Published May, 1982
Design of Experiments in Growth Chambers -- Uniformity
Ithe North Carolina State University Phytotron
2Chong-soon Lee and J. O. Rawlings
ABSTRACT
Experimentsconductedin growthchambersfrequently are defi-
cient with respect to one or moreaspects of experimentaldesign,
primarily, properuse of randomization and replication. To provide
informationwith whichto counter the usual argumentsassociated
withthe failure to use properdesigns andto improvethe efficiency
of growthchamber experiments,extensive uniformitytrials (using
soybeans) were conductedin the NorthCarolinaState University
Phytrotronin three sizes of chambers.
Theresults confirmedthe existence of sizeable, patternedplant-
to-plant variation within chambersand amongchambersof all
sizes. Patternsof plant growthwithin chambers tendedto be char-
acteristic of chamber type and to persist over time. Bothchamber
and timeeffects werevery important.
Correlationsamongspatially related observationsconfirmedthe
potential for seriousbias in estimatesof both treatmenteffects and
experimentalerror in experimentswhichignore either randomiza-
tion or properreplication. Properlydesignedexperimentsare as
importantin growth chambersas in greenhouseor field experi-
ments. Properblocking both within chambers(for within-chamber
experiments) and with respect to time and/or chambers(for
between-chamber experiments)appearsto be effective in control-
ling a majorportionof the variation.
Additional index words:Experimentaldesign, Uniformitytrials,
Randomization,Truereplication, Growthchamberstudies, Intra-
class correlations.
W HILE controlled-environment growth chambers have
proven to be a valuable asset to plant research, there
is a continuing tendency for some researchers to ignore
good experimental design practices in growth chamber
experiments. Discussion of design of experiments within
growth chambers must recognize two levels of experimen-
tation in terms of what constitutes an experimental unit.
On the one hand, there is the class of treatments which
may be applied to individual pots, or sets of contiguous
pots, within each chamber; e.g., levels of various
nutrients, moisture levels, cultivars, or plant densities. In
this case, the individual pot, or set of pots, is the experl-
mental unit and each chamber will contain many such
experimental units. On the other hand, there is the class
of treatments, e.g., irradiance levels, day/night tempera-
ture regimes, or CO2levels, which by their nature require
that all plants within the chamber be exposed to the same
treatment. Hence, the chamber is the experimental unit
for these treatments and even a single replicate of an
’Paper No. 7044 of the Journal Series of the North Carolina ARS
Raleigh,N.C.This paper presents part of the results of a Ph.D.Diss.
preparedby the senior author while enrolled at NorthCarolina State
Univ.Theauthors wishto thankDr. R. J. Downs,director, NorthCar-
olina State Univ. Phytotron,Dr. V. P. Bonaminio,extension assistant
professor of horticultural science, NorthCarolinaState Univ. and the
phytotron staff for extensiveassistance andcooperationduringthe con-
duct of these studies. Received10 Aug.1981.
~Research assistant, presentlysenior researchofficer, MalaysianAgri-
cultural Researchand Development Institute, Serdang,Selangor, West
Malaysia, and professor of statistics, NorthCarolina State Univ.,
Raleigh, N.C.
55~
Trials in
experiment requires several chambers or at least several
runs over time in one chamber. The former will be
referred to as within-chamber experiments; the latter, as
between-chamber experiments. If both levels of experi-
mentation are involved in a single study, the arrangement
is the classical split-plot experiment with the individual
chambers being the whole-plot experimental units and the
individual plants, or sets of plants as the case may be,
within chambers being the sub-plot experimental units.
Two fundamental experimental design concepts, repli-
cation and randomization, are frequently ignored in phy-
totron experiments. In some cases they are ignored
because of a commonmisconception that the highly tech-
nical control of the growth chamber environments insures
uniform conditions, at least for all practical purposes, both
within and among chambers (with the same nominal set-
tings) and, therefore, placement of the plants and assign-
ment of treatments is not that critical. In other cases, the
problem arises because of misconception of what consti-
tutes replication in the experimental design context. Both
proper replication and proper use of randomization
require a clear understanding of what constitutes an
experimental unit. Absence of replication means that the
experiment does not contain the information needed to
estimate the experimental error; absence of randomiza-
tion means that the estimate of experimental error, even if
there is proper replication, may be seriously biased.
