In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Article (PDF Available)inEuphytica 91(3):341-349 · May 1996with 122 Reads
DOI: 10.1007/BF00033096
Abstract
Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R3. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R0) than the non-selected ones. Among 18 R1 lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R3 lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R3 selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.
Euphytica
57: 251-257, 1991.
© 1991
Kluwer Academic Publishers. Printed in the Netherlands.
In vitro
tec iques for selecting wheat
(Triticum aestivum
L.)
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
Summary
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.
Introduction
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.
252
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
R 2
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
controls.
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.
Table 1.
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
F. graminearum
Callus weight, g
MS medium Double-layer
(control I)
without with
F. graminearum
(control II)
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
F. graminearum
and both control
calluses are significant, but between control I and control II
calluses are not significant (T-test, P0.1)
253
Fig. 2.
Wheat calluses kept for 4 weeks on MS medium (upper
two rows) and calluses grown in double-layer culture with
Fusa-
rium
(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
254
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-
ed
in vitro
previously, Maddock & Semple (1986)
also found a line with improved
Septoria
resistance.
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
Table 2.
Callus growth and plant regeneration of various wheat genotypes in the double-layer culture technique
Fusarium
spp. and isolate Genotype Explant Total no. of calluses Number of calluses Number of regenerants
N S M G
F. graminearum
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
F. culmorum
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
F. culmorum
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
in vitro
processes can also be
involved, induced and non-induced somaclonal
variation can or cannot be of similar value for re-
sistance against
Fusarium
spp. Because of partial
or complete sterility, R2 lines of Siete Cerros, those
of Lerma Rojo 64 from unselected calluses and of
255
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.
Table 3.
Reaction of R: plants selected by the double-layer culture technique and from unselected calluses as compared to that of the
original genotypes to
F. graminearum
and
F. culmorurn
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
256
Table 4.
Disease reaction of regenerated R2 lines selected by the double-layer culture technique for resistance to
F. graminearurn
and F.
culmorum
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
Fusarium-resist-
ant calluses of wheat and is definitely cheaper than
the
in vitro
selection methods using commercially
available
Fusarium
toxins. Nevertheless, its effec-
tivity does not reflect in the number of resistant
regenerants. It is possible that the
Fusarium
toxins
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
in vitro
selection for
fusaric acid-resistance, Wenzel & Foroughi-Wehr
(1990) obtained barley plants resistant to fusaric
acid, but their
Fusarium-resistance
was not
checked.
Acknowledgements
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
work.
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-
rum,
F2/18-B
+ F. graminearum,
Tobari 66
+ F. culmorum,
F2/18-B
+ F. culmorurn
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Supplementary resources

  • ... The tissue culture technique is the most widely applied method for improvement of plant genotype tolerance to abiotic stress such as drought, high salinity, heavy metal toxicity, acid soil, as well as biotic stress herbicides and disease through in vitro selection of somaclonal variants (Ahmed et al., 1996;Jain et al., 1998;Yusnita et al., 2005). The selection agent in the culture media brings about a number of physiological, biochemical, metabolic and genetic changes in the cells of explanted tissues, callus and regenerated shoots resulting in the somaclonal variations (Biswas et al., 2002). ...
    ... In vitro selection of heat-tolerant clonal lines from tussock plantlets and callus of Senecio cruentus was carried out. The results showed that [1] 40ºC for 16- 20 h and 30 mmol·L -1 HYP were optimal direct and indirect selection stress pressure, respectively; [2] Two heat-tolerant clones (Z 1-1-1 and N 1-2-1 ) were obtained by using high temperature direct selection, HYP and high temperature selection; [3] The regenerated plants of Z 1-1-1 were different significantly from their mother plants(Z 1 ) in leaf thickness, TPT/TST and stomata index. The fruit setting rate of adult plant of Z 1-1-1 was only 1.0 per cent after artificial pollination, and the fruit setting rate of Z 1 was over 85 per cent, [4]After heat stress (40ºC 24 h), the heat injury index, content of MDA and electrolyte leakage of the regenerated plants of Z 1-1-1 and N 1-2-1 were lower than their mother plants, but the activity of POD and SOD were higher than their mother plants (Z 1 and N 1 ). ...
