Changes in leaf organisation, photosynthetic performance and wood formation during ex vitro acclimatisation of black mulberry (Morus nigra L.)

Changes in anatomical organisation of the leaf, photosynthetic performance and wood formation were examined to evaluate the temporal and spatial patterns of acclimatisation of micropropagated slow-growing black mulberry ( Morus nigra L.) plantlets to the ex vitro environment. Leaf structure differentiation, the rates of net photosynthesis (P_n), transpiration (E) and stomatal conductance (g_s), and secondary xylem growth were determined in the course of a 56-day acclimatisation. Differentiation of palisade parenchyma was observed 7 days after transfer. At this stage, the rates of P_n, E and g_s reached maximum values, after which the rates of all three gas exchange parameters gradually decreased. The highest proportion of woody area occupied by vessels was also observed 7 days after transfer. An important feature of developing woody tissue is the difference in patterns of vessel distribution from the characteristic differentiation patterns of earlywood and latewood vessels in mature wood of ring-porous trees. Vessels with lumen areas over 3000 μm² were only differentiated in acclimatised plantlets, whereas vessels in stems sampled on days 0 and 7 had very small lumen areas of up to 560 μm². Full acclimatisation, observed 56 days after transfer to the ex vitro environment, was associated with the rapid growth of new in vivo formed leaves, very low rates of E and g_s, and much increased secondary xylem tissue within the stem area.     

Wood ontogeny during ex vitro acclimatization in micropropagated hybrid poplar clones

Wood ontogeny patterns were determined during the ex vitro acclimatization period in micropropagated plantlets of hybrid poplar clones T-14 [Populus tremula × (Populus × canescens)] and T-50 [(Populus × canescens) × Populus tremula]. The temporal course of developmental changes in the woody tissue was characterized on a weekly basis starting from the day of transfer to the ex vitro environment until full acclimatization was achieved on day 28. In vitro rooted plantlets had already initiated lignification of secondary xylem cells. The greatest increase in the amount of woody tissue was observed on days 21 and 28. At the end of the acclimatization period, T-14 plantlets contained on average 41.4 % of secondary xylem tissue compared to 30.3 % found in T-50 plantlets. During the course of acclimatization, both clones displayed identical patterns of vessel lumen size distribution from small vessel lumen area to large vessel lumen area. This pattern differs from the characteristic diffuse-porous pattern of approximately evensized vessel lumen area distribution typical of mature wood. At the end of acclimatization, the differences in vessel lumen area and relative conductivity between the clones were negligible. Development of secondary xylem tissue during ex vitro acclimatization promotes the establishment of vigorous regenerants with stems that show increased bending strength and stiffness.     

Arbuscular mycorrhizal fungi influence water relations, gas exchange, abscisic acid and growth of micropropagated chile ancho pepper (Capsicum annuum) plantlets during acclimatization and post-acclimatization

Little is known about the role of arbuscular mycorrhiza fungi (AMF) on physiological changes of micropropagated plantlets during acclimatization and post-acclimatization. Using chile ancho pepper (Capsicum annuum L. cv. San Luis), measurements were made of water relations, gas exchange, abscisic acid (ABA), plantlet growth and AMF development. Plantlets had low photosynthetic rates (A) and poor initial growth during acclimatization. Relative water content (RWC) decreased during the first days after transfer from tissue culture containers to ex vitro conditions. Consequently, transpiration rates (E) and stomatal conductance (gs) declined, confirming that in vitro formed stomata were functional and able to respond ex vitro to partial desiccation--thus avoiding excessive leaf dehydration and plant death. Colonization by AMF occurred within 3 days after inoculation. Colonized plantlets had lower leaf ABA and higher RWC than noncolonized (NonAMF) plantlets during peak plant dehydration (6 days after plant transfer)--and a higher A and gs as early as days 5 and 7. During post-acclimatization [after day 8, when RWC increased and stabilized], A increased in all plantlets; however, more dramatic changes occurred with AMF plantlets. Within 48 days, 45% of the roots sampled of inoculated plantlets were colonized and had extensive arbuscule development. At this time, AMF plantlets also had greater E, A, leaf chlorophyll, leaf elemental N, P and K, leaf dry biomass and leaf area, fruit production and differences in carbon partitioning [lower root/shoot ratio and higher leaf area ratio] compared with NonAMF plantlets. Rapid AMF colonization enhanced physiological adjustments, which helped plantlets recover rapidly during acclimatization and obtain greater growth during post-acclimatization.     