Several authors have issued warnings that growth
chamber environments are not spatially uniform.
Hruschka and Koch (1964-) reported "strong gradients"
in growth in all directions in a temperature and humidity
controlled storage room and warned, "... heterogeneity
is inevitable in environmentally controlled experimental
chambers .... " Collip and Acock (1967), using lettuce,
and Hammer and Langhans (1972), using maize, both
reported large patterned within-chamber variation.
Measures et al., (1973) presented results from cucumbers
(Cucumis sativus L.), corn (Zea mays L.), oats (Avena sativa
L.), and wheat (Triticurn aestivum L.) discounting the con-
cept of environmental homogeneity within growth cham-
bers. They indicated that heterogeneity within chambers
was so great that choice of position within the chambers
could critically bias the results. All of these authors rec-
ommended reliance on randomization, replication, and
blocking to increase precision and eliminate bias. With
respect to heterogeneity between chambers, Hammer et
al., (I 978) reported large experimental variation over time
within laboratories and emphasized the necessity of rep-
licating growth chamber experiments over time to obtain
a proper estimate of experimental error.
The lack of spatial uniformity within growth chambers
of the physical environment, primarily temperature and
552 CROPSCIENCE, VOL. 22, MAY-JUNE
radiance, has been reported by many authors; e.g.,
Measures et al., (1973), Tibbitts et al., (1976), Hammer
and Langhans (1972). Knievel (1973) and Tibbitts et
(1977) reported on other environmental factors which
might be contributing to environmental variation within
and between chambers.
Hammer and Langhans (1978) reiterate the impor-
tance of using proper replication and experimental designs
in an introductory manual on the use of controlled envi-
ronment growth chambers, and Hammer and Urquhart
(1979) present a more complete discussion of the applica-
tions of experimental design to growth chamber experi-
ments. In spite of the many references to lack of
uniformity within and between chambers and in spite of a
general acceptance of the need for proper experimental
design in both field and greenhouse experiments, there
continue to be rather frequent occurrences of failure to use
proper experimental design in controlled environment
experiments.
In the context of the within-chamber experiment, the
experimental unit implicitly includes the space within the
chamber assigned to the pot or set of pots. It is not
uncommon, for example, to allocate one treatment to all
the plants on one truck, another treatment to all plants on
a second truck, and to place the trucks wherever space
permits within the given chamber. In the event environ-
mental conditions are not uniform, it has long been rec-
ognized that such procedures introduce unknown biases
in the estimation of treatment effects and an underesti-
mation of experimental error, if variation among plants on
a truck is used as the error. Even if the growth chamber
provides a perfectly uniform environment, the mere
grouping together of all plants for a given treatment and
the inescapable tendency to handle the group as a unit,
such as in thinning, watering, and data collection will tend
to introduce positive correlations among the plants within
a treatment with the net result that variation amongplants
on a truck will underestimate experimental error. The
truck or group of plants, in effect, becomes the experi-
mental unit so that the experiment has no true replica-
tion. Variation among plants within treatments is really
sampling variation within experimental units.
The same problem occurs, and is probably much more
serious in nature, with between-chamber experiments.
Because of the nature of the treatment, all plants within a
chamber must be subject to the same treatment. It is com-
mon practice with between-chamber experiments for a
series of standard environments involving day/night tem-
perature and light regimes to be provided on a continuous
basis by designated chambers, with individual researchers
using the chambers providing the treatments of interest
when space permits. Operationally, this is much more
convenient than allowing random assignment of treat-
ments to chambers and it is convenient to leave unchanged
over relatively long periods the assignment of standard
environments to chambers. In such case, if replication in
time is used, it means repeated use of the same growth
chamber for a given treatment. As before, the failure to
use randomization in the assignment of treatments
experimental units, the chambers, and the associated
potential for biases in the estimation of treatment effects
I982
and experimental error must be recognized. Of even more
potential seriousness in between-chamber experiments is
the very strong desire, for obvious economic reasons, to
use only one chamber (one experimental unit) for each
treatment and to regard plants within chambers as proper
replication for the estimation of experimental error. This
is analogous to the previous situation of using plants
within trucks as an estimate of experimental error and
provides the potential for gross underestimation of the.
experimental error.
