利用P5CS基因转化白三叶的研究

为了培育抗旱白三叶,构建了植物表达载体pBPC-P5CS,利用基因枪法转化白三叶的愈伤组织,PCR检测和Southern blot鉴定证实白三叶中已导入P5CS基因。对转P5CS基因白三叶植株的不同抗旱指标进行了分析。结果表明,与对照相比,转P5CS基因株系的抗旱能力得到了较大的提高。干旱胁迫下,与对照相比,转P5CS基因植株的脯氨酸含量和相对含水量分别比对照高20.0%-21.2%和5.6%-8.5%。     

Contribution from Stolons and Roots to Estimates of the Total Amount of N2Fixed by White Clover (Trifolium repensL.)

Growth and N-accumulation rates in leaves, stolons and rootsof individual white clover plants were studied in three experimentsusing two methods. In a growth chamber experiment, the relativedifferences between tissues were found to be almost constantfor a wide range of clover plant sizes. The stolon dry matter(DM) production was 56% and the root DM production 40% of theDM production in leaves. The N yield of stolons was 30% whileN yield in roots was 34% of N yield in leaves. The effect ofN application on these relations was investigated in a glasshouseexperiment. Application of N reduced the root:shoot N ratiofrom 0.50 to 0.28, whereas the stolon+root:leaf N ratio (i.e.for abovevs.below cutting-height tissues) was only reduced from0.97 to 0.80. In a field trial with two contrasting N regimes,growth and N accumulation were measured on individual cloverplants. Dinitrogen fixation was estimated by¹⁵N isotope dilutionbased on analysis of leaves-only or by including stolons. Usingleaves-only did not affect the calculation of percentage ofclover N derived from N₂fixation (% Ndfa) since the¹⁵N enrichmentwas found to be uniform in all parts of the clover. A correctionfactor of 1.7 to account for N in below cutting-height tissueis suggested when N₂fixation in white clover is estimated byharvesting the leaves only.Copyright 1997 Annals of Botany Company Leaves; N accumulation; N₂fixation; ¹⁵N isotope dilution; pastures; roots; root/shoot ratio; stolons; Trifolium repensL.; white clover     

Changes in Stolon Carbohydrates During the Winter in Four Varieties of White Clover (Trifolium repensL.) with Contrasting Hardiness

Miniswards of white clover (Trifolium repensL.) varieties Menna,Huia, AberCrest and AberHerald, with contrasting winter hardiness,were established from cloned material in flat boxes. The miniswardswere placed in an unheated glasshouse with natural lightingfor the winter of 1995/96. During this time there were severalperiods of cold weather. The plants experienced temperaturesat least as severe as those experienced by unprotected plantsduring preceding mild winters. Growth measurements carried outmonthly suggested the cloned genotypes were representative ofthe varieties from which they were derived. Growth continuedthroughout the experiment, particularly when expressed as appearanceof new internodes. During the coldest weather these internodeswere short, and the amount of tissue produced was small. Starchconcentration showed a complex pattern of changes during thewinter. Older internodes were the major site of starch storagein the autumn; starch was lost from these internodes duringthe winter and early spring. Starch synthesis also occurred,even during the coldest periods. Starch accumulated in all newstolon tissue. There were significant differences between varieties.AberCrest and AberHerald retained higher concentrations of starchin older stolon tissue, synthesized higher concentrations ofstarch in young tissue and retained these for longer. Therewere few differences in soluble sugar concentrations betweenvarieties, but there were significant changes during the winter.In mid-winter, sucrose and pinitol were at their highest concentrations,whereas reducing sugars were at their lowest concentrations.This study further supports the important role of starch reservesin supporting winter survival.Copyright 1998 Annals of BotanyCompany Trifolium repens; white clover; winter hardiness; starch; sugar; pinitol.     

