Introgression of disease resistance genes from Arachis kempff-mercadoi into
N. Mallikarjuna, S. Pande, D. R. Jadhav, D. C. Sastri and J. N. Rao
International Crops Research Institute for the Semi-Arid Tropics, Patancheru 502 324, Andhra Pradesh, India; E-mail:
Received May 16, 2003/Accepted June 4, 2004
Communicated by A. Ashri
Arachis kempff-mercadoi is a wild species from the section Arachis. All
kempff-mercadoi accessions originate from the Santa Cruz province of
Bolivia and they represent Arachis species with the A genome. From
molecular analysis it was found that although cultivated A. hypogaea
is made up of A and B genomes, A. kempff-mercadoi may not be as
closely related to it as are some of the other A genome species. Arachis
kempff-mercadoi is of interest because it has multiple disease
resistance. It was crossed with a Spanish A. hypogaea cultivar which
is susceptible to foliar diseases and to the insect pest Spodoptera litura.
The success rate of the cross A. hypogaea (2n ¼ 40) · A. kempff-
mercadoi (2n ¼ 20) was very low, but it could be increased by
culturing immature seeds in vitro. Although the hybrids were triploids,
a few fertile pollen grains were obtained due to the formation of
restitution nuclei in the F1 plants. Interspecific derivatives at the
BC2F2generation were scored for early leaf spot, late leaf spot and to
Spodoptera damage. Screening results showed that 29% of the
derivatives had both early and late leaf spot resistance and that less
than 5% of the derivatives had resistance to both the foliar diseases
and to Spodoptera.
Key words: Arachis hypogaea — Arachis kempff-mercadoi —
Cercospora arachidicola — Phaeoisariopsis personata —
Spodoptera litura — interspecific hybrids
The genus Arachis, a native of the Brazil-Paraguay region of
South America (Simpson et al. 2001), is made up of nine
sections (Krapovickas and Gregory 1994). The cultivated
species Arachis hypogaea L. belongs to the section Arachis.
Many wild species from the section Arachis such as A. villosa,
A. correntina, A. diogoi (¼A. chacoense), A. stenosperma,
A. cardenasii, A. duranensis and A. batizocoi have been
successfully crossed with cultivated species (Stalker 1985,
Singh 1986, Stalker and Simpson 1995) with pod formation
ranging from 0 to 30%. It is not known if all the pods were
mature and the seeds germinated under in vivo conditions.
Transfer of rust resistance to the cultivated species has been
reported from A. duranensis (Singh 1986). Simpson et al.
(1993) reported the successful transfer of nematode resistance
from A. cardenasii and A. diogoi. Stalker et al. (2002) reported
a leaf spot-resistant population from the cross A. hypogaea ·
A. cardenasii. The population also had resistance to root knot
nematode and southern corn root worm. Milla (2003) has
reported the transfer of tomato spotted wilt virus resistance
from A. cardenasii.
Arachis kempff-mercadoi (2n ¼ 20, PI 468331; ICG 8959;
Coll no. 30085) is a perennial species and a native of the Santa
Cruz province in Bolivia, South America. It belongs to the
section Arachis with the A genome (Milla 2003). Based on
molecular and cytogenetic analysis, A. hypogaea is made up of
A and B genomes (Singh and Moss 1984, Gimenes et al. 2002).
Based on RAPD-molecular analysis it was found that
A. kempff-mercadoi may not be as closely related to
A. hypogaea as some of the other A genome species
(Mallikarjuna et al. 2003). Although A. kempff-mercadoi has
published report of transfer of disease resistance and the nature
of seed set in the crosses using A. kempff-mercadoi.
Surveys at ICRISAT have shown that A. kempff-mercadoi
has resistance to foliar diseases (Subrahmanyam and Moss
1983, Subrahmanyam et al. 1985, Pande and Narayana Rao
2001) and to the insect pest, Spodoptera litura (Stevenson et al.
1993). Although wild species from the section Arachis have
been recognized by Stalker and Simpson 1995) as cross-
compatible with cultivated groundnut (A. hypogaea L.), not all
wild species from this section cross readily with A. hypogaea
(Ozias-Akins et al. 1992). The success rate of the cross
A. hypogaea · A. kempff-mercadoi is very low. Very few mature
seeds (2%) are produced and a large number of them contain
well-developed embryos that have still not reached maturity.
These seeds do not germinate in vivo but can do so in vitro. A
small number of pods also had small aborted seeds (3–4 mm).
