Proposed dates for lifting Sitka spruce planting stock for fresh planting or cold storage,based on physiological indicators

The relationship between physiological status and theability to withstand the stresses of lifting, coldstoring and planting was evaluated from 1992–1995 inSitka spruce (Picea sitchensis (Bong.) Carr.)2 + 1 transplants of Washington/northern Oregon originsgrowing in Ireland. Needle primordium initiationcontinued until later and cold hardiness developedmore slowly in the mild lifting season of 1994/95. The seasonal decline in shoot mitotic activity wasgenerally similar each year, with apices becominginactive in December. Cell divisions in roots ceasedin November or December. Shoots began to deharden inearly February each year, but the rates of dehardeningwere slow until March. Cell divisions in root apicesresumed in February each year, compared with March forshoot apices. The vitality of the seedlings followingcold storage was high for most lift dates except theOctober and April dates. The period of highest stressresistance (as indicated by cold hardiness levels) forlifting for immediate planting was from about lateNovember/early December to early March; shoots werecold hardy to about –20 °C (LT₅₀) at thistime. Seedlings could be lifted and cold stored (1–2 °C)until May/June when shoots were cold hardyto about –30 °C or lower, which coincided withthe period from mid- to late December until March.     

The effect of short day treatments on containerized Douglas-fir morphology,physiology and phenology

The effect of short day treatments (blackout) on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) container seedlings at the time of lift and following cold storage was investigated. Variables measured included height, root collar diameter (RCD), root growth capacity (RGC), photosynthetic efficiency after –18 °C freezing (PEF), and days to terminal bud break (DBB). From one to four blackout dormancy induction treatments were started on three dates (July 12, July 26, and August 10) with 10 or 20 d between multiple blackouts. Increasing the number of blackout treatments resulted in lower RCD, lower DBB in the late winter/early spring, and higher PEF in the early fall. Later blackout start dates decreased PEF in the early fall, and increased overall height and late fall RGC as compared to earlier blackout start dates. Nurseries growing Douglas-fir seedlings from coastal Pacific Northwest provenances should be aware that blackout regimes can decrease RGC in the late fall, and cause quicker dormancy release in the early spring. Coastal Douglas-fir can be lifted and planted in the early fall, when RGC and DBB are relatively high. If planting between February and April is necessary, seedlings given blackout should be cold stored in January to maintain an adequate level of dormancy, RGC and PEF.     

Effects of storage conditions on dormancy and vigor of Picea abies seeds

The dormancy and vigor of Picea abies seeds were studied after five months of storage at 25 and 75% relative humidity (RH) and 5 and 12 °C. Dormancy was evaluated by studying germination response to light and moist chilling at 12 and 21 °C. Dormancy causing germination reduction in the dark was induced in seeds during storage at 25% RH (5–6% moisture content, MC) in darkness. The dormancy was greater when seeds were germinated at 12 than at 21 °C and after storage at 12 than at 5 °C. The effective dormancy relief by light indicates that germination was under phytochrome control. Moist chilling could partly replace light. According to accelerated aging and leachate conductivity tests, dry seeds could be stored at 12 °C for five months without affecting their vigor. After storage at 75% RH (11% MC) and 5 °C, the seeds germinated slowly and incompletely. The decreased germination response to light indicates that other processes than those mediated directly by phytochrome restricted germination of these seeds. The positive germination response to moist chilling suggested that secondary dormancy was induced in the seeds. However, vigor tests gave some evidence of simultaneous decrease of vigor. Storage at 75% RH and 12 °C decreased germination nearly to 0%. Germination of seeds stored at 75% RH could be stimulated by a short accelerated aging period.     

Influence of nutrient supply on spring frost hardiness and time of bud break in Norway spruce (Picea abies (L.) Karst.) seedlings

When spring frosts occur on recently planted forest sites, severe damage may occur to the seedlings. The aim of the present study was to test how different low levels of nutrient concentrations in Norway spruce (Picea abies (L.) Karst.) seedlings affected spring frost hardiness and time of bud break. Seedlings were grown in a greenhouse for one season and supplied with fertiliser containing 22, 43 and 72 mg N l^–1, respectively. The treatments resulted in needle nitrogen concentrations ranging from 0.9 to 1.8% in autumn. After winter storage at 0 °C, bud break was recorded on seedlings growing in the greenhouse, outdoors and in growth chambers at 12 °C and at 17 °C. Freezing tests were performed on seedlings directly removed from winter storage and following one week growth in the greenhouse. Seedlings receiving fertiliser with 43 mg N l^–1 had less freezing injury than the two other fertilisation treatments in the present study. The earliest bud break occurred in seedlings receiving 72 mg N l^–1.     