Comparison of Horizontal Spread of White Clover (Trifolium repensL.) Grown Under Two Artificial Light Sources Differing in their Content of Blue Light

The effects of blue light on the growth and development of differentbranches were studied to determine if variations in the bluelight supply modified the horizontal spread of white clover.Cuttings of white clover (‘Huia’) were grown for56 d in two controlled chambers under conditions similar exceptfor level of blue light (BL). The horizontal spread of whiteclover was characterised by the phyllochron, internode lengthand branching or flowering of different axes. The responsesto a reduction in BL were different according to axis degree:(1) on the main axis, lowering BL slightly decreased the phyllochronand had no effect on internode length or stolon length; (2)on older primary branches, it decreased their phyllochron, butreduced stolon length due to a reduction in internode length;(3) on secondary branches, it increased the phyllochron anddecreased internode length, triggering a strong reduction instolon length (up to 54%). Moreover, an increase in BL reversedthese effects. The switch to lower BL or higher BL had no effecton internode length of primary branches, but triggered changesin internode length of the main axis. These differential effectsof variations in BL on the morphogenesis of different branchesare discussed in relation to the clonal integration of variationsin light composition. Changes in the horizontal spread of whiteclover induced by BL could play a role, combined with responsesto low red:far-red ratio and low photosynthetic photon flux(PPF) in the strategy of horizontal colonisation and shade-avoidanceof white clover.Copyright 1998 Annals of Botany Company Blue light, branching, horizontal spread, morphogenesis, phyllochron, plant development, plant morphology, stolon growth,Trifolium repensL., white clover.     

Evidence For Root Contraction In White Clover (Trifolium repens L.)

White clover (Trifolium repens L.) stolons become buried inthe field. It was observed that this also occurred in the greenhousewhere the accepted mechanisms of burial, treading by livestockand earthworm casting, did not occur. It was also observed thatthe crown of seedling T. repens plants become closely appressedto the soil. Experiments showed that, regardless of varietyof T. repens or depth of planting, all seedling hypocotyls firstlift the cotyledons clear of the soil, then ‘ contract’towards the soil until the cotyledons are in contact with orbelow the soil surface. Auxanometers were used to measure therate and extent of this contraction and were also attached tostolon nodes in experiments which showed that stolons move downwardsrelative to the soil surface and that the speed and extent ofthis duration varied with soil type. A further experiment showedthat only rooted nodes show this behaviour. The force exertedby the contraction of nodal roots was estimated experimentallyas 0.21 N g^-1fresh root. A mechanism for the root contraction,based on examination of root anatomy of seedling tap-roots andnodal roots, is suggested. These experiments provide evidencefor root contraction in T. repens which may lead to stolon burial.The importance of this to T. repens as a pasture species andas a means of further improving T. repens varieties is discussed.Copyright 1999 Annals of Botany Company White clover, Trifolium repens, L., stolon, seedling, burial, root, nodes, nodal, force, contractile, soil resistance, pasture, phloem, fibres.     

Phosphate Uptake and Transport by Roots and Stolons of Intact White Clover (Trifolium repens L.)

The extent to which phosphate can be absorbed directly fromthe outer medium by stolon internodes and contribute to thetotal accumulation of phosphate by intact plants of white clover(Trifolium repens L. cv. Blanca) was assessed in hydroponicexperiments in a controlled environment room. The uptake ofphosphate by intact roots or stolons was measured by sealinga segment (6-0 mm long) across a flow-cell in which ³²P-labellednutrient solution was circulated for 24 h, the rest of the rootsystem receiving unlabelled nutrient solution. The rate of uptakeof phosphate (µmol g^–1 d^–1 dry wt. basis)by roots was more than 300 times that by intact stolons. Pretreatmentof stolons by gentle abrasion to remove cuticle, so as to simulatethe condition of stolons in the field, increased the uptakeof phosphate 7-fold compared with that of intact stolons. However,the potential of stolons to contribute to the P status of whitedover in the field was calculated to be small (5%). When an incision was made through the hypodermal layer of stolons,the rate of phosphate uptake greatly increased, attaining 71%of that by root segments. This increase, which was greater athigher phosphate concentrations, indicates that the suberi.zedhypodermis constitutes a major barrier to the influx of phosphatein the stolon. After withholding phosphate for different time intervals, thesubsequent rate of phosphate uptake by roots was increased 2-3-foldafter 2 d phosphate deprivation and 3-4-fold after 6 d or 13d phosphate deprivation. A higher proportion of absorbed phosphatewas transported to shoots in phosphate-deprived plants. After1 d of uptake following restoration of the phosphate supply,the concentrations of labelled phosphate in shoots were greaterthan in control plants, although the concentrations of labelin roots was less. However, the rate of uptake of phosphateby stolons, following deprivation, was not significantly increased.These results suggest that the mechanism regulating the enhancedrate of phosphate loading into the xylem, initiated by a periodof phosphate deprivation, is specific to roots and is not inducedin stolons. The results are discussed in relation to the growth and acquisitionof phosphate by white clover in the field. Key words: Nutrient deficiency, phosphate, stolons, transport (ions), Trifolium repens     

