Plant Pathology (2006) 55, 679–689
Doi: 10.1111/j.1365-3059.2006.01433.x
Response of interspecific Brassica juncea/Brassica rapa
hybrids and their advanced progenies to Albugo candida
(white blister)
Blackwell Publishing Ltd
K. Guptaa, D. Prema, N. I. Nashaatc and A. Agnihotrib*
a
Centre for Bioresources and Biotechnology, TERI School of Advanced Studies; bPlant Biotechnology, TERI, Darbari Seth Block,
IHC complex, Lodhi Road, New Delhi, 110003, India; and cPlant–Pathogen Interactions Division, Rothamsted Research, Harpenden,
Hertfordshire AL5 2JQ, UK
Transfer of factors for resistance to white blister disease caused by Albugo candida between Brassica species involving
two genotypes each of B. juncea and B. rapa was studied in hybrids. More hybrids were obtained by in vivo than in vitro
techniques, although an in vitro phase was a prerequisite for the establishment of in vivo hybrids. Hybrids were identified
by PCR-based inter-simple sequence repeat (ISSR) markers with both male and female species-specific bands being identified. There was a positive correlation between disease severity and number of days after sowing (r > 0·93), the highest
being towards pod formation and plant maturity at 110 days after sowing. The plants from F2 and BC1 progeny showed
higher resistance to A. candida than either of the parents. Plants of B. juncea and B. rapa with high field resistance (disease
index < 1·0) were selected from BC2 and F2BC1 generations. The frequency of plants classified as resistant in BC2 progeny
ranged from 4·5 to 39·0% in cross-combinations involving B. juncea genotypes as female parent, compared with 100%
in the reciprocal cross involving B. rapa as female parent.
Keywords: ISSR, resistance breeding, white blister
Introduction
Brassica juncea (AABB = 36) is an oilseed species in
Southeast Asia. It occupies almost 80% of the total area
of oilseed Brassica cultivation in India. Brassica juncea is
highly susceptible to white blister caused by Albugo candida, resulting in yield losses up to 60% (Saharan et al.,
1984). Severe infection culminates in systemic ‘staghead’
symptoms in the inflorescence often in association with
the downy mildew Peronospora parasitica (Awasthi et al.,
1997). Thirteen races of A. candida have been reported
from different Brassica species (Verma et al., 1999) with
isolates of A. candida from different Brassica species
normally most pathogenic on their host genotype or
species of origin. They are nevertheless able to grow,
although generally not as well, on other Brassica species
(Liu et al., 1996). Genotypes of B. juncea (Townsend
et al., 2003) and B. rapa (unpublished data) have been
identified under controlled environmental conditions to
have differential resistance responses to a range of Indian
isolates of A. candida derived from these two species.
Combining factors for resistance to A. candida in B. juncea
and B. rapa through interspecific crossing may therefore
*E-mail: abhagni@teri.res.in
Accepted 11 February 2006
© 2006 The Authors
Journal compilation © 2006 BSPP
result in breeding lines of both host species with resistance
that can be expressed under field conditions. Hybrids
have previously been obtained from crosses involving
B. napus/Raphano brassica, using an in vitro embryo rescue
technique, to investigate important traits such as compatibility, shattering resistance, disease resistance and
morphological variations (Agnihotri et al., 1990a). The
generation of hybrids under normal field conditions and
with the aid of in vitro embryo rescue technique has also been
reported from crosses involving B. juncea/B. rapa genotypes (Mohapatra & Bajaj, 1987; Choudhary et al., 2002).
The aim of the present research was to combine factors for
resistance to A. candida from B. juncea and B. rapa, through
interspecific hybridization using both in vivo, i.e. normal
sexual reproduction under field conditions, and in vitro
embryo rescue techniques. Resultant progenies were evaluated for white blister response under epiphytotic conditions.
Materials and methods
Parent material, disease assessment and plant growth
Interspecific crosses involved two genotypes of B. juncea,
RESBJ-837 (J7) and RESBJ-830 (J0), and two of B. rapa,
RESBR-219 (R2) and RESBR-350 (R3). The former two
genotypes were selected from the F4 generation of a cross
involving selected S3 lines from cv. Kranti and cv. Krishna.
679
680
K. Gupta et al.
Brassica rapa genotype R2 was the S3 progeny of a line
selected from PYSLP-2, whereas R3 was the S3 progeny
of a line selected from YST-151. Both B. juncea (Nashaat
et al., 2004) and B. rapa (unpublished data) genotypes
possessed resistance to a wide range of Indian and UK
isolates of P. parasitica. Brassica juncea genotypes were
also resistant to a field isolate of A. candida from B. rapa
cv. Pusa Gold at Pantnagar (northern India) but susceptible
to two field isolates from B. juncea cv. Varuna, one from
Delhi and the other from Pantnagar. Brassica rapa
genotypes were moderately resistant to two B. juncea
isolates, but moderately susceptible to B. rapa isolate.
All tests were done at the cotyledonary leaf growth stage
under optimum controlled environmental conditions
(unpublished data).
The seeds of all parental species, the hybrids and their
subsequent advanced progenies were grown in The
Energy and Resources Institute (TERI) field experimental
station near New Delhi, India between October to April
over a period of three successive years. The plants were
grown as single plant progeny with 30 cm between rows
and 10 cm within row spacing following standard local
agricultural practices (Reddi & Reddy, 1980).
Generation and establishment of hybrids
Hybridization and pollen–pistil interaction
Young flower buds of the female parents were emasculated 1 day prior to anthesis, enclosed in paper bags and
the emasculated buds pollinated the following day with
freshly dehisced pollen of the male donor and re-bagged.
