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Key words: factor screening, factorial treatments, fractional factorial design, PlackettBurman design,
prioritizing factors, screening designs
Abstract
The majority of plant tissue culture experiments are set up as factorial experiments in completely randomized designs (CRD), randomized complete block designs (RCBD) or split-plot designs (SPD) that
require a great number of stabilized cultures. For various reasons, an insucient number of explants may
prevent the employment of CRD, RCBD or SPD with factorial treatments and hinder the optimization of
factors aecting culture response. To prioritize the optimization of the most important factors aecting
culture response, we explored the applicability of PlackettBurman Design (PBD) and Fractional Factorial
Design (FFD) with a limited number of explants. To test the eects of 8 factors (genotype, 6-benzyladenine
(BA), CuSO4 5H2O, Fe source, agar, pH, myo-inositol and cold treatment following inoculation of explants) at 2 levels on response of single-node grape vine cultures, 12 treatment combinations were generated
according to the PBD and 16 treatment combinations were generated according to the FFD. These designs
require many fewer explants since a typical experiment with eight factors, each at two levels of will require
256 (28) treatment combinations. However, the costs of the PBD and FFD are that there is limited, if any
information, on interactions and if available may be more dicult to interpret. Of the factors tested,
cultivar, BA and agar concentrations were found to be the most important factors aecting culture response. The types of culture response were in agreement with previous reports indicating that PBD and
FFD can eectively be employed with limited number of explants to test eects of several factors and
prioritize the most important factors.
Abbreviations: BA 6-benzyladenines CRD - completely randomized designs; IBA - indole - 3 butyric acid;
FFD - Fractional Factorial Design PBD - Plackett - Burman designs; NMR - nas and Read (2004) medium
RCBD - randomized complete block designs; SPD - split-plot designs
Introduction
The use of appropriate experimental designs and
statistical analyses in plant cell and tissue culture
studies is necessary to ensure unbiased and precise
estimates of treatment eects and to provide
proper interpretation of results. The design and
214
and are set up as factorial experiments in completely randomized designs (CRD), randomized
complete block designs (RCBD) or split-plot designs (SPD) (Compton, 1994; Compton and Mize,
1999). These types of experimental designs are
useful when the researcher has previously identied a few factors to study and there is a sucient
amount of explant material to properly replicate.
However, in some tissue culture research, these
types of experimental designs may not be appropriate, or even feasible. In the initial stages of a
research program, typically there are a large
number of factors of interest and a limited number
of available explants. Therefore, the objectives
should be prioritized and experimental components should be selected according to budget and
time available (Compton and Mize, 1999). During
the establishment of aseptic cultures (Stage I),
where for various reasons many explants are lost,
using a factorial experiment with a CRD, RCBD
or SPD may not be possible since these designs
would require too many explants. For instance, an
experiment with eight factors, each with only two
levels will result in 256 (28) treatment combinations and will require 512 explants when two replicates are used per treatment. In these situations,
there is a need for special types of designs that
require a minimal number of explants and can aid
with screening a large number of factors for those
with major eects which will be further studied in
more detail using classical types of experimental
designs.
Fractional factorial designs can be used in
plant cell and tissue culture research to eectively
identify important factors and interactions while
using a minimal number of explants. These designs
have been used for many years in industrial research where it is critical to minimize the amount
of needed experimental material (Box et al., 1978).
A very simple example is an experiment that has
four factors called A, B, C and D where each
factor has two levels. The full factorial will have a
total of 24 16 treatment combinations, while a
fraction would have 24 8 treatment combinations. The obvious advantage of these types of
experiments is that they reduce the required
number of experimental units while providing
information on all factors, however, information
on one or more eects is lost and information on
the remaining eects maybe more dicult to
interpret. In the fraction example, the particular
set of treatment combinations are chosen to provide the maximum amount of information on the
main eects of the four factors, while foregoing
any information on the four way interaction
(ABCD). In addition, information on each twoway interaction is confounded (or aliased) with
another two-way interaction. More specically,
the AB interaction is aliased with CD, AC is aliased with BD, and AD is aliased with BC. This
means that based on the data analysis, it is not
possible to separate the AB interaction eects
from the CD eects etc.
Aliasing and loss of information on some effects are important considerations with FFD in
tissue culture research, but may not be very
important depending on the particular objectives
of the research. Often with tissue culture experiments, higher order interactions are small or negligible and the researcher knows which factors are
likely to interact. In such cases, fractional factorials may be constructed to obtain information on
eects that are likely to be present and forego
information on those that are not. For example,
with the 24 fraction described above, if the
major objective of the research is to obtain information on the main eects of the factors (A, B, C
and D), with less interest on the interactions, and if
it is likely that the interactions CD, BD, BC and
ABCD will be small, then this design could be
quite useful. However, if the focus of the research
is on interactions, then a full factorial design as
described above should be used.