To obtain hybrid plants from aborted seeds, embryo rescue
techniques have had to be used (Mallikarjuna and Sastri
Early leaf spot (ELS) caused by Cercospora arachidicola
Hori and larly leaf spot (LLS) caused by Phaeoisariopsis
personata (Berk and MA Curtis) are economically significant
and widely distributed diseases of groundnut (Waliyar 1991).
The incidence and severity of the diseases vary with location,
year and cultivar and both diseases cause severe yield losses.
Although management options are available, they may not
be the best options because of the high costs of fungicides,
the possibility of obtaining fungicide-resistant strains and
environmental degradation due to the use of chemicals. Hence,
the best option to combat these diseases is to obtain disease-
The tobacco caterpillar, Spodoptera litura (Fab.) is a
polyphagous noctuid moth of high reproductive capacity, with
the ability to migrate over long distances. These characteristics
have resulted in its becoming a pest of many agricultural crops
throughout South Asia and South East Asia. Groundnut yield
losses up to 71% have been reported in India (Amin 1988).
High yield losses of groundnuts have been directly associated
with increased larval density of S. litura, and the intensity of
defoliation (Panchbhavi and Nethradani Raj 1987). With the
Plant Breeding 123, 573—576 (2004)
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development of insecticide resistance to the major chemical
groups, including synthetic pyrethroids, the focus has shifted
to exploit the genetic diversity of wild Arachis species for
resistance to S. litura has been initiated as an important aspect
of integrated pest management (Wightman and Amin 1988).
The objective of this work was to cross the wild species
A. kempff-mercadoi, which has multiple disease resistance,
with A. hypogaea and to transfer disease resistance, and thus
broaden the genetic base of the crop.
Materials and Methods
Arachis kempff-mercadoi (accession ICG 8959; Coll no. 30085; PI
468331) a wild species from the section Arachis, was obtained from the
Genetic Resources Unit, ICRISAT, India and an A. hypogaea cultivar
ICGS 44, a Spanish type cultivar released from ICRISAT, were
maintained in a glasshouse. Pollinations were carried out in the
morning before 10.00 AM, followed by gibberellic acid (GA; 87.5 mg/l)
application to the base of pollinated pistils. Pods were harvested
30–40 days after pollination, or were left on the plant to mature
(50–60 days). For embryo germination, pods were thoroughly washed
in tap water and sterilized in commercial bleach (Clorox). After
repeated washes in sterilized distilled water, the embryos were dissected
out of the seeds and cultured on MS (Murashige and Skoog’s) basal
medium with sucrose (2%), agar (0.7%), NAA (0.1 mg/l) and BAP
(1.0 mg/l). Cultures were incubated in the dark at 26?C for the first
7 days. They were later incubated with a 16-h light and an 8-h dark
photoperiod at 26?C. After 4 weeks of culture, seedlings with well-
developed shoot systems and long taproots were transferred to the
rooting medium to induce secondary roots. The rooting medium
consisted of 1/10 MS basal medium with NAA (2.0 mg/l) and IBA
(1.0 mg/l). Seedlings with robust root systems were transferred to soil.
The F1hybrid plants were triploids but had some fertile pollen and
were selfed. The F2 plants were used as the female parents and
pollinated with A. hypogaea pollen. BC2plants were scored for ELS,
LLS and S. litura.
The ploidy of the derivatives was determined by pollen diameter
analysis. Three classes of pollen diameter were observed. Diploids had
a diameter of 25–27 lM, triploid sterile pollen grains were 25–29 lM,
and tetraploids had a diameter of 45–47 lM (Singsit and Ozias-Akins
1992). The pollen diameter of A. hypogaea was between 45 and 47 lM
and that of A. kempff-mercadoi was between 25 and 29 lM. Triploid
fertile (2n restitution) grains were 43–45 lM, which was comparable
with the pollen grains of tetraploid plants.
Disease screening for ELS and LLS was carried out under simulated
conditions by a detached leaf technique (Pande and Narayana Rao
2001). Plastic trays with autoclaved sand were used to place the tetra-
foliate leaves in a randomized block design with four replications. The
spores for the two fungi were harvested with a cyclone spore collector.
The concentration of the inoculum was 20 000 spores/ml. A few drops
of surfactant Tween 80 (polyoxyethylene sorbitan mono-oleate) were
added. Immediately after inoculation, leaves were placed in a dew
chamber at 23?C to ensure wetness of the leaf surface during the night.
