57: 251-257, 1991.
Kluwer Academic Publishers. Printed in the Netherlands.
tec iques for selecting wheat
for Fusarium-resistance. I. Double-layer culture technique
Kasem Z. Ahmed, A. Mesterhfizy & F. Sfigi
Cereal Research Institute, P.O. Box 391, H-6701 Szeged, Hungary
Received 25 June 1991; accepted 17 October 1991
Key words: Fusarium resistance, Fusarium spp., wheat, Triticum aestivum, double-layer technique,
in vitro selection, somaclonal variation
Calluses of spring and winter wheats (Triticum aestivum L.) were selected for Fusarium resistance in vitro,
using the double-layer culture technique. Potato-dextrose agar medium in vials was inoculated with mycelia
of Fusarium graminearum and F. culmorum. After one week, fungal cells were killed by autoclaving and the
agar medium containing the thermostable toxic metabolites was overlayered with MS callus-growing
medium. Later, wheat calluses were placed on the upper medium for 4-5 weeks, and from the surviving
calluses plants were regenerated. R2 seedling populations from self-fertilized R~ plants of 4 varieties were
tested for Fusarium resistance by artificial infections in the greenhouse, and 3% of the regenerated R2 plants
have been found to be more resistant than the original cultivars.
Fusarium spp. are serious pathogens causing seed-
ling blight, root rot and head blight in wheat and
other cereal crops worldwide. Wheat can be in-
fected by Fusarium spp. in all developmental stages
(Atanasoff, 1920; Snijders, 1987). These fungi pro-
duce a number of toxic compounds that can affect
both human health and animal productivity (Aust-
wick, 1984; Wang & Miller, 1988). Breeding for
Fusarium resistance is difficult, although efficient
minor resistance genes are known (Yu, 1982; Go-
cho, 1985; Mesterh~y, 1989). Mielke (1988)found
no absolute resistance or tolerance in wheat to F.
culmorum. Immunity was also not found, but this
does not mean that highly significant differences in
resistance and tolerance do not exist (Mesterhhzy,
1989; Snijders, 1990). Therefore, in vitro selection
of somaclonal variants insensitive to toxic metabo-
lites produced by these pathogens seems to be a
viable approach of obtaining resistant or tolerant
plants as experienced already by various authors on
different plant species (Daub, 1984; Hartman et
al., 1984; Wenzel, 1985; Arcioni et al., 1987; Chaw-
la & Wenzel, 1987; Pauly et al., 1987; Latunde-
Dada & Lucas, 1988; Binarov~i et al., 1990). In
most cases the toxin resistance expressed in the
regenerated plants correlated with the level of their
disease resistance. Furthermore, the resistance has
been transmitted to the progeny of selected plants
in all investigated cases (Daub, 1986).
This paper describes the in vitro selection of
wheat calluses for insensitivity to toxic metabolites
of F. graminearum and F. culmorum via a double-
layer culture technique, and the results of testing
the R 2 seedlings for Fusarium resistance.
Fig. 1. The double-layer culture. From left to right: double-layer
callus culture without Fusarium, the same with Fusarium and
single-layer callus culture on MS medium
Materials and methods
a. Plant materials and callus cultures
Callus cultures of 9 spring and winter wheat (Trit-
icum aestivum L.) cultivars (Lerma Rojo 64, Sakha
8, Sakha 69, Siete Cerros, Tobari 66 and GK
Bence, GK Kincs6, GK Mini Man6, GK S~igv~iri,
respectively) were established from immature in-
florescences, immature and mature embryos on
solidified MS medium (Murashige & Skoog, 1962)
supplemented with 1 or 2 mg/L 2,4-D, or meso-
cotyls in 2,4-D solution (5-8 mg/L, Bart6k & S~igi,
1990). Cultures were grown at 26°C in darkness.
b. Double-layer culture technique
The double layer technique classically utilized in
microbiology (Lepoivre et al., 1986) was modified
as follows: vials containing 33 ml of potato-dex-
trose agar medium (consisting of extract of 200 g
potato tubers, 20 g glucose and 7 g agar per liter,
thickness: 12 mm), were inoculated with mycelium
of F. graminearum (isolate No. 12216) or F. culmo-
rum (isolates No. 12375 or 12551). These cultures
were grown at 26 ° C for 4 days in a 16/8 h light-dark
cycle, then at 5°C for 3 days in continuous light.