The Growth and Carbon Allocation Patterns of White Clover (Trifolium repens L.) Plants of Contrasting Branching Structure

The growth, morphology and carbon allocation patterns of F1progeny white clover (Trifolium repens L.) plants selected foreither low (‘LBF’) or high (‘HBF’) frequencyof stolon branching were compared in two controlled-environmentexperiments. Selections from within both a small-leaved (‘GrasslandsTahora’) and a large-leaved (‘Grasslands Kopu’)clover cultivar were compared, and plants were grown under arelatively lenient defoliation treatment (expt 1) or under threelevels of defoliation seventy (expt 2). Carbon allocation patternswere measured by ¹⁴CO₂ pulse-chase labelling using fully unfoldedleaves on the main (parent) stolon. LBF and HBF displayed consistent differences in the selectedcharacter though, within cultivars, the difference between selectionswas most pronounced for Kopu. The selections developed fundamentallydifferent branching structures resulting from differences inbranching frequency, with total branch weight per plant averaging122 mg for LBF and 399 mg for HBF (mean of both experiments).More C moved from parent stolon leaves to branches in HBF thanin LBF (mean 22.6% vs. 15.1% respectively of the ¹⁴C exportedfrom source leaves). More C also moved to stolon tissue in HBF,but, counterbalancing this and the difference in allocationto branches, less moved to developing leaves and roots on theparent stolon itself compared to LBF. However, the total weightof developing leaves and roots per parent stolon was generallygreater in HBF than in LBF, probably reflecting greater C importby these sinks from the higher number of branches present perplant in the former selection. HBF plants were consistentlylarger at harvest than LBF plants. There were no defoliationtreatment x selection interactions in C allocation patternsin expt 2. The implications of the results for plant performancein grazed pastures are discussed. Branching, carbon translocation, defoliation, growth, morphology, Trifolium repens, white clover     

Photosynthesis of White Clover (Trifolium Repens L.) Germplasms with Contrasting Leaf Size

In a growth chamber experiment, we determined net photosynthetic rate (PN) and leaf developmental characteristics of cultivars of a relatively small-, intermediate-, and a large-leaf genotype grown under irradiance of 450-500 µmol(photon) m-2 s-1 (HI), shade [140-160 µmol(photon) m-2 s-1] (LI), and after a shade-to-irradiation (LI »HI) transfer. Differences in physiological responses of the genotypes were more pronounced in HI and LI»HI plants than in LI plants. The small- and intermediate-leaf sizes had greater PN in the first measured leaf than the large-leaf type by 70 and 63 % in HI plants, and by 23 and 18 % in LI»HI plants, respectively. Similar relationships were observed in the next developed leaf. The LI plants did not differ significantly in PN. Greater PN in the small- and intermediate-leaf size genotypes were not associated with greater total dry matter of the plant. Under irradiation, the large-leaf genotype accumulated more total nonstructural saccharides (TNS) and starch than the small- or intermediate-leaf size plants. TNS and starch concentrations in LI plants were about one-half those of HI and LI»HI plants. These results should help to develop management practices that capitalize upon the competitive features of white clover in mixed-species swards.     

Floral Development in Sonja White Clover (Trifolium repens) a Papilionoid Legume

Floral development in Sonja white clover was examined usingscanning electron microscopy. Florets and bracts were foundto arise from common primordia initiated as protuberances fromthe apical meristematic area of the inflorescence. The patternof floret initiation on the inflorescence was acropetal, theoldest florets resting basally. Floral organ initiation withineach floret was acropetal, petals being initiated before stamens.Floret development was zygomorphic, each whorl of floral organsdeveloping unidirectionally from the abaxial side. There wasfound to be overlapping in the timing of initiation and developmentof these organs. Antesepalous stamens were found initially tooutgrow their antepetalous counterparts. Early petal developmentwas synpetalous. Eglandular hairs were found basally on thecalyx cup and on the pedicel. Procumbent hairs were found tobe more numerous and randomly distributed on the abaxial surfacesof the mature calyx cup. Trifolium repens L., Sonja cultivar, white clover, scanning electron microscopy, floral development, inflorescence     

Floral Initiation of White Clover (Trifolium repens L.) in Response to Daylength and Temperature

The response to different combinations of daylength (14, 15and 16 h) and day/night temperature (20/10°C, 30/15°C)of four white clover (Trifolium repens) varieties in terms offloral initiation and development was studied. More plants flowered under long days (16 h) at 30°C thanin any other combination. Varietal differences in response todaylength were large but temperature was the factor controllinginitiation. Daylength was more important in regulating peduncle length,floret and ovule numbers than was temperature. The amount ofnectar produced was lowest at the higher temperature althoughnectar concentration was unaffected by temperature. Nectar concentrationwas lowest in the shortest daylength (14 h). White clover, floral initiation, temperature, daylength     

Influence of Shade on the Growth and Sink Activity of Young Flower Heads and Peduncles of White Clover (Trifolium repens L.)