Of the total ovaries pollinated, 25% were left on the
plants until maturity to study seed set under field conditions, i.e. in vivo, while 75% were cultured in laboratory
under controlled conditions, i.e. in vitro, in the form of
ovary, ovule and sequential embryo culture. Some of the
pollinated pistils were fixed in 70% v/v ethanol, 24– 48 h
after pollination. Pollen germination and pollen tube
growth were observed in the fixed pistils using aniline blue
fluorescence (Shivanna & Rangaswamy, 1992).
In vitro culture and embryo rescue
Pistils were excised 4–6 days after pollination (DAP),
surface-sterilized and cultured on MS medium (Murashige
& Skoog, 1962) for ovary culture; the medium was supplemented with 1·0 mg L−1 kinetin, 0·1 mg L−1 naphthalene
acetic acid, 1·0 mg L−1 gibberellic acid and 50 mg L−1
casein hydrolysate (Agnihotri, 1993). The swollen pistils
were subcultured onto fresh medium after 2–3 weeks, dissected after 4 –5 weeks of initial culture, and the excised
ovules cultured again on fresh medium. For ovule culture,
surface-sterilized pollinated pistils were dissected 15–20
DAP and the developing ovules excised and cultured on
fresh medium. The cultured ovules, obtained from the
ovary/ovule cultures either germinated directly or were
dissected after 1 week. The developing embryos were cultured with 10% concentrations of growth regulators.
Surface-sterilized ovaries were cultured 4 –6 DAP and
dissected for ovule and embryo culture following the
method described by Agnihotri (1993). The regenerated
embryos were transferred on MS basal medium (without
growth regulators) and grown into plantlets with regular
subculturing every 2–3 weeks. All in vitro cultures were
maintained at 25 ± 2°C at 16 h photoperiod with a photosynthetic photon flux of 170 µmol m−2 s−1.
Raising of plantlets
Plantlets obtained in vitro were multiplied through
axillary bud proliferation and apical meristem culture on
MS medium supplemented with 1·0 mg L−1 kinetin. Plantlets at the three- to four-leaf growth stage were rooted
on MS medium with 20 g L−1 sucrose supplemented with
0·1 mg L−1 indole butyric acid and then hardened by
transferring to 10 cm pots containing sterilized compost
[two parts soil to one part Soilrite (80:10:10 peatmoss:
vermiculite:perlite) to one part Agropeat (cocopeat;
Varsha Enterprises)] in a Conviron PGV-36 growth cabinet at 22 ± 2°C with 8 h of darkness initially followed by
a 16 h photoperiod, with a photosynthetic photon flux of
290 µmol m−2 s−1. The plantlets were covered with glass
jars for an initial 3– 4 days and watered with half-strength
MS medium without sucrose after which the jars were
removed gradually and the hardened plantlets subsequently transplanted into the field after 10–15 days at the
four- to five-leaf growth stage.
Seeds obtained in vivo were divided into equal numbers
and raised in the field; being either sown directly in soil
along with parent lines or initially germinated on MS
medium containing 10 g L−1 sucrose. The rooted seedlings, obtained by germinating seeds on medium, were
hardened at three- to four-leaf growth stages and transplanted to the field at four- to five-leaf growth stage. The
plantlets obtained through in vitro embryo rescue, and the
seedlings obtained from seeds raised in vitro were transplanted into the field at similar growth stages.
Characterization of hybrids
Morphology
Comparative morphological features of the hybrids were
recorded, including leaf, floral and inflorescence characters. The percentage of pollen fertility was evaluated by
staining pollen grains from freshly dehisced anthers using
1 g /100 mL acetocarmine and 2 mg mL−1 fluorescein-diacetate (Shivanna & Rangaswamy, 1992).
DNA analysis
A modified cetyl-trimethyl ammonium bromide method
was used for DNA extraction (Weising et al., 1995).
Qualitative and quantitative assessments of total genomic
DNA were performed by subjecting the samples to
agarose gel electrophoresis followed by staining with
ethidium bromide. Serial dilution of uncut lambda DNA
(25–150 ng µL−1) was used as a standard molecular size
marker to quantify genomic DNA.
ISSRs were performed using the modified protocol of
Kumar et al. (2001). A total of eight University of British
Plant Pathology (2006) 55, 679–689
White blister resistance in Brassica interspecific hybrids
Columbia ISSR primers (synthesized by Microsynth) were
used for DNA amplification. Polymerase chain reaction
(PCR) amplification used in a Perkin-Elmer thermocycler
programmed for 35 cycles as: one cycle of 94°C for 5 min;
35 cycles of 94°C for 45 s, 50°C for 45 s, and 72°C for
1 min 30 s followed by one cycle of 72°C for 7 min. The
amplification products were observed on 15 g L−1 agarose
gel by staining with ethidium bromide.
Disease screening
Zoosporangial suspensions and inoculation
Plants were sprayed with a mixture of field isolates
derived from leaves of B. juncea cv. Varuna and B. rapa cv.
Pusa Gold. Infected leaves of B. juncea were collected
from field experimental stations of the Indian Agricultural
Research Institute (IARI) in New Delhi (35 km west of
TERI field station) and GB Pant University of Agriculture
and Technology (GBPUAT)-Pantnagar 250 km northeast
of TERI field station, whereas those of B. rapa were collected from GBPUAT. Three zoosporangial suspensions
were prepared separately in isolation. The first and second
were from white blister-affected leaves of B. juncea
collected from IARI and GBPUAT, respectively; the third
suspension was from diseased leaves of B. rapa from
GBPUAT. The infected segments of the leaves were cut
with the aid of a sterile scalpel, placed into a beaker containing sterilized distilled water (SDW) and shaken thoroughly to dislodge the zoosporangia from the white blister
pustules. Extraneous material was removed by filtering
twice through three layers of muslin cloth (Singh et al.,
1999). The density of isolate suspensions was adjusted
with SDW to 5 × 104 zoosporangia mL−1. Zoosporangia
suspensions from B. juncea leaves from IARI and
GBPUAT were then mixed together. Individual plants
were inoculated in the evening, when the temperature was
12 ± 2°C and humidity > 90%, by simultaneous spraying
with 20 mL of the mixture of B. juncea zoosporangia
suspension and an equal volume of B. rapa zoosporangia
suspension to minimize the possibility, if any, of induced
resistance or susceptibility (Singh et al., 1999).