    ... In vitro selection for acid soil and Al toxicity tolerance could be applied with AlCl 3 .6H 2 O as the selection agent on the low acid media as much as pH 4 (Short et al., 1987). The Al toxicity to selection media could be emerged by modifying macro nutrient MS, i.e. by increasing NH 4 NO 3 , CaCl 2 .2H 2 O and decreasing KH 2 PO 4 and the application of Fe which was not chelated by EDTA (Purnamaningsih et al., 2001). ...
    Chapter
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    The food grain production needs to be enhanced constantly to feed the increasing World population on a decreasing fertility of cultivable land under environmental stresses. Both abiotic and biotic stresses are the major threat to agriculture productivity. Sexual hybridization, induced mutation and genetic transformation approaches have demonstrated success in developing stress tolerance in the limited plant species. In the past two decades, cell and tissue culture based in vitro selection system has emerged as a simple, fast and economically feasible tool for developing stress-tolerant plants. The method exploits the capability to manipulate the variation for obtaining the desired characteristic of plant to the expected result. Therefore, by applying the selection agent into the media, plant tolerance to both abiotic and biotic stresses could be acquired. Chemical reagents such as NaCl (for salt tolerance), PEG and mannitol (for drought tolerance, osmotic stress tolerance), A1Cl3.6H2O (for Al tolerance) have been incorporated in the tissue culture media as selection agents. Only the explants capable of sustaining such environments survive in the long run and are selected. In vitro selection is based on the induction of genetic variation among cells, tissues and/or organs in cultured and regenerated plants. The selection of somaclonal variations appearing in the regenerated plants may be genetically stable and useful in crop improvement. This chapter describes a common strategy for regeneration of plants tolerant to various abiotic environmental stresses through tissue culture based in vitro selection methods. Characterization of the selected callus line, determination of callus growth rate, callus survival efficiency and frequency of regeneration in response to selection agents in the medium has been presented. Plants have evolved many physiological, biochemical and molecular defense mechanisms to survive under stress conditions by bringing certain structural and functional changes. Further, the methods for characterization of in vitro selected plants for these structural and functional changes in response to stress condition has been described for the measurements of stomatal dynamics, photosynthetic assay, leaf water loss, leaf relative water content, chlorophyll content, assays for carbohydrates, proteins, proline, ABA, glycine betain, electrolytic leakage, malondialdehyde, determination of elements such as sodium, potassium, calcium and phosphorus, detection of reactive oxygen species such as superoxide anion and hydrogen peroxide content, antioxidant enzyme activity for superoxide dismutase, gtutathione reductase, peroxidase and catalase,. Procedure for screening of salinity and drought tolerance, plantlet growth and seed germination assays has been described for greenhouse and field growing plants. The common mechanisms of tolerance and the success of in vitro selection methods for drought, salinity, oxidative, osmotic, low and high temperature, and Al toxicity stress tolerance have also been reviewed. The chapter is concluded with suggestion for the application of in vitro method to select plants tolerant to abiotic environmental stresses for improvement of various crop species in conjunction with the molecular biological approaches and functional genomics and proteomics for future research.
  • ... Since the late 1970s, the process of in vitro selection has been applied to several cell culture systems, to generate mutants with useful agronomic traits, such as disease resistance (Jayasankar and Gray, 2003). In vitro selection was considered as a supplementary tool to the classical selection in breeding disease resistant cultivars, because of several advantages, such as faster testing of large numbers of individuals in a small space, easier manipulation of mutants, and the production of somaclones and haploids with higher genome variability (Ahmed et al., 1996). The objective of this study was to establish a suitable tissue culture protocol for plant regeneration and in vitro selection of resistance to Allium white rot (Sclerotium cepivorum) in commercial Egyptian onion varieties. ...