The influence of shade on the growth and sink activity of youngflower heads and peduncles of white clover (Trifolium repensL.) plants was measured in controlled environments. Measurementof the peduncle elongation rate using a linear voltage displacementtransducer showed that when the inflorescence alone was shaded,peduncle elongation was higher than in the light. Translocationstudies using ¹¹C-labelling techniques showed that shading theinflorescence alone had little effect on translocation of assimilatesinto the inflorescence but induced a major change in partitioningof assimilates within it. Peduncle elongation induced by shadeaccompanied an increase in partitioning of photoassimilatesto the peduncle, at the expense of the flower head. Key words: White clover, partitioning, translocation     

The Extent and Pattern of Osmotic Adjustment in White Clover (Trifolium repens L.) During the Development of Water Stress

White clover plants were subjected to water stress followingthe cessation of watering. As a water deficit developed, waterand osmotic potentials were measured in stolon tips, leavesfrom the stolon tip and leaves from the plant crown. Pressurepotentials were calculated. Pressure potential was maintainedin stolon tips even when water potential fell to around –2·0MPa. In contrast, pressure potential in leaves fell rapidlyas water stress developed. Total amino acid and potassium levels were largely unaffectedin both stolon tips and leaves. Water-soluble carbohydratesand proline accumulated during water stress. The increase inproline level in leaves did not follow the same pattern as thatin stolon tips, although toward the end of the water stressperiod the level had increased by a similar extent in both partsof the plant. Additionally, pressure potential and osmotic potentialappeared to be significantly related to proline content in stolontips. No such relationship was found for leaves. The role ofproline in osmotic adjustment is discussed. Trifolium repens L. cv. Olwen, white clover, water stress, osmotic adjustment, proline     

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.)

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.     

3D Architectural Modelling of Aerial Photomorphogenesis in White Clover (Trifolium repens L.) using L-systems

The objective of this work was to construct a model of aerialdevelopment of clover that takes into account morphogeneticresponses to the light environment, and to use it to analyseand understand these processes in terms of signal perceptionand integration. The plant model was interfaced with a MonteCarlo model that determines photosynthetically active radiation(PAR) and red/far-red ratio (R/FR) throughout the canopy, takinginto account the absorption, reflection and transmission oflight by individual leaves. Light intensity and quality weresensed by the plant model at discrete time intervals and atdiscrete sites of perception: apices, emerging internodes andpetiole tips. This input regulated the final size of internodesand leaves, the vertical positioning of leaves, and the branchingdelay. The empirical relations (regression functions) quantifyingthis regulation were derived from data reported in the literatureand original measurements. Simulations produced realistic visualizationsand quantitative characterizations of the modelled plants fordifferent light treatments. These results were in general agreementwith observations of real plants growing under similar conditions,suggesting that the dependence of organ size and position onlight treatments can be regarded as an integration of the responsesof individual plant organs to their local light environment.The model is used to describe the regulation of branch appearanceand the impact of self-shading on plant morphogenesis as a functionof local light environment. Copyright 2000 Annals of BotanyCompany Clover, Trifolium repens L, photomorphogenesis, plant architecture, L-system, modelling, Monte-Carlo method, competition for light, red : far-red ratio, irradiance, light quality, leaf size, self-shading     

Calcium and Nodulation in Subterranean Clover (Trifolium subterraneum L.)

From a study of the effects of Ca ions on the nodulation of subterranean clover in flowing culture solutions it is concluded that root infection or nodule initiation has a higher Ca requirement than either nodule development or host plant growth in the presence of fixed nitrogen.     