Of course, one does not expect complete uniformity of
plant development even under highly controlled environ-
mental conditions. The critical point, however, in the
event randomization has not been used is whether the
variation is random or patterned in some way. If the var-
iation is nonrandom, then the magnitude of the variation
will affect the biases in the estimation of treatment effects
and experimental error. The discussion in Cochran and
Cox (1957) of randomization as an insurance "against
disturbances that may or may not occur and that may or
may not be serious if they do occur" is relevant but is, in
the authors’ opinions, an understatement of the serious-
ness of lack of randomization.
To quantify the seriousness of the failure to make proper
use of randomization and true replication in growth
chamber studies and to obtain basic information relevant
to the design of experiments in growth chambers, a series
of uniformity studies was run in the North Carolina State
University Phytotron (Lee, 1977). The objective was
assess the magnitude and nature of the within-chamber
and between-chamber variation under uniform condi-
tions in the three sizes of growth chambers in the NCSU
Phytotron. With this information, the magnitude of the
bias from misspecification of experimental error and fail-
ur.e to use randomization can be ascertained and efficient
experimental designs identified.
MATERIALS AND METHODS
Since the objective was to detect what, a priori, were expected
to be relatively minor environmental variations, it was decided
that a relatively sensitive plant species would be used in a tem-
perature regime that would favor detection of environmental
variation. Soybeans (Glycine max.), ’Bragg’, was chosen as the
test species and 35 of the 47 uniformity studies were conducted
with a day/night temperature regime of 22/18 C. To check the
effect of temperature, the other 12 studies were conductedat 26/
22 C. The distribution of the studies over the three sizes of
chambers (A, B, and C) in the North Carolina State University
Phytotron, the dates and the temperatures at which they were
done are shown in Table 1.
All seeds were pregerminated. The seedlings were selected for
uniformity and transplanted three to each pot. At 7 days of
growth the two extreme plants were eliminated leaving one plant
per pot. All studies were terminated at 20 days and the following
five measurementstaken on each plant:
1. plant height (ram) from the cotyledonary node to the tip
the apical bud,
2. total leaf area (era~) of all fully opehedleaves,
3. petiole length (ram) of the first trifoliate leaf,
4. fresh weight (rag) and,
to 5. dry weight (rag) of the plant above the cotyledonary node.
Each A chamber (3.66 × 2.44 m) held 24 movable trucks in
6 × 4 configuration; each truck held 16 plants in a 4 × 4 config-
 LEE & RAWLINGS:DESIGNOF EXPERIMENTS
IN GROWTH
CHAMBERS 553

Table 1. Dates, chambers,and temperatures of the uniformity Table 2. Means,standarddeviations {within studies), and coef-
studies with soybeans in the NCSUPhytotron. ficients of variationaveragedover all studies for a given cham-
ber type.
Trial Date{1975} Chamber
Variable
A Chambers
1 7/8 -7/30 A8 A9 A10 Plant Leaf Petiole Fresh Dry
2 7/29-8/20 A4 A9 A10 ht. area length wt. wt.
3 8/19-9/10 A4 A8 A9 mm cm* mm mg mg
4 9/9 -10/1 A4 A8 A9
Achambers
Ill studiesat 22/18CI~
B Chambers~f
1 3/14-4/3 B2 B3 B8 Mean 77 133 27 318 49
4/26-5/16 B1 SD 12 24 5.5 53 8.5
3 B2~: B85 C.V. 16 18 20 17 17
4 5/17-6/6 B2 B3 B8
5 6/7 -6/27 B1 B3 B8 B chambers
(12studiesat 22/18C)
6 6/28-7/18 B1 B2 B3§ Mean 119 155 39 386 54
C Chambers SD 27 33 10.3 86 10.9
C12 C15 C.V. 23, 22 27 22 20
1 5/31-6/20 C3:]: C8:~ C9
2 6/21-7/11 C3 C8 C9:~ C12~: C15~/ Cchambers
(10 studiesat 22/18CI
3 7/12-8/1 C3~t C8 C9 C12~t C15 Mean 99 110 24 254 37
4 8/2 -8/22 C3 C8:~ cg~t C12 C15~ SD 17 23 4.6 46 6.2
C.V. 17 21 19 18 17
Trial 2 wasdiscardeddueto atypicalplant growthin all three chambers.