Plants were removed from the dew chamber the next morning and
returned to the glasshouse during the day. The alternating wet and dry
period treatments were repeated for 5 days. Plants were then held in
the glasshouse until the end of the experiment. The experiment was
terminated at the end of 50 days following inoculation. The percentage
of defoliation was recorded for ELS and LLS. The leaf area damaged
by ELS and LLS was assessed by comparing each leaf with standard
diagrams depicting leaves with known percentages of leaf area affected.
Disease assessment was scored on a rating scale of 1–100, where a
score of 1–10 was rated as highly resistant (HR), 10–20 as resistant (R),
20–50 as moderately resistant (MR) and 50–100 as susceptible (S).
Data were collected at 10, 20 and 30 days after inoculation.
Spodoptera litura egg masses were collected on a groundnut crop
grown in the experimental block at the ICRISAT research farm. In the
laboratory, on hatching, the neonate larvae were reared on a semi-
synthetic diet based on chickpea flour and dried sorghum leaves
(G. V. Ranga Rao, personal communication, 1999) at 25 ± 2?C and a
14-h light and 10-h dark regime. Three to four pairs of adult moths
were released in a cylindrical cage and provided with 10% sucrose
solution. The hatched neonate larvae from the egg masses collected
from the cages were used. The experiment was replicated with leaflets
on each interspecific derivative, which were observed for larval survival
The basal first, third and seventh leaflets of the interspecific
derivatives were excised and arranged in a circle in a round plastic
pot (4¢¢ diameter), which was kept moist. Ten neonate larvae of
S. litura were released inside the pot, and sealed with a transparent
polythene cover to test their survival on each leaflet. The experimental
pots were then transferred to an incubator maintained at 25 ± 2?C, a
14-h light and 10-h dark regime, and with 70% RH. The experiment
was replicated with three leaflets per pot of each interspecific
derivative. Leaflets were observed for recording survival and develop-
ment of larvae on each species at 24, 48, 72 and 96 h after release.
Two F1hybrid plants were obtained by germination of mature
seeds in vivo and 11 plants were obtained by germinating well-
developed but immature embryos in vitro. Hybrid plants from
in vitro embryo germination and mature seeds from in vivo seed
germination did not show any morphological difference.
Pollen fertility in the F1 plants ranged from 0 to 5%. F1
plants were chosen as the female parent and A. hypogaea as the
pollen donor. In spite of hundreds of pollinations, pod
formation was below 10% and pod set was not observed in
the reciprocal crosses. Twenty two per cent of the pollinations
formed pods, of which 2% of the seeds were mature and the
rest were shrivelled seeds. Mature seeds were germinated
in vivo and BC1F2plants were obtained. The BC1F2plants had
5–7% pollen fertility; again, these hybrid plants were used as
the female parents and crossed with A. hypogaea. A large
number of pollinations induced pegs and many of these pegs
later had set pods. Seed set on BC2F2plants was mature and
was tetraploids, which was confirmed by pollen diameter
analysis of the BC2F2plants.
A total of 105 BC2F2plants were scored for foliar diseases.
The results for ELS screening showed that 33% of the plants
had resistant reactions to the disease, judging by the leaf tests.
There was a direct relationship between defoliation and disease
resistance. All the leaf samples that had 0–10% defoliation
were highly resistant. Those samples with <20% defoliation
were in the resistant category. The two categories made up
approximately 22% of the derivatives screened. On many of
the resistant leaf samples small spots less than 1.00 mm were
observed, which did not enlarge to form regular fungal
Twelve per cent of the BC2F2 plants showed resistant
reaction to LLS. Five plants did not have any defoliation of
the leaflets or disease spots on them. These were highly
resistant to LLS. More than half of the derivatives showed
50% or more defoliation with moderately resistant to suscept-
ible reactions to the disease. It was observed that 10% leaf area
damage could cause 50–100% defoliation. Here the latent time
of leaf area damage was important.
Among 105 plants scored for S. litura damage, 65% showed
less than 45% neonate mortality. About 30% of derivatives
showed moderate to high mortality >50–69%, with less than
5% of the plants having more than 75% mortality, showing
574MALLIKARJUNA, PANDE, JADHAV, SASTRI and RAO
high levels of antibiosis. A very small percentage of plants
showed 100% pest mortality, thus showing highly resistant
reactions. The neonate larvae had taken a bite of the leaf
nearer to the chewed portion on the adaxial surface of the leaf
and regurgitated smears on to the excised leaves. The mortality
of larvae on the first, third and seventh leaflets was 25, 31 and
45%, respectively, indicating that feeding on the third leaf
conferred a high level of resistance. Even when some of the
larvae survived on the leaves, either they did not pupate
normally or only abnormal adults were observed.