Chlorophyll fluorescence,root growth potential,and stomatal conductance as estimates of field performance potential in conifer seedlings

After cold storage, conifer seedlings in British Columbia are tested for field growth potential before planting. We compared three tests of performance potential using container-grown seedlings of Douglas-fir, interior spruce, lodgepole pine, and western larch (14 seedlots total). On several autumn dates, seedlings were lifted and stored at −2°C. The following spring we tested stored seedlings for root growth potential (RGP), chlorophyll fluorescence (CF), and stomatal conductance (Gs), and then planted seedlings in nursery beds. We assessed survival and shoot dry weight (SDW) after one growing season. Performance test results were significantly correlated with each other (r ≥ 0.47) but showed different relationships with field performance, which varied with lift date. The best performance predictor was the sum of CF and RGP (R ² = 0.79 for 78 seedlot by lift-date combinations), which minimized the risk of planting poor seedlings and not planting good seedlings. A sum of 83 for CF (Fv/Fm %) and RGP (new roots >1 cm) provided a threshold above which survival and growth were good. For evergreen conifers, Gs was a good performance predictor, but required extra time to measure leaf area. We recommend a combination of CF and RGP to assess vigor of shoot and root systems before planting. Wolfgang D. Binder––Scientist Emeritus.     

Overwinter Storability of Conifer Planting Stock: Operational Testing of Fall Frost Hardiness

Operational stock-testing facilities that estimate overwinter storability of seedlings (ability to survive and grow after storage) need a reliable method that provides fast results to forest nurseries. We compared three methods using container-grown seedlings of Douglas-fir, interior spruce, lodgepole pine, and western larch from forest nurseries in British Columbia. On three to nine dates in autumn, frost hardiness at −18°C was estimated using visible injury of foliage or stems (VI), electrolyte leakage from needles or stems (EL), and chlorophyll fluorescence of shoots (CF). Seedlings were placed into overwinter cold storage (−2°C). In the spring, stored seedlings were planted in nursery beds; survival and growth were assessed after one growing season. There were close correlations (r ≥ 0.93) between the assessment methods. Seedlings lifted after they reached thresholds of 69% or higher for CF and 25% or lower for EL and VI had over 90% survival at harvest and doubled shoot dry weight compared with seedlings lifted earlier. Measuring CF was the fastest and most easily replicated method to estimate successful storability, and reduced testing time by 6 days relative to VI tests.     

Slash pine bud dormancy as affected by lifting date and root wrenching in the nursery

Bud dormancy of root wrenched and unwrenched slash pine (Pinus elliottii Engelm.) seedlings growing in a forest nursery was measured on five lifting dates. Determination of bud dormancy was based on days to budbreak (DBB) under optimal growing conditions, mitotic activity in the apical meristem, chilling hours accumulated, and bud morphology. Based on DBB, seedlings were most dormant at Lift 2 on November 24 after exposure to 189 hours below 10 degrees C and 93 hours below 6.7 degrees C. Mitotic activity in the apical meristem was at its lowest 23 days later at Lift 3, possibly indicating the period when seedlings are most resistant to transplanting stresses. Multiple wrenching resulted in a slight shift in the dormancy cycle as wrenched seedlings set bud sooner in the nursery and broke bud sooner at the planting site in the spring than control seedlings. This implies that wrenched seedlings can be successfully lifted from the nursery earlier and will initiate spring shoot growth earlier than control seedlings.     