The Influence of Irradiance Distribution on the Growth of White Clover (Trifolium repens L.) in Differently Managed Canopies of Permanent Grassland

Growth of white clover was investigated in permanent grasslandcut three or five times per year. The influence of cutting frequencyand nitrogen fertilization on dry-matter yield, leaf-area distributionand the distribution of photosynthetically active radiationwithin the canopy were examined. In the five cut treatments, total dry-matter yield was nearlyequal, with and without nitrogen. However, nitrogen practicallyeliminated white clover. Leaf-area distribution showed characteristicpatterns for the different treatments. The small proportionof white clover in the treatment with nitrogen fertilizationwas thought to be due to the large leaf area of the other speciesat heights which white clover could not attain. This conclusionwas supported additionally by the radiation measurements withinthe canopy. The sunlit fractional area within canopy layers was measuredwith ‘quantum sensors’ and calculated from canopytransmission measured with tube solarimeters. The leaf areaindex of white clover was highly correlated (r² = 0.68) withthe sunlit fractional area above the canopy layers where whiteclover was present. This response of white clover leaf growth to the light regimeis discussed in relation to the potential petiole growth. White clover, Trifolium repens L., permanent grassland, irradiance distribution, sunlit fractional area, petiole extension, leaf area, dry matter, stratified clipping     

Accumulation and Loss of Nitrogen in White Clover (Trifolium repens L.) Plant Organs as Affected by Defoliation Regime on Two Sites in Norway

In order to improve the basis for utilising nitrogen (N) fixed by white clover (Trifolium repens L.) in northern agriculture, we studied how defoliation stress affected the N contents of major plant organs in late autumn, N losses during the winter and N accumulation in the following spring. Plants were established from stolon cuttings and transplanted to pots that were dug into the field at Apelsvoll Research Centre (60°42′ N, 10°51′ E) and at Holt Research Centre (69°40′ N, 18°56′ E) in spring 2001 and 2002. During the first growing season, the plants were totally stripped of leaves down to the stolon basis, cut at 4 cm height or left undisturbed. The plants were sampled destructively in late autumn, early spring the second year and after 6 weeks of new spring growth. The plant material was sorted into leaves, stolons and roots. Defoliation regime did not influence the total amount of leaf N harvested during and at the end of the first growing season. However, for intensively defoliated plants, the repeated leaf removal and subsequent regrowth occurred at the expense of stolon and root development and resulted in a 61–85% reduction in the total plant N present in late autumn and a 21–59% reduction in total accumulation of plant N (plant N present in autumn + previously harvested leaf N). During the winter, the net N loss from leaf tissue (N not recovered in living nor dead leaves in the spring) ranged from 57% to 74% of the N present in living leaves in the autumn, while N stored in stolons and roots was much better conserved. However, the winter loss of stolon N from severely defoliated plants (19%) was significantly larger than from leniently defoliated (12%) and non-defoliated plants (6%). Moreover, the fraction of stolon N determined as dead in the spring was 63% for severely defoliated as compared to 14% for non-defoliated plants. Accumulation in absolute terms of new leaf N during the spring was highly correlated to total plant N in early spring (R² = 0.86), but the growth rates relative to plant N present in early spring were not and, consequently, were similar for all treatments. The amount of inorganic N in the soil after snowmelt and the N uptake in plant root simulator probes (PRS^TM) during the spring were small, suggesting that microbial immobilisation, leaching and gas emissions may have been important pathways for N lost from plant tissue.     

Assessing the Geometric Structure of a White Clover (Trifolium repens L.) Canopy using3-D Digitising

A method was developed for assessing the three dimensional (3-D)geometric structure of white clover canopies. 3-D co-ordinatesof pre-defined points on leaves, petioles and stolons were measuredusing a Polhemus Fastrak electromagnetic 3-D digitiser. Digitisingprogressed downwards from the top of the canopy and plant partswere removed after they have been digitised. Leaflets were treatedas four quarter-ellipses, and petiole and stolons were treatedas cylinders. Leaf dimensions and areas calculated from 3-Dco-ordinates were within about 5% and 20% of direct measurementsmade with a ruler and a planimeter, respectively. Special softwareand freeware POV-Ray were used to reconstruct a virtual canopyfrom digitiser records and to calculate canopy characteristicssuch as leaf area index (LAI), petiole intersection area, andprofiles of leaflet areas and inclinations with height. It tookbetween 3 and 7 h to digitise 10 x 10 cm stands of clover andthe resulting information was considerably more comprehensiveand accurate than could have been obtained by the alternative‘point quadrat’ or ‘stratified clipping’methods.Copyright 2000 Annals of Botany Company White clover, Trifolium repens, geometric structure, leaf area, leaf angle, 3-D digitising     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.