The onion (Allium cepa L.) is one of the important crops grown
in India amongst vegetables and spices. At present, breeding for resistance
to biotic and abiotic stresses is a high priority. Conventional breeding
programmes including extensive intermating and screening campaign helps
breeder to improve cultivars, however, this is limited by inherent difficulties,
open pollination, high level of heterozygosity and poor fertility of F1
hybrids. Moreover, unavailability of disease/pest resistance genes in
gene pool makes breeder task more difficult. In vitro selection
against disease causing phytotoxin offers an alternative means of screening
disease resistant cell lines.
In vitro selection offers an immense potential for the quick and
comprehensive generation of useful somaclones or mutants for resistance
against various biotic and abiotic factors. These plants may serve as
an excellent donor of the resistance gene(s) in breeding programmes. Selection
in plant cell cultures using pathotoxins as a screening agent for resistance
have been reported in Brassica napus to Phoma lignum (Sacristan
1985), in potato to Phytophthora infestans (Behnke 1979)
and Fusarium oxysporum (Behnke 1980, Schuchmann 1985), in tobacco
to Pseudomonas syringae and Alternaria alternata (Thanutong
et al., 1983), in alfalfa to Fusarium oxysporum (Hartman
et al., 1984), in maize to culture filtrate of Helminthosporium
maydis (Gengenbach and Green 1975) and in groundnut (Venkatachalam
et al., 1998, Ashok et al., 2004).
In practice involvement of in vitro selection techniques in the
crop improvement programme is very limited. Except for few, most of the
reports suggest negative correlation between in vitro and in
vivo responses for resistance. Reason for this lies with several factors
such as plant species, genotypes, source of the explant and nutritional
and environmental factors. Moreover, expression of resistance or susceptibility
may require specific cell types/tissue arrangements or physiological function
not present in the cultures.
Although, a few studies have been conducted to obtain prolific in
vitro selection, no work has been reported so far on onion in the
world. During the present investigations an effort has been made for selection
against purple blotch disease of onion.
MATERIALS AND METHODS
Plant material: Two purple blotch susceptible onion cultivars
namely Agrifound Dark Red (ADR) and Agrifound Light Red (ALR) were selected
to carry out the present study. An efficient in vitro plant regeneration
protocol from callus and cell suspension cultures were optimized before
conducting the in vitro cell line selection experiments using toxic
culture filtrate. Callus and suspension cultures were established from
mature embryos (Tiwari et al., 2004, 2007).
For raising embryogenic callus and suspension culture a series of growth
regulators were supplemented to basal MS (Murashige and Skoog, 1962) medium.
All culture media combinations were short-listed on the basis of previous
work conducted by various scientists and preliminary experiment of this
laboratory. The basal MS media supplemented with different concentrations
of plant growth regulators in various combinations (NAA, 2,4-D, picloram,
kinetin, zeatin, TDZ and BA alone as well as kinetin, zeatin, TDZ and
BA in combination with NAA, 2,4-D and picloram). Unless specified, all
the media contained MS macro and micronutrients, vitamins, 30.0 g L-1
sucrose and 7.5 g L-1 agar only for semi-solid medium. All
culture medium were autoclaved at 121°C under 1.1 kg cm-2
for 20 min after adjusting the pH to 5.6±0.1 with 1N KOH. MS basal
medium and all other add-ons were procured from HiMedia†7 Laboratories,
For callus culture, mature seeds were surface sterilized with 70% (v/v)
ethanol for 1 min followed by a treatment with 5% (w/v) sodium hypochlorite
for 15 min and were finally rinsed 3 times with sterile double distilled
water under aseptic conditions. Surface sterilized seeds were soaked for
24 h in sterile double distilled water. Mature embryos from pre-soaked
seed were excised and cultured on the explant inoculation medium in 100x17
mm glass petridishes. For raising suspension cultures, six to eight weeks
old embryogenic calli obtained from mature embryo culture were transferred
in to MS liquid medium. The cultures were agitated on a horizontal shaker
at 140 rpm, at 25±2°C, under the complete darkness. Every 15
days the old medium was replaced by an equal volume of the fresh medium.
Isolation and purification of pathogen: A. porri
isolates from infected plants were collected from different locations.