Studies conductedat 26/22C. All otherstudieswereconducted at 22/18 (10studies
C chambers at26/22 C)
C. All pooledresultsincludeonlytrials conducted
at 22/18C. Mean 295 257 76 565 75
A faulty temperature controlrequiredmovement of the plants to cham- SD 40 32 8.3 74 9.8
ber A15for 3 days.Datafromthis trial are not includedin the combined C.V. 14 12 11 13 13
analyses.
Malfunction
of the light controlin chamber
A8in trial 3 wasrecorded
for
threeconsecutivedays.Datafromthis trial areexcluded.
uration giving a total of 384 plants per A chamber, 16 columns
and 24 rows of plants. Each B chamber (1.22 × 2.44 m) held Alternatively, large intraclass correlations identify classifica- "
trucks in a 2 × 4 configuration, 16 plants each, giving a total of tions that would constitute effective blocks for experimental
128 plants per B chamber, 16 columnsand eight rows of plants. design purposes, assuming treatments are randomly assigned to
Each C chamber (0.91 × 1.22 m) held 15 racks in a 3 × 5 con- individual plants within the classification. Therelative efficiency
figuration, four plants per rack, giving 60 plants per C chamber, of the blocked design to a completely randomdesign is (approx-
10 columnsand six rows of plants. For a detailed description of imately) 1/(1-~), ignoring adjustments for loss of degrees
the North Carolina State University Phytotron facility, see freedom, where~ is the appropriate intraclass correlation.
Downs and Bonaminio (1976). As a second test for randomnessof variation within chambers
All analyses of the data were done by chamber type, and and to characterize the pattern of the variation, polynomial
within temperature levels for the C chambers. Nested analyses response surfaces were fit to the data for each plant variable in
of variance were used to test and quantify the importance of dif- each study using row and columncodes as the coordinates for the
ferences amongstudies (= chamber-trial combinations), posi- response surface. Similarities of the response surfaces over
tions in studies, and plants in positions, wherepositions were chambersand trials were investigated.
defined, in turn, by carts, rows, or columnswithin chambers.If
the variation in plant growth is random,all meansquares will be RESULTS AND DISCUSSION
estimating the same variance and should be approximately
equal. Equivalently, all intraclass correlations for different clas- The means, standard deviations (within study), and
sifications of spatially (or temporarily) related plants should coefficients of variation, based on the pooled within-study
zero. The results of the analysis of variance are summarizedin individual plant variation, averaged over all studies in a
terms of different types of intraclass correlations on the bases of given chamber type arc shown in Table 2. Plant-to-plant
within-chamber variation, relevant to within-chamber experi- variation within chambers was large in all experiments.
ments, and on the basis of total variation over studies, relevant Average coefficients of variation at 22/18 C ranged from
to between-chamberexperiments. The intraclass correlations is 16 to 27 %. In terms of extremes in plant performance, the
the average pairwise covariance between observations within
classes relative to the total variance in the reference base. Sig- largest measurement for any variable in a study was two
to three times the smallest. The average coefficients of
nificance of intraclass correlations (different from zero)
inferred from variance ratio tests in the appropriate analyses of variation for the higher temperature wcrc smaller but still
variance. For simplicity of presentation, and because of the sim- ranged from 11 to 14%. Inspection of the variance showed
ilarity of results for all plant variables, all intraclass correlations significant heterogeneity of variances associated with the
are presented as averages over all plant variables and all studies mean level of performance. Amongseveral transforma-
within chamber type. tions tried, the square root transformation was the most
The intraclass correlations are useful for two purpsoes. The consistent in stabilizing the variance and all subsequent
impact of misusing a within experimental unit variance as analyses arc in terms of square root transformed varia-
experimental error, rather than variance among experimental bles.
units, is quantified by the "error bias ratio" = 1 + (n-1)~/
The analyses of variance, using trucks to identify posi-
(1-~), where ~ is an estimate of the appropriate intraclass cor-
relation and n is the numberof plants per experimental unit. The tions in chambers (Table 3) shows a very significantly
error bias ratio estimates the size of the F-ratio for the test of increasing hierarchy of mean squares for every variable in
treatment effects under the null hypothesis of no treatment effects every chamber type. Clearly, all components of variance
whenthe incorrect within experimental unit variance is used as are greater than zero (or the intraclass correlations appro-
experimental error. priate to this nested system are greater than zero).