FFD are generally characterized by the number
of factors being evaluated and the size of the
fraction. These two features will generally determine which eects will be aliased and which eects
will be lost. Large fractions, such as or
fractions with six or more factors, will provide
sound information on all main eects and some of
the lower order interactions since main eects and
lower order interactions are aliased with higher
order interactions, which are assumed to be negligible. As the size of the fraction becomes smaller,
main eects become aliased with lower order
interactions and information on more eects is
lost. PlackettBurman designs (PBD) are a special
type of fractional factorial where up to n)1 factors
can be evaluated in n runs and when n is a multiple
of four (Box et al., 1978). PBD have some main
eects aliased with two-way interactions and
information on a number of eects is totally lost.
215
Yet if the primary objective is to determine the
important factors to study in further experiments,
small fractions such as PBD can be quite eective
in plant cell and tissue culture research.
To the best of our knowledge, PBD and Fractional Factorial Design (FFD) have not been reported for the plant cell and tissue culture literature.
The objectives of this study are to demonstrate the
applicability of the PBD and FFD in identifying the
most important medium and treatment factors
aecting culture response in vitro with axillary buds
of two grape cultivars and to compare results from
these two dierent designs.
Experimental designs
Two dierent experiments were conducted, each
with a dierent design. One experiment was set up
as a PBD where twelve treatment combinations
were generated to examine the eects of the eight
factors according to a PBD (Table 1; Box et al.,
1978). Since the design allows up to eleven factors
to be included with twelve treatment combinations, we did not use the last three factors given in
Cultivar
BA
(mg l)1)
CuSO4 5H2O
(mg l)1)
Sequestrene
Fe source
Agar
(g l)1)
pH
Myo-inositol
(mg l)1)
Cold (h)
1
2
3
4
5
6
7
8
9
10
11
12
MN 1047
MN 1047
Chancellor
MN 1047
MN 1047
MN 1047
Chancellor
Chancellor
Chancellor
MN 1047
Chancellor
Chancellor
0.2
0.4
0.4
0.2
0.4
0.4
0.4
0.2
0.2
0.2
0.4
0.2
2.5
0.025
2.5
2.5
0.025
2.5
2.5
2.5
0.025
0.025
0.025
0.025
138
330
138
330
330
138
330
330
330
138
138
138
6
6
8
6
8
8
6
8
8
8
6
6
5
5
5
6
5
6
6
5
6
6
6
5
300
100
100
100
300
100
300
300
100
300
300
100
48
48
0
0
0
48
0
48
48
0
48
0
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
216
Table 2. Schematic illustration of fractional factorial design for 16 combinations of 8 factors (1/16 of 28 factorial combinations) at 2
levels
Run
Cultivar
BA
(mg l)1)
CuSO4 5H2O
(mg l)1)
Sequestrene Agar
Fe source
(g l)1)
pH
Myo-inositol
(mg l)1)
Cold
(h)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Chancellor
Chancellor
Chancellor
Chancellor
Chancellor
Chancellor
Chancellor
Chancellor
MN 104
MN 104
MN 104
MN 104
MN 104
MN 104
MN 104
MN 104
0.2
0.2
0.2
0.2
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.2
0.4
0.4
0.4
0.4
0.025
0.025
2.5
2.5
0.025
0.025
2.5
2.5
0.025
0.025
2.5
2.5
0.025
0.025
2.5
2.5
138
330
138
330
138
330
138
330
138
330
138
330
138
330
138
330
5
6
6
5
5
6
6
5
6
5
5
6
6
5
5
6
100
300
100
300
300
100
300
100
300
100
300
100
100
300
100
300
0
0
48
48
48
48
0
0
48
48
0
0
0
0
48
48
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
6
8
8
6
8
6
6
8
6
8
8
6
8
6
6
8
217
Results and discussion
The results obtained from the PBD and FFD were
similar. Genotype, BA and agar were found to be
the most important factors aecting culture
response (Tables 3 and 4). In general, with respect
to proliferation rates (number of shoots produced
per cultured explant), potential multiplication
rates (number of nodes produced per cultured explant) and the amount of callus produced per
cultured explant, cultivar Chancellor was superior
to cultivar MN104. The use of 0.4 mg BA l)1 or
6 g agar l)1 in the culture medium resulted in
higher number of shoot and/or nodes per cultured
explant compared to the use of 0.2 mg BA l)1 or
8 g agar l)1 (Tables 5 and 6), respectively.