Leaves with infected lesions were surface sterilized with HgCl2
solution and thoroughly washed with sterile double distilled water before
inoculation. Inoculated plates with Potato Dextrose Agar (PDA) medium
were kept for incubation at 25°C. The fungal colonies appearing on
plates were subcultured, purified and maintained at 25°C.
Pathogenicity test: The virulence of the isolate of A. porri
was tested by seedling leaf injury method. Leaves from 25 cm tall seedlings
were gently injured by sterilized needle and mycelial mat was spread on
the injured leaves. Inoculated seedlings were incubated in plastic bell
jars under high humidity. After 10 to 15 days, virulent strains revealed
typical purple to brown blotches were observed on the inoculated leaf
Preparation of culture filtrate: For the extraction of toxic culture
filtrate, 5 mm discs from 30 days old fungal cultures grown on PDA were
transferred to a fresh PDA medium every 4 weeks. Ten to fifteen pieces
of PDA cut from a two-week-old culture of Alternaria porri
were inoculated in 250 mL Erlenmeyer flask containing 50 mL liquid MS
medium. Two weeks later medium was divided into five equal portions of
10 mL. Cultures were incubated under dim light at 22°C for six weeks.
After 5-7 weeks small mycelium balls emerged in a uniform suspension.
The suspension was passed through filter paper Number1 (Whatman) and culture
filtrate was then subjected to centrifugation at 10,000xg for 20 min.
Supernatant was sterilized using nitrocellulose filter (0.22 μm)
and stored at -20°C.
Determining the LD50 of A. porri toxic culture filtrate:
To determine the selection concentration of phytotoxin, calluses were
separated into small pieces and were placed on varying concentrations
of toxin. LD50 was established with reference to approximately
50% retarded growth of callus. Fresh weight and relative growth rates
of callus and suspension culture on seven different levels of phytotoxin
added in initial culture medium were determined after 4 weeks ( Fig.
1A-D and Table 1).
In vitro selection and regeneration procedures: For
initiation and selection of disease tolerance, small pieces of embryogenic
calluses and suspension cultures were subjected to fortified MS culture
medium supplemented with varying levels of toxic culture filtrate and
0.5 mg L-1 each of NAA, kinetin and BA. After continuous and
discontinuous selections, tolerant/resistant calli were grown on MS medium
with 7.8 mL-1 toxic culture filtrate to select the disease
tolerant cell lines. After two weeks of plating, A. porri culture
filtrate toxin tolerant colonies with considerable growth were selected
(Fig. 1E ). Following selection on toxic medium, surviving
callus obtained from embryo cultures and embryoids acquired from suspension
cultures were grown on MS maintenance medium without growth regulators
and parts of survived callus were placed on MS regeneration medium fortified
with 0.5 mg L-1 each of NAA, Kn, BA and 20 g L-1
sucrose (Fig. 1F -G). Regenerates
were planted in 2.5 cm root trainers filled with 1:1:1 sand, soil and
FYM sterilized mixture. Root trainers with transplanted plants were transferred
under 30±2°C and 60±5% RH for 15-20 days in greenhouse
|| In vitro selection in onion cv ADR and ALR: A-D. Effect
of varying concentration of Alternaria porri culture filtrate on onion
callus cultures; E. Callus proliferated on tolerant/resistant callus;
F-G. Plant regeneration from cultures tolerant to A. porri; H-J. In
vivo testing of putative tolerant plants: H. Uninoculated leaf; I.
Inoculated leaves J. putative tolerant and susceptible plants against
A. porri; K-L. Field trial of putative resistant/tolerant plants of
cv Agrifound Dark Red and Agrifound Light Red.
In vivo testing of regenerated plants against pathogen:
Leaves of regenerated putative disease tolerant and control plants were
sprayed with 70% ethanol. After approximately 30 min dried leaves were
inoculated with 10 μL spore suspension and kept under 60±5%
RH. In susceptible and control plants, the purple blotches appeared on
the surface of leaves within 3-4 days of inoculation. The plants regenerated
from in vitro cultures were categorized as resistant, intermediate
and susceptible (Fig. 1H-J).
Evaluation of onion plants, developed by in vitro selection
against purple blotch disease: The two onion varieties ADR and ALR
either developed by embryo culture or by suspension culture were selected
at LD50 of A. pori toxic filtrate. To observe their
performance, plants were placed under field conditions (Fig.