    ... Since the late 1970s, the process of in vitro selection has been applied to several cell culture systems, to generate mutants with useful agronomic traits, such as disease resistance ( Jayasankar and Gray, 2003). In vitro selection was considered as a supplementary tool to the classical selection in breeding disease resistant cultivars, because of several advantages, such as faster testing of large numbers of individuals in a small space, easier manipulation of mutants, and the production of somaclones and haploids with higher genome variability ( Ahmed et al., 1996). The objective of this study was to establish a suitable tissue culture protocol for plant regeneration and in vitro selection of resistance to Allium white rot (Sclerotium cepivorum) in commercial Egyptian onion varieties. ...
    Article
    Full-text available
    In vitro selection is one of the most effective and efficient techniques for plant improvement. This is due to its ability to isolate plants with the desired character(s), either by applying a selection agent on the culture media to drive the selection of somaclones with the required character(s), or by establishing particular conditions that change in the genomes of somaclones toward the required character. The objective of this study was to identify a suitable protocol for in vitro selection of Allium white rot disease (Sclerotium cepivorum) tolerance in commercial Egyptian onion varieties, namely Giza 20, Giza 6 and Beheri Red. Oxalic acid (OA), the phytotoxin produced by Sclerotium cepivorum, was used as the selective agent. Seeds of the three Egyptian varieties were germinated on four concentrations (0.0, 0.02, 0.2, 2 and 20 mM) of Oxalic acid. Among the tested cultivars, Beheri Red had the highest germination frequency (52%) at all concentrations tested, followed by Giza 20 (42.6%), and Giza 6 at (32%). Cotyledon explants from the varieties were cultured on toxic MSBDK medium, supplemented with 0, 3, 6 and 12 mM OA. The survival of calli on MSBDK free toxic medium was 70.7% for all tested cultivars; however, MSBDK-stressed medium, with 3 mM OA reduced the viable calli to 42.1%. The highest OA concentration (12 mM) completely inhibited calli induction from cotyledons explants. A medium supplement with 3 mM OA retarded 80% of calli growth. Among 156 tested calli of Beheri Red, only 23 calli (14.7%) survived on toxic medium for 45 days. Similarly, there was 15.6% survival for Giza 20 calli, while 40.1% of the Giza 6 calli survived. Plantlets were regenerated from surviving calli and transplanted onto ex vitro, and formed bulb after acclimatisation. Key Words: Allium cepa, oxalic acid, Sclerotium cepivorum
  • ... Gengenbach & Green (1975) produced maize calli resistant to Helminthosporium maydis, the first report of the use of an in vitro protocol to develop disease resistant lines. Since that report, in vitro selection technologies had been applied to several cell culture systems to generate mutants with useful agronomic traits such as disease resistance. Ahmed et al. (1996) successfully regenerated resistant wheat plants against Fusarium fungi and when the R 3 selfed generations were tested in the field, 35.7% of the lines were found to be more resistant than the original cultivars. However, the promise of genetic engineering technology and some early failures of some in vitro selected plants stifled resea ...