Polar auxin transport is implicated in vessel differentiation and spatial patterning during secondary growth in Populus

Premise of the Study

Dimensions and spatial distribution of vessels are critically important features of woody stems, allowing for adaptation to different environments through their effects on hydraulic efficiency and vulnerability to embolism. Although our understanding of vessel development is poor, basipetal transport of auxin through the cambial zone may play an important role.

Methods

Stems of Populus tremula ×alba were treated with the auxin transport inhibitor N‐1‐naphthylphthalamic acid (NPA) in a longitudinal strip along the length of the lower stem. Vessel lumen diameter, circularity, and length; xylem growth; tension wood area; and hydraulic conductivity before and after a high pressure flush were determined on both NPA‐treated and control plants.

Key Results

NPA‐treated stems formed aberrant vessels that were short, small in diameter, highly clustered, and angular in cross section, whereas xylem formed on the untreated side of the stem contained typical vessels that were similar to those of controls. NPA‐treated stems had reduced specific conductivity relative to controls, but this difference was eliminated by the high‐pressure flush. The control treatment (lanolin + dimethyl sulfoxide) reduced xylem growth and increased tension wood formation, but never produced the aberrant vessel patterning seen in NPA‐treated stems.

Conclusions

These results are consistent with a model of vessel development in which basipetal polar auxin transport through the xylem‐side cambial derivatives is required for proper expansion and patterning of vessels and demonstrate that reduced auxin transport can produce stems with altered stem hydraulic properties.     

Sap flow rates and sapwood density are critical factors in within- and between-tree variation in CO2 efflux from stems of mature Dacrydium cupressinum trees

Measurements of CO2 efflux from stems and branches, sap velocity, and respiratory activity of excised wood cores were conducted in Dacrydium cupressinum trees that differed in diameter, age, and canopy emergence. The objective of this study was to determine if consistent linkages exist among respiratory production of CO2 within stems, xylem transport of CO2, and the rate of CO2 diffusing from stem surfaces. Stem CO2 efflux was depressed during periods of sap flow compared with the efflux rate expected for a given stem temperature and was positively correlated with sapwood density. By contrast, no significant relationships were observed between CO2 efflux and the respiratory activity of wood tissues. Between 86 and 91% of woody tissue respiration diffused to the atmosphere over a 24-h period. However, at certain times of the day, xylem transport and internal storage of CO2 may account for up to 13-38% and 12-18%, respectively, of woody tissue respiration. These results demonstrate that differences in sap flow rates and xylem anatomy are critically important for explaining within- and between-tree variation in CO2 efflux from stems.     

SOME OBSERVATIONS ON THE MECHANISM OF ORIENTATION MOVEMENT OF WOODY STEMS

The force that induces orientation movement of inclined or bent woody stems is generated in reaction wood even in young terminal stems with a large proportion of soft tissues to secondary xylem. Compression wood formed in pine as a response to inclination expands longitudinally after the stresses are released by sawing it from the stem. The increment of length of compression wood when sawed is equal to the decrement of its length which occurs during drying. This suggests that stresses developed by compression wood in the stem are related to imbibition of water by its cell walls. Not all compression wood develops tensile forces in the stem. Neutral compression wood was observed in the lower portion of inclined stems of pine. Tension wood in poplar develops contractile forces in the stem during its aestival maturation. However, when harvested before developing contractile forces in situ, it develops such forces during drying. This suggests that in poplar the mechanism which produces forces responsible for orientation bending also involves changes in cell wall hydration.     

Regenerative Xylem in Inflorescence Stems of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

By inserting entomological needles into the lower parts of young inflorescence stems of three-month-old Arabidopsis thaliana (L.) Heynh var. Colombia plants, we studied the process of regenerative xylem production. Regenerative xylem was formed only in one- to two-day-old inflorescence stems but not in older ones. The regenerative vessels originated from re-differentiation of cortical parenchyma. To characterize the process of regenerative xylem formation, we conducted a histological study from the time of wounding to day 30 after wounding. In the first day after wounding the tissues showed no structural responses except for the wounding itself. After six days, regenerative vessel members were already differentiating in a basipetal pattern, forming a vascular bypass around the wound. Regenerative vessel member formation reached a maximal level on the twelfth day after wounding. Sixteen days after wounding the pith parenchyma started to become loose as if indicating tissue senescence. Altogether, vascular regeneration following wounding in inflorescence stems of Arabidopsis thaliana is similar to that in other dicotyledon plants. These findings provide the basis for the use of Arabidopsis thaliana as a model system to study the genetics, physiology and cell biology of wound healing and regenerative vascular tissue formation.     