554 CROPSCIENCE, VOL. 22, MAY-JUNEI982

Table 3. Meansquares in the nested analyses of variance over Table 5. Coefficients of determination, averaged over all
experimentsfor each chambertype using trucks to define posi- studies, of polynomial response surfaces fit to uniformity
tions within chambers. Analyses are on square root trans- data.
formeddata.
Chamber
Degree of Plant Leaf Petiole Fresh Dry
Variable type polynomial ht. area length wt. wt.
Plant Leaf Petiole Fresh Dry A 6th 0.51 0.46 0.57 0.42 0.46
Source~ df ht. area length wt. wt. B 4th 0.70 0.52 0.69 0.53 0.51
C 4th 0.64 0.48 0.53 0.46 0.42
A chambers
Amongexperiments 10 93.7 236.0 48.9 384.2 43.8
Amongtrucks in
experiments 253 3.6 6.9 2.1 11.9 2.2 the A chambers. (All plant variables showed similar pat-
Amongplants in terns.) Note in particular the pattern characteristic to each
trucks 3960 0.3 0.7 0.2 1.6 0.2
chamber. A4 tends to have a pattern of its own with peaks
B chambers (P) near the left rear and right front of the chamber.
Amongexperiments 11 153.7 204.3 45.6 525.8 43.3
Amongtrucks in has a similar pattern to A4 but the valley displaced closer
experiments 84 14.4 14.9 6.7 36.6 4.0 to the right front. A9 and A10 patterns appear to be
Amongplants in almost mirror images of A8 and A4. Similar consistency
trucks 1440 0.7 1.2 0.3 2.8 0.4
of patterns over time is exhibited by the other variables.
C chambers
Among experiments Figure 2 shows the response surfaces for plant height for
in temperatures 18 33.2 33.1 14.2 96.5 9.6 the B chambers. In this case the major features of the con-
Among racks in tour surface seem to be characteristic of all chambers.
experiments 280 2.7 2.4 0.5 4.6 0.5
Amongplants in Closer examinaton suggests some chamber-to-chamber
racks 900 0.6 0.7 0.1 1.7 0.2 differences. Particular attention should be paid to B2 and
In the absenceof experiment andtruckeffects andwithindependent B8 in trial 3, where the pattern holds up very well at the
errors, the threemeansquareswithineachanalysishavethe sameex- 26/22 C temperature regime, and to B3 in trial 6, where
pectation.In a correlatederrorsmodel,withQ, beingthe correlationbe- the pattern is quite different, possibly disrupted by the
tweenplants on the sametruckandQ2beingthe correlationbetween
plantsondifferenttrucksbut in the samechamber, the meansquareex- movement of all plants to A15 for 3 days when the tem-
pectationsarea~[l+ (n-I)~i + n{k-I)~i],
a~[l+ {n-I)~i], ando~{I ~) perature control in B3 malfunctioned.
respectively, where n = number ofplants pertruck andk = number of
trucks perchamber. The response surfaces in the C chambers, (not shown)
fail to show the same high degree of pattern consistency
Table4. Estimatedintraclass correlations for various classifica- over time. There is suggestion of a tendency for the peak
tions within chambers,averagedover five plant variables. to be near the left-rear of the chamber but the patterns
shift considerably both with chambers and time (and with
Chamber
type
temperature).