Sitka Spruce and Douglas fir Seedlings in the Nursery and in Cold Storage: Root Growth Potential, Carbohydrate Content, Dormancy, Frost Hardiness and Mitotic Index

Seedlings (transplants) of 2+1 Sitka spruce (Picea sitchensis(Bong.) Carr.) and 1 + 1 Douglas fir (Pseudotsuga menziesii(Mirb.) Franco) were grown in a nursery at the Bush Estate,Scotland. Batches were lifted and cold stored at 0.5°C inNovember, December and January. Changes in growth, shoot apicalmitotic index, root growth potential (RGP), carbohydrate content,bud dormancy and shoot frost hardiness were monitored throughoutthe winter by taking samples at intervals from the nursery andfrom cold storage. Frost hardening occurred during the later stages of bud development(as mitotic indices decreased); autumn hardening was arrestedwhen seedlings were put in cold store, and some dehardeningoccurred in cold storage, especially in spring. Bud dormancystarted, and was greatest, just after bud growth (mitotic activity)virtually ceased; chilling in cold store was almost as effectivein releasing dormancy as natural chilling. The concentrationof total nonstructural carbohydrates stayed more or less constantat 100–150mg g^–1 from September to April in thenursery; in cold storage carbohydrates were depleted at 0.4–0.6mgg^–1 d^–1 (corresponding to respiration at 0.03–0.05mgCO₂ g^–1 h^–1) until there was only 40–50mgg^–1. Root growth potentials in the nursery increased in December,once the buds ceased growth, became dormant and had receivedsome chilling. Sitka spruce was ‘storable’ in November,before RGPs increased, but they then failed to achieve maximalfrost hardiness or ROP. Winter RGPs were high in Sitka spruceand were increased or maintained in cold storage, whereas RGPswere low in Douglas fir and decreased immediately after storage(except when stored in January). By the end of April, the RGPof cold stored Sitka spruce was much higher than that of directlifted plants. ROP changes in the nursery and in cold storagewere not consistently related to changes in seedling carbohydratecontents, shoot frost hardiness or bud dormancy. In practical terms, it was concluded that (1) the optimum dateto start lifting bare- rooted conifer transplants in the autumnis when their shoot apical mitotic indices have decreased tonear zero, and their RGPs have risen sharply; (2) high RGPsmay depend as much on the morphology of the roots (e.g. numberof undamaged root apices) as on the physiology of the shoots(e.g. carbohydrate status, dormancy and frost hardiness); and(3) in spring, transplants kept in cold storage since November,December or January are more frost hardy, slightly more dormant,and (in May) have higher RGPs than transplants lifted from thenursery.     

The Influence of Desiccation, Rough Handling and Cold Storage on the Quality and Establishment of Sitka Spruce Planting Stock

In January 3-year-old Sitka spruce were lifted and treated eithercarefully or roughly. Plants in each treatment were either transferredto the laboratory for testing, or transferred to cold storageat –1°C. In the laboratory, half of the plants ineach treatment were desiccated to shoot water potentials ofless than –2.0 MPa. Plants were then tested for root growthpotential (RGP), bud dormancy, frost hardiness and carbohydratecontent before transplanting at a field site, Additional plantswere removed from the nursery and cold store at approximatelymonthly intervals until late April, and then treated as above.In April, plants in each treatment were transferred to on-sitefield storage (sheughs), for 2 or 4 weeks prior to testing asabove. Carefully handled plants maintained large RGP which was positivelycorrelated with plant water potential, root water content, shootrelative growth rate and field survival. Overall RGP was reduced: 59 per cent by desiccation; 85 percent by rough handling; and 98 per cent by desiccation and roughhandling in combination. On site storage for 4 weeks in April/May reduced RGP. Whereplants produced more than 30 new roots > 1 cm long in RGPtests, field survival exceeded 90 per cent. Cold storage at–1°C maintained RGP, (of carefully handled plants),bud dormancy and shoot frost hardiness at approximately pre-storagevalues. In contrast, total nonstructural carbohydrate contentdeclined in cold store but increased in March/April for plantsin the nursery.     

Germination and seedling growth of Quercus vulcanica: effects of stratification, desiccation, radicle pruning, and season of sowing

Effects of stratification, desiccation, radicle pruning, and season of sowing on Quercus vulcanica germination and growth were studied to identify optimum nursery procedures for artificial regeneration of this species. Following stratification (0, 2, 4 and 8 weeks), acorns were germinated, and acorn moisture content and germination performance were also determined at various times during desiccation. In early December unsprouted acorns were planted in containers, and in early April sprouted acorns, which had been stored in polyethylene bags at 4 °C, with radicles left intact or with radicles pruned back to 1.0 cm were planted in containers.Stratification for 2 and 4 weeks did not increase germination percentage but significantly increased germination rate. Germination percentage of the seeds dropped when the moisture content of the seeds was reduced by desiccation, and the critical minimum moisture content of the recalcitrant Q. vulcanica acorns was found to be 11–16%. Spring sowing of sprouted acorns altered the morphology the containerized seedlings and caused the formation of significantly more main roots, but resulted in significantly less shoot height and shoot dry weight. Spring-sown sprouted acorns had also a significant advantage over the fall-sown unsprouted acorns in seedling survival, and thus nursery personnel should not be concerned if acorns sprout before sowing.     