1 K-L). The fungal inoculum was artificially sprayed during the initial
vegetative phase. The data on disease incidence was recorded after 30
days by rating the size of the spots in to different grades. The Percent
Disease Intensity (PDI) was calculated as under.
RESULTS AND DISCUSSION
In vitro selection is often hampered by difficulties in regenerating
plants in long culture phages from in vitro selected calli against
toxins and lower expressibility of selected traits in the regenerated
plants in culture systems. The present experimental evince that in
vitro regeneration of rather recalcitrant species like onion can be
improved upon and can become amenable to in vitro selection.
During embryo and suspension culture, Tiwari et al. (2004, 2007)
observed significant differences among different growth regulator type
concentrations and their interactions with each genotype for callus induction,
embryoid formation and plantlet regeneration. For callus culture, MS medium
fortified with 2.0 mg L-1 2, 4-D and 0.5 mg L-1
BA substantiated superior for all culture phases. In case of suspension
cultures, the liquid medium containing 4.0 mg L-1 2, 4-D in
combination with 0.5 mg L-1 BA was found the most competent.
For subsequent subculturing reduced concentration of 2,4-D (2.0 mg L-1
2, 4-D in combination with 0.5 mg L-1 BA) was supported faster
development of embryos. Frequent plantlets were regenerated on solid MS
medium supplemented with 0.5 mg L-1 NAA, 0.5 mg L-1
BA, 0.5 mg L-1 kinetin, 20 g L-1 sucrose and 7.5g
L-1 agar. Cultivar ADR was found more responsive than ALR for
the both culture systems.
During the present investigation, the culture filtrate of the pathogenic
fungus was used as the source of crude toxin preparations. At higher toxin
concentration (8.1 mL L-1), almost mortality was observed.
At minimum level of toxic culture filtrate (below to 7.5 mL L-1),
significant effect on callus growth was not observed (Fig.
1A-D and Table 1). Thus, LD50 (7.8 mL
L-1) of toxin concentration was used for selection criteria.
The control callus and the one grown at the lowest concentration of fungal
toxin looked healthy and survived considerably, whereas, the calli treated
with the highest toxin level showed a brown colour and eventually died.
This regular decrease in the callus survival rate with the toxic culture
filtrate in the medium was probably due to the presence of toxic metabolites
in the toxic culture filtrate.
Toxin-induced leakage of electrolytes from tissues suggests plasmalemma
as the site of action and toxin induced electrolyte leakage from leaves
(Rudolf, 1976; Yoder, 1983). During the present investigation, with the
dilution of the toxin, there was a gradual decrease in electrolyte leakage
from calli and leaves. The identical response of calli and leaf tissues
to toxic culture filtrate indicates that physiological and molecular testing
and selection for disease resistance is feasible at callus phase (Fig.
During continuous method, after the first cycle of selection of callus
cultures on the toxic medium, onion cultivars ADR and ALR showed 37.2
and 33.3 % survival rate respectively (Table 2). However,
during the second cycle about 5% of the calli still died but thereafter
calli subjected to further selections exhibited insensitivity to medium
with toxic culture filtrate, since smaller number of calli died (2-3%).
Selection by continuous method resulted in 27.7% callus survival for cv
ADR and 23.6% for cultivar ALR after 4 cycles of selections. During discontinuous
method, 29.3% calli of cv: ADR and 26.4 % calli of cv: ALR survived.
With the continuous method applied with cell suspension cultures, like
callus culture selections resulted in 34.61% surviving embryoid of ADR
and 31.26% of ALR after the first cycle of selection on the medium with
toxic culture filtrate (Table 3). Similarly, 4-5% of
the calli still died during the second cycle before acquiring insensitivity
during the third cycle. Thereafter only few embryoids (2-3%) died due
to toxic culture filtrate present in medium during the fourth round of
selection. Total of embryoid 25.38% from ADR and 22.09% from ALR survived
after 4 cycles of selection by continuous method. Whereas, selection by
discontinuous method finally survived 27.26% and 24.28% embryoids of cv
ADR and ALR, respectively.