Classification A B C These two methods of summarization clearly show that
Trucks(or racks) 0.37 0.47’ 0.36 the variation within chambers is not random; that as much
Rows 0.02~ 0.05 0.08 as 50 to 70% of the plant-to-plant variation within a
Columns 0.17 0.43 0.21
chamber can be ascribed to position differences within
Two of the five correlations
averagedto give this valueweresignificant-
ly differentfromzero,p = 0.05. In all othercases,all intraclasscorre- chambers. These results are surprising only in their mag-
lationsweresignificantlydifferentfromzero. nitude. They verify that the basic principle in field exper-
iments, that experimental units adjacent in time or space
Considering first the within chamber variation, the tend to be positively correlated, extends to the highly con-
large intraclass correlations (Table 4) for trucks (racks trolled environments in growth chambers. Hence, failure
the C chambers) and for columns clearly indicate nonran- to utilize proper design techniques for within-chamber
domness of the variation within chambers. The smaller experiments will lead to biased estimation of treatment
intraclass correlations for rows simply reflect the fact that effects and underestimation of the true error.
the individual rows span most of the variation within each The seriousness of failing to use proper design princi-
chamber, since we know from the other correlations that ples is demonstrated by noting that the error bias ratio
there are strong patterns to the variation within cham- resulting from using the within truck variance as an esti-
bers. mate of error, when the truck, due to lack of randomiza-
The polynomial response surfaces fit to each plant var- tion, is the experimental unit, is 10, 14, and 3 for the A,
iable in each study confirm that there is strong patterning B, and C chambers, respectively (computed from Table
in the plant-to-plant variation within chambers. In all 4). It is also clear that blocking by trucks or columns
cases, many terms of the polynomials were significant. A would be very efficient, relative efficiency, 1/(1-~), var-
sixth degree polynomial seemed to adequately character- ies from 1.56 to 1.89 for trucks and from 1.20 to 1.75 for
ize the surfaces in the A chambers, a fourth degree poly- columns as blocks. Randomization is imperative and
nomial in the B and C chambers. The coefficients of blocking within chambers would be beneficial. In addi-
determination (R2’s) for these models, averaged over tion, to the extent that the response surface in a chamber
studies, varied from 0.42 to 0.70 (Table 5). is consistent over time, prior information on the response
Inspection of the response surfaces reveals a fairly high surface would be a potentially useful covariate for control
consistency of chamber patterns over time. Figure 1 shows of within-chamber variation.
contours of the fitted response surface for plant height in The preceding has been concerned primarily with the
 LEE & RAWLINGS: DESIGN OF EXPERIMENTS IN GROWTH CHAMBERS
Trial I
^8
,I. "",,.’’"’1’... ~"~’:~~"’~
Fig. 1. Gontours of individual experiments, arranged by trial, of a sixth degree response surface for square root of plant height. Each contour
covers an interval t~ units. Chamber identification
of 0.5 mm
trial ~ with a defective light control is shown with ?. The bold-face letters
the contour surface.
effects of nonrandom within-chamber variations on
within-chamber experimental designs. It is clear from M1
the nested anMysesof variance, see Table 3, that the posi-
tive intraclass correlations of observations related spa-
tially (and temporarily) extend across chambersand trims;
the among experiments mean squares are much larger
than the within experiments mean squares.
The intraclass correlations (Table 6) are far from zero
at MIlevels and, consequently, the use of any within-clas-
sification (within-chamber, within-truck) sampling vari-
ance as the experimental error grossly underestimates the
true experimentMerror. For example, the average intra-
chambercorrelations were 0.31, 0.42, and 0.38 in the A, B,
and C chambers, respectively. Using these averages, and
assuming full chambers for each treatment in an inter-
chamber experiment, the error bias ratios from inappro-
priately using a within chambervariance as experimentM
error would be 175, 91, and 37, respectively (Table 7).
The other lines in Table 7 showthe bias resulting from
using only part of each chamber.For example, if one truck
(or rack) within a chamberis used for each treatment, the
error bias ratios are 90, 32, and 5, for the A, B, and C
chambers, respectively.
Each of the sources of variation in the anMysisshownin
Table 3 was partitioned further; variation amongexperi-
,~,~.~
is shown at the left. Trial identification is shown at the top. Chamber A8 in
P and V on each diagram show locations of peaks and valleys in
ments was partitioned into that due to chambersand trials
(or time) differences and the "amongtrucks" variation
was partitioned into average position effects and interac-
tions of positions with chambersand with trims (to show
the consistencyor lack of consistency of the position effects
over chamber and trims).
First, with respect to the anMysisof position effects, the
following conclusions might be made:
1. In all chambertypes the position maineffects (trucks
or racks) were highly significant indicating at least
some consistency of the pattern over trials and
chambers.