    Conference Paper
    Full-text available
    In vitro selection is one of the most effective and efficient methods for plant improvement. This is due to its ability to isolate plants with the desired character(s), either by applying a selection agent on the culture media to drive the selection of somaclones with the required character(s) or by establishing particular conditions that favor a change in the genomes of somaclones toward the required character(s). In the present work, a suitable protocol for in vitro selection for Allium white rot disease (Sclerotium cepivorum) tolerance in several commercial Egyptian onion varieties (Giza 20, Giza 6 and Beheri Red) was established. Oxalic acid (the phytotoxin produced by Sclerotium cepivorum) was used as selective agent. Seed of the 3 different Egyptian varieties were germinated on four concentrations (0.0, 0.02, 0.2, 2 and 20 mM) of Oxalic acid (OA). Among tested genotypes, Beheri Red had the highest germination frequency (60%) at all concentrations tested, followed by Giza 20 (58.2%), and Giza 6 at (53.3%). The response of onion explants to OA were also studied. Cotyledon explants from the 3 Egyptian varieties were cultured on toxic MS BDK medium supplemented with 0, 3, 6 and 12 mM OA. The mean percentage survival of calli on MS BDK free toxic medium was 70.7 % for all tested onion genotypes, however, MS BDK stressed medium with 3 mM OA reduced the percentage of viable calli to 42.1%, and the highest OA concentration (12 mM) completely inhibited calli induction from cotyledons explants. Toxicity of OA to onion calli was calculated as an LD 80 where a medium supplement with 3 mM OA retarded 80% of calli growth which delivered a suitable in vitro selection protocol. Among 156 tested calli of Beheri Red only 23 calli (14.7%) survived on toxic medium for 45 days. Similarly there was 15.6% survival for Giza 20 calli, while 40.1% of the Giza 6 calli survived. Plantlets were regenerated from surviving calli and transplanted to ex vitro and formed bulb after acclimatization. The results presented here offer a useful method for the production of germplasm with potential resistance to white rot disease, an important pathogen for onion production in Egypt.
  • ... Plant improvement through somaclonal variation and in vitro selection is some techniques of in vitro culture for obtaining plant genotype tolerance to the biotic or abiotic stress, such as drought, high salinity, Al stress, acid soil, and disease tolerance (Ahmed et al., 1996; Yusnita et al., 2005). In addition, the plants are expected to have some desired characters such as having bigger fruit size, more interesting flower texture, more delicious taste and higher production (Pedrieri, 2001; Ahloowalia and Maluszynski, 2001; Witjaksono, 2003). ...
    Article
    Full-text available
    As an alternative technology, plant improvement through somaclonal variation is expected to support conventional breeding. New superior variants with a better performance and more attractive texture could be obtained through this method. To enhance genetic variation, both physical and chemical treatments such as gamma ray (Co 60) and Ethyl Methane Sulphonate (EMS) compound could be applied. In particular for vegetative propagated plants, in vitro induced mutation is the most effective method to improve variation. For obtaining the desired characteristic of plant, in vitro selection is the best method due to its capability to manipulate the variation to the expected result. Therefore, by applying the selection agent to the media, plant tolerance to both abiotic and biotic could be acquired. Generally, the tolerance at the callus level at the specific selection agent is positively correlated with the tolerance at the plant level. At this point, PEG (polyethylene glycol) and manitol is chemical compound useful for drought tolerance, fusaric or filtrate is for fusarium wilt, A1Cl 3. 6H 2 O is for Al tolerance.
  • ... Bouizgarne et al. (2004, 2006) reported on the differential sensitivity of the diseaseresistant and disease-susceptible cultivars of date palm, which could be attributed to the differential sensitivity of H + ATPases to FA, because nanomolar concentrations of FA induced an early transient membrane hyperpolarization of root hairs. Accordingly, there were successful attempts to obtain tolerant plants by in vitro selection with FA or culture filtrate of Fusarium (Chawla and Wenzel 1987; Matsumoto et al. 1995, Ahmed et al. 1996; Sva´bovaánd Lebeda 2005). On the other hand, Wenzel and Foroughi- Wehr (1990) could not gain significant improvement in disease resistance by in vitro selection. ...
    Article
    Fusarium head blight and rot root are among the most devastating plant diseases in modern agriculture. The causal pathogen, Fusarium spp., reduces plant yield and food quality in part because of mycotoxins, suggesting that breeding for resistance to Fusarium is an important control strategy. A simple and low-cost tactic in plant resistance breeding is testing the cultivars for their sensitivity to fungal metabolites and secretion products. We analysed barley cultivars with differential resistance to Fusarium culmorum KF350 for their sensitivity to 5-butylpicolinic acid [syn. fusaric acid (FA)], a product synthesized by Fusarium isolates of the Liseola section of the Gibberella fujikuroi species complex. We found similar sensitivity of first and second leaves of the cultivars to KF350 and to FA, as well as to head blight in the literature. Upon FA treatment, more resistant cultivars, Jolante and Chevron, compared with susceptible cultivars, Carola and Uschi, showed less necrosis and ion leakage in primary leaves and roots, suggesting that FA can possibly be used in breeding programs as a screening compound for improved cultivars. Importantly, sensitivity to H2O2 was not correlated with resistance to KF350 although it accumulated at penetration sites and in lesions associated with Fusarium infection in all cultivars.