Effects of in vitro treatments on leaf area growth of potato transplants during acclimatisation

The importance of leaf area of in vitro propagated potato (Solanum tuberosum L.) plantlets for further growth during acclimatisation and the after-effects of in vitro treatments on growth were examined. The in vitro treatments included different levels of alar, nitrogen or mannitol or different temperatures during the last in vitro phase, the rooting phase. Leaf area or ground cover was recorded one day after planting to soil and at the end of the first phase of ex vitro growth, the acclimatisation phase. Regression analysis showed that leaf area of a transplant at the end of acclimatisation phase was positively influenced by leaf area of the same plantlet at the beginning of the phase. The relative increase in leaf area during acclimatisation (increase/early leaf area) was linearly related to the inverse of the early leaf area, indicating almost comparable relative increases for plantlets having larger early leaf areas, but more variable responses for plantlets having smaller early leaf areas. In vitro treatments mainly affected leaf area of transplants through their effects on early leaf area. Adding alar, reducing nitrogen and reducing temperature increased leaf area. Reducing mannitol increased ground cover. A lower nitrogen concentration and higher temperature in some cultivars had slight negative effects on the relative increase in leaf area after acclimatisation. For nitrogen these negative effects were less significant than the positive effects through early leaf area. Results stress the importance of manipulation of leaf area in vitro to enhance plant performance in later stages of growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of<Emphasis Type="Italic"> Acer caudatifolium</Emphasis> Hayata plantlets from juvenile explants

Juvenile and fully mature Acer caudatifolium Hayata explants were assayed for their organogenic capacity. A protocol for multiple shoot culture formation and in vitro plant regeneration was developed for juvenile axillary bud cultures. Mature explants failed in shoot regeneration. Shoot multiplication was achieved by releasing apical dominance of the single elongated shoot on woody plant medium (WPM) supplemented with 0.7 mg l(-1) 6-benzylaminopurine and 0.05 mg l(-1) alpha-naphthaleneacetic acid. The highest rooting percentage was recorded on half-strength WPM containing 1.0 mg l(-1) indole-3-butyric acid. Regenerated plantlets were successfully hardened to ex vitro conditions and continued to grow after transfer to soil. No morphological aberrations were observed in the regenerates.     

A comparative study on growth and morphology of wasabi plantlets under the influence of the micro-environment in shoot and root zones during photoautotrophic and photomixotrophic micropropagation

The growth of wasabi (Wasabia japonica Matsumura) plantlets under different micro-environments inside culture vessels in photoautotrophic micropropagation (PA) and photomixotrophic micropropagation (PM) conditions were compared. After 28 days of culture, dry weight, relative growth rate, leaf area, and leaf chlorophyll contents of plantlets in PA were greater than those in PM. The number of leaves did not differ significantly between PA and PM conditions. PA promoted root growth and development with a greater number of roots, root length, root diameter, root fresh weight, root dry weight, and root xylem vessel system. Dissolved oxygen concentration in PA culture medium sharply decreased after 7 days of culture and then recovered. In PM culture medium, no significant fluctuation of dissolved oxygen concentration was apparent. The net photosynthetic rates of plantlets in PA were much higher than those in PM and increased with culture time. In contrast, the net photosynthetic rates of wasabi plantlets in PM kept a low and constant value during the culture period. With the presence of gas exchange membranes attached to the vessel lids, the detected vapor pressure deficit was higher in PA than in PM conditions. Higher stomatal density and larger stomatal aperture on the abaxial and adaxial surfaces of the leaves in PM medium promoted leaf water loss following ex vitro conditions. Thus, PA is applicable for producing healthy wasabi transplants.     

Enhancement of phenylpropanoid enzymes and lignin in Phalaenopsis orchid and their influence on plant acclimatisation at different levels of photosynthetic photon flux

Effects of three levels of photosynthetic photon flux (PPF: 60, 160 and 300 μmol m⁻²s⁻¹) were investigated in one-month-old Phalaenopsis plantlets acclimatised ex vitro. Optimal growth, chlorophyll and carotenoid concentations, and a high carotenoid:chlorophyll a ratio were obtained at 160 μmol m⁻²s⁻¹, while net CO₂ assimilation (A), stomatal conductance (g), transpiration rate (E) and leaf temperature peaked at 300 μmol m⁻²s⁻¹, indicating the ability of the plants to grow ex vitro. Adverse effects of the highest PPF were reflected in loss of chlorophyll, biomass, non-protein thiol and cysteine, but increased proline. After acclimatisation, glucose-6-phosphate dehydrogenase, shikimate dehydrogenase, phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) increased, as did lignin. Peroxidases (POD), which play an important role in lignin synthesis, were induced in acclimatised plants. Polyphenol oxidase (PPO) and β-glucosidase (β-GS) activities increased to a maximum in acclimatised plants at 300 μmol m⁻²s⁻¹. A positive correlation between PAL, CAD activity and lignin concentration was observed, especially at 160 and 300 μmol m⁻²s⁻¹. The study concludes that enhancement of lignin biosynthesis probably not only adds rigidity to plant cell walls but also induces defence against radiation stress. A PPF of 160 μmol m⁻²s⁻¹was suitable for acclimatisation when plants were transferred from in vitro conditions.     