Subsequently, the vials were autoclaved at 120°C
for 15 min to kill the fungal cells (Joffe, 1974). After
2-3 h the cooled agar medium containing the ther-
mostable toxic compounds (Patey & Gilbert, 1989)
was overlayered with 33 ml of MS callus-growing
medium supplemented with lmg/L 2,4-D (thick-
ness: 12 mm). After diffusion of the toxic materials
from the fungal culture into the upper nutrient
layer (7 days), 5-weeks-old wheat calluses were
placed on the upper medium (5 calluses/vial) for
4-5 weeks at 26°C in 16 h photoperiods (Fig. 1),
and toxin-sensitivity was assessed on the basis of
callus weight or necrosis rating. The surviving cal-
luses were transferred to MS regeneration medium
(0 or 0.1mg/L 2,4-D added). The regenerated
plants (R0) were transplanted in pots and grown to
maturity in the greenhouse.
c. Evaluation of
plants for resistance
The 2nd selfed generation (R2 progeny) of Sakha
69, Sakha 8, Lerma Rojo 64 and Tobari 66 was
evaluated for reaction to F. graminearum and F.
culmorum separately by a greenhouse seedling test
based upon seed germination, number of killed
plants, plant height and dry matter production cal-
culated for 60 seeds, each in 6 replication according
to Mesterh~izy (1978). Two resistant genotypes
(74-2, 84-42) and the original cultivars were used as
Results and discussion
An initial test indicated that callus growth on the
upper medium layer was strongly inhibited (Fig. 2).
Effect of Fusarium toxins on callus weight of the
susceptible variety GK S~igv~iri was assessed after 4
weeks and the level of inhibition is given in Table 1.
Majority of the calluses ceased growth, turned
brown and died within 6 weeks after being trans-
ferred on the upper layer, similarly to the calluses
of other varieties. Consequently, the double-layer
technique seems to be suitable for selection of
wheat calluses resistant to toxic Fusarium metabo-
lites. Table 2 demonstrates the growth of calluses
on toxic medium as affected by the genotype. Most
calluses of the resistant GK Bence, the susceptible
GK S~igv~iri and of the very susceptible GK Mini
Man6 exhibited good early growth on the upper
layer, independently on Fusarium spp., while cal-
luses of the intermediate GK Kincs6 and Sakha 8
tolerated F. graminearum and those of the resistant
Sakha 69 F. culmorum isolate No. 12551 only. Dou-
ble-layer callus cultures without the fungus (con-
trols) exhibited maximum growth in all cases. The
different Fusarium-sensitivity of the varieties can
probably influence their in vitro selection for resist-
ance. Dependence of in vitro selection results on
the genotype has been mentioned by Chawla &
Wenzel (1987) and M6gn6gneau & Branchard
(1988) also. However, somaclonal variation may
have an effect, too (Daub, 1986; Latunde-Dada &
Lucas, 1988; Kaleikau et al., 1989).
Regenerating ability of the resistant calluses
from all genotypes was lower than that of the unse-
lected calluses as found by Arcioni et al. (1987) as
well. From the 9 genotypes tested, plants were
regenerated from in vitro selected calluses of 5
only, i.e. Lerma Rojo 64, Sakha 8, Sakha 69, Toba-
ri 66 and Siete Cerros (Table 2), mostly from imma-
ture inflorescences and young embryo-derived cal-
luses. In the cultivars GK Kincs6, GK Bence, GK
Mini Man6 and GK S~igv~iri no plants were ob-
tained from selected calluses. Toxin treatment re-
duced the regenerative capacity of calluses in the
experiments of Latunde-Dada & Lucas (1988) also.
Changes of fresh weight of mesocotyl-derived calluses
of GK S~gv~iri wheat in the double-layer technique after 4 weeks
with and without
Callus weight, g
MS medium Double-layer
InitiaP 1.18 1.10 1.31
After 4 weeks ~ 1.73 1.48 I. 11
Difference h 0.55 0.38 - 0.20
Difference in per cent 46.61 34.55 - 15.27
"Total fresh weight of 30 calluses each. bDifferences between
double-layer calluses with
and both control
calluses are significant, but between control I and control II
calluses are not significant (T-test, P0.1)
Wheat calluses kept for 4 weeks on MS medium (upper
two rows) and calluses grown in double-layer culture with
(lower three rows)
Plant regeneration in cereals is known to be geno-
type and explant specific (Rengel, 1987; Kaleikau
et al., 1989), and its seems that callus growth and
plant regenerating ability is not equally sensitive to
the toxic Fusarium-metabolites. Reaction of R2
progeny to F. graminearum and F. culmorum in the
seedling test revealed that large variation exists
within and between the lines from selected and
unselected callus cultures in most resistance par-
ameters, for instance 0 to 71.6% (Sakha 8, shoot
length) or 8.3 to 78.9% (Sakha 69, germination,
Table 3). Resistance of the spring wheat cultivar
Sakha 69 is equal to or better than that of the
reference cultivars. On this basis, Sakha 8 can be
regarded as less resistant, and Lerma Rojo 64 and
Tobari 66 as susceptible. Resistance differences
between lines from selected and unselected callus
cultures, on the one hand, and between lines from
selected calluses and the original varieties on the
other hand, are significant in many cases (Table 3).