Disease assessment
Field-transplanted hybrids were inoculated with A.
candida 20 days after transplanting; disease reaction was
scored twice, 40 and 60 days after inoculation. In the case
of seed-raised progenies, plants were inoculated 50 days
after sowing (DAS) and disease reaction was scored three
times: at peak flowering stage (PFS) 75 DAS, end of flowering stage (EFS) 90 DAS, and pod-bearing stage (PBS)
110 DAS. All plants were tagged and numbered. In all
cases, disease reaction was scored on the third, fourth and
fifth leaves from the base of the plant on a 0–5 scale, following the modified method of Conn et al. (1990): 0, no
visible disease symptoms; 1, 1–5% leaf area covered with
white blisters (WB); 2, 6–10% leaf area covered with WB;
3, 11–25% leaf area covered with WB; 4, 26 –50% leaf
area covered with WB; 5, > 50% leaf area covered with
WB. Host material was classified into three categories:
Plant Pathology (2006) 55, 679–689
681
resistant (disease reaction 0–1), partially resistant (2–3),
and susceptible (4–5).
Selection of plants for white blister resistance and
progeny advancement
At flower bud formation, some inflorescences of all
hybrids were covered with butter-paper bags to prevent
cross-pollination (self-pollination), while others were
backcrossed to their respective female parents by bud pollination of emasculated flowers; the remaining inflorescences were left uncovered (open pollination). F2 seeds
were obtained from the protected as well as the unprotected inflorescences and BC1 seeds were obtained from
the racemes backcrossed to the female parents. The seeds
were sown in single plant progeny (SPP) rows with their
respective female and male parent genotypes. Infector
rows of B. juncea cv. Varuna and B. rapa cv. Pusa Gold
were sown at intervals of every 10 lines as controls. The
SPP rows were screened and plants classified as resistant
were selected and further selfed by covering the inflorescence with bags to prevent cross-pollination and backcrossed to obtain F3 and F2BC1 as well as BC1F2 and BC2
seeds, respectively. Seeds harvested from selected plants
were forwarded as five SPP rows sown together in adjacent blocks. The screening and selection procedure were
repeated for BC1F2 and BC2 progenies to select plants with
the least disease score.
Data analysis
DNA-based characterization
A binary matrix was constructed by scoring the data for
the presence (1) and absence (0) of bands across genotypes. On the basis of this, a similarity matrix was
constructed using Jaccard’s coefficient. The similarity
matrix was subjected to sequential agglomerative hierarchical nested clustering (SAHN) and a phenotypic
dendrogram constructed employing the unweighted pair
group method of arithmetic averages (upgma) to group
individuals into discrete clusters. The above analysis was
performed using a software package ntsys pc version 2·0
(Rohlf, 1998).
Disease evaluation
Disease index (DI) of each plant was obtained by averaging the disease reaction recorded on third, fourth and fifth
leaves at the base of a plant. Overall mean of the disease
reaction of all individual plants in each progeny was calculated to give the DI for that progeny. Differences among
the SPP rows of an individual hybrid, differences among
the individual hybrids of a given cross of the same parents
and differences among the crosses involving different parents were analysed by single factor analysis of variance
(anova) (Gomez & Gomez, 1984) using the DI of an individual plant. Since most of the comparisons involved an
unequal number of plants, the least significant difference
(LSD) was calculated at P = 0·05 as:
LSD = [error mean square(1/nith + 1/mjth)0·5]t0·05
682
K. Gupta et al.
Table 1 Morphological variation in hybrids between Brassica juncea and B. rapa
Cross
B. juncea/B. rapa
RESBJ-830/RESBR-219 (J0/R2)
J0R2-1
RESBJ-830/RESBR-350 (J0/R3)
J0R3-1
J0R3-2
J0R3-3
J0R3-4
J0R3-5
RESBJ-837/RESBR-219 (J7/R2)
J7R2-1
J7R2-2
J7R2-3
J7R2-4
J7R2-5
B. rapa/ B. juncea
RESBR-350/RESBJ-830 (R3/J0)
R3J0-1
RESBR-219/RESBJ-837 (R2/J7)
R2J7-1
Parents/and control cultivar
RESBR-219 (R2)
RESBR-350 (R3)
RESBJ-830 (J0)
RESBJ-837 (J7)
Varuna (control)
Percent pollen fertilitya
(mean ± SEM)
Siliqua lengthb
(mean ± SEM)
0·83 ± 0·37
2·14 ± 0·08
0·0
7·07 ± 1·77
3·62 ± 1·57
0·0
0·0
2·66 ± 0·09
5·28 ± 0·60
5·07 ± 0·08
1·93 ± 0·04
No pod formation
1·12 ± 0·47
0·0
0·63 ± 0·63
0·0
0·0
3·15 ± 0·09
2·14 ± 0·08
2·56 ± 0·08
3·46 ± 0·15
2·20 ± 0·07
0·9 ± 0·23
0·3 ± 0·15
0·6 ± 0·21
0·3 ± 0·15
0·3 ± 0·15
5·24 ± 0·81
1·9 ± 0·04
9·8 ± 1·51
0·0
No pod formation
> 90·0
> 90·0
> 90·0
> 90·0
> 90·0
5·11 ± 0·08
5·14 ± 0·08
5·70 ± 0·07
5·06 ± 0·08
5·76 ± 0·02
Seed set per siliquac
(mean ± SEM)
0·7 ± 0·15
0·0
13·3 ± 1·86
9·4 ± 2·12
0·0
31·50 ± 0·32
19·90 ± 0·19
16·10 ± 0·09
15·50 ± 0·22
15·70 ± 0·09
a
Percentage pollen fertility is the average of six replicates. For each replicate, eight observations were taken from each slide prepared using six
anthers from one flower at different 10× microscopic fields.
b
Siliqua length is the average of 10 randomly selected pods from main branch.
c
Seed set per siliqua is the average number of seeds harvested from 10 randomly selected pods from the main branch through open pollination.
where n and m are the numbers of plants in ith and jth
progeny/cross, respectively, and t0·05 denotes the tabulated
value at P = 0·05.