9. In the A chambers, the chamber × truck interaction
meansquares were nearly as large as the truck main
effects mean squares indicating that the position
effects are quite different from chamberto chamber
but, were reasonably consistent over time for a given.
chamber. That is, the within-chamber pattern of
variation for the A chambers seems to be relatively
stable over the duration of these studies but the pat-
tern in one chamber is not transferable to another
without testing.
3. In the B chambers the truck × chamber mean
square is again the next largest meansquare but this
time muchsmMler, relatively, than in the A chain-
556 CROP SCIENCE, VOL. 92, MAY-JUNE ,982

Trial Trial

B1

B2

B3

88

Fig. 2. Contours of individual experiments, arranged by chambers, of a fourth degree response surface for square root of plant height. Each
contour shows a range of 0.5 mm ~ units. Chamber identification is shown at the left. Trial identification is shown at the top. Chambers B2
and B8 in trial 3 conducted at 26/22 C temperature are shown with $. Chamber B3 in trial 6 with a defective temperature control is shown
with $. The bold-face letters P and V on each diagram show locations of peaks and valleys in the contour surface.

Table 6. Estimatedintraclass correlations for between-chamber Table 7. Error bias ratios~ resulting fromu~ing various within
experiments, averagedover variables and studies, where the experimental unit plant-to-plant variances as experimental
class, or experimentalunit, is the indicated portion of the error for testing between-chamber treatments. Theresults are
chamber. computedfrom the appropriate observed intraclass correla-
tions averagedover all variables and studies within a chamber
Chamber type type.
Class(experimental
unit) A B C Chamber type
Full chamber 0.31~ 0.42 0.38 Experimental unit A B C
One truck~chamber 0.56 0.67 0.60
One row/chamber 0.33 0.45 0.44 Full chamber 175 91 37
One column/chamber 0.42 0.67 0.52 One truck/chamber 20 32 5
One row/chamber 8 13 8
~fAll intraclass correlations involved in all averages are significantly One column/chamber 18 15 6
greater than zero (p = 0.05).
~fTheerrorbiasratiois computedas 1 + (n-l)(~ll-~)wheren is
bers. Thus, the large position effects are showing a numberof plantswithineachexperimental
unitand ~ is the appropriate
intraclass
correlation.
great deal of consistency over chambers as well as
time. This is a confirmation of the general appear-
ance of greater consistency in the response surface
contours for the B chambers shown earlier. may change with the plant species used and with the con-
4. In the C chambers, none of the interactions seem to ditions under which studies are conducted, it is evident
dominate although most are signifiant. Thus, the that use of proper within-chamber experimental designs
position effects show some consistency over cham- should increase the efficiency of experimentation. These
bers and time but the changes in pattern that do data suggest that simple blocking by trucks (or racks), for
occur are, more or less, unpredictable. example, in a similar experiment involving the entire
To summarize, there are distinct patterns to the within- chamber would reduce the error sum of squares by an
chamber variation in these studies that, to a sizeable average of 36% in A chambers, 43% in the B chambers
degree, are characteristic of the chamber type, i.e., and 52% in the C chambers. It is equally clear that ran-
repeatable over time and space. At the same time, there domization must be used to insure unbiased estimation of
are significant components of these patterns which "are experimental error.
unique to given chambers, particularly in the A cham- Shifting now to the analyses of the chamber-trial differ-
bers, and which change with time. While it must be rec- ences, the following conclusions are relevant:
ognized that the specific patterns of variation observed 1. A chambers: The large variation among experi-
 LEE & RAWLINOS: DESIGN OF EXPERIMENTS IN GROWTH CHAMBERS
ments (Table 3) is not ascribable to either chambers
or trials; that is, no consistent chamber effect nor
time effect is responsible for the large differences
among experiments. The implication is that blocking
either by chamber or time would be largely ineffec-
tive in controlling the experiment-to-experiment
variation.
2. B chambers: The major contributor to the variance
among experiments is trial effects; that is, changes
associated with time. It might be noted that the B
chamber studies spanned a greater length of time
than the studies in the other two types of chambers.
3. C Chambers: There was again some indication that
trial effects (time) was of some importance in
accounting for the experiment-to-experiment varia-
tion but the chamber effects were nearly as impor-
tant.