Mean comparisons demonstrated that one unse-
lected R~ line of Tobari 66 was significantly more
resistant than the original cultivar. This superior
somaclone (F2/18-B) had a resistance similar to
that of the genotypes 74-2 and 84-42 (Tables 3, 4,
Fig. 3). However, many R2 lines were significantly
more susceptible than the cultivars from which they
were derived. In an R2 wheat population not select-
previously, Maddock & Semple (1986)
also found a line with improved
Complete resistance in R2 tomato plants was ob-
tained from non-selected callus tissue to bacterial
wilt, too (Toyoda et al., 1989).
Resistance differences of R2 lines derived from
selected and unselected calluses seemingly does
Callus growth and plant regeneration of various wheat genotypes in the double-layer culture technique
spp. and isolate Genotype Explant Total no. of calluses Number of calluses Number of regenerants
N S M G
7C I 15 5 5 3 2
No. 12216 LR YE 16 5 2 0 9 8
T66 YE 12 6 1 5 0
K6 ME 61 32 9 4 16
$8 I 16 0 0 2 14 8
$69 I 18 8 7 3 0 1
B ME 36 0 10 8 18
MM ME 12 4 2 1 5
SV ME 16 0 0 2 14
LR I 21 16 2 3 0 3
No. 12375 T66 I, YE 12 7 2 1 2 1
K6 ME 48 35 11 0 2
$8 I 17 14 3 0 0
$69 I 18 15 0 3 0 3
B ME 24 0 3 0 21
MM ME 30 3 0 4 23
SV ME 16 0 0 2 14
7C I 13 12 0 0 1 1
No. 12551 LR YE 13 0 9 4 0
T66 I, YE 14 8 3 1 2 1
K6 ME 60 50 4 6 0
$8 I 12 6 6 0 0
$69 I 24 7 5 6 6 4
B ME 36 1 2 13 20
MM ME 24 5 18 1 0
SV ME 12 0 0 6 6
Control LR I 10 0 0 0 10 2
T66 YE 15 0 0 0 15 5
K6 ME 30 0 12 4 14
$8 I 12 0 0 0 12 6
$69 I 18 2 1 0 15 9
B ME 18 0 0 1 17
MM ME 22 2 3 6 11
SV ME 16 0 0 3 13
Total: 737 243 120 92 282 52
Abbreviations. Genotypes: 7C = Siete Cerros, LR = Lerma Rojo 64, T66 = Tobari 66. K6 = GK Kincs6, $8 = Sakha 8, $69 = Sakha
69, B = GK Bence, MM = GK Mini Man6, SV = GK S~gv~ri. Explants: I = immature inflorescence, YE = immature embryo, ME =
mature embryo. Callus growth: N = no growth, S -- slight growth, M = moderate growth, G = good growth
not depend on the resistance of the genotype.
However, since the
processes can also be
involved, induced and non-induced somaclonal
variation can or cannot be of similar value for re-
spp. Because of partial
or complete sterility, R2 lines of Siete Cerros, those
of Lerma Rojo 64 from unselected calluses and of
Tobari 66 from selected calluses could not be in-
cluded into this comparison.
As summarized in Table 4, from the 30 R2 lines
tested, only one (Tobari 66, F2/18-B, unselected,
3%) showed an improved resistance over the origi-
nal cultivar, 9 (30%) possessed a similar character
and 20 (66,7%) proved to be more susceptible.