Progenies of BC1F2 and BC2 generation were analysed
collectively in one anova to study the differences between
the progenies, since they were grown in the same year. The
segregation of resistant and susceptible plants in BC1
progeny was also assessed using chi-squared analysis (χ 2).
Pearson’s coefficient analysis was used to study the relationship between the progressions of disease with the
increasing DAS in all generations.
Data for the F1 generation were obtained in year 1,
those for the BC1 generation in year 2, and those for
the generations BC1F2 and BC2 in year 3. In all cases,
the different generations were compared with parental
genotypes.
Results
Production of B. juncea/B. rapa hybrids
Germ tubes produced by the pollen grains successfully
reached the ovular region through the stigma and style,
indicating an absence of a prefertilization barrier. Limited
seed set was observed, however, in pods left in vivo. The
seeds were shrivelled, did not germinate in soil, and
required an in vitro culture for germination and seedling
establishment. Twelve hybrids were obtained from the
straight crosses involving B. juncea/B. rapa genotypes
through both seed set in vivo and in vitro techniques. Of
these, five were from crosses involving RESBJ-830/
RESBR-350 (J0/R3), six from RESBJ-837/RESBR-219
(J7/R2) and one from RESBJ-830/RESBR-219 (J0/R2).
All hybrids survived to produce seeds except one from the
cross involving J7/R2 (Table 1). Among the surviving
hybrids, two were obtained through in vitro embryo rescue technique from each of the crosses involving J0/R3
(J0R3-2 and J0R3-3) and J7/R2 (J7R2-3 and J7R2-4); the
remaining hybrids were obtained through germination in
vitro of seeds (Table 1). In the reciprocal crosses, one
hybrid was obtained from the cross involving R3/J0
(R3J0-1) and one from R2/J7 (R2J7-1) through seeds germinated on MS medium. One putative hybrid from the
cross involving J7/R3 (J7R3-1) was deduced to be a
matromorph resembling the female parent with more
than 90% pollen fertility.
Out of the three in vitro techniques used (ovary culture,
ovule culture and sequential embryo rescue), ovule culture
gave the highest number of hybrids followed by sequential
embryo culture, averaging 0·72 and 0·62%, respectively.
No hybrid was obtained through ovary culture. The
hybrids were successfully multiplied through auxiliary
Plant Pathology (2006) 55, 679–689
White blister resistance in Brassica interspecific hybrids
683
Table 2 Characterization of Brassica interspecific hybrids using UBC-ISSR (inter-simple sequence repeat) primers
Primer
Sequencea
No. of
amplified
bands
ISSR-UBC 812
ISSR-UBC 814
ISSR-UBC 818
ISSR-UBC 840
ISSR-UBC 842
ISSR-UBC 843
ISSR-UBC 848
ISSR-UBC s2
5′GA-GA-GA-GA-GA-GA-GA-GA-A-3′
5′CT-CT-CT-CT-CT-CT-CT-CT-A-3′
5′CA-CA-CA-CA-CA-CA-CA-CA-G-3′
5′GA-GA-GA-GA-GA-GA-GA-GA-YT-3′
5′GA-GA-GA-GA-GA-GA-GA-GA-YG-3′
5′CT-CT-CT-CT-CT-CT-CT-CT-RA-3′
5′CA-CA-CA-CA-CA-CA-CA-CA-RG-3′
5′CTC-TC-TC-TC-GT-GT-GT-GTG3′
5
7
9
–b
–
7
8
5
No. of
polymorphic
bands
%
polymorphism
No. of bands
specific to
male parent
3
6
7
–
–
5
5
3
60
85·7
77·7
–
–
71·4
62·5
60
0
1
2
–
–
0
1
1
a
Y, pyramidine; R, purine.
–, no amplification product observed.
b
Figure 1 Banding profile generated through
inter-simple sequence repeat (ISSR) primer
UBC-818 for Brassica juncea genotypes J7
(RESBJ-837) and J0 (RESBJ-830), Brassica
rapa genotypes R2 (RESBR-219) and R3
(RESBR-350) and their hybrids. M, 1 kb
standard DNA marker. Bands A and B were
monomorphic to all genotypes. Bands a1 and a2
were specific to female parent (B. juncea) and
the hybrids only, whereas bands b1 and b2 were
specific to the male donor (B. rapa), present in
hybrids but absent in female parent.
buds and apical meristems. A short-duration incubation
of embryos in MS liquid medium without growth regulators
was a prerequisite for proper development and establishment of plantlets (Agnihotri, 1993). The plantlets were
rooted with a frequency of 80%.
Characterization
Morphology and percentage pollen fertility
The putative hybrids were raised to maturity under field
conditions. The plantlets were dwarf phenotypes with
intermediate morphological traits such as plant type
(branching pattern, main raceme and height), leaf (size,
shape, texture, colour, venation, tip, margins) and floral
morphology. Leaves were glabrous, petiolate, lyrately pinnatifid, resembling the female parent B. juncea, and ovate
with obtuse tip showing variable marginal shapes, some
of which were dentate – resembling B. juncea, with conspicuous midrib and veins. Hybrids from the reciprocal
crosses had clasping or petiolate leaves with dark green
colour coupled with entire or sinuate margins.