WOOD FORMATION IN PROSOPIS: EFFECT OF 2,4-D, 2,4,5-T,AND TIBA

Treatment of erect stems of Prosopis with near phytotoxic levels of 2,4-D or 2,4,5-T causes the formation of an unusual wood with narrow, thick-walled vessels and axial parenchyma in which cell wall thickening is inhibited. Although reduced in diameter, the vessels formed during 2,4-D and 2,4,5-T treatment are so numerous that there is no significant difference between phenoxyacetic acid and control seedling groups with regard to total area of xylem occupied by vessels. The preferential maturation of xylem vessels over parenchyma and the transformation of fusiform initials into septate parenchyma strands in phenoxyacetic acid-treated Prosopis resemble the structural changes reported to occur after girdling in the cambial tissue of other arborescent angiosperms. Bending experiments indicate that tension-wood fibers of Prosopis differentiate in response to an auxin deficiency. However, xylogenesis in erect stems treated with TIBA is affected such that a significantly higher proportion of the cambial cell population becomes axial xylem parenchyma.     

Allometric neoteny and the evolution of succulence in cacti

With the objective of analysing the role of heterochrony in the evolution of succulence in the cactus family, a comparative study of xylem development in six species with contrasting morphologies was carried out. Two woody leaf-bearing cacti and four succulent cactus species belong to different subdivisions within the family were analysed. In each species and for different ages, vessel-element length was measured, vessel-element lateral wall-pitting described and the percentage of xylem and parenchyma in the stem quantified. In the succulent species it was found that vessel element length did not change between juvenile and adult wood, that wall-pitting in adult plants was similar to that of seedlings, and that the woody tissue in adult plants was organized in vascular bundles as in the primary tissue of seedlingS. Leaf-bearing cacti, in contrast, changed in both vessel element length and wall-pitting when secondary wood was produced, and the secondary woody tissue of adult plants was organized in a continuous cambial cylinder as in most dicotyledonS. An allometric analysis suggests that a retardation in the developmental rate of woody tissues (allometric neoteny) is the main mechanism in the development of succulence in cacti.     

Wood ontogeny during the first year of hybrid poplar development

During the first year of hybrid poplar development, we assessed radial growth dynamics quantified by the proportion of secondary xylem tissue within the stem area, the vessel area percentage, the content of both lignin and cellulose, the lignin monomeric composition, and the macromolecular properties of cellulose. The intraannual radial growth dynamics in the proportion of secondary xylem tissue was fitted by the Gompertz regression line whereas changes in the vessel area percentage were fitted maximally by a cubic regression line. Under constant temperature and photoperiod, this study revealed that nonlinear patterns of radial growth dynamics are the result of a developmental programme which drives cambial activity and ageing. The increased proportion of guaiacyl units found may be important for the greater stability of the lignin structure in the first year of hybrid poplar development. The tensile strength of juvenile wood was ensured by the trade-off between a slight increase in the degree of polymerization of cellulose and a slight decrease in the content of cellulose during ageing.     

How to become a tree without wood – biomechanical analysis of the stem of Carica papaya L

Carica papaya L. does not contain wood, according to the botanical definition of wood as lignified secondary xylem. Despite its parenchymatous secondary xylem, these plants are able to grow up to 10‐m high. This is surprising, as wooden structural elements are the ubiquitous strategy for supporting height growth in plants. Proposed possible alternative principles to explain the compensation for lack of wood in C. papaya are turgor pressure of the parenchyma, lignified phloem fibres in the bark, or a combination of the two. Interestingly, lignified tissue comprises only 5–8% of the entire stem mass. Furthermore, the phloem fibres do not form a compact tube enclosing the xylem, but instead form a mesh tubular structure. To investigate the mechanism of papaya's unusually high mechanical strength, a set of mechanical measurements were undertaken on whole stems and tissue sections of secondary phloem and xylem. The structural Young's modulus of mature stems reached 2.5 GPa. Since this is low compared to woody plants, the flexural rigidity of papaya stem construction may mainly be based on a higher second moment of inertia. Additionally, stem turgor pressure was determined indirectly by immersing specimens in sucrose solutions of different osmolalities, followed by mechanical tests; turgor pressure was between 0.82 and 1.25 MPa, indicating that turgor is essential for flexural rigidity of the entire stem.     

