancy Level and Light Requirement in Weed Seeds During Stratification

The rate of change in dormancy level and light requirement,induced during stratification at 3.2 °C, was investigatedin seeds ofStellaria media, Cerastium fontanum, Veronica agrestisandTaraxacumofficinale.Two stratification environments, soil and wet filterpaper in petri-dishes, were used. On nine occasions during a6 week stratification period, germination was tested under threelight conditions at 3.5/18.5 °C: (1) darkness; (2) light;and (3) short-term light exposure followed by darkness. Priorto stratification, germination in all species was 89% in lightand 53% in darkness. Within 2 weeks of stratification, germinabilityinV. agrestisandS. mediaseeds decreased in all treatments. InC.fontanumandT. officinaleseeds, germinability also decreasedafter 2 weeks but only in the dark treatment, indicating inductionof a light requirement. After two more weeks of stratification,the induced dormancy inS. mediaseeds stratified in soil becameweaker and the light requirement inT. officinaleseeds was lost.Differences between the two stratification environments and/orinteractions between light conditions, stratification environmentsand time were found for all species. These results suggest that:(1) the dormancy level and the light requirement of seeds maychange dramatically over relatively short-time periods duringstratification; and (2) germinability depends on the stratificationenvironment experienced by the seeds. Predicting the dormancylevel or light requirement in a seed batch is difficult andrequires a thorough knowledge of the effect of the stratificationconditions used.Copyright 1997 Annals of Botany Company Burial; Cerastium fontanumBaumg.; chickweed; cold stratification; common mouse-ear; dandelion; dormancy; light requirement; seed; Stellaria media(L.) Vill.; speedwell; Taraxacum officinalegroup; Veronica agrestisL.; weed