The inflorescence resembled the female parent in both
crosses. Anther conditions ranged from rudimentary and
shrivelled to partially sterile. Pollen grains responded similarly to both acetocarmine and FDA stains. The average
pollen fertility of hybrids from the straight and reciprocal
crosses involving B. juncea/B. rapa ranged from completely
Plant Pathology (2006) 55, 679–689
sterile to 7·07 and 5·24%, respectively (Table 1). Female
fertility was also reduced and reflected by limited seed set
per siliqua. Seed set in all hybrids was achieved through
backcrossing and open pollination. But no seeds were
developed following self-pollination. Pods in hybrids
from unprotected inflorescences were normal and showed
an undulating surface over seed development; seeds were
either small or shrivelled with less than one seed per pod.
DNA analysis using ISSR markers
Of eight ISSR primers employed for characterization, six
primers produced a total of 41 bands with 70·1% polymorphism (Table 2). The primers resulted in bands specific to either male or female parent or both. Two primers,
UBC-840 and UBC-842, did not amplify any band. The
banding pattern of all hybrids and their parents with
primer UBC-818 is shown in Fig. 1.
Genetic similarity and cluster analysis
The genotypes were clustered into three main groups:
B. juncea (BJ), B. rapa (BR) and hybrids (BH) (Fig. 2). The
parental genotypes were distinctly placed in two separate
clusters. The hybrids were shown to be of a relatively
intermediate nature as indicated by their genetic similarity
(GS) values of 0·71 and 0·55, resembling the B. juncea
and B. rapa genotypes, respectively. All hybrids from
crosses involving B. juncea/B. rapa were grouped together
684
K. Gupta et al.
Figure 2 A dendrogram showing clustering
pattern between the Brassica juncea (BJ) and
B. rapa (BR) parents and their hybrids (BH)
using the UPGMA clustering method. Groups
HJ and HR represent hybrids obtained from
crosses involving B. juncea/B. rapa and its
reciprocal, respectively. Subclusters HJ1,
HJ2 and HJ3 represent hybrids obtained
from crosses involving genotypes J7/R2
(RESBJ-837/RESBR-219), J0/R3 (RESBJ-830/
RESBR-350) and J0/R2 (RESBJ-830/
RESBR-219), respectively.
Table 3 Response of F1, BC1, BC1F2 and BC2 generations of Brassica hybrids, parental genotypes and control cultivars to white blister at 110 days
after sowing
Disease index (mean ± SEM)a
Cross
RESBJ-830/RESBR-219 (J0/R2)
J0R2-1
RESBJ-830/RESBR-350 (J0/R3)
J0R3-1
J0R3-2
J0R3-3
J0R3-4
J0R3-5
RESBR-350/RESBJ-830 (R3/J0)
R3J0-1
RESBJ-837/RESBR-219 (J7/R2)
J7R2-1
J7R2-2
J7R2-3
J7R2-4
J7R2-5
RESBR-219/RESBJ-837 (R2/J7)
R2J7-1
Parents/and control cultivarsc
RESBJ-837 (J7)
RESBJ-830 (J0)
RESBR-350 (R3)
RESBR-219 (R2)
Pusa Gold (control)
Varuna (control)
F1
BC1
BC1F2
BC2
1·0 ± 0·6 (5)
No germination
0·47 ± 0·13 (5)
0·60 ± 0·13 (5)
0·47 ± 0·13 (5)
1·0 ± 0·0 (5)
1·0 ± 0·0 (5)
1·10 ± 0·16 bb (10)
1·84 ± 0·13 de (17)
1·49 ± 0·09 c (17)
No germination
No pod formation
2·09 ± 0·28 de (7)
1·79 ± 0·18 cd (28)
1·87 ± 0·19 cd (30)
2·15 ± 0·15 de (25)
2·69 ± 0·12 e (12)
2·59 ± 0·08 e (50)
0·67 ± 0·13 (5)
1·11 ± 0·11 bc (16)
1·77 ± 0·12 cd (57)
0·53 ± 0·11 a (17)
1·0 ± 0·0 (5)
1·4 ± 0·13 (5)
0·2 ± 0·11 (5)
0·2 ± 0·11 (5)
1·0 ± 0·11 (5)
2·44 ± 0·33 f (9)
3·33 ± 0·33 g (2)
1·06 ± 0·13 b (5)
0·24 ± 0·19 a (11)
2·17 ± 0·17 ef (2)
1·75 ± 0·27 cd (15)
Not forwarded
1·06 ± 0·21 b (26)
No germination
No germination
1·88 ± 0·21 cd (12)
1·0 ± 0·0 (5)
No pod formation
3·66 ± 0·25
4·00 ± 0·14
1·73 ± 0·26
0·99 ± 0·09
2·35 ± 0·05
4·66 ± 0·15
3·06 ± 0·10 g
3·28 ± 0·05 g
1·46 ± 0·06 c
1·06 ± 0·06 b
2·18 ± 0·04 ef
4·57 ± 0·09 h
1·62 ± 0·17 c (21)
No germination
No germination
3·17 ± 0·08 f
3·41 ± 0·08 f
1·75 ± 0·09 cd
1·15 ± 0·06 b
2·40 ± 0·06 e
4·66 ± 0·09 g
Values in parentheses are numbers of plants used to calculate the disease index of the crosses.
Disease index is the average of three leaves (third, fourth and fifth leaves from the base of the plant)/five plants/hybrid scored on a 0–5 scale point;
0, no visible disease symptoms; 1, 1–5% leaf area covered with white blisters (WB); 2, 6–10% area covered with WB; 3, 11–25% leaf covered with
WB; 4, 26–50% leaf covered with WB; 5, > 50% leaf covered with WB.
b
Values with same letter in a column indicate nonsignificant differences at P = 0·05.
c
Five randomly selected plants from each of the parents and the control genotypes were used for screening.
a
(HJ) and subsequently subgrouped into three clusters: HJ1
(J7/R2), HJ2 (J0/R3) and HJ3 (J0/R2). The two hybrids
from the reciprocal crosses, R2J7-1 and R3J0-1, were separated from the hybrids from straight crosses in group HR,
but resembled each other at a GS value of 0·88 (Fig. 2).