Anatomical Characteristics of Cherry Rootstocks as Possible Preselecting Tools for Prediction of Tree Vigor

An anatomical study of roots and stems of five self-rooted cherry rootstocks with different growth control potentials was performed to compare their structure and xylem anatomy. The aim was to correlate anatomical parameters with rootstock dwarfing potential and theoretical hydraulic conductance (k _h), and to evaluate the potential application of anatomical characteristics in the preselection process for prediction of ultimate tree vigor. One of the mechanisms of water transport efficiency reduction in dwarfing rootstock stems is from the rootstock xylem anatomy. Anatomical parameters of ??Gisela 5?? and ??Mazzard?? were typical for dwarfing and vigorous rootstocks, respectively, and were thus suggested as reference rootstocks. Significantly greater vessel diameter and frequency were found in invigorating and dwarfing rootstocks, respectively. Higher k _h was obtained in roots, compared to stems, due to significantly larger vascular elements. Dwarfing rootstocks had lower k _h due to small vessel lumens and percentage and, to a lesser extent, because of low wood/cortex ratios or percentage of wood. A higher percentage of wood or xylem in cherry roots and stems was not always positively correlated with their conductivity and vigor. Thus, these parameters cannot be reliably used in prediction of the ultimate vigor, although this method was previously suggested for some other fruit tree species. The most reliable anatomical parameters for that purpose proved to be vessel frequency, vessel lumen area, and percentage of vessels on wood cross section. These characteristics could thus be an effective way to estimate dwarfing capacity and could be applied in rootstock selection and breeding programs.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

Wood anatomy and wood density in shrubs: Responses to varying aridity along transcontinental transects

Wood density plays a key role in ecological strategies and life history variation in woody plants, but little is known about its anatomical basis in shrubs. We quantified the relationships between wood density, anatomy, and climate in 61 shrub species from eight field sites along latitudinal belts between 31° and 35° in North and South America. Measurements included cell dimensions, transverse areas of each xylem cell type and percentage contact between different cell types and vessels. Wood density was more significantly correlated with precipitation and aridity than with temperature. High wood density was achieved through reductions in cell size and increases in the proportion of wall relative to lumen. Wood density was independent of vessel traits, suggesting that this trait does not impose conduction limitations in shrubs. The proportion of fibers in direct contact with vessels decreased with and was independent of wood density, indicating that the number of fiber-vessel contacts does not explain the previously observed correlation between wood density and implosion resistance. Axial and radial parenchyma each had a significant but opposite association with wood density. Fiber size and wall thickness link wood density, life history, and ecological strategies by controlling the proportion of carbon invested per unit stem volume.     

Variation in wood density and anatomy in a widespread mangrove species

Wood density is an important plant trait that influences a range of ecological processes, including resistance to damage and growth rates. Wood density is highly dependent on anatomical characteristics associated with the conductive tissue of trees (xylem and phloem) and the fibre matrix in which they occur. Here, we investigated variation in the wood density of the widespread mangrove species Avicennia marina in the Exmouth Gulf in Western Australia and in the Firth of Thames in New Zealand. We assessed how variation in xylem vessel size, fibre wall thickness and proportion of phloem within the wood contributed to variation in wood density and how these characteristics were linked to growth rates. We found the wood density of A. marina to be higher in Western Australia than in New Zealand and to be higher in taller seaward fringing trees than in scrub trees growing high in the intertidal. At the cellular level, high wood density was associated with large xylem vessels and thick fibre walls. Additionally, wood density increased with decreasing proportions of phloem per growth layer of wood. Tree growth rates were positively correlated with xylem vessel size and wood density. We conclude that A. marina can have large xylem vessel sizes and high growth rates while still maintaining high wood density because of the abundance and thickness of fibres in which vessels are found.     

Stomatal Morphology during Acclimatization of Tobacco Plantlets to ex vitro Conditions

Image analysis was used in studying stomatal morphology during acclimatization of tobacco plantlets to ex vitro conditions, 45 d after transfer leaf area was 15 times, and total number of stomata per leaf four times increased. During acclimatization stomatal density was decreased considerably on both leaf sides, and was compensated by an increase in stomatal sizes, e.g., in stomatal length and in stomatal area (both guard cells and pore). Elongation of stomata was increased indicating that the originally circular stomata of in vitro plantlets were changed into elliptical ones in ex vitro acclimatized plants. This revised version was published online in June 2006 with corrections to the Cover Date.