Seedling growth and establishment in natural stands of yellow-cedar (Chamaecyparis nootkatensis) seedlings derived from the use of modified seed dormancy-breaking treatments

Following dispersal from the parent tree, seeds of yellow-cedar (Chamaecyparis nootkatensis[D. Don] Spach) exhibit low germination, primarily as a result of coat-imposed dormancy. Dormancy of the mature (intact) seed is effectively terminated by traditional warm/cold treatments. A chemical treatment using the anaesthetic 1-propanol combined with a three day warm water soak (30 °C), a two day GA₃ treatment and 60 d of moist chilling not only promotes high germinability of yellow-cedar seeds, but also elicits vigorous post-germinative growth following seedling emergence under nursery greenhouse conditions. Here we compare the effectiveness of the more traditional warm/cold treatments with the chemical treatment in terms of their capacity to elicit vigorous growth and establishment in natural stands following transplant of seedlings from a nursery greenhouse environment. Two seed lots (42313 and 43697) and open-pollinated seed from parent trees 13-6 and 19-8 showed equivalent seedling growth in natural stands following the chemical treatment and two traditional warm/cold treatments typically used for dormancy breakage by the forest industry and by the Ministry of Forests in British Columbia. The chemical protocol offers the advantage of reducing the time required to break seed dormancy. We have now demonstrated that it yields seedlings that exhibit vigorous growth and are capable of withstanding the vagaries of the environment.     

Seedling cold hardiness, bud set, and bud break in nine provenances of Pinus greggii Engelm.

Cold hardiness and timing of bud set and bud break are important processes that provide protection of nursery seedlings against low temperatures. Seedlings of 9 provenances of Pinus greggii from two different regions of Mexico were tested to determine cold hardiness, bud set, and bud break timing differences. Needle sections were exposed to freezing temperatures to determine an injury index of each provenance. In addition, bud set and bud break timing were recorded through the fall, winter and spring. There were significant differences in cold hardiness between seedlings from northern and southern provenances. At the maximum cold hardiness, the index of injury (LT₅₀) for northern provenances was LT₅₀ = −18 °C, compared to −12 °C for southern provenances. There was a considerable variation among the provenances in the proportion of seedlings that set terminal buds. Seedlings from northern provenances had greater proportions of seedlings that set a terminal bud than seedlings from southern provenances. There were also significant differences in the bud break timing in the following spring among the 9 provenances. Seedlings from northern provenances broke bud earlier than southern provenances. Cold hardiness, bud set, and bud break timing results may be useful to determine how far a specific seed source can be moved from its natural environment.     

Storage of non-dormant seeds of Fraxinus angustifolia Vahl

The effects of drying and storage on the quality of Fraxinus angustifolia non-dormant seeds have been investigated. Mature seeds subjected to a 2-week warm stratification (20 °C) followed by a 2-week cold stratification (3 °C), were exposed to a gentle air stream at room temperature to dry them to 9.5% moisture content. After 4, 8 and 12 months of storage at –3 °C, in hermetically sealed containers, germination tests were conducted in darkness at 30/20 °C and 25/5 °C to evaluate possible changes in seed quality. The results obtained suggest the feasibility of drying non-dormant seeds. In fact, though causing a slight decrease in seed quality, such a process enables storage of ready to sow seeds for at least 12 months without any further loss. The temperature regime of 30/20 °C, prescribed by the International Seed Testing Association (ISTA), was found to be suboptimal for the particular conditions tested in this trial, the 25/5 °C cycle being more stimulating for germination.     