The amenability of many plants to in vitro culture is reported to be genotype dependent (Henry
et al., 1994) and the eect of genotype on in vitro
culture response has been realized to the extent that
many researchers have focused on understanding
the inheritance of genes responsible for culture response (Carputo et al., 1995; Machii et al., 1998;
McLean and Nowak 1998; Hansen et al., 1999;
Ozgen et al., 2001) and selection for more responsive or even breeding donor plants for an improved
culture response (Rosati et al., 1994). Reports
indicate that the culture response may be controlled
by nuclear (McLean and Nowak 1998) and/or
cytoplasmic genes (Ozgen et al., 2001) and reorganization of nuclear and cytoplasmic genomes may
occur during the culture period (Rani et al., 2000).
The eect of cytokinins on shoot morphogenesis is a long known phenomenon (Skoog and
Miller, 1957). Cytokinins (BA) are commonly used
to promote shoot regeneration and shoot proliferation via axillary budbreak. BA in the multiplication medium increased the total number of
shoots produced per three-node explants of
strawberry tree (Mereti et al., 2002) and singleshoot explants of Cryptocoryne wendtii (Kane
et al., 1999). The balance of auxin and cytokinins
in the culture medium plays an important role in
the morphogenic fate of cultures. A relatively high
level of auxin to cytokinin favors rooting, a
low level leads to shoot formation and an intermediate level stimulate callus proliferation (Skoog
and Miller, 1957). In the current study,
0.4 mg BA l)1 resulted in higher number of shoots
compared to 0.2 mg BA l)1. Although not significant, a high ratio of IBA (0.01 mg l)1) to BA
(0.2 mg l)1) produced more amount of callus
compared to the lower ratio of IBA (0.01 mg l)1)
to BA (0.4 mg l)1) (Tables 3 and 4).
Agar is the most commonly used medium gelling agent. To determine the inuence of agar
concentration on the in vitro growth and development of Maranta leuconeura, Ebrahim and
Ibrahim (2000) added Difco-Bacto agar into the
culture medium at 0, 3, 5, 7 and 9 g l)1. With respect to growth and development at pH of 5.7,
liquid medium (control) was superior to solidied
media and, although statistically not signicant, a
decrease in explant response was observed as the
Table 3. Analysis of variance for the PBD for dependent variables: number of shoots produced per cultured explant, number of nodes
per cultured explant and callus fresh weight per cultured explant (Callus W.)
Source
Cult.
BA
Cu
Seq.
Agar
pH
Myo.
Cold
Error
DF
1
1
1
1
1
1
1
1
15
Shoot/explant
Node/explant
Callus W. explant
SS
F value
SS
F value
SS
F value
140
485
2.0
2.1
16.4
1.0
17.4
30
97.0
21.7**
74.9**
0.3
0.3
2.5
0.1
2.7
4.6*
6.5+
773
2530
41
40
215
12
10
95
390
30**
97**
1.6
1.5
8.3*
0.5
0.4
3.7*
26+
16.4
0.2
0.0
0.2
1.8
0.9
0.3
0.2
11.8
21**
0.3
0.0
0.2
2.3
1.2
0.4
0.3
0.8+
Cult., cultivar; BA, 6-benzyldenine; Cu, CuSO4 5H2O; Seq., Sequestrene Fe source; Myo., Myo-inositol; **, *: Signicant at 0.005,
0.05; +: Mean square error, respectively.
218
Table 4. Analysis of variance of the fractional factorial design for dependent variables: number of shoots produced per cultured
explant, number of nodes per cultured explant and callus fresh weight per cultured explant (Callus W.)
Source
Cult.
BA
Cu
Seq.
Agar
PH
Myo.
Cold
Cult.
Cult.
Cult.
Cult.
Cult.
Cult.
Cult.
Error
DF
BA
Cu
Seq.
Agar
pH
Myo.