  • Chapter
    Resistance to Fusarium head blight appears to be long lasting, race and species nonspecific, and durable. In the 1990s Fusarium head blight has reemerged and devastated millions of acres of wheat and barley. This disaster increased research efforts to control this disease with numerous papers published. One goal was to determine the role of breeding in decreasing mycotoxin contamination. This chapter evaluates how the breeding process should be modified to reach this goal, and which methodical approaches seem to be most suitable. There are several important conditions for successful breeding. One of them is without significant differences in resistance, no method will yield good results. Additionally, reliable inoculation methods are essential, so that differences in resistance can be replicated and consistently observed. The method used depends on the purpose of the evaluation.
  • Chapter
    Genetic modifications using contemporary biotechnological tools hold immense potential for improvement of the quality of crops. The copious paraphernalia of genetic engineering has been adopted to identify functions of various genes with an eventual aim of crop improvement. Among these, RNA interference (RNAi), a double-stranded RNA (dsRNA)-based gene silencing technology, serves as an extending platform for genetic interventions intended at the improvement of crops, by modifying these for important agronomical traits. RNAi is a gene silencing process initiated by the presence of dsRNA molecules which are ulti- mately responsible for complementary mRNA-specific degradation through a series of steps. Two important enzymatic complexes of RNAi are named as Dicer and RNA-induced silencing complex (RISC). Dicer is a multidomain enzyme complex belonging to ribonuclease (RNase) III class. It plays an important role in the cleaving of dsRNA to produce small interfering RNA molecules. Another complex, RISC, is composed of Argonaute proteins, which uses interfering RNA as guide molecules for complementary mRNA degradation. A huge number of practical applications of RNAi technology reflect the usefulness and importance for the crop improvement. This review focuses on the RNA interference (RNAi) technology and its impending role in the crop improvement.
  • Article
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    Toxins are compounds that are produced by the pathogens and cause part or all of the symptoms of a disease. Genetic and biochemical studies revealed that at least in part of the plant-pathogen interactions toxins are the determinants of specificity. In such cases, resistance or susceptibility to the fungus correlates with insensitivity or sensitivity to the toxin. Based oh these studies, the utilization of phytotoxins as selective agents in in vitro selection for improved disease resistance is reviewed in this study. The principal steps in establishing the in vitro screening scheme are indicated. Practical applications of this method are pointed out in an attempt to summarize some of the successful achievements in ABI. The potential of in vitro culturing is discussed as an alternative strategy for breeding disease resistant lines and cultivars In addition some examples of transgenic plants in which introduced transgene activates detoxification of the pathogen toxin or avoiding its destroyed function are considered.
  • Article
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    Fusarium oxysporum Link is the cause of basal rotting in cardamom. The present article evaluates the phytotoxical effect of of the fungus crude filtrate,for selecting somaclonal variations of cardamom that showed resistance in greenhouse pathogenicity tests. According to mortality test results clone 5 and the isolated 4 were pointed with the most susceptibility-virulence interaction. From accelerated growth media seedlings there were selected 25 and inoculated with different concentrations of filtrate, causing mortality on the initial weeks. By the sixth week only a 32% resisted the concentration increment. By the tenth cycle of inoculation 100% of seedlings remained in the crude filtrate without dilutions. The more time the seedlings remain in accelerated growth media and the more selection pressure increasing filtrate concentration show increased probability of getting somaclonal variants with in vitro resistance of Fusarium oxysporum.