Seasonal changes in chlorophyll fluorescence of white spruce seedlings from different latitudes in relation to gas exchange and winter storability

Variable chlorophyll fluorescence (F_var) was investigated as a tool in detection of distinct seasonal physiological changes in 1+0 intact white spruce seedlings. The loss of the characteristic F_var peak (Fp) between 0.8 and 1.0 s after illumination of dark adapted seedlings is an indication of regulation of photosynthetic activity in August. The peak represents excess photochemical water-splitting of photosystem II. We interpret its loss as a physiological indicator of the process of dormancy induction. Three dimensional (i.e. X[0-300 s], Y[rfu], Z[time of year] axis) F_var curves of non-stressed seedlings measured over 300 s followed a three phase change over the growing season. In actively growing seedlings, the portion of the Kautsky induction curve between 60 and 300 s was 0.4 relative fluorescence units (rfu) in northern (i.e. >56° latitude) seedlots and 0.6 in the southern (i.e. <56° latitude) range seedlot from August until early September. About mid-September curve features between 60 and 300 s decreased sharply to approximately zero (rhu) by October. Freeze test data indicated seedlings became frost hardy during this time. The third, or inactive phase was seen as flat line from 40 to 100 s. The portion of the curve after 100 s was responsive to short term temperature changes. White spruce seedlots of northern and southern B.C. latitudes having curve fluorescence peak (F_p) values at about 1 s of 0.6, and 0.8 (rhu) respectively, plus curve minimum (F_min) values at about 60 s which do not decrease further over a 2–3 week period represent stock which can safely be lifted for cold storage. The F_var attribute at 5 s after the actinic light is turned on (F_5s) correlates well with net photosynthesis (r² =0.61) during the growing season.     

Effect of warm storage and date of lifting on the quality of Douglas-fir seedlings

The effect of warm storage (15 °C)for up to 21 days on the vitality of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings lifted on four occasions from October to January wasassessed using fine root electrolyte leakage (REL). After 0, 7 and 21 days warm storage, additionalseedlings were potted and placed in a controlledgreenhouse environment to determine root growthpotential (RGP) after 4 weeks and survival after 8weeks. REL was a poor indicator of survival potential;REL increased after 7 days storage, despite the factthat RGP and survival were unaffected. REL increased further during the remainder of the 21-days storage period, but values were often not greatly different from those following 7days storage. Storage for up to 21 days greatlydecreased RGP and survival. Seedlings lifted inOctober and November were most affected by storagetreatments, but differences among lifting dates wererelatively small.     

Inoculation of fumigated nursery soil increases nodulation and yield of bare-root red alder (Alnus rubra Bong.)

To determine if inoculation increases nodulation and yield of bare-root red alder (Alnus rubra Bong.), fumigated nursery plots were treated with inoculum and ammonium sulfate (28 kg N ha^–1) in a factorial experiment. Inoculum was alder soil with 100 infective units of Frankia g^–1. Seedlings were evaluated for nodulation at age 10 wk and when lifted, at age 9 mo. Inoculation produced earlier and more extensive nodulation and increased seedling root collar diameter, height, and dry weight. Fertilization decreased seedling height, but did not decrease nodulation. No interaction of fertilization with inoculation was found. Inoculated unfertilized plots had the highest yield of packable seedlings (257 m^–2), and uninoculated fertilized seedlings had the lowest yield (126 m^–2).     

Cold hardiness and transplant response of Juglans nigra seedlings subjected to alternative storage regimes

? Effects of overwinter storage regimes on seedling cold hardiness and physiological vigor are relatively unexplored, particularly for temperate deciduous forest tree species.

? We evaluated influence of storage duration (0, 66, 119, or 175 d) on electrolyte leakage of stem and root collar tissues following exposure to a series of freeze-test temperatures in black walnut (Juglans nigra L.) seedlings sampled from cold (3 °C) or freezer (?2 °C) storage. Seedlings were subsequently transplanted into a controlled growth chamber environment for two months.

? Regardless of storage temperature, mean LT₅₀ was lowest for seedlings stored for 66 d (≤ ?34 °C) and increased dramatically after 119 d (≥ ?13 °C).

? Root collar tissue had lower LT₅₀ than stem tissue after 119 d for cold-stored seedlings, reflecting importance of evaluative tissue type. Days to bud break shortened with increasing storage duration up to 119 d and stabilized thereafter for both storage regimes. Root growth potential was maximized after 119 d of storage, and subsequently declined for cold-stored seedlings. Height growth increased following storage, regardless of duration.