Cold
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
16
Shoot/explant
Node/explant
Callus W. explant
SS
F value
SS
F value
SS
F value
71.4
572.3
15.3
27.5
78.5
2.9
0.5
25.7
32.1
34.1
0.8
1.3
38.0
22.8
6.8
106.8
10.7**
85.8**
2.3
4.1
11.8**
0.4
0.0
3.9
4.8*
5.1*
0.1
0.2
5.7*
3.4
1.0
6.7+
28.4
1912.2
32.6
3.1
186.4
20.8
2.8
2.8
12.1
0.6
1.7
12.0
117.8
5.0
61.3
249.7
1.8
122.5**
2.0
0.2
11.9**
1.3
0.1
0.1
0.7
0.0
0.1
0.7
7.5*
0.3
3.9
15.6+
18.7
1.5
0.1
0.1
0.1
0.4
0.0
0.0
5.0
1.2
0.0
1.2
1.0
1.3
0.0
10.5
28.5**
2.3
0.1
0.1
0.4
0.8
0.0
0.0
7.7*
1.8
0.0
1.9
1.7
1.9
0.0
0.6+
Alias structure of two-way interactions (assume 3-way interactions and higher zero)
Cult.
Cult.
Cult.
Cult.
Cult.
Cult.
Cult.
Cult, cultivar; BA, 6-benzyldenine; Cu, CuSO4 5H2O; Seq., sequestrene Fe source; Myo., Myo-inositol; **, *: Signicant at 0.005,
0.05; +, Mean square error, respectively.
did not, while PBD found cold to be slightly signicant (p 0.049) while FFD did not. The reasons for these dierences in results are likely due to
large error variances of the experiments. Coecients of variation ranged from approximately 30
for nodes with FFD to approximately 102 for
callus with PBD.
Due to the nature of these designs, FFD provided tests for some two-way interaction while
PBD did not. Based on FFD, Cultivar BA was
signicant for shoots/explant and callus weight;
Cultivar pH was signicant for shoots per explant and nodes/explant while Cultivar Cu was
signicant for shoots/explant. However, these
interactions should be interpreted with care since
they are aliased with other interactions (Table 4).
For example, Cultivar BA was aliased with
219
Table 5. The eects of eight factors on culture responses
obtained using PBD
Factor level
Genotype
Chancellor
MN1047
BA (mg l)1)
0.2
0.4
CuSO4 5H2O
(mg l)1)
0.025
2.5
Fe source
(6 mg Fe l)1)
Sequestrene 138
Sequestrene 330
Agar (mg l)1)
6
8
pH
5 0.02
6 0.02
Myo-inositol
(mg l)1)
100
300
Storage of explants at 4 C for
48 h
48 h
0h
Shoot
/explant
Node
/explant
6.9a
3.4b
19.7a
11.5b
1.5a
0.3b
2.0b
8.3a
8.4b
22.9a
0.9a
0.8a
5.4a
4.8a
16.7a
14.3a
0.9a
0.9a
5.4a
4.8a
14.8a
16.2a
0.8a
0.9a
5.6a
4.6a
17.4a
13.7b
0.7a
1.0a
5.0a
5.1a
16.3a
14.7a
1.0a
0.7a
4.6a
5.6a
15.3a
16.0a
1.0a
0.8a
5.8a
4.4b
16.7a
14.4b
0.8a
0.9a
Genotype
Chancellor
MN1047
BA (mg l)1)
0.2
0.4
CuSO4 5H2O (mg l)1)
0.025
2.5
Fe source (6 mg Fe l)1)
Sequestrene 138
Sequestrene 330
Agar (mg l)1)
6
8
pH
5 0.02
6 0.02
Myo-inossitol (mg l)1)
100
300
Storage of explants at
4 C for 48 h
48 h
0h
Shoot
/explant
6.2a
4.0b
14.a
12.7a
1.5a
0.5b
2.0b
8.1a
7.8b
18.8a
1.1a
0.8a
5.6a
4.6a
14.1a
12.5a
0.9a
1.0a
5.8a
4.4a
13.6a
13.0a
1.0a
1.0a
6.2a
4.0b
15.1a
11.6b
1.0a
1.1a
5.3a
4.9a
14.0a
12.7a
0.9a
1.1a
5.2a
5.0a
13.1a
13.5a
1.0a
1.0a
4.4a
5.7a
13.1a
13.5a
1.0a
1.0a
Cultivar pH) aects shoots and nodes per explants. Overall, since pH, Myo and Cold main
eects are insignicant the three factors Cultivar,
BA and Agar would appear to be the most
important factors to study further.
In the current study, that the eects of cultivar,
BA and agar concentration on the culture responses were signicant and the type of culture
responses were in agreement with previous reports
indicate that PBD and FFD can eectively be
employed to test eects of several factors with a
limited number of explants and prioritize the most
important factors. The next logical step would be
the optimization of the prioritized factors followed
by that of less signicant ones when sucient
numbers of explants become available for larger
experiments.
220
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