Reaction of R: plants selected by the double-layer culture technique and from unselected calluses as compared to that of the
original genotypes to
in the seedling test. Data are averages presented as % of the controls
Cultivar Original cv. or R2 line Isolate Germination % Mortality % Shoot length % Dry matter %
Sakha 69 original cv. 97.5 17.5 94.2 92.5
sel. F7/1-A F.g.12216 78.9 44.7 76.4 58.3
F8/38-B F.c.12551 69.5 38.9 73.1 71.3
F8/2-B F.g.12216 52.6 57.9 46.8 46.4
F4/9-B F.c.12375 44.5 72.2 64.5 39.3
F8/2-C F.g. 12216 35.3 70.6 32.4 44.3
F8/2-A F.g.12216 20.0 85.0 15.0 15.3
F4/9-A F.c. 12375 8.3 91.7 16.6 11.7
F8/38-A F.c. 12551 12.0 93.8 15.7 10.0
unsel. F4/23-H 80.0 35.0 97.5 86.0
F4/23-F 23.4 80.0 18.5 13.7
F4/23-I 20.6 82.4 17.8 31.7
F4/23-C 20.6 88.2 16.5 9.3
Sakha 8 original cv. 82.5 52.5 94.4 72.7
sel. F8/4-A F.g.12216 47.1 52.9 71.6 61.5
F8/4-B F.g. 12216 55.6 55.6 53.3 42.1
F8/10-A F.g.12216 27.8 80.6 36.7 31.5
F8/10-B F.g.12216 21.1 84.2 18.5 16.4
F8/20-A F.c. 12375 6.3 93.8 6.0 6.5
F4/3-A F.g. 12216 0.0 100.0 0.0 0.0
F8/10-C F.g. 12216 0.0 100.0 0.0 0.0
unsel. F8/48-B 9.4 96.9 6.0 1.7
F8/48-A 6.3 96.9 0.9 1.7
F4/22-D 0.0 100.0 0.0 0.0
F4/22-C 0.0 100.0 0.0 0.0
Lerma Rojo original cv. 13.3 93.4 7.3 4.2
64 sel. F2/8-B F.c.12375 33.3 72.2 28.0 30.6
F2/8-D F.c.12375 25.0 75.0 29.8 30.8
F2/3-A F.g.12216 23.6 85.3 22.0 35.7
F2/3-C F.g.12216 7.9 92.1 3.5 8.6
F2/8-C F.c. 12375 0.0 100.0 0.0 0.0
Tobari 66 original cv. 32.2 75.0 22.7 23.0
unsel. F2/18-B 73.7 32.9 94.1 103.3
F2/17-A 31.6 71.1 30.9 35.6
74-2 reference cv. 92.5 12.5 77.2 91.7
84-42 92.5 15.0 120.2 90.2
LSD 5% 23.3 24.1 33.2 31.5
Disease reaction of regenerated R2 lines selected by the double-layer culture technique for resistance to
as compared to that of the original cultivars (based upon the significant differences in Table 3)
Cultivar Type a R2 line No. of lines Disease reaction
similar susceptible resistant
Sakha 69 R selected 8 1 7 0
unselected 4 1 3 0
total 12 2 10 0
Sakha 8 R/I selected 7 1 6 0
unselected 4 0 4 0
total 11 1 10 0
Lerma Rojo S selected 5 5 0 0
64 unselected b ....
Tobari 66 S selected u ....
unselected 2 1 0 1
gross total 30 9 20 1
% 100 30.0 66.7 3.3
a Disease reaction, compared to that of the reference cultivars. R = resistant, R/I = resistant/intermedier, S = susceptible, b Sterile or
semisterile regenerants, not tested
Thus, the double-layer culture technique seems to
be an useful method of selecting
ant calluses of wheat and is definitely cheaper than
selection methods using commercially
toxins. Nevertheless, its effec-
tivity does not reflect in the number of resistant
regenerants. It is possible that the
do not play a significant role in the blight pathoge-
nesis, the resistance at seedling stage will be lost
during regeneration or has not been transmitted to
the progeny. By means of
fusaric acid-resistance, Wenzel & Foroughi-Wehr
(1990) obtained barley plants resistant to fusaric
acid, but their
The senior author (K.Z.A.) acknowledges with
thanks the scholarship offered by the Hungarian
Academy of Sciences, Mrs. H. Lomniczi and Mrs.
F. Elawadi for their kind help in the experimental
Fig. 3. Fusarium
resistance of Tobari 66 control and R2 wheat
seedlings derived from unselected calluses. From left to the
right: Tobari 66, F2/18-B (controls), Tobari
66 + F. graminea-
+ F. graminearum,
+ F. culmorum,
+ F. culmorurn
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