? To promote stress resistance and transplant growth response, we recommend that black walnut seedlings from this genetic source be outplanted after approximately 66–119 d of storage.

     

Root exposure effects on water relations of Eucalyptus nitens nursery stock

Bare-root seedlings of Eucalyptus nitens frequently exhibit water stress after planting resulting in leaf lamina damage and reduced leaf area. Two trials examined effects of root exposure and desiccation between lifting and transplanting on post-planting water relations, leaf retention and root growth. Plants with roots exposed on a glasshouse bench initially lost water rapidly. In one trial ₁ declined to around –2.0 MPa within 2.5 h, after which there was no further change with exposure up to 7.5 h. In the second trial, the initial decline in ₁ was more rapid, reaching below –2.0 MPa in the first hour, before remaining stable with continuing exposure up to 4.5 h. A further decline then continued to –4.0 MPa after 7.5 h.Two days after transplanting into potting mix, day – time leaf water potentials in all desiccation treatments had declined to near –2.0 MPa. Hydraulic resistivity, measured as leaf specific resistivity two days after transplanting, increased following exposure for greater than 2.5 h, but there was no further increase between 4.5 and 7.5 h. The increase in resistivity corresponded with leaf water potential declining below –2.0 MPa during exposure.In the second trial, increasing root exposure time resulted in decreased leaf area due to lamina necrosis. Root growth, measured three weeks after planting, was also reduced. and there was also a positive curvilinear relationship between leaf area remaining at three weeks and new root growth. The results are discussed in terms of hardiness and the management of E. nitens seedlings from nursery to plantation.     

Stock-type patterns of phosphorus uptake,retranslocation, net photosynthesis and morphological development in interior spruce seedlings

One-year-old interior spruce (Picea glauca (Moench) Voss × Picea engelmannii Parry) spring-stock and summer-stock were grown under two phosphorus (P) fertility regimes, with (+P) or without (–P), followed by a simulated winter, and a second growing period under an adequate fertility regime in a controlled environment room. The two stock-types differed in their response to low P availability. For spring-stock, morphological development, phosphorus-use efficiency (PUE) and P specific absorption rate (SAR) were similar between –P and +P seedlings. For summer-stock, –P seedlings compared to +P seedlings had lower (p 0.05) morphological development, but greater PUE and SAR. For both stock-types, P content increased in +P seedlings, remained low in –P seedlings, and P concentration decreased in nursery-needles (i.e., formed in the nursery) of –P seedlings. The difference in stock-type response to low phosphorus availability (–P) was attributed to internal supply of P and it's retranslocation. Assimilation (A) of CO₂ in nursery-needles was similar between –P and +P seedlings for both stock-types. For spring-stock, +P seedlings had greater A in new-needles (i.e., needles formed during the trial) than –P seedlings. It was recommended that the spring-stock be selected over summer-stock for sites low in P availability.     

Effect of Temperature on the Induction of Bud Dormancy in Ecotypes of Betula pubescens and Betula pendula

The purpose of this study was to determine the influence of temperature applied during short day-induced budset on induction of dormancy in six ecotypes of Betula pubescens Ehrh. and two ecotypes of Betula pendula Roth. Seedlings were grown in a phytotron at constant temperatures of 9–21°C under a 12 h photoperiod (SD) during dormancy induction. Induction of dormancy was monitored by following bud flushing and shoot growth after transfer to long photoperiod conditions (24 h) at 18°C. Chilling requirement was studied in seedlings exposed to 10 weeks of SD. In both species induction of bud dormancy developed most rapidly at 15–18°C, and both 9–12°C and 21°C delayed the induction of dormancy. Raising the temperature (from 9 to 21°C) applied during induction of dormancy significantly increased the chilling requirement. These responses were noted for all ecotypes tested, but in general the northern ecotypes entered dormancy more quickly than the southern ones. No such trend was recorded for chilling requirement, although a B. pubescens ecotype from Iceland and another from the coast of northern Norway appeared to require a longer chilling treatment than the other ecotypes. In conclusion, induction and depth of bud dormancy in birch are significantly affected by temperature conditions and these effects may explain some of the annual variation in dormancy and chilling requirement observed in nature.