||Comparison of growth rates 1 of mature embryo derived
calli from callus and cell suspension of onion on different levels
of toxin concentration in growing medium
Mean was obtained from weights of five inoculum/
treatments after 4 weeks, Callus and cell suspension cultures were
cultured on MS medium fortified with 2.0 mg L-1 2, 4-D,
0.5 mg L-1 BA and different levels of toxic culture filtrate,
Callus and cell growth appeared average fresh weight (mg) of 5 calli
||Response of mature embryo derived calli of onion to
the toxin preparation of Alternaria porri
||Response of clumps obtained from embryogenic suspension
culture of onion to the toxin preparation of Alternaria porri
Similar to experiment at issue, Chawla and Wenzel (1987) performed in
vitro selection of wheat and barley by applying intense initial selection
pressure of toxin to kill about 70% of the calli and subsequently increased
the concentration of toxin in the media. Similarly during the present
investigation, first cycle selection resulted in 65-70% callus mortality
in both the cultivars. A discontinuous method of selection was experimented
in onion to prolong regeneration capability of cultures on toxic medium.
Results after completing four cycles of selection by continuous and discontinuous
methods were almost similar however, discontinuous method exhibited apparent
advantage of high regeneration ability over the continuous method.
In vivo testing of 51 regenerated plants of ADR to the pathogen
have revealed 4 resistant plants, 29 intermediately tolerant plants with
very few brown spots and 18 susceptible plants. Likewise, 32 regenerated
plants of ALR have revealed 102 plants resistant to the pathogen, 21 intermediate
and 9 susceptible (Fig. 1H-J and Table
4). Exposure and selection of callus cultures to toxin resulted in
plants that were tolerant to the pathogen. Variation in reaction to the
pathogen observed during present experiment indicates that the tolerance
to Alterneria porri in onion is probably due to changes in nuclear
genome however, confirmation at molecular level and subsequent generation
The response of regenerated plants was recorded as the size of the spot
on the leaves the plants were then categorized into different grades after
calculating the PDI as described earlier. Based upon the PDI value, the
plants were categorized and counted, as resistant (<10), moderately
resistant (11-25), moderately susceptible (26-40) and susceptible (>40).
Thirty days after transplanting
||Regeneration frequency of onion calli tolerant/resistant
to phytotoxin and in vivo reaction of regenerated plants to
Alternaria porri phytotoxin
|† R: Resistant; I: Intermediate; S: susceptible
||Performance of the onion plants against purple blotch
disease under field conditions
transplanting, final stand of the plants was counted 402 and 201 for
ALR during embryo and suspension culture respectively and 318 and 180
for ADR. Data on PDI is presented in the Table 5 and
Fig. 1K-L indicates that embryo culture of cv ALR on
culture medium with toxic culture filtrate regenerated only one resistant
plant and 03 plants raised from suspension culture confirmed resistance,
whereas remaining plants have shown varying degree of reaction against
purple blotch disease. Similarly, from cv ADR, only 02 plants raised by
suspension culture exhibited tolerance whereas, none of the plants raised
from embryos revealed tolerance against purple blotch disease. Such plants
will be grown in the next season to confirm tolerance. The seeds of these
single plants have been collected so as to confirm performance under natural
This approach of generating disease tolerant/resistance raises question
weather the trait acquired is due to the mutation or somaclonal variation.
Many plant species earlier have been selected for disease tolerant/resistance
using culture filtrate or partially purified toxins (Song et al.,
1994; Gangaotti et al., 1985, Sunlihua and Lu, 1997). Selection
of disease resistant plants without other apparent mutations has been
accomplished in rice by short-term exposure of calli to Helmenthosporium
oryzae toxin followed by regeneration on toxin free medium (Vidyasekaran
et al., 1990). It can be concluded from the present study that
the sensitivity of the cultured cells to the toxic culture filtrate of
A. porri is related to the susceptibility of the onion regenerants
to the pathogen and the selection protocol using the toxic culture filtrate
of A. porri produced purple blotch disease resistant plants.
Similar results in groundnut for C. personatum (Venkatachalam et
al., 1998) and tobacco for A. alternate (Thanutong et al.,
1983) have proved useful in identifying resistant plants for corresponding
pathogens using culture filtrate strategy. Hence, it could be a method
of choice for developing fungal disease resistant genotypes in onion and
Authors are thankful to the Indian Council of Agricultural Research,
New Delhi, India for funding and to the National Horticultural Research
and Development Foundation, Nashik, India for experimental materials.