Disease assessment
In general, the maximum disease severity was recorded at
110 DAS and was higher on B. juncea than on B. rapa
genotypes (Table 3).
Plant Pathology (2006) 55, 679–689
White blister resistance in Brassica interspecific hybrids
F1 progenies
All 11 hybrids obtained were resistant to A. candida with
a lower DI than the female parent, B. juncea (Table 3).
The average DI of the five hybrids from each of the crosses
involving J0/R3 (J0R3-1 to J0R3-5) and J7/R2 (J7R2-1 to
J7R2-5) were 0·77 ± 0·06 SEM and 0·70 ± 0·12 SEM,
respectively, whereas the DI from the two hybrids of the
reciprocal crosses R3/J0 (R3J0-1) and R2/J7 (R2J7-1)
were 0·67and 1·0, respectively.
F2 progeny
F2 seeds were produced from the inflorescences that were
left unprotected, but not from those which were covered
with bags to prevent cross-pollination. One of the plants,
raised from the F2 seeds of the cross involving J7/R2
(J7R2-3), was selected for its resistance to white blister.
The inflorescences of the selected plant, which resembled
a B. juncea type, were self-pollinated and backcrossed
to the female parent (J7) to obtain F3 and F2BC1 seeds,
respectively. Symptoms of white blister on the leaves of
plants raised from these seeds were not observed when
screened under field conditions.
BC1 progenies
Only nine SPPs were successfully raised from the 11 BC1
seed sets, which were generated from crosses involving B.
juncea/B. rapa//B. juncea. BC1 seeds from two hybrids
obtained from the crosses involving J0R3//J0 failed to
germinate (Table 3). Significant differences were observed
among the BC1 progenies obtained from individual
hybrids of a particular cross and their respective parents.
Resistance of BC1 progenies was significantly higher than
either one or both parents. The resistance of BC1 progeny
of hybrid J0R3-3 was equal with the male parent, whereas
that of J0R3-2 showed a significantly higher resistance
than the female parent. Resistance of BC1 progeny of
J0R3-1, however, was significantly higher than both
female and male parents as well as the control cv. Varuna.
A similar trend was observed with the BC1 progenies from
the crosses J7R2//J7 and R3J0//R3 (Table 3).
Among the five BC1 progenies from J7R2//J7, the progeny from J7R2-4//J7 showed higher resistance than the
progeny from J7R2-3//J7, followed by those from J7R25//J7. Only plants with a DI ≤ 1·0 were selected. Due to
poor seed set, the total number of plants screened in
the BC1 progeny from backcrosses involving J0R3//J0,
J7R2//J7 and R3J0//R3 were limited to 44, 29 and 16,
respectively. The BC1 plants from J7R2//J7 and R3J0//R3
segregated in the ratio of 1 resistant (R):1 susceptible (S)
(χ2 = 0·035 and 0·31, respectively), whereas segregation
of BC1 plants from J0R3//J0 was 1R:3S (χ2 = 11·02).
BC1F2 and BC2 progenies
Based on the disease reaction of BC1 progenies, a total
of 11 plants were selected with DI of ≤ 1·0; five from
the crosses involving J0R3-1//J0, two from J0R3-2//J0
and four from J0R3-3//J0. The selected plants were selfpollinated and backcrossed again to their respective
female parents. The generated BC1F2 and BC2 seeds were
Plant Pathology (2006) 55, 679–689
685
sown as SPPs along with their respective parents and the
infector rows. Plants of BC1F2 and BC2 progenies raised
from these seeds were screened and showed similar resistance to the male parent, B. rapa or expressed higher resistance than both parents. Significant differences in overall
DI were observed among the three BC2 progenies from
crosses involving (J0R3-1//J0)//J0 (J0R3-2//J0)//J0 and
(J0R3-3//J0)//J0. Differences between or among SPPs
descended from each of these crosses were not significant
(Table 3). Similarly, 13 BC1 plants were selected, one from
the crosses involving J7R2–1//J7, three from J7R2-3//J7
and nine from J7R2-4//J7. The selected plants were also
selfed and backcrossed again to their respective female
parents to generate BC1F2 and BC2 seeds, but in this
instance, all but four plants failed to generate seed. Significant differences in DI were also observed between plants
from the BC2 progenies from crosses involving (J7R2-1//
J7)//J7 and (J7R2-3//J7)//J7. The DI of BC1F2 progenies
followed a similar trend to that of BC2. The DI of BC1F2
progenies from crosses involving J0R3//J0, J7R2//J7 were
either similar to, or lower than, BC2 progenies, whereas the
DI of the progeny from the cross involving R3J0//R3 was
higher than its BC2 progeny (Table 3). Significant variation was observed in DI of BC2 progenies from the
crosses involving J0R3//J0 and J7R2//J7; the percentage
of resistant plants in progenies from the later crosses was
much higher than those from the former crosses (39 and
4·5%, respectively).
Correlation of disease response of advanced progenies
with plant growth stages
Pearson’s coefficient analysis showed a positive correlation between different growth stages of plants and disease
development in BC1 progeny as the disease progressed
from 75 to 90 DAS (r = 0·93, P = 0·007) and from 90 to
110 DAS (r = 0·98, P = 0·0002). Disease symptoms on the
control cultivar Varuna and the parent lines became
visible 15 days after inoculation (65 DAS), whereas symptoms on BC1 plants appeared 5 days later (70 DAS). The
severity of disease at 75 DAS was higher on B. juncea parents
(DI = 0·33–1·66) and the control B. juncea cv. Varuna
(DI = 1·0–2·66) than on B. rapa parents (DI = 0·0–1·0). In
general, disease severity on plants at the end of flowering
stage (90 DAS) was higher than at the peak flowering
stage (75 DAS), with the exception of those from crosses
involving R3/J0. The DI of plants from crosses involving
J7/R2 was increased from 0·58 at end of flowering stage
to 1·41 at pod-bearing stage (110 DAS) [n = 29, SE
(difference) ± 0·22, P = 0·05]; no significant difference
between these two growth stages was observed in the case
of parents and the control cv. Varuna. The DI of BC1 progenies from crosses involving J0/R3 and R3/J0 followed a
similar trend to those from the cross J7/R2 (Fig. 3).
A positive correlation was also observed between DI
and DAS as the disease severity progressed from 90 to
110 DAS in BC1F2 progenies of all hybrids (r = 0·97, P =
0·000001). No disease symptoms were observed at 75
DAS in BC1F2 and BC2 plants. Symptoms of white blister
were first observed on the control 80 DAS. Out of the
686
K. Gupta et al.
Figure 3 Response of Brassica juncea and B.
rapa genotypes to white blister in BC1 progeny
from crosses involving J0/R3 (RESBJ-830/
RESBR-350), R3/J0 (RESBR-350/RESBJ-830)
and J7/R2 (RESBJ-837/RESBR-219) along with
the parent lines and the controls B. juncea cv.
Varuna and B. rapa cv. Pusa Gold. Bars
represent average disease response at 75, 90
and 110 days after sowing (DAS). Lines on
bars represent the standard error of the mean.
Different letters on bars indicate significant
differences at P = 0·05.
Figure 4 Frequency of plants in successive generations with respect to disease score at 110 days after sowing. The frequency of plants in each
category has been calculated as the proportion of plants out of total plants scored on 0–5 scale; where 0 represents no visible disease symptoms,
whereas, 1, 2, 3, 4 and 5 represent < 1·0%, 1–5%, 6–10%, 11–25%, 26–50% and > 50% of leaf area covered with white blister, respectively.
crosses involving J7/R2, R3/J0 and J0/R3, increase in
disease severity in BC2 plants was observed only on those
from the cross involving J0/R3 (DI = 2·08 ± 0·16 SEM at
90 DAS vs. 2·48 ± 0·16 SEM at 110 DAS, n = 87).
Disease reaction of plants from backcross generations
The resistance of BC1, BC2 and BC1F2 generations from
crosses involving B. juncea/B. rapa genotypes was similar
to the male parents and higher than the female parents,
whereas the resistance of plants from the reciprocal cross
was either similar to or higher than the female parent
(Table 3). Significant difference was observed among the
DI of BC1 (1·53 ± 0·08 SEM), BC2 (2·48 ± 0·16 SEM) and
BC1F2 (1·86 ± 0·12 SEM) generations from crosses involving J0/R3. No significant difference was observed among
DI of BC1, BC2 and BC1F2 generations from crosses
involving J7/R2 (DI = 1·41 ± 0·24 SEM, 1·75 ± 0·14 SEM
and 1·28 ± 0·20 SEM, respectively). The DI of plants from
the reciprocal cross R3/J0 was significantly lower in BC2
than BC1 and BC1F2 generations (Table 3). The percentage
of plants with DI ≤ 1·0 in the generations from the crosses
involving J0/R3 and J7/R2 was the highest at BC1F2, followed by BC1 and BC2, whereas in the reciprocal cross,
the percentage of resistant plants was highest in the BC2
generation (Fig. 4). Plants resembling B. juncea and B.
rapa types with DI ≤ 1·0 were selected from crosses
involving J0/R3, J7/R2 and R3/J0, respectively.
White blister-affected plants are also often attacked by
P. parasitica, which was also scored in this study on a
0–5 scale, where 0 indicates resistance and 5 indicates
high susceptibility (Conn et al., 1990). Three leaves per
plant and a total of 15 plants per genotype were scored.
Plant Pathology (2006) 55, 679–689
White blister resistance in Brassica interspecific hybrids
Peronospora parasitica was only observed at the beginning of the flowering stage in association with A. candida
on B. rapa parents, R2 (RESBJ-219) and R3 (RESBJ-350).
In the case of B. juncea, very little infection was observed
on the parents J0 (RESBJ-830) and J7 (RESBJ-837), but it
gradually intensified at the beginning of the flowering
stage in association with white blister (downy mildew
DI = 1·77 ± 0·05 SEM). More downy mildew occurred
on B. juncea cv. Varuna (DI = 2·62 ± 0·09 SEM) than on
B. juncea parent genotypes and, similarly, was higher on
B rapa cv. Pusa Gold (DI = 0·97 ± 0·07 SEM) than on B.
rapa parents (DI = 0·51 ± 0·07 SEM). Downy mildew was
also observed in association with A. candida on BC1F2 and
BC2 progenies where the downy mildew DI on individual
plants ranged between 0·0 and 2·66.
Discussion
The pollen–pistil interaction studies in this work provided
evidence of effective fertilization. Difficulties associated
with the production of hybrids in diploid and amphidiploid Brassica species have previously been reported
(Nishiyama et al., 1991). Sequential embryo rescue was
previously considered to be the most efficient technique
for a successful interspecific hybridization between different Brassica species (Agnihotri et al., 1990b; Chrungu
et al., 1999) but ovule culture was the most successful
technique in producing hybrids in this study, perhaps due
to the delayed effect of a postfertilization barrier. The
enhancement of the regeneration of viable embryos, following treatment in liquid medium, was probably due to
leaching of extra endogenous hormones into the medium
(Agnihotri et al., 1990b). The frequency of hybrid plantlets was higher in vivo (3·57%) than in vitro (0·65%) irrespective of the genotype of the female parent. Failure of
seed germination in soil and the requirement of an in vitro
phase for the establishment of hybrid seedlings may be
attributed to the development of partially degenerated
endosperm (Shivanna, 1996). The frequency of hybrids in
this work was higher in the straight (5·82%) than in the
reciprocal (1·03%) crosses involving the amphidiploid
B. juncea and diploid B. rapa species. This is in agreement
with Nishiyama et al. (1991), who observed a higher frequency of hybrids when genotypes of amphidiploid species
were used as female parents. Most hybrids previously
obtained from crosses involving B. rapa/B. juncea exhibited
poor growth and did not survive to maturity (Choudhary
& Joshi, 1999). The matromorph observed in the present
work is a common phenomenon in interspecific/intergeneric
hybridization involving Brassica species (Shivanna, 1996).
Previous results obtained using RAPD markers for characterization of hybrids are nonreproducible (Marshall
et al., 1994; Chrungu et al., 1999), whereas ISSRs are
reproducible and have therefore been preferred to study
the genetic diversity, clonal fidelity and phylogenetic relationships in Brassica species (Sarla et al., 2001). In this
work, ISSR markers were successfully used for the first
time to study hybridity of interspecific crosses involving
Brassica species. The ISSR markers exhibited a strong
Plant Pathology (2006) 55, 679–689
687
genotypic influence, and clustering of hybrids into different groups was observed, depending on the species and
genotypes involved.
The intermediate parental morphological characteristics shown by hybrids in the present work have frequently
been reported (Choudhary & Joshi, 1999; Choudhary
et al., 2002). Hybrids may also bear more resemblance to
one or other parent (Das et al., 1984; Mamatha, 1989).
Complete male sterility or partial pollen fertility with no
self-seed formation in the hybrids may be expected as a
result of likely genetic imbalance and meiotic irregularities
associated with interspecific hybridization. Seed formation through open pollination provides an opportunity for
recombination between genetically balanced gametes.
This is possible in B. juncea where cross-pollination was
estimated to be 5–15% (Asthana & Singh, 1973).
The observed variations in the response of hybrids to
white blister under epiphytotic conditions may indicate
the possibility of transfer/recombination of factors/genes
for resistance across the two species. The differential
response expressed by B. juncea (cv. Varuna, J0 and J7) in
comparison with B. rapa (cv. Pusa Gold, R2 and R3)
genotypes to the mixture of field isolates from B. juncea
and B. rapa indicates a gene-for-gene relationship in the
Brassica/Albugo interaction, but the possibility of additional infection with other sources of inoculum cannot
be ruled out in these field-based assessments. Resistance
observed in the advanced progenies of B. juncea-type
plants in F2 generations may be due to the possible existence of partially dominant heterozygous gene(s) in the F1
progeny, which as a result of recombination were later
expressed in the homozygous condition in the successive
generations.
The delayed onset of disease in the artificially inoculated BC1F2 and BC2 progenies as well as parents and
control cultivars when they were tested in year 3 may
be attributed to unfavourable environmental conditions
(Gadre et al., 2002). The higher severity of white blister
observed at 110 DAS in comparison to 75 and 90 DAS
was most likely due to factors determined by the population dynamics of the pathogen associated with the
progression of the disease and increasing sporulation
potential coupled with favourable environmental conditions. However, plant response to white blister can also
vary at different growth stages (Kumar & Saharan, 2002;
Yadav & Kumar, 2003).
The interpretation of variation in the segregation ratio
into resistant and susceptible categories of BC1 progenies
from the crosses involving J0/R3 (1R:3S), J7/R2 (1R:1S)
and R3/J0 (1R:1S) is complicated by the genomic differentiation between B. juncea (AABB, 2n = 36) and B. rapa
(AA, 2n = 20).
A comparison of BC1 with BC1F2 and BC2 generations
grown in two successive years was validated by the similar
disease response of parents and controls recorded over the
same period (Table 3). The number of plants with DI ≤ 1·0
was highest in BC1F2, followed by BC1 and BC2 progenies.
This may be attributed to the increasing genomic component of B. juncea as a result of successive backcrossing.
688
K. Gupta et al.
The importance of backcrossing in interspecific hybridization to produce improved plants of the parent types was
reflected by the resemblance in the morphological traits
between BC1F2 progeny and the male donor. The higher
resistance observed in BC2 progenies compared with their
respective female parents was probably due to the introgression of resistant factors/genes in the advanced progenies (Chauhan & Sharma, 2001). The percentage of
resistant plants in BC2 progeny from the cross involving
R3/J0, which amounted to 100% (Fig. 4), may be due to
the increased level of homozygosity of resistance genes as
a result of genetic recombination.
The B. juncea and B. rapa parent lines used in the
present work were primarily selected/developed for
resistance at the cotyledonary leaf stage to a number
of P. parasitica isolates (Nashaat et al., 2004). This may
explain the absence or low incidence of this disease
recorded on these genotypes. The appearance of downy
mildew associated with white blister on some of the
genotypes at flowering stage was probably due to the susceptibility induced to P. parasitica by the precolonization
with A. candida (Awasthi et al., 1997).
Brassica napus and B. carinata have previously been
used as likely sources of resistance to A. candida through
interspecific hybridization with B. juncea under natural
field conditions, but progression of the disease and plant
phenotypes of the advanced progenies were not reported
(Chauhan & Sharma, 2001; Kumar et al., 2002). As far as
is known, this is the first study reporting on the utilization
of B. rapa as a disease resistance donor and evaluating the
response of advanced progenies from crosses involving
B. juncea/B. rapa genotypes to A. candida at different stages
of plant growth under epiphytotic conditions. Plants of
B. juncea and B. rapa types with high or complete resistance to white blister have been selected. Further work is
in progress to select plants with good agronomic characteristics associated with resistance to white blister and
probably downy mildew.
Acknowledgements
We gratefully acknowledge the financial support of the
UK Department for International Development (DFID).
The views expressed here are not necessarily those of
DFID. We are grateful to M. S. Negi and Professors V. P.
Gupta, N. B. Singh, K. R. Shivanna, R. P. Awasthi, S. J.
Kolte, and John Lucas for their critical and valuable
suggestions.
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