Thus, it is not clear whether blocking by chamber or
time would be more effective for between-chamber stud-
ies. It is clear, however, that the chamber and the time
(trial) components of variance are sizable and need to be
taken into account in the between-chamber experimental
designs. The same nominal chamber setting is not pro-
ducing the same plant response over time over chambers.
This suggests that the practice of using standard cham-
bers, i.e., certain chambers maintained continuously at
prescribed settings, may be yielding biased estimates of
treatment effects and, therefore, is an undesirable prac-
tice.
The final point to be emphasized is that, because of the
large components of variance for chambers and trials and
the very important spatial correlations among plants
within chambers, it is an absolute requirement that exper-
imental error for between-chamber experiments be esti-
mated via proper replication of the experiment. The
chamber-trial combination is, in such cases, the experi-
mental unit and each treatment must be replicated by
using several chambers and/or repeating the experiment
over time. Experimental error estimated from within-
chamber "replication" is inappropriate for testing
between-chamber differences. The error bias ratios (Table
7) show that within-chamber estimates of error, com-
pared to proper between-chamber estimates, are too small
by a factor (on the average in these studies) of from 6 to
175. Recalling that an F-ratio of 2.5 to 3.0 is declared sig-
nificant (a — 0.05) for any reasonable number of degrees
of freedom, it is clear that even the smallest of error bias
ratios completely invalidates any tests of significance.
While these results were obtained from an extensive set
of 39 uniformity trials, over 13 different growth cham-
bers, only the soybean cv. Bragg was grown and, for the
most part, in one standardized environmental regime.
Consequently, one does not expect the quantitative results
such as estimates of intraclass correlations, error bias
ratios, and response surface patterns to extrapolate with-
out modification to other conditions in this or other labo-
ratories. However, it is the mere presence of nonrandom
variation that is critical and the results have clearly shown
nonrandom variation in plant growth within and between
chambers in the NCSU Phytotron which, presumably, is
the result of nonrandom environmental variation, envi-
557
ronment used in the broadest sense. Several other studies
(see the References) have shown similar results. It is
unlikely that such environmental variations would not
impact to some extent on the growth of other species under
other conditions.
SUMMARY AND CONCLUSIONS
The uniformity data using soybeans in the NCSU Phy-
totron vividly demonstrate that all of the conventional
experimental design principles must be considered in
experiments conducted in growth chambers. In spite of
highly technical control of the environments and stand-
ardization of daily operating procedures, the variation in
plant growth both within chambers and between trials
(chambers and/or time) was neither trivial nor random.
Failure to use randomization and proper replication can
lead to biased treatment effects and gross underestimation
of experimental error.
Experiments involving treatments which require an
entire chamber for a given level must use chambers as
experimental units and, consequently, replication must
involve the use of several chambers (possibly over time).
Experiments which use both between-chamber and
within-chamber treatments belong to the classical split-
plot experiments and, as in nearly all split-plot field
experiments, the sub-plot error (the within-chamber
error) is much smaller than the whole plot error and totally
inappropriate for testing between-chamber treatments;
the whole-plot error must be estimated via replication of
the between-chamber treatments. Such replication is very
expensive, a fact which only increases the importance of
using proper and efficient experimental designs.
Even in experiments which involve only within-cham-
ber treatments, the nonrandom nature of the within-
chamber variation dictates that proper randomization
techniques be used to avoid bias in the estimation of
experimental error and treatment effects and that the
experimental unit (a plant, a truck, etc.) be properly
identified so that true replication will be used. In addi-
tion, the magnitude of the patterned within-chamber var-
iation shows that efficiency of experimentation can be
increased by paying proper attention to use of blocking
techniques and covariates.
While within-chamber experiments can be completed,
including within-chamber replication, in a single trial in
one chamber, it should be borne in mind that some uni-
dentified environmental and/or experimenter variables are
changing with time and chamber even in the same phy-
totron, as evidenced by the large chamber and trial effects.
Thus, it may be desirable, in order to broaden the base of
inference, to replicate even the within-chamber experi-
ments over chambers and/or time (or possibly over phy-
totrons).
558 CROP SCIENCE, VOL. 22, MAY-JUNE 1982