Among 105 BC2F2plants, one showed highly resistant (zero
defoliation with no disease spots) reaction to ELS and LLS.
Two derivatives showed a highly resistant reaction to ELS but
a moderately resistant reaction to LLS. Three derivatives
showed a highly resistant reaction to LLS and a moderately
resistant reaction to ELS. About 29% of the derivatives
showed different levels of resistance to ELS and LLS. Five
derivatives showed resistance to ELS, LLS and Spodoptera, of
which one derivative was highly resistant to ELS and LLS and
moderately resistant to Spodoptera.
Tremendous progress has been made to improve groundnut as
a crop (Holbrook and Stalker 2002). It is well known that
groundnut as a crop rests on a narrow genetic base. Isozyme,
molecular and pedigree analysis has shown limited diversity in
the cultivated species (Grieshammer 1989, Knauft and Gorbet
1989, Kochert et al. 1991). One of the best ways to introduce
resistance to biotic constraints (such as foliar diseases and
insects) and to introduce genetic variation is by using wild
species from the compatible gene pool.
In the present investigation seed set was observed when
triploid interspecific hybrids were used as the female parents
and crossed with the tetraploid A. hypogaea. Triploids
obtained as a result of crossing diploid wild species with
cultivated A. hypogaea have been previously found to be
partially fertile (Singh and Moss 1984). The F1plants were
able to set seeds when used as the female parent but failed to
produce seeds when used as the pollen parent. This meant that
the discrepancy in the number of chromosomes or loss of a few
segments of a chromosome in the egg was tolerated and limited
seed set was observed. Although most of the pollen grains were
sterile, female fertility was not greatly affected. This was
evidenced by formation of 99 seeds as a result of 452
pollinations when F1triploids were used as female parents.
But only nine of these seeds were filled and well developed.
In earlier investigations, a triploid was treated with colch-
icine to double the chromosome number (Singh 1986). Hexa-
ploids so obtained were backcrossed to the tetraploid
groundnut to obtain mixoploids and, later, tetraploids. This
process of obtaining tetraploid derivatives took over 3 years.
At every generation, screening for target diseases had to be
carried out. This route was time-, labour- and resource-
consuming. In the present investigation, tetraploidy was
achieved in one step and the time taken was reduced by
Instead of the conventional method of ploidy determination
by chromosome counts, pollen diameter is a good indicator of
the ploidy of the hybrid. The presence of 2n gametes obtained
as a result of restitution nuclei is common in triploids obtained
in crosses involving diploid wild species and the tetraploid
cultivated groundnut (Singh and Moss 1984, Singsit and
Crosses with A. kempf-mercadoi yielded derivatives resistant
to ELS and LLS. Many of these derivatives also showed
resistant reactions to rust under simulated conditions. Resist-
ance to Spodopterawas successfully
A. kempff-mercadoi into the derivatives. Many of the deriva-
tives showed either resistance to ELS or LLS. Combined
resistance to both diseases was observed in 30 derivatives with
five derivatives showing disease resistance to all three con-
The mortality of neonate larvae of S. litura on hybrids
indicates high levels of resistance (Lynch et al. 1981, Stevenson
et al. 1993). Some neonates were also found dead on the stems
of the excised leaves due to the hairiness of the plant. The
larval mortality on the leaf surface suggests the involvement of
chemicals, and poor larval growth on some excised leaf
material also contributed to resistance against S. litura larvae.
Stevenson et al. (1993) noticed high mortality of neonate
larvae and their retarded development on excised leaves of wild
species of Arachis: results similar to those obtained in the
present investigation. Similarly, several species of wild Arachis
had incomplete larval development and a very high larval
mortality of S. frugiperda (Lynch et al. 1981). Apart from leaf
chemistry, the toughness of leaves in the derivatives may have
played a role in conferring resistance.
It is evident that the ELS-, LLS- and Spodoptera-resistant
genes in A. kempf-mercadoi are not linked and probably their
loci are in different regions or on different chromosomes.
Hence, to bring multiple disease resistance genes into deriv-
atives, resistant derivatives may have to be crossed, thus
pyramiding the resistance genes can be achieved. The present
investigation opens up vistas in groundnut improvement by
accessing resistance genes to foliar diseases and Spodoptera
from A. kempff-mercadoi and transfer to cultivated groundnut.
The results show that wild species from